Julien Sage

All Publications

• GCN2 is a determinant of the response to WEE1 kinase inhibition in small-cell lung cancer. Cell reports Drainas, A. P., Hsu, W. H., Dallas, A. E., Poltorack, C. D., Kim, J. W., He, A., Coles, G. L., Baron, M., Bassik, M. C., Sage, J. 2024; 43 (8): 114606

  Abstract

  Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a dependency on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M checkpoint kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here, using CRISPR-Cas9 knockout screens in vitro and in vivo, we identified multiple factors influencing the response of SCLC cells to the WEE1 kinase inhibitor AZD1775, including the GCN2 kinase and other members of its signaling pathway. Rapid activation of GCN2 upon AZD1775 treatment triggers a stress response in SCLC cells. Pharmacological or genetic activation of the GCN2 pathway enhances cancer cell killing by AZD1775. Thus, activation of the GCN2 pathway represents a promising strategy to increase the efficacy of WEE1 inhibitors in SCLC.

  View details for DOI 10.1016/j.celrep.2024.114606

  View details for PubMedID 39120974

• Small-cell lung cancer neuronal features and their implications for tumor progression, metastasis, and therapy. Molecular cancer research : MCR Hartmann, G. G., Sage, J. 2024

  Abstract

  Small-cell lung cancer (SCLC) is an epithelial neuroendocrine form of lung cancer for which survival rates remain dismal and new therapeutic approaches are greatly needed. Key biological features of SCLC tumors include fast growth and widespread metastasis, as well as rapid resistance to treatment. Similar to pulmonary neuroendocrine cells, SCLC cells have traits of both hormone-producing cells and neurons. Here we specifically discuss the neuronal features of SCLC. We consider how neuronal G-protein-coupled receptors (GPCRs) and other neuronal molecules on the surface of SCLC cells can contribute to the growth of SCLC tumors and serve as therapeutic targets in SCLC. We also review recent evidence for the role of neuronal programs expressed by SCLC cells in the fast proliferation, migration, and metastasis of these cells. We further highlight how these neuronal programs may be particularly relevant for the development of brain metastases, and how they can assist SCLC cells to functionally interact with neurons and astrocytes. A greater understanding of the molecular and cellular neuronal features of SCLC is likely to uncover new vulnerabilities in SCLC cells, which may help develop novel therapeutic approaches. More generally, the epithelial-to-neuronal transition (ENT) observed during tumor progression in SCLC and other cancer types can contribute significantly to tumor development and response to therapy.

  View details for DOI 10.1158/1541-7786.MCR-24-0265

  View details for PubMedID 38912893

• Genetically-engineered mouse models of small cell lung cancer: the next generation. Oncogene Oser, M. G., MacPherson, D., Oliver, T. G., Sage, J., Park, K. S. 2024

  Abstract

  Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.

  View details for DOI 10.1038/s41388-023-02929-7

  View details for PubMedID 38191672

  View details for PubMedCentralID 3715060

• Small cell lung cancer plasticity enables NFIB-independent metastasis. Cancer research Ko, J. H., Lambert, K. E., Bhattacharya, D., Lee, M. C., Colon, C. I., Hauser, H., Sage, J. 2023

  Abstract

  Metastasis is a major cause of morbidity and mortality in patients with cancer, highlighting the need to identify improved treatment and prevention strategies. Previous observations in pre-clinical models and tumors from patients with small cell lung cancer (SCLC), a fatal form of lung cancer with high metastatic potential, identified the transcription factor NFIB as a driver of tumor growth and metastasis. However, investigation into the requirement for NFIB activity for tumor growth and metastasis in relevant in vivo models is needed to establish NFIB as a therapeutic target. Here, using conditional gene knockout strategies in genetically engineered mouse models of SCLC, we found that upregulation of NFIB contributes to tumor progression, but NFIB is not required for metastasis. Molecular studies in NFIB wild-type and knockout tumors identified the pioneer transcription factors FOXA1/2 as candidate drivers of metastatic progression. Thus, while NFIB upregulation is a frequent event in SCLC during tumor progression, SCLC tumors can employ NFIB-independent mechanisms for metastasis, further highlighting the plasticity of these tumors.

  View details for DOI 10.1158/0008-5472.CAN-23-1079

  View details for PubMedID 37963187

• Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest. Cell chemical biology Rodencal, J., Kim, N., He, A., Li, V. L., Lange, M., He, J., Tarangelo, A., Schafer, Z. T., Olzmann, J. A., Long, J. Z., Sage, J., Dixon, S. J. 2023

  Abstract

  Ferroptosis is a non-apoptotic form of cell death that can be triggered by inhibiting the system xc- cystine/glutamate antiporter or the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). We have investigated how cell cycle arrest caused by stabilization of p53 or inhibition of cyclin-dependent kinase 4/6 (CDK4/6) impacts ferroptosis sensitivity. Here, we show that cell cycle arrest can enhance sensitivity to ferroptosis induced by covalent GPX4 inhibitors (GPX4i) but not system xc- inhibitors. Greater sensitivity to GPX4i is associated with increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids (PUFA-PLs). Higher PUFA-PL abundance upon cell cycle arrest involves reduced expression of membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) and epithelial membrane protein 2 (EMP2). A candidate orally bioavailable GPX4 inhibitor increases lipid peroxidation and shrinks tumor volumes when combined with a CDK4/6 inhibitor. Thus, cell cycle arrest may make certain cancer cells more susceptible to ferroptosis in vivo.

  View details for DOI 10.1016/j.chembiol.2023.10.011

  View details for PubMedID 37963466

• Crosstalk between small-cell lung cancer cells and astrocytes mimics brain development to promote brain metastasis. Nature cell biology Qu, F., Brough, S. C., Michno, W., Madubata, C. J., Hartmann, G. G., Puno, A., Drainas, A. P., Bhattacharya, D., Tomasich, E., Lee, M. C., Yang, D., Kim, J., Peiris-Pagès, M., Simpson, K. L., Dive, C., Preusser, M., Toland, A., Kong, C., Das, M., Winslow, M. M., Pasca, A. M., Sage, J. 2023

  Abstract

  Brain metastases represent an important clinical problem for patients with small-cell lung cancer (SCLC). However, the mechanisms underlying SCLC growth in the brain remain poorly understood. Here, using intracranial injections in mice and assembloids between SCLC aggregates and human cortical organoids in culture, we found that SCLC cells recruit reactive astrocytes to the tumour microenvironment. This crosstalk between SCLC cells and astrocytes drives the induction of gene expression programmes that are similar to those found during early brain development in neurons and astrocytes. Mechanistically, the brain development factor Reelin, secreted by SCLC cells, recruits astrocytes to brain metastases. These astrocytes in turn promote SCLC growth by secreting neuronal pro-survival factors such as SERPINE1. Thus, SCLC brain metastases grow by co-opting mechanisms involved in reciprocal neuron-astrocyte interactions during brain development. Targeting such developmental programmes activated in this cancer ecosystem may help prevent and treat brain metastases.

  View details for DOI 10.1038/s41556-023-01241-6

  View details for PubMedID 37783795

  View details for PubMedCentralID 6602095

• An engineered interleukin-1 decoy cytokine inhibits receptor signaling and proliferation in lung adenocarcinoma BIOENGINEERING & TRANSLATIONAL MEDICINE McIntosh, B. J., Hartmann, G. G., Yamada-Hunter, S. A., Liu, P., Williams, C. F., Sage, J., Cochran, J. R. 2023

  View details for DOI 10.1002/btm2.10573

  View details for Web of Science ID 001031916000001

• Monitoring the Cell Cycle of Tumor Cells in Mouse Models of Human Cancer. Cold Spring Harbor perspectives in medicine Hammond, T., Sage, J. 2023

  Abstract

  Cell division is obligatory to tumor growth. However, both cancer cells and noncancer cells in tumors can be found in distinct stages of the cell cycle, which may inform the growth potential of these tumors, their propensity to metastasize, and their response to therapy. Hence, it is of utmost importance to monitor the cell cycle of tumor cells. Here we discuss well-established methods and new genetic advances to track the cell cycle of tumor cells in mouse models of human cancer. We also review recent genetic studies investigating the role of the cell-cycle machinery in the growth of tumors in vivo, with a focus on the machinery regulating the G1/S transition of the cell cycle.

  View details for DOI 10.1101/cshperspect.a041383

  View details for PubMedID 37460156

• Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer Pearsall, S. M., Williamson, S. C., Humphrey, S., Hughes, E., Morgan, D., García Marqués, F. J., Awanis, G., Carroll, R., Burks, L., Shue, Y. T., Bermudez, A., Frese, K. K., Galvin, M., Carter, M., Priest, L., Kerr, A., Zhou, C., Oliver, T. G., Humphries, J. D., Humphries, M. J., Blackhall, F., Cannell, I. G., Pitteri, S. J., Hannon, G. J., Sage, J., Dive, C., Simpson, K. L. 2023

  Abstract

  Vasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.We analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs and patient biopsies. VM-proficient cell subpopulations in ex vivo cultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions.We show that VM vessels are present in 23/25 CDX models, 2 GEMMs and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin β1-dependent process.We identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in the future.

  View details for DOI 10.1016/j.jtho.2023.07.012

  View details for PubMedID 37455012

• Unified tumor growth mechanisms from multimodel inference and dataset integration. PLoS computational biology Beik, S. P., Harris, L. A., Kochen, M. A., Sage, J., Quaranta, V., Lopez, C. F. 2023; 19 (7): e1011215

  Abstract

  Mechanistic models of biological processes can explain observed phenomena and predict responses to a perturbation. A mathematical model is typically constructed using expert knowledge and informal reasoning to generate a mechanistic explanation for a given observation. Although this approach works well for simple systems with abundant data and well-established principles, quantitative biology is often faced with a dearth of both data and knowledge about a process, thus making it challenging to identify and validate all possible mechanistic hypothesis underlying a system behavior. To overcome these limitations, we introduce a Bayesian multimodel inference (Bayes-MMI) methodology, which quantifies how mechanistic hypotheses can explain a given experimental datasets, and concurrently, how each dataset informs a given model hypothesis, thus enabling hypothesis space exploration in the context of available data. We demonstrate this approach to probe standing questions about heterogeneity, lineage plasticity, and cell-cell interactions in tumor growth mechanisms of small cell lung cancer (SCLC). We integrate three datasets that each formulated different explanations for tumor growth mechanisms in SCLC, apply Bayes-MMI and find that the data supports model predictions for tumor evolution promoted by high lineage plasticity, rather than through expanding rare stem-like populations. In addition, the models predict that in the presence of cells associated with the SCLC-N or SCLC-A2 subtypes, the transition from the SCLC-A subtype to the SCLC-Y subtype through an intermediate is decelerated. Together, these predictions provide a testable hypothesis for observed juxtaposed results in SCLC growth and a mechanistic interpretation for tumor treatment resistance.

  View details for DOI 10.1371/journal.pcbi.1011215

  View details for PubMedID 37406008

• Taking it up a notch: a promising immunotherapy against small cell lung cancer. Translational lung cancer research Apaydin, A. A., Sage, J. 2023; 12 (5): 948-952

  View details for DOI 10.21037/tlcr-23-230

  View details for PubMedID 37323167

  View details for PubMedCentralID PMC10261866

• Integrative analysis of a large real-world cohort of small cell lung cancer identifies distinct genetic subtypes and insights into histological transformation. Cancer discovery Sivakumar, S., Moore, J. A., Montesion, M., Sharaf, R., Lin, D. I., Colon, C. I., Fleischmann, Z., Ebot, E. M., Newberg, J. Y., Mills, J. M., Hegde, P. S., Pan, Q., Dowlati, A., Frampton, G. M., Sage, J., Lovly, C. M. 2023

  Abstract

  Small cell lung cancer (SCLC) is a recalcitrant neuroendocrine carcinoma with dismal survival outcomes. A major barrier in the field has been the relative paucity of human tumors studied. Here we provide an integrated analysis of 3,600 "real-world" SCLC cases. This large cohort allowed us to identify new recurrent alterations and genetic subtypes, including STK11-mutant tumors (1.7%) and TP53/RB1 wild-type tumors (5.5%), as well as rare cases that were human papillomavirus-positive. In our cohort, gene amplifications on 4q12 are associated with increased overall survival while CCNE1 amplification is associated with decreased overall survival. We also identify more frequent alterations in the PTEN pathway in brain metastases. Finally, profiling cases of SCLC containing oncogenic drivers typically associated with NSCLC demonstrates that SCLC transformation may occur across multiple distinct molecular cohorts of NSCLC. These novel and unsuspected genetic features of SCLC may help personalize treatment approaches for this fatal form of cancer.

  View details for DOI 10.1158/2159-8290.CD-22-0620

  View details for PubMedID 37062002

• A multiplexed in vivo approach to identify driver genes in small cell lung cancer. Cell reports Lee, M. C., Cai, H., Murray, C. W., Li, C., Shue, Y. T., Andrejka, L., He, A. L., Holzem, A. M., Drainas, A. P., Ko, J. H., Coles, G. L., Kong, C., Zhu, S., Zhu, C., Wang, J., van de Rijn, M., Petrov, D. A., Winslow, M. M., Sage, J. 2023; 42 (1): 111990

  Abstract

  Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

  View details for DOI 10.1016/j.celrep.2023.111990

  View details for PubMedID 36640300

• DLL3 regulates Notch signaling in small cell lung cancer. iScience Kim, J. W., Ko, J. H., Sage, J. 2022; 25 (12): 105603

  Abstract

  Tumor heterogeneity plays a critical role in tumor development and response to treatment. In small-cell lung cancer (SCLC), intratumoral heterogeneity is driven in part by the Notch signaling pathway, which reprograms neuroendocrine cancer cells to a less/non-neuroendocrine state. Here we investigated the atypical Notch ligand DLL3 as a biomarker of the neuroendocrine state and a regulator of cell-cell interactions in SCLC. We first built a mathematical model to predict the impact of DLL3 expression on SCLC cell populations. We next tested this model using a single-chain variable fragment (scFv) to track DLL3 expression in vivo and a new mouse model of SCLC with inducible expression of DLL3 in SCLC tumors. We found that high levels of DLL3 promote the expansion of a SCLC cell population with lower expression levels of both neuroendocrine and non-neuroendocrine markers. This work may influence how DLL3-targeting therapies are used in SCLC patients.

  View details for DOI 10.1016/j.isci.2022.105603

  View details for PubMedID 36483011

  View details for PubMedCentralID PMC9722452

• Radiotherapy in combination with CD47 blockade elicits a macrophage-mediated abscopal effect. Nature cancer Nishiga, Y., Drainas, A. P., Baron, M., Bhattacharya, D., Barkal, A. A., Ahrari, Y., Mancusi, R., Ross, J. B., Takahashi, N., Thomas, A., Diehn, M., Weissman, I. L., Graves, E. E., Sage, J. 2022

  Abstract

  Radiation therapy is a mainstay of cancer treatment but does not always lead to complete tumor regression. Here we combine radiotherapy with blockade of the 'don't-eat-me' cell-surface molecule CD47 in small cell lung cancer (SCLC), a highly metastatic form of lung cancer. CD47 blockade potently enhances the local antitumor effects of radiotherapy in preclinical models of SCLC. Notably, CD47 blockade also stimulates off-target 'abscopal' effects inhibiting non-irradiated SCLC tumors in mice receiving radiation. These abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by radiation and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with radiation and CD47 blockade. The systemic activation of antitumor macrophages following radiotherapy and CD47 blockade may be particularly important in patients with cancer who suffer from metastatic disease.

  View details for DOI 10.1038/s43018-022-00456-0

  View details for PubMedID 36411318

• Spatial epitope barcoding reveals clonal tumor patch behaviors. Cancer cell Rovira-Clave, X., Drainas, A. P., Jiang, S., Bai, Y., Baron, M., Zhu, B., Dallas, A. E., Lee, M. C., Chu, T. P., Holzem, A., Ayyagari, R., Bhattacharya, D., McCaffrey, E. F., Greenwald, N. F., Markovic, M., Coles, G. L., Angelo, M., Bassik, M. C., Sage, J., Nolan, G. P. 2022

  Abstract

  Intratumoral heterogeneity is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are still largely unsuitable to accurately track phenotypes and clonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitopes for imaging using combinatorial tagging (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using EpicMIBI, we dissected the spatial component of cell lineages and phenotypes in xenograft models of small cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of clonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in these models. In a tumor model harboring a fraction of PTEN-deficient cancer cells, we observed a non-autonomous increase of clonal patch size in PTEN wild-type cancer cells. EpicMIBI facilitates in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.

  View details for DOI 10.1016/j.ccell.2022.09.014

  View details for PubMedID 36240778

• Archetype tasks link intratumoral heterogeneity to plasticity and cancer hallmarks in small cell lung cancer. Cell systems Groves, S. M., Ildefonso, G. V., McAtee, C. O., Ozawa, P. M., Ireland, A. S., Stauffer, P. E., Wasdin, P. T., Huang, X., Qiao, Y., Lim, J. S., Bader, J., Liu, Q., Simmons, A. J., Lau, K. S., Iams, W. T., Hardin, D. P., Saff, E. B., Holmes, W. R., Tyson, D. R., Lovly, C. M., Rathmell, J. C., Marth, G., Sage, J., Oliver, T. G., Weaver, A. M., Quaranta, V. 2022

  Abstract

  Small cell lung cancer (SCLC) tumors comprise heterogeneous mixtures of cell states, categorized into neuroendocrine (NE) and non-neuroendocrine (non-NE) transcriptional subtypes. NE to non-NE state transitions, fueled by plasticity, likely underlie adaptability to treatment and dismal survival rates. Here, we apply an archetypal analysis to model plasticity by recasting SCLC phenotypic heterogeneity through multi-task evolutionary theory. Cell line and tumor transcriptomics data fit well in a five-dimensional convex polytope whose vertices optimize tasks reminiscent of pulmonary NE cells, the SCLC normal counterparts. These tasks, supported by knowledge and experimental data, include proliferation, slithering, metabolism, secretion, and injury repair, reflecting cancer hallmarks. SCLC subtypes, either at the population or single-cell level, can be positioned in archetypal space by bulk or single-cell transcriptomics, respectively, and characterized as task specialists or multi-task generalists by the distance from archetype vertex signatures. In the archetype space, modeling single-cell plasticity as a Markovian process along an underlying state manifold indicates that task trade-offs, in response to microenvironmental perturbations or treatment, may drive cell plasticity. Stifling phenotypic transitions and plasticity may provide new targets for much-needed translational advances in SCLC. A record of this paper's Transparent Peer Review process is included in the supplemental information.

  View details for DOI 10.1016/j.cels.2022.07.006

  View details for PubMedID 35981544

• OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage. Nature Wu, X. S., He, X. Y., Ipsaro, J. J., Huang, Y. H., Preall, J. B., Ng, D., Shue, Y. T., Sage, J., Egeblad, M., Joshua-Tor, L., Vakoc, C. R. 2022

  Abstract

  Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft cell-like human tumors2,3, particularly as a variant form of small cell lung cancer (SCLC). Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F32,4, although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1 and OCA-T2, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogs of the B cell-specific coactivator OCA-B, which are encoded in a gene cluster and harbor a conserved peptide that binds to class II POU transcription factors and octamer motif DNA in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft cell-like SCLC. In addition, we generated OCA-T1 knockout mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal the POU2F3-OCA-T complex as the master regulator of tuft cell identity and a prominent molecular vulnerability of tuft cell-like SCLC.

  View details for DOI 10.1038/s41586-022-04842-7

  View details for PubMedID 35576971

• A conserved YAP/Notch/REST network controls the neuroendocrine cell fate in the lungs. Nature communications Shue, Y. T., Drainas, A. P., Li, N. Y., Pearsall, S. M., Morgan, D., Sinnott-Armstrong, N., Hipkins, S. Q., Coles, G. L., Lim, J. S., Oro, A. E., Simpson, K. L., Dive, C., Sage, J. 2022; 13 (1): 2690

  Abstract

  The Notch pathway is a conserved cell-cell communication pathway that controls cell fate decisions. Here we sought to determine how Notch pathway activation inhibits the neuroendocrine cell fate in the lungs, an archetypal process for cell fate decisions orchestrated by Notch signaling that has remained poorly understood at the molecular level. Using intratumoral heterogeneity in small-cell lung cancer as a tractable model system, we uncovered a role for the transcriptional regulators REST and YAP as promoters of the neuroendocrine to non-neuroendocrine transition. We further identified the specific neuroendocrine gene programs repressed by REST downstream of Notch in this process. Importantly, we validated the importance of REST and YAP in neuroendocrine to non-neuroendocrine cell fate switches in both developmental and tissue repair processes in the lungs. Altogether, these experiments identify conserved roles for REST and YAP in Notch-driven inhibition of the neuroendocrine cell fate in embryonic lungs, adult lungs, and lung cancer.

  View details for DOI 10.1038/s41467-022-30416-2

  View details for PubMedID 35577801

• Succinate Accumulation Is Not Sufficient for Tumorigenesis in Mouse Chromaffin Cells But Dual Loss of SDHB and NF1 Yields SDHx-Like Pheochromocytomas Armstrong, N., Storey, C. M., Noll, S. E., Margulis, K., Soe, M. H., Xu, H., Yeh, B., Fishbein, L., Kebebew, E., Howitt, B. E., Zare, R. N., Sage, J., Annes, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2022: E32-E33

  View details for Web of Science ID 000819123700022

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• The cell cycle inhibitor RB is diluted in G1 and contributes to controlling cell size in the mouse liver. Frontiers in cell and developmental biology Zhang, S., Zatulovskiy, E., Arand, J., Sage, J., Skotheim, J. M. 2022; 10: 965595

  Abstract

  Every type of cell in an animal maintains a specific size, which likely contributes to its ability to perform its physiological functions. While some cell size control mechanisms are beginning to be elucidated through studies of cultured cells, it is unclear if and how such mechanisms control cell size in an animal. For example, it was recently shown that RB, the retinoblastoma protein, was diluted by cell growth in G1 to promote size-dependence of the G1/S transition. However, it remains unclear to what extent the RB-dilution mechanism controls cell size in an animal. We therefore examined the contribution of RB-dilution to cell size control in the mouse liver. Consistent with the RB-dilution model, genetic perturbations decreasing RB protein concentrations through inducible shRNA expression or through liver-specific Rb1 knockout reduced hepatocyte size, while perturbations increasing RB protein concentrations in an Fah -/- mouse model increased hepatocyte size. Moreover, RB concentration reflects cell size in G1 as it is lower in larger G1 hepatocytes. In contrast, concentrations of the cell cycle activators Cyclin D1 and E2f1 were relatively constant. Lastly, loss of Rb1 weakened cell size control, i.e., reduced the inverse correlation between how much cells grew in G1 and how large they were at birth. Taken together, our results show that an RB-dilution mechanism contributes to cell size control in the mouse liver by linking cell growth to the G1/S transition.

  View details for DOI 10.3389/fcell.2022.965595

  View details for PubMedID 36092730

• RB depletion is required for the continuous growth of tumors initiated by loss of RB. PLoS genetics Doan, A., Arand, J., Gong, D., Drainas, A. P., Shue, Y. T., Lee, M. C., Zhang, S., Walter, D. M., Chaikovsky, A. C., Feldser, D. M., Vogel, H., Dow, L. E., Skotheim, J. M., Sage, J. 2021; 17 (12): e1009941

  Abstract

  The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

  View details for DOI 10.1371/journal.pgen.1009941

  View details for PubMedID 34879057

• Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature Kamber, R. A., Nishiga, Y., Morton, B., Banuelos, A. M., Barkal, A. A., Vences-Catalan, F., Gu, M., Fernandez, D., Seoane, J. A., Yao, D., Liu, K., Lin, S., Spees, K., Curtis, C., Jerby-Arnon, L., Weissman, I. L., Sage, J., Bassik, M. C. 2021

  Abstract

  Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

  View details for DOI 10.1038/s41586-021-03879-4

  View details for PubMedID 34497417

• The AMBRA1 E3 ligase adaptor regulates the stability of cyclinD. Nature Chaikovsky, A. C., Li, C., Jeng, E. E., Loebell, S., Lee, M. C., Murray, C. W., Cheng, R., Demeter, J., Swaney, D. L., Chen, S., Newton, B. W., Johnson, J. R., Drainas, A. P., Shue, Y. T., Seoane, J. A., Srinivasan, P., He, A., Yoshida, A., Hipkins, S. Q., McCrea, E., Poltorack, C. D., Krogan, N. J., Diehl, J. A., Kong, C., Jackson, P. K., Curtis, C., Petrov, D. A., Bassik, M. C., Winslow, M. M., Sage, J. 2021

  Abstract

  The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclinD) couple these inputs to the initiation of DNA replication1. Increased levels of cyclinD promote cell division by activating cyclin-dependent kinases4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclinD-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclinD are incompletely understood4,5. Here we show that autophagy and beclin1 regulator1 (AMBRA1) is the main regulator of the degradation of cyclinD. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclinD in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclinD as a substrate receptor for the cullin4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclinD, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

  View details for DOI 10.1038/s41586-021-03474-7

  View details for PubMedID 33854239

• Small-cell lung cancer. Nature reviews. Disease primers Rudin, C. M., Brambilla, E. n., Faivre-Finn, C. n., Sage, J. n. 2021; 7 (1): 3

  Abstract

  Small-cell lung cancer (SCLC) represents about 15% of all lung cancers and is marked by an exceptionally high proliferative rate, strong predilection for early metastasis and poor prognosis. SCLC is strongly associated with exposure to tobacco carcinogens. Most patients have metastatic disease at diagnosis, with only one-third having earlier-stage disease that is amenable to potentially curative multimodality therapy. Genomic profiling of SCLC reveals extensive chromosomal rearrangements and a high mutation burden, almost always including functional inactivation of the tumour suppressor genes TP53 and RB1. Analyses of both human SCLC and murine models have defined subtypes of disease based on the relative expression of dominant transcriptional regulators and have also revealed substantial intratumoural heterogeneity. Aspects of this heterogeneity have been implicated in tumour evolution, metastasis and acquired therapeutic resistance. Although clinical progress in SCLC treatment has been notoriously slow, a better understanding of the biology of disease has uncovered novel vulnerabilities that might be amenable to targeted therapeutic approaches. The recent introduction of immune checkpoint blockade into the treatment of patients with SCLC is offering new hope, with a small subset of patients deriving prolonged benefit. Strategies to direct targeted therapies to those patients who are most likely to respond and to extend the durable benefit of effective antitumour immunity to a greater fraction of patients are urgently needed and are now being actively explored.

  View details for DOI 10.1038/s41572-020-00235-0

  View details for PubMedID 33446664

• NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis. Molecular cell Sengupta, D., Zeng, L., Li, Y., Hausmann, S., Ghosh, D., Yuan, G., Nguyen, T. N., Lyu, R., Caporicci, M., Morales Benitez, A., Coles, G. L., Kharchenko, V., Czaban, I., Azhibek, D., Fischle, W., Jaremko, M., Wistuba, I. I., Sage, J., Jaremko, Ł., Li, W., Mazur, P. K., Gozani, O. 2021

  Abstract

  The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.

  View details for DOI 10.1016/j.molcel.2021.08.034

  View details for PubMedID 34555356

• A Call to Action: Dismantling Racial Injustices in Preclinical Research and Clinical Care of Black Patients Living with Small Cell Lung Cancer. Cancer discovery Thomas, P. L., Madubata, C. J., Aldrich, M. C., Lee, M. M., Owonikoko, T. K., Minna, J. D., Rudin, C. M., Sage, J., Lovly, C. M. 2020

  Abstract

  Small cell lung cancer (SCLC) is an aggressive disease with dismal survival rates and limited therapeutic options. SCLC development is strongly associated with exposure to tobacco carcinogens. However, additional genetic and environmental risk factors that contribute to SCLC pathogenesis are beginning to emerge. Here, we specifically assess disparities pertaining to SCLC in Black populations. In contrast to non-small cell lung cancer, preliminary data suggest that Black individuals may actually be at a lower risk of developing SCLC relative to white individuals. This difference remains unexplained but urgently needs to be verified in larger data sets, because it could provide important new insights and approaches to understanding this recalcitrant tumor. Importantly, little biological information exists on SCLC in Black individuals, and few patient-derived preclinical SCLC models from diverse ancestries are available in the laboratory. Unfortunately, we note strikingly low numbers of Black participants in clinical trials testing new treatments for SCLC. Evidence further indicates that care for patients with SCLC may vary between communities with a large fraction of Black patients and those without. Together, these observations underscore the need to better investigate genetic, environmental, and socioeconomic factors associated with SCLC development, preclinical research, clinical care, and outcomes.

  View details for DOI 10.1158/2159-8290.CD-20-1592

  View details for PubMedID 33318034

• Mechanisms of small cell lung cancer metastasis. EMBO molecular medicine Ko, J., Winslow, M. M., Sage, J. 2020: e13122

  Abstract

  Metastasis is a major cause of morbidity and mortality in cancer patients. However, the molecular and cellular mechanisms underlying the ability of cancer cells to metastasize remain relatively poorly understood. Among all solid tumors, small cell lung cancer (SCLC) has remarkable metastatic proclivity, with a majority of patients diagnosed with metastatic disease. Our understanding of SCLC metastasis has been hampered for many years by the paucity of material from primary tumors and metastases, as well as the lack of faithful pre-clinical models. Here, we review recent advances that are helping circumvent these limitations. These advances include methods that employ circulating tumor cells from the blood of SCLC patients and the development of diverse genetically engineered mouse models of metastatic SCLC. New insights into the cellular mechanisms of SCLC metastasis include observations of cell fate changes associated with increased metastatic ability. Ongoing studies on cell migration and organ tropism promise to expand our understanding of SCLC metastasis. Ultimately, a better molecular understanding of metastatic phenotypes may be translated into new therapeutic options to limit metastatic spread and treat metastatic SCLC.

  View details for DOI 10.15252/emmm.202013122

  View details for PubMedID 33296145

• Cells of origin of lung cancers: lessons from mouse studies. Genes & development Ferone, G., Lee, M. C., Sage, J., Berns, A. 2020; 34 (15-16): 1017–32

  Abstract

  As one of the most common forms of cancer, lung cancers present as a collection of different histological subtypes. These subtypes are characterized by distinct sets of driver mutations and phenotypic appearance, and they often show varying degrees of heterogenicity, aggressiveness, and response/resistance to therapy. Intriguingly, lung cancers are also capable of showing features of multiple subtypes or converting from one subtype to another. The intertumoral and intratumoral heterogeneity of lung cancers as well as incidences of subtype transdifferentiation raise the question of to what extent the tumor characteristics are dictated by the cell of origin rather than the acquired driver lesions. We provide here an overview of the studies in experimental mouse models that try to address this question. These studies convincingly show that both the cell of origin and the genetic driver lesions play a critical role in shaping the phenotypes of lung tumors. However, they also illustrate that there is far from a direct one-to-one relationship between the cell of origin and the cancer subtype, as most epithelial cells can be reprogrammed toward diverse lung cancer fates when exposed to the appropriate set of driver mutations.

  View details for DOI 10.1101/gad.338228.120

  View details for PubMedID 32747478

• Investigating Tumor Heterogeneity in Mouse Models. Annual review of cancer biology Tammela, T., Sage, J. 2020; 4 (1): 99-119

  Abstract

  Cancer arises from a single cell through a series of acquired mutations and epigenetic alterations. Tumors gradually develop into a complex tissue comprised of phenotypically heterogeneous cancer cell populations, as well as noncancer cells that make up the tumor microenvironment. The phenotype, or state, of each cancer and stromal cell is influenced by a plethora of cell-intrinsic and cell-extrinsic factors. The diversity of these cellular states promotes tumor progression, enables metastasis, and poses a challenge for effective cancer treatments. Thus, the identification of strategies for the therapeutic manipulation of tumor heterogeneity would have significant clinical implications. A major barrier in the field is the difficulty in functionally investigating heterogeneity in tumors in cancer patients. Here we review how mouse models of human cancer can be leveraged to interrogate tumor heterogeneity and to help design better therapeutic strategies.

  View details for DOI 10.1146/annurev-cancerbio-030419-033413

  View details for PubMedID 34164589

  View details for PubMedCentralID PMC8218894

• The MEK5-ERK5 kinase axis controls lipid metabolism in small cell lung cancer. Cancer research Cristea, S., Coles, G. L., Hornburg, D., Gershkovitz, M., Arand, J., Cao, S., Sen, T., Williamson, S. C., Kim, J. W., Drainas, A. P., He, A., Le Cam, L., Byers, L. A., Snyder, M. P., Contrepois, K., Sage, J. 2020

  Abstract

  Small cell lung cancer (SCLC) is an aggressive form of lung cancer with dismal survival rates. While kinases often play key roles driving tumorigenesis, there are strikingly few kinases known to promote the development of SCLC. Here we investigated the contribution of the MAP kinase module MEK5/ERK5 to SCLC growth. MEK5 and ERK5 were required for optimal survival and expansion of SCLC cell lines in vitro and in vivo. Transcriptomics analyses identified a role for the MEK5-ERK5 axis in the metabolism of SCLC cells, including lipid metabolism. In-depth lipidomics analyses showed that loss of MEK5/ERK5 perturbs several lipid metabolism pathways, including the mevalonate pathway that controls cholesterol synthesis. Notably, depletion of MEK5/ERK5 sensitized SCLC cells to pharmacological inhibition of the mevalonate pathway by statins. These data identify a new MEK5-ERK5-lipid metabolism axis that promotes the growth of SCLC.

  View details for DOI 10.1158/0008-5472.CAN-19-1027

  View details for PubMedID 31969375

• Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells. Cancer cell Coles, G. L., Cristea, S. n., Webber, J. T., Levin, R. S., Moss, S. M., He, A. n., Sangodkar, J. n., Hwang, Y. C., Arand, J. n., Drainas, A. P., Mooney, N. A., Demeter, J. n., Spradlin, J. N., Mauch, B. n., Le, V. n., Shue, Y. T., Ko, J. H., Lee, M. C., Kong, C. n., Nomura, D. K., Ohlmeyer, M. n., Swaney, D. L., Krogan, N. J., Jackson, P. K., Narla, G. n., Gordan, J. D., Shokat, K. M., Sage, J. n. 2020

  Abstract

  Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

  View details for DOI 10.1016/j.ccell.2020.05.003

  View details for PubMedID 32531271

• Integrating Old and New Paradigms of G1/S Control. Molecular cell Rubin, S. M., Sage, J. n., Skotheim, J. M. 2020

  Abstract

  The Cdk-Rb-E2F pathway integrates external and internal signals to control progression at the G1/S transition of the mammalian cell cycle. Alterations in this pathway are found in most human cancers, and specific cyclin-dependent kinase Cdk4/6 inhibitors are approved or in clinical trials for the treatment of diverse cancers. In the long-standing paradigm for G1/S control, Cdks inactivate the retinoblastoma tumor suppressor protein (Rb) through phosphorylation, which releases E2F transcription factors to drive cell-cycle progression from G1 to S. However, recent observations in the laboratory and clinic challenge central tenets of the current paradigm and demonstrate that our understanding of the Rb pathway and G1/S control is still incomplete. Here, we integrate these new findings with the previous paradigm to synthesize a current molecular and cellular view of the mammalian G1/S transition. A more complete and accurate understanding of G1/S control will lead to improved therapeutic strategies targeting the cell cycle in cancer.

  View details for DOI 10.1016/j.molcel.2020.08.020

  View details for PubMedID 32946743

• Investigating Tumor Heterogeneity in Mouse Models ANNUAL REVIEW OF CANCER BIOLOGY, VOL 4 Tammela, T., Sage, J., Jacks, T., Sawyers, C. L. 2020; 4: 99–119

  View details for DOI 10.1146/annurev-cancerbio-030419-033413

  View details for Web of Science ID 000518890900006

• Immune receptor inhibition through enforced phosphatase recruitment. Nature Fernandes, R. A., Su, L. n., Nishiga, Y. n., Ren, J. n., Bhuiyan, A. M., Cheng, N. n., Kuo, C. J., Picton, L. K., Ohtsuki, S. n., Majzner, R. G., Rietberg, S. P., Mackall, C. L., Yin, Q. n., Ali, L. R., Yang, X. n., Savvides, C. S., Sage, J. n., Dougan, M. n., Garcia, K. C. 2020

  Abstract

  Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

  View details for DOI 10.1038/s41586-020-2851-2

  View details for PubMedID 33087934

• Axon-like protrusions promote small cell lung cancer migration and metastasis. eLife Yang, D., Qu, F., Cai, H., Chuang, C., Lim, J. S., Jahchan, N., Gruner, B. M., S Kuo, C., Kong, C., Oudin, M. J., Winslow, M. M., Sage, J. 2019; 8

  Abstract

  Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC.

  View details for DOI 10.7554/eLife.50616

  View details for PubMedID 31833833

• Manipulating a tumour suppressor NATURE Rubin, S. M., Sage, J. 2019; 569 (7756): 343–44

  View details for Web of Science ID 000468123700027

  View details for PubMedID 31076732

• Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer CANCER DISCOVERY Sen, T., Rodriguez, B., Chen, L., Della Corte, C. M., Morikawa, N., Fujimoto, J., Cristea, S., Thuyen Nguyen, Diao, L., Li, L., Fan, Y., Yang, Y., Wang, J., Glisson, B. S., Wistuba, I. I., Sage, J., Heymach, J., Gibbons, D. L., Byers, L. A. 2019; 9 (5): 646–61

  View details for DOI 10.1158/2159-8290.CD-18-1020

  View details for Web of Science ID 000466758900025

• Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix. Molecular cell Topacio, B. R., Zatulovskiy, E., Cristea, S., Xie, S., Tambo, C. S., Rubin, S. M., Sage, J., Koivomagi, M., Skotheim, J. M. 2019

  Abstract

  The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in theRb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

  View details for PubMedID 30982746

• Molecular subtypes of small cell lung cancer: a synthesis of human and mouse model data. Nature reviews. Cancer Rudin, C. M., Poirier, J. T., Byers, L. A., Dive, C., Dowlati, A., George, J., Heymach, J. V., Johnson, J. E., Lehman, J. M., MacPherson, D., Massion, P. P., Minna, J. D., Oliver, T. G., Quaranta, V., Sage, J., Thomas, R. K., Vakoc, C. R., Gazdar, A. F. 2019

  Abstract

  Small cell lung cancer (SCLC) is an exceptionally lethal malignancy for which more effective therapies are urgently needed. Several lines of evidence, from SCLC primary human tumours, patient-derived xenografts, cancer cell lines and genetically engineered mouse models, appear to be converging on a new model of SCLC subtypes defined by differential expression of four key transcription regulators: achaete-scute homologue 1 (ASCL1; also known as ASH1), neurogenic differentiation factor 1 (NeuroD1), yes-associated protein 1 (YAP1) and POU class 2 homeobox 3 (POU2F3). In this Perspectives article, we review and synthesize these recent lines of evidence and propose a working nomenclature for SCLC subtypes defined by relative expression of these four factors. Defining the unique therapeutic vulnerabilities of these subtypes of SCLC should help to focus and accelerate therapeutic research, leading to rationally targeted approaches that may ultimately improve clinical outcomes for patients with this disease.

  View details for PubMedID 30926931

• Road map for fibrolamellar carcinoma: progress and goals of a diversified approach. Journal of hepatocellular carcinoma Kastenhuber, E. R., Craig, J., Ramsey, J., Sullivan, K. M., Sage, J., de Oliveira, S., Riehle, K. J., Scott, J. D., Gordan, J. D., Bardeesy, N., Abou-Alfa, G. K. 2019; 6: 41-48

  Abstract

  Fibrolamellar carcinoma is a rare liver cancer, which primarily afflicts adolescents and young adults worldwide and is frequently lethal. Given the rarity of this disease, patient recruitment for clinical trials remains a challenge. In November 2017, the Second Fibrolamellar Cancer Foundation Scientific Summit (Stamford, CT, USA) provided an opportunity for investigators to discuss recent advances in the characterization of the disease and its surrounding liver and immune context. The Fibrolamellar Cancer Foundation has thus set out a road map to identify and test therapeutic targets in the most efficient possible manner.

  View details for DOI 10.2147/JHC.S194764

  View details for PubMedID 30951568

  View details for PubMedCentralID PMC6362920

• E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family. Nature communications Hsu, J. n., Arand, J. n., Chaikovsky, A. n., Mooney, N. A., Demeter, J. n., Brison, C. M., Oliverio, R. n., Vogel, H. n., Rubin, S. M., Jackson, P. K., Sage, J. n. 2019; 10 (1): 2939

  Abstract

  E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.

  View details for DOI 10.1038/s41467-019-10901-x

  View details for PubMedID 31270324

• Systems-level network modeling of Small Cell Lung Cancer subtypes identifies master regulators and destabilizers. PLoS computational biology Wooten, D. J., Groves, S. M., Tyson, D. R., Liu, Q. n., Lim, J. S., Albert, R. n., Lopez, C. F., Sage, J. n., Quaranta, V. n. 2019; 15 (10): e1007343

  Abstract

  Adopting a systems approach, we devise a general workflow to define actionable subtypes in human cancers. Applied to small cell lung cancer (SCLC), the workflow identifies four subtypes based on global gene expression patterns and ontologies. Three correspond to known subtypes (SCLC-A, SCLC-N, and SCLC-Y), while the fourth is a previously undescribed ASCL1+ neuroendocrine variant (NEv2, or SCLC-A2). Tumor deconvolution with subtype gene signatures shows that all of the subtypes are detectable in varying proportions in human and mouse tumors. To understand how multiple stable subtypes can arise within a tumor, we infer a network of transcription factors and develop BooleaBayes, a minimally-constrained Boolean rule-fitting approach. In silico perturbations of the network identify master regulators and destabilizers of its attractors. Specific to NEv2, BooleaBayes predicts ELF3 and NR0B1 as master regulators of the subtype, and TCF3 as a master destabilizer. Since the four subtypes exhibit differential drug sensitivity, with NEv2 consistently least sensitive, these findings may lead to actionable therapeutic strategies that consider SCLC intratumoral heterogeneity. Our systems-level approach should generalize to other cancer types.

  View details for DOI 10.1371/journal.pcbi.1007343

  View details for PubMedID 31671086

• Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin. Cancer discovery Yang, D., Denny, S. K., Greenside, P. G., Chaikovsky, A. C., Brady, J. J., Ouadah, Y., Granja, J. M., Jahchan, N. S., Lim, J. S., Kwok, S., Kong, C. S., Berghoff, A. S., Schmitt, A., Reinhardt, H. C., Park, K., Preusser, M., Kundaje, A., Greenleaf, W. J., Sage, J., Winslow, M. M. 2018

  Abstract

  The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms.SIGNIFICANCE: We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. Cancer Discov; 8(10); 1-16. ©2018 AACR.See related commentary by Pozo et al., p. 1216.

  View details for PubMedID 30228179

• Non-canonical functions of the RB protein in cancer NATURE REVIEWS CANCER Dick, F. A., Goodrich, D. W., Sage, J., Dyson, N. J. 2018; 18 (7): 442–51

  Abstract

  The canonical model of RB-mediated tumour suppression developed over the past 30 years is based on the regulation of E2F transcription factors to restrict cell cycle progression. Several additional functions have been proposed for RB, on the basis of which a non-canonical RB pathway can be described. Mechanistically, the non-canonical RB pathway promotes histone modification and regulates chromosome structure in a manner distinct from cell cycle regulation. These functions have implications for chemotherapy response and resistance to targeted anticancer agents. This Opinion offers a framework to guide future studies of RB in basic and clinical research.

  View details for PubMedID 29692417

• Human hepatic organoids for the analysis of human genetic diseases. JCI insight Guan, Y., Xu, D., Garfin, P. M., Ehmer, U., Hurwitz, M., Enns, G., Michie, S., Wu, M., Zheng, M., Nishimura, T., Sage, J., Peltz, G. 2017; 2 (17)

  Abstract

  We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.

  View details for DOI 10.1172/jci.insight.94954

  View details for PubMedID 28878125

  View details for PubMedCentralID PMC5621886

• CD47 is not Over-Expressed in Fibrolamellar Hepatocellular Carcinoma ANNALS OF CLINICAL AND LABORATORY SCIENCE Cooney, T., Wei, M. C., Rangaswami, A., Xu, L., Sage, J., Hazard, F. K. 2017; 47 (4): 395–402

  Abstract

  CD47 is a transmembrane receptor that inhibits phagocytosis. Over-expression of CD47 is associated with an increased risk of tumor growth and metastasis. Clinical trials based on anti-CD47 therapy in adults are underway in a variety of malignancies. CD47 has been shown to be over-expressed in conventional hepatocellular carcinoma (HCC), a common liver tumor in adults. To our knowledge, there have been no studies to evaluate CD47 expression in the fibrolamellar subtype of HCC (FL-HCC), common in children and young adults. This study will evaluate CD47 expression in FL-HCC and shed light on its suitability for anti-CD47 therapy.Using immunohistochemistry, 10 samples of FL-HCC from 8 patients were evaluated for CD47 (anti-phagocytic) and calreticulin (pro-phagocytic) expression. By direct comparison, CD47 and calreticulin expression were evaluated in 21 samples of conventional HCC. Additionally, transcriptome sequencing to detect CD47 mRNA expression was performed on fresh tissue from 1 FL-HCC institutional patient and previously published sequencing data from 20 additional samples was reviewed.Immunohistochemistry showed only weak CD47 expression in 20% of FL-HCC samples. In contrast, 57% of conventional HCC samples showed CD47 expression. All (100%) FL-HCC samples showed moderate or strong calreticulin expression. The difference between CD47 and calreticulin expression in FL-HCC is statistically significant (p=0.0007). Transcriptome sequencing revealed no difference in CD47 expression between FL-HCC and normal liver samples.CD47 is not over-expressed in FL-HCC. Our studies provide no support for expanding ongoing clinical trials in adults to include children and young adults with FL-HCC.

  View details for PubMedID 28801364

• Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease. The Journal of clinical investigation Li, L. X., Fan, L. X., Zhou, J. X., Grantham, J. J., Calvet, J. P., Sage, J., Li, X. 2017; 127 (7): 2751-2764

  Abstract

  Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longer than Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early- and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-κB, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-α in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Akt signaling, via PTPN13-mediated phosphorylation.

  View details for DOI 10.1172/JCI90921

  View details for PubMedID 28604386

  View details for PubMedCentralID PMC5490754

• Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer NATURE Lim, J. S., Ibaseta, A., Fischer, M. M., Cancilla, B., O'Young, G., Cristea, S., Luca, V. C., Yang, D., Jahchan, N. S., Hamard, C., Antoine, M., Wislez, M., Kong, C., Cain, J., Liu, Y., Kapoun, A. M., Garcia, K. C., Hoey, T., Murriel, C. L., Sage, J. 2017; 545 (7654): 360-?

  Abstract

  The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

  View details for DOI 10.1038/nature22323

  View details for PubMedID 28489825

• CHK1 inhibition in small cell lung cancer produces single-agent activity in biomarker-defined disease subsets and combination activity with cisplatin or olaparib. Cancer research Sen, T., Tong, P., Stewart, C. A., Cristea, S., Valliani, A., Shames, D. S., Redwood, A., Fan, Y., Li, L., Glisson, B. S., Minna, J., Sage, J., Gibbons, D. L., Piwnica-Worms, H., Heymach, J., Wang, J., Byers, L. A. 2017

  Abstract

  Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 1-15. ©2017 AACR.

  View details for DOI 10.1158/0008-5472.CAN-16-3409

  View details for PubMedID 28490518

• Combining immune checkpoint inhibition and DNA damage repair (DDR) targeted therapy in small cell lung cancer (SCLC) Sen, T., Chen, L., Rodriguez, B., Yang, Y., Fan, Y., Stewart, C., Glisson, B., Piwnica-Worms, H., Sage, J., Heymach, J. V., Gibbons, D. L., Byers, L. A. AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/2326-6074.TUMIMM16-B72

  View details for Web of Science ID 000419245100114

• G1 cyclins protect pluripotency. Nature cell biology Arand, J., Sage, J. 2017; 19 (3): 149-150

  Abstract

  G1 cyclins are considered essential for DNA replication and cell division. A recent report now shows that some cells can cycle in the absence of G1 cyclins. In embryonic stem cells and cancer cells, G1 cyclins are required to activate cyclin-dependent kinases to phosphorylate core pluripotency factors and maintain pluripotency.

  View details for DOI 10.1038/ncb3480

  View details for PubMedID 28248303

• Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis. Cancer cell Gardner, E. E., Lok, B. H., Schneeberger, V. E., Desmeules, P., Miles, L. A., Arnold, P. K., Ni, A., Khodos, I., de Stanchina, E., Nguyen, T., Sage, J., Campbell, J. E., Ribich, S., Rekhtman, N., Dowlati, A., Massion, P. P., Rudin, C. M., Poirier, J. T. 2017; 31 (2): 286-299

  Abstract

  Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.

  View details for DOI 10.1016/j.ccell.2017.01.006

  View details for PubMedID 28196596

  View details for PubMedCentralID PMC5313262

• Relationship between anti-depressant use and lung cancer survival. Cancer treatment and research communications Zingone, A., Brown, D., Bowman, E. D., Vidal, O., Sage, J., Neal, J., Ryan, B. M. 2017; 10: 33–39

  Abstract

  OBJECTIVES: In recent years, the anti-cancer properties of several commonly used drugs have been explored, with drugs such as aspirin and beta-blockers associated with improved cancer outcomes. Previous preclinical work demonstrated that tricyclic anti-depressants have antitumor efficacy in lung cancer. Our goal was to examine the association between anti-depressant use and survival in lung cancer.MATERIALS AND METHODS: We examined the association between use of common anti-depressants and survival in 1,097 lung cancer patients from the NCI-Maryland lung cancer study. The types of anti-depressants included in the study were norepinephrine and dopamine reuptake inhibitors, serotonin reuptake inhibitors, selective serotonin reuptake inhibitors, non-selective serotonin reuptake inhibitors, and tricyclic anti-depressants. Anti-depressant use was extracted from the medical history section of a detailed interviewer-administered questionnaire. Specific use in the three months before a lung cancer diagnosis was determined. Cox portioned hazards modeling was used to estimate the association between anti-depressant use with lung cancer-specific death with adjustment for potential confounding co-factors.RESULTS: Anti-depressant use was associated with extended lung cancer-specific survival. In an analysis of specific classes of anti-depressant use, NDRIs and TCAs were associated with improved survival. Importantly, the extended survival associated with anti-depressants was maintained after adjustment for the clinical indications for these drugs, suggestive of a direct effect on lung cancer biology.CONCLUSIONS: Considering the manageable and largely tolerable side effects of anti-depressants, and the low cost of these drugs, these results indicate that evaluation of anti-depressants as adjunct therapeutics with chemotherapy may have a translational effect for lung cancer patients.

  View details for PubMedID 28944316

• Novel functions for the transcription factor E2F4 in development and disease. Cell cycle Hsu, J., Sage, J. 2016: 1-8

  Abstract

  The E2F family of transcription factors is a key determinant of cell proliferation in response to extra- and intra-cellular signals. Within this family, E2F4 is a transcriptional repressor whose activity is critical to engage and maintain cell cycle arrest in G0/G1 in conjunction with members of the retinoblastoma (RB) family. However, recent observations challenge this paradigm and indicate that E2F4 has a multitude of functions in cells besides this cell cycle regulatory role, including in embryonic and adult stem cells, during regenerative processes, and in cancer. Some of these new functions are independent of the RB family and involve direct activation of target genes. Here we review the canonical functions of E2F4 and discuss recent evidence expanding the role of this transcription factor, with a focus on cell fate decisions in tissue homeostasis and regeneration.

  View details for PubMedID 27753528

• Essential role for the planarian intestinal GATA transcription factor in stem cells and regeneration. Developmental biology Flores, N. M., Oviedo, N. J., Sage, J. 2016; 418 (1): 179-188

  Abstract

  The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.

  View details for DOI 10.1016/j.ydbio.2016.08.015

  View details for PubMedID 27542689

  View details for PubMedCentralID PMC5055475

• Is the Canonical RAF/MEK/ERK Signaling Pathway a Therapeutic Target in SCLC? Journal of thoracic oncology Cristea, S., Sage, J. 2016; 11 (8): 1233-1241

  Abstract

  The activity of the RAF/MEK/ERK signaling pathway is critical for the proliferation of normal and cancerous cells. Oncogenic mutations driving the development of lung adenocarcinoma often activate this signaling pathway. In contrast, pathway activity levels and their biological roles are not well established in small cell lung cancer (SCLC), a fast-growing neuroendocrine lung cancer subtype. Here we discuss the function of the RAF/MEK/ERK kinase pathway and the mechanisms leading to its activation in SCLC cells. In particular, we argue that activation of this pathway may be beneficial to the survival, proliferation, and spread of SCLC cells in response to multiple stimuli. We also consider evidence that high levels of RAF/MEK/ERK pathway activity may be detrimental to SCLC tumors, including in part by interfering with their neuroendocrine fate. On the basis of these observations, we examined when small molecules targeting kinases in the RAF/MEK/ERK pathway may be useful therapeutically in patients with SCLC, including in combination with other therapeutic agents.

  View details for DOI 10.1016/j.jtho.2016.04.018

  View details for PubMedID 27133774

  View details for PubMedCentralID PMC5391975

• Identification and Targeting of Long-Term Tumor-Propagating Cells in Small Cell Lung Cancer. Cell reports Jahchan, N. S., Lim, J. S., Bola, B., Morris, K., Seitz, G., Tran, K. Q., Xu, L., Trapani, F., Morrow, C. J., Cristea, S., Coles, G. L., Yang, D., Vaka, D., Kareta, M. S., George, J., Mazur, P. K., Nguyen, T., Anderson, W. C., Dylla, S. J., Blackhall, F., Peifer, M., Dive, C., Sage, J. 2016; 16 (3): 644-656

  Abstract

  Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all clinical features of SCLC are linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors and a means to target them in this most fatal form of lung cancer.

  View details for DOI 10.1016/j.celrep.2016.06.021

  View details for PubMedID 27373157

• Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility CELL Denny, S. K., Yang, D., Chuang, C., Brady, J. J., Lim, J. S., Gruner, B. M., Chiou, S., Schep, A. N., Baral, J., Hamard, C., Antoine, M., Wislez, M., Kong, C. S., Connolly, A. J., Park, K., Sage, J., Greenleaf, W. J., Winslow, M. M. 2016; 166 (2): 328-342

  Abstract

  Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

  View details for DOI 10.1016/j.cell.2016.05.052

  View details for PubMedID 27374332

• CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer JOURNAL OF CLINICAL INVESTIGATION Weiskopf, K., Jahchan, N. S., Schnorr, P. J., Cristea, S., Ring, A. M., Maute, R. L., Volkmer, A. K., Volkmer, J., Liu, J., Lim, J. S., Yang, D., Seitz, G., Thuyen Nguyen, T., Wu, D., Jude, K., Guerston, H., Barkal, A., Trapani, F., George, J., Poirier, J. T., Gardner, E. E., Miles, L. A., de Stanchina, E., Lofgren, S. M., Vogel, H., Winslow, M. M., Dive, C., Thomas, R. K., Rudin, C. M., van de Rijn, M., Majeti, R., Garcia, K. C., Weissman, I. L., Sage, J. 2016; 126 (7): 2610-2620

  Abstract

  Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

  View details for DOI 10.1172/JCI81603

  View details for Web of Science ID 000379094800024

  View details for PubMedID 27294525

  View details for PubMedCentralID PMC4922696

• Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Krampitz, G. W., George, B. M., Willingham, S. B., Volkmer, J., Weiskopf, K., Jahchan, N., Newman, A. M., Sahoo, D., Zemek, A. J., Yanovsky, R. L., Nguyen, J. K., Schnorr, P. J., Mazur, P. K., Sage, J., Longacre, T. A., Visser, B. C., Poultsides, G. A., Norton, J. A., Weissman, I. L. 2016; 113 (16): 4464-4469

  Abstract

  Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

  View details for DOI 10.1073/pnas.1600007113

  View details for PubMedID 27035983

• Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer GENES & DEVELOPMENT Reynoird, N., Mazur, P. K., Stellfeld, T., Flores, N. M., Lofgren, S. M., Carlson, S. M., Brambilla, E., Hainaut, P., Kaznowska, E. B., Arrowsmith, C. H., Khatri, P., Stresemann, C., Gozani, O., Sage, J. 2016; 30 (7): 772-785

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

  View details for DOI 10.1101/gad.275529.115

  View details for Web of Science ID 000373194300003

  View details for PubMedCentralID PMC4826394

• Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer. Genes & development Reynoird, N., Mazur, P. K., Stellfeld, T., Flores, N. M., Lofgren, S. M., Carlson, S. M., Brambilla, E., Hainaut, P., Kaznowska, E. B., Arrowsmith, C. H., Khatri, P., Stresemann, C., Gozani, O., Sage, J. 2016; 30 (7): 772-785

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

  View details for DOI 10.1101/gad.275529.115

  View details for PubMedID 26988419

  View details for PubMedCentralID PMC4826394

• Control of Proliferation and Cancer Growth by the Hippo Signaling Pathway. Molecular cancer research Ehmer, U., Sage, J. 2016; 14 (2): 127-140

  Abstract

  The control of cell division is essential for normal development and the maintenance of cellular homeostasis. Abnormal cell proliferation is associated with multiple pathological states, including cancer. Although the Hippo/YAP signaling pathway was initially thought to control organ size and growth, increasing evidence indicates that this pathway also plays a major role in the control of proliferation independent of organ size control. In particular, accumulating evidence indicates that the Hippo/YAP signaling pathway functionally interacts with multiple other cellular pathways and serves as a central node in the regulation of cell division, especially in cancer cells. Here, recent observations are highlighted that connect Hippo/YAP signaling to transcription, the basic cell-cycle machinery, and the control of cell division. Furthermore, the oncogenic and tumor-suppressive attributes of YAP/TAZ are reviewed, which emphasizes the relevance of the Hippo pathway in cancer. Mol Cancer Res; 14(2); 127-40. ©2015 AACR.

  View details for DOI 10.1158/1541-7786.MCR-15-0305

  View details for PubMedID 26432795

  View details for PubMedCentralID PMC4755889

• Loss of Pten Disrupts the Thymic Epithelium and Alters Thymic Function. PloS one Garfin, P. M., Nguyen, T., Sage, J. 2016; 11 (2)

  Abstract

  The thymus is the site of T cell development and selection. In addition to lymphocytes, the thymus is composed of several types of stromal cells that are exquisitely organized to create the appropriate environment and microenvironment to support the development and selection of maturing T cells. Thymic epithelial cells (TECs) are one of the more important cell types in the thymic stroma, and they play a critical role in selecting functional T cell clones and supporting their development. In this study, we used a mouse genetics approach to investigate the consequences of deleting the Pten tumor suppressor gene in the TEC compartment of the developing thymus. We found that PTEN deficiency in TECs results in a smaller thymus with significantly disordered architecture and histology. Accordingly, loss of PTEN function also results in decreased T cells with a shift in the distribution of T cell subtypes towards CD8+ T cells. These experiments demonstrate that PTEN is critically required for the development of a functional thymic epithelium in mice. This work may help better understand the effects that certain medical conditions or clinical interventions have upon the thymus and immune function.

  View details for DOI 10.1371/journal.pone.0149430

  View details for PubMedID 26914657

  View details for PubMedCentralID PMC4767252

• Novel insights into the oncogenic function of the SMYD3 lysine methyltransferase. Translational cancer research Mazur, P. K., Gozani, O. n., Sage, J. n., Reynoird, N. n. 2016; 5 (3): 330–33

  View details for PubMedID 30713830

• Crosstalk between stem cell and cell cycle machineries CURRENT OPINION IN CELL BIOLOGY Kareta, M. S., Sage, J., Wernig, M. 2015; 37: 68-74

  Abstract

  Pluripotent stem cells, defined by an unlimited self-renewal capacity and an undifferentiated state, are best typified by embryonic stem cells. These cells have a unique cell cycle compared to somatic cells as defined by a rapid progression through the cell cycle and a minimal time spent in G1. Recent reports indicate that pluripotency and cell cycle regulation are mechanistically linked. In this review, we discuss the reciprocal co-regulation of these processes, how this co-regulation may prevent differentiation, and how cellular reprogramming can re-establish the unique cell cycle regulation in induced pluripotent stem cells.

  View details for DOI 10.1016/j.ceb.2015.10.001

  View details for Web of Science ID 000367112800010

  View details for PubMedID 26520682

• Pancreatic cancer takes its Toll. The Journal of experimental medicine Mazur, P. K., Sage, J. 2015; 212 (12): 1988

  View details for DOI 10.1084/jem.21212insight1

  View details for PubMedID 26573583

  View details for PubMedCentralID PMC4647270

• Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma. Nature medicine Mazur, P. K., Herner, A., Mello, S. S., Wirth, M., Hausmann, S., Sánchez-Rivera, F. J., Lofgren, S. M., Kuschma, T., Hahn, S. A., Vangala, D., Trajkovic-Arsic, M., Gupta, A., Heid, I., Noël, P. B., Braren, R., Erkan, M., Kleeff, J., Sipos, B., Sayles, L. C., Heikenwalder, M., Heßmann, E., Ellenrieder, V., Esposito, I., Jacks, T., Bradner, J. E., Khatri, P., Sweet-Cordero, E. A., Attardi, L. D., Schmid, R. M., Schneider, G., Sage, J., Siveke, J. T. 2015; 21 (10): 1163-1171

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.

  View details for DOI 10.1038/nm.3952

  View details for PubMedID 26390243

• Comprehensive genomic profiles of small cell lung cancer NATURE George, J., Lim, J. S., Jang, S. J., Cun, Y., Ozretic, L., Kong, G., Leenders, F., Lu, X., Fernandez-Cuesta, L., Bosco, G., Mueller, C., Dahmen, I., Jahchan, N. S., Park, K., Yang, D., Karnezis, A. N., Vaka, D., Torres, A., Wang, M. S., Korbel, J. O., Menon, R., Chun, S., Kim, D., Wilkerson, M., Hayes, N., Engelmann, D., Puetzer, B., Bos, M., Michels, S., Vlasic, I., Seidel, D., Pinther, B., Schaub, P., Becker, C., Altmueller, J., Yokota, J., Kohno, T., Iwakawa, R., Tsuta, K., Noguchi, M., Muley, T., Hoffmann, H., Schnabel, P. A., Petersen, I., Chen, Y., Soltermann, A., Tischler, V., Choi, C., Kim, Y., Massion, P. P., Zou, Y., Jovanovic, D., Kontic, M., Wright, G. M., Russell, P. A., Solomon, B., Koch, I., Lindner, M., Muscarella, L. A., la Torre, A., Field, J. K., Jakopovic, M., Knezevic, J., Castanos-Velez, E., Roz, L., Pastorino, U., Brustugun, O., Lund-Iversen, M., Thunnissen, E., Koehler, J., Schuler, M., Botling, J., Sandelin, M., Sanchez-Cespedes, M., Salvesen, H. B., Achter, V., Lang, U., Bogus, M., Schneider, P. M., Zander, T., Ansen, S., Hallek, M., Wolf, J., Vingron, M., Yatabe, Y., Travis, W. D., Nuernberg, P., Reinhardt, C., Perner, S., Heukamp, L., Buettner, R., Haas, S. A., Brambilla, E., Peifer, M., Sage, J., Thomas, R. K. 2015; 524 (7563): 47-U73

  Abstract

  We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

  View details for DOI 10.1038/nature14664

  View details for Web of Science ID 000359002300029

  View details for PubMedID 26168399

  View details for PubMedCentralID PMC4861069

• Comprehensive genomic profiles of small cell lung cancer. Nature George, J., Lim, J. S., Jang, S. J., Cun, Y., Ozretic, L., Kong, G., Leenders, F., Lu, X., Fernández-Cuesta, L., Bosco, G., Müller, C., Dahmen, I., Jahchan, N. S., Park, K., Yang, D., Karnezis, A. N., Vaka, D., Torres, A., Wang, M. S., Korbel, J. O., Menon, R., Chun, S., Kim, D., Wilkerson, M., Hayes, N., Engelmann, D., Pützer, B., Bos, M., Michels, S., Vlasic, I., Seidel, D., Pinther, B., Schaub, P., Becker, C., Altmüller, J., Yokota, J., Kohno, T., Iwakawa, R., Tsuta, K., Noguchi, M., Muley, T., Hoffmann, H., Schnabel, P. A., Petersen, I., Chen, Y., Soltermann, A., Tischler, V., Choi, C., Kim, Y., Massion, P. P., Zou, Y., Jovanovic, D., Kontic, M., Wright, G. M., Russell, P. A., Solomon, B., Koch, I., Lindner, M., Muscarella, L. A., la Torre, A., Field, J. K., Jakopovic, M., Knezevic, J., Castaños-Vélez, E., Roz, L., Pastorino, U., Brustugun, O., Lund-Iversen, M., Thunnissen, E., Köhler, J., Schuler, M., Botling, J., Sandelin, M., Sanchez-Cespedes, M., Salvesen, H. B., Achter, V., Lang, U., Bogus, M., Schneider, P. M., Zander, T., Ansén, S., Hallek, M., Wolf, J., Vingron, M., Yatabe, Y., Travis, W. D., Nürnberg, P., Reinhardt, C., Perner, S., Heukamp, L., Büttner, R., Haas, S. A., Brambilla, E., Peifer, M., Sage, J., Thomas, R. K. 2015; 524 (7563): 47-53

  Abstract

  We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

  View details for DOI 10.1038/nature14664

  View details for PubMedID 26168399

• Inhibition of pluripotency networks by the rb tumor suppressor restricts reprogramming and tumorigenesis. Cell stem cell Kareta, M. S., Gorges, L. L., Hafeez, S., Benayoun, B. A., Marro, S., Zmoos, A., Cecchini, M. J., Spacek, D., Batista, L. F., O'Brien, M., Ng, Y., Ang, C. E., Vaka, D., Artandi, S. E., Dick, F. A., Brunet, A., Sage, J., Wernig, M. 2015; 16 (1): 39-50

  Abstract

  Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.

  View details for DOI 10.1016/j.stem.2014.10.019

  View details for PubMedID 25467916

  View details for PubMedCentralID PMC4389904

• Genomic analysis of fibrolamellar hepatocellular carcinoma. Human molecular genetics Xu, L., Hazard, F. K., Zmoos, A., Jahchan, N., Chaib, H., Garfin, P. M., Rangaswami, A., Snyder, M. P., Sage, J. 2015; 24 (1): 50-63

  Abstract

  Pediatric tumors are relatively infrequent but are often associated with significant lethality and lifelong morbidity. A major goal of pediatric cancer research has been to identify key drivers of tumorigenesis to eventually develop targeted therapies to enhance cure rate and minimize acute and long-term toxic effects. Here we used genomics approaches to identify biomarkers and candidate drivers for fibrolamellar hepatocellular carcinoma (FL-HCC), a very rare subtype of pediatric liver cancer for which limited therapeutic options exist. In-depth genomics analyses of one tumor followed by immunohistochemistry validation on seven other tumors showed expression of neuroendocrine markers in FL-HCC. DNA and RNA sequencing data further showed that common cancer pathways are not visibly altered in FL-HCC but identified two novel structural variants, both resulting in fusion transcripts. The first, a 400kb deletion, results in a DNAJ1-PRKCA fusion transcript, which leads to increased PKA activity in the index tumor case and other FL-HCC cases compared to normal liver. This PKA fusion protein is oncogenic in HCC cells. The second gene fusion event, a translocation between the CLPTML1 and GLIS3 genes, generates a transcript whose product also promotes cancer phenotypes in HCC cell lines. These experiments further highlight the tumorigenic role of gene fusions in the etiology of pediatric solid tumors and identify both candidate biomarkers and possible therapeutic targets for this lethal pediatric disease.

  View details for DOI 10.1093/hmg/ddu418

  View details for PubMedID 25122662

• In vivo disruption of an Rb-E2F-Ezh2 signaling loop causes bladder cancer. Cancer research Santos, M., Martínez-Fernández, M., Dueñas, M., García-Escudero, R., Alfaya, B., Villacampa, F., Saiz-Ladera, C., Costa, C., Oteo, M., Duarte, J., Martínez, V., Gómez-Rodriguez, M. J., Martín, M. L., Fernández, M., Viatour, P., Morcillo, M. A., Sage, J., Castellano, D., Rodriguez-Peralto, J. L., de la Rosa, F., Paramio, J. M. 2014; 74 (22): 6565-6577

  Abstract

  Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb-E2F-Ezh2 axis in bladder whose disruption can promote tumor development.

  View details for DOI 10.1158/0008-5472.CAN-14-1218

  View details for PubMedID 25252918

  View details for PubMedCentralID PMC4233185

• Cancer: The origin of human retinoblastoma. Nature Bremner, R., Sage, J. 2014; 514 (7522): 312-3

  View details for DOI 10.1038/nature13748

  View details for PubMedID 25252972

  View details for PubMedCentralID PMC5438154

• Organ Size Control Is Dominant over Rb Family Inactivation to Restrict Proliferation In Vivo. Cell reports Ehmer, U., Zmoos, A., Auerbach, R. K., Vaka, D., Butte, A. J., Kay, M. A., Sage, J. 2014; 8 (2): 371-381

  Abstract

  In mammals, a cell's decision to divide is thought to be under the control of the Rb/E2F pathway. We previously found that inactivation of the Rb family of cell cycle inhibitors (Rb, p107, and p130) in quiescent liver progenitors leads to uncontrolled division and cancer initiation. Here, we show that, in contrast, deletion of the entire Rb gene family in mature hepatocytes is not sufficient for their long-term proliferation. The cell cycle block in Rb family mutant hepatocytes is independent of the Arf/p53/p21 checkpoint but can be abrogated upon decreasing liver size. At the molecular level, we identify YAP, a transcriptional regulator involved in organ size control, as a factor required for the sustained expression of cell cycle genes in hepatocytes. These experiments identify a higher level of regulation of the cell cycle in vivo in which signals regulating organ size are dominant regulators of the core cell cycle machinery.

  View details for DOI 10.1016/j.celrep.2014.06.025

  View details for PubMedID 25017070

  View details for PubMedCentralID PMC4128252

• SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature Mazur, P. K., Reynoird, N., Khatri, P., Jansen, P. W., Wilkinson, A. W., Liu, S., Barbash, O., Van Aller, G. S., Huddleston, M., Dhanak, D., Tummino, P. J., Kruger, R. G., Garcia, B. A., Butte, A. J., Vermeulen, M., Sage, J., Gozani, O. 2014; 510 (7504): 283-287

  Abstract

  Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

  View details for DOI 10.1038/nature13320

  View details for PubMedID 24847881

• From Fly Wings to Targeted Cancer Therapies: A Centennial for Notch Signaling CANCER CELL Ntziachristos, P., Lim, J. S., Sage, J., Aifantis, I. 2014; 25 (3): 318-334

  Abstract

  Since Notch phenotypes in Drosophila melanogaster were first identified 100 years ago, Notch signaling has been extensively characterized as a regulator of cell-fate decisions in a variety of organisms and tissues. However, in the past 20 years, accumulating evidence has linked alterations in the Notch pathway to tumorigenesis. In this review, we discuss the protumorigenic and tumor-suppressive functions of Notch signaling, and dissect the molecular mechanisms that underlie these functions in hematopoietic cancers and solid tumors. Finally, we link these mechanisms and observations to possible therapeutic strategies targeting the Notch pathway in human cancers.

  View details for DOI 10.1016/j.ccr.2014.02.018

  View details for Web of Science ID 000333233400010

  View details for PubMedCentralID PMC4040351

• A Drug Repositioning Approach Identifies Tricyclic Antidepressants as Inhibitors of Small Cell Lung Cancer and Other Neuroendocrine Tumors CANCER DISCOVERY Jahchan, N. S., Dudley, J. T., Mazur, P. K., Flores, N., Yang, D., Palmerton, A., Zmoos, A., Vaka, D., Tran, K. Q., Zhou, M., Krasinska, K., Riess, J. W., Neal, J. W., Khatri, P., Park, K. S., Butte, A. J., Sage, J. 2013; 3 (12): 1364-1377

  Abstract

  Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. We used a systematic drug repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate U.S. Food and Drug Administration (FDA)-approved drugs to treat SCLC. We found that tricyclic antidepressants and related molecules potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine survival signals involving neurotransmitters and their G protein-coupled receptors. The candidate drugs inhibit the growth of other neuroendocrine tumors, including pancreatic neuroendocrine tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs.Our work shows the power of bioinformatics-based drug approaches to rapidly repurpose FDA-approved drugs and identifies a novel class of molecules to treat patients with SCLC, a cancer for which no effective novel systemic treatments have been identified in several decades. In addition, our experiments highlight the importance of novel autocrine mechanisms in promoting the growth of neuroendocrine tumor cells.

  View details for DOI 10.1158/2159-8290.CD-13-0183

  View details for Web of Science ID 000328257500023

  View details for PubMedID 24078773

  View details for PubMedCentralID PMC3864571

• Defining a new vision for the retinoblastoma gene: report from the 3rd International Rb Meeting. Cell division Rubin, S. M., Sage, J. 2013; 8 (1): 13

  Abstract

  The retinoblastoma tumor suppressor (Rb) pathway is mutated in most, if not all human tumors. In the G0/G1 phase, Rb and its family members p107 and p130 inhibit the E2F family of transcription factors. In response to mitogenic signals, Cyclin-dependent kinases (CDKs) phosphorylate Rb family members, which results in the disruption of complexes between Rb and E2F family members and in the transcription of genes essential for S phase progression. Beyond this role in early cell cycle decisions, Rb family members regulate DNA replication and mitosis, chromatin structure, metabolism, cellular differentiation, and cell death. While the RB pathway has been extensively studied in the past three decades, new investigations continue to provide novel insights into basic mechanisms of cancer development and, beyond cancer, help better understand fundamental cellular processes, from plants to mammals. This meeting report summarizes research presented at the recently held 3rd International Rb Meeting.

  View details for DOI 10.1186/1747-1028-8-13

  View details for PubMedID 24257515

  View details for PubMedCentralID PMC3866465

• Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression. journal of experimental medicine Garfin, P. M., Min, D., Bryson, J. L., Serwold, T., Edris, B., Blackburn, C. C., Richie, E. R., Weinberg, K. I., Manley, N. R., Sage, J., Viatour, P. 2013; 210 (6): 1087-1097

  Abstract

  Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow-independent mechanism, identifying a novel pathway to target to increase thymic function in patients.

  View details for DOI 10.1084/jem.20121716

  View details for PubMedID 23669396

• RB goes mitochondrial. Genes & development Attardi, L. D., Sage, J. 2013; 27 (9): 975-979

  Abstract

  The retinoblastoma tumor suppressor RB is well known for its capacity to restrict cell cycle progression at the G1/S transition of the cell cycle by controlling the transcription of cell cycle genes. In this issue of Genes & Development, Hilgendorf and colleagues (pp. 1003-1015) have identified a novel tumor suppressor function for RB independent of its role as a transcriptional regulator, in which RB directly activates the apoptosis regulator Bax at the mitochondria to promote cell death.

  View details for DOI 10.1101/gad.219451.113

  View details for PubMedID 23651852

  View details for PubMedCentralID PMC3656326

• IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors. Nature medicine Jameson, K. L., Mazur, P. K., Zehnder, A. M., Zhang, J., Zarnegar, B., Sage, J., Khavari, P. A. 2013; 19 (5): 626-630

  Abstract

  Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif-containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.

  View details for DOI 10.1038/nm.3165

  View details for PubMedID 23603816

• The RB family is required for the self-renewal and survival of human embryonic stem cells NATURE COMMUNICATIONS Conklin, J. F., Baker, J., Sage, J. 2012; 3

  Abstract

  The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood, limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest, differentiation and cell death. Conversely, inactivation of the entire RB family (RB, p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells, mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally, loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells, uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells.

  View details for DOI 10.1038/ncomms2254

  View details for Web of Science ID 000316356700012

  View details for PubMedID 23212373

• Inactivating All Three Rb Family Pocket Proteins Is Insufficient to Initiate Cervical Cancer CANCER RESEARCH Shin, M., Sage, J., Lambert, P. F. 2012; 72 (20): 5418-5427

  Abstract

  Human papillomavirus-16 (HPV-16) is associated etiologically with many human cervical cancers. It encodes 3 oncogenes E5, E6, and E7. Of these oncogenes, E7 has been found to be the dominant driver of cervical cancer in mice. More than 100 cellular proteins have been reported to associate with HPV-16 E7, which is thought to dysregulate the cell cycle in part by binding and inducing the degradation of pRb and its related pocket protein family members, p107 and p130. The ability of E7 to inactivate the pRb family correlates with its ability to induce head and neck cancers in mice. We previously showed that the inactivation of pRb is itself not sufficient to recapitulate the oncogenic properties of E7 in cervical carcinogenesis. In this study, we evaluated mice that were deficient in multiple pocket proteins, including mice that lacked pRb, p107, and p130. Strikingly, combined loss of two or all 3 pocket proteins resulted in development of high-grade cervical intraepithelial neoplasia, but not frank cervical carcinoma. These findings strongly argue that the oncogenic properties of HPV-16 E7 in human cervical carcinogenesis may involve disruption of E7 binding proteins beyond simply the pRb family members.

  View details for DOI 10.1158/0008-5472.CAN-12-2083

  View details for Web of Science ID 000309972700030

  View details for PubMedID 22942253

• Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer NATURE GENETICS Peifer, M., Fernandez-Cuesta, L., Sos, M. L., George, J., Seidel, D., Kasper, L. H., Plenker, D., Leenders, F., Sun, R., Zander, T., Menon, R., Koker, M., Dahmen, I., Mueller, C., Di Cerbo, V., Schildhaus, H., Altmueller, J., Baessmann, I., Becker, C., De Wilde, B., Vandesompele, J., Boehm, D., Ansen, S., Gabler, F., Wilkening, I., Heynck, S., Heuckmann, J. M., Lu, X., Carter, S. L., Cibulskis, K., Banerji, S., Getz, G., Park, K., Rauh, D., Gruetter, C., Fischer, M., Pasqualucci, L., Wright, G., Wainer, Z., Russell, P., Petersen, I., Chen, Y., Stoelben, E., Ludwig, C., Schnabel, P., Hoffmann, H., Muley, T., Brockmann, M., Engel-Riedel, W., Muscarella, L. A., Fazio, V. M., Groen, H., Timens, W., Sietsma, H., Thunnissen, E., Smit, E., Heideman, D. A., Snijders, P. J., Cappuzzo, F., Ligorio, C., Damiani, S., Field, J., Solberg, S., Brustugun, O. T., Lund-Iversen, M., Saenger, J., Clement, J. H., Soltermann, A., Moch, H., Weder, W., Solomon, B., Soria, J., Validire, P., Besse, B., Brambilla, E., Brambilla, C., Lantuejoul, S., Lorimier, P., Schneider, P. M., Hallek, M., Pao, W., Meyerson, M., Sage, J., Shendure, J., Schneider, R., Buettner, R., Wolf, J., Nuernberg, P., Perner, S., Heukamp, L. C., Brindle, P. K., Haas, S., Thomas, R. K. 2012; 44 (10): 1104-?

  Abstract

  Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

  View details for DOI 10.1038/ng.2396

  View details for Web of Science ID 000309550200009

  View details for PubMedID 22941188

• The retinoblastoma tumor suppressor and stem cell biology GENES & DEVELOPMENT Sage, J. 2012; 26 (13): 1409-1420

  Abstract

  Stem cells play a critical role during embryonic development and in the maintenance of homeostasis in adult individuals. A better understanding of stem cell biology, including embryonic and adult stem cells, will allow the scientific community to better comprehend a number of pathologies and possibly design novel approaches to treat patients with a variety of diseases. The retinoblastoma tumor suppressor RB controls the proliferation, differentiation, and survival of cells, and accumulating evidence points to a central role for RB activity in the biology of stem and progenitor cells. In some contexts, loss of RB function in stem or progenitor cells is a key event in the initiation of cancer and determines the subtype of cancer arising from these pluripotent cells by altering their fate. In other cases, RB inactivation is often not sufficient to initiate cancer but may still lead to some stem cell expansion, raising the possibility that strategies aimed at transiently inactivating RB might provide a novel way to expand functional stem cell populations. Future experiments dedicated to better understanding how RB and the RB pathway control a stem cell's decisions to divide, self-renew, or give rise to differentiated progeny may eventually increase our capacity to control these decisions to enhance regeneration or help prevent cancer development.

  View details for DOI 10.1101/gad.193730.112

  View details for Web of Science ID 000306175400002

  View details for PubMedID 22751497

• Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation EPIGENETICS Van Aller, G. S., Reynoird, N., Barbash, O., Huddleston, M., Liu, S., Zmoos, A., McDevitt, P., Sinnamon, R., Le, B., Mas, G., Annan, R., Sage, J., Garcia, B. A., Tummino, P. J., Gozani, O., Kruger, R. G. 2012; 7 (4): 340-343

  Abstract

  Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

  View details for Web of Science ID 000302493300004

  View details for PubMedID 22419068

  View details for PubMedCentralID PMC3368817

• GENOMICS The path to retinoblastoma NATURE Sage, J., Cleary, M. L. 2012; 481 (7381): 269-270

  View details for Web of Science ID 000299210600023

  View details for PubMedID 22258599

• A crucial requirement for Hedgehog signaling in small cell lung cancer NATURE MEDICINE Park, K., Martelotto, L. G., Peifer, M., Sos, M. L., Karnezis, A. N., Mahjoub, M. R., Bernard, K., Conklin, J. F., Szczepny, A., Yuan, J., Guo, R., Ospina, B., Falzon, J., Bennett, S., Brown, T. J., Markovic, A., Devereux, W. L., Ocasio, C. A., Chen, J. K., Stearns, T., Thomas, R. K., Dorsch, M., Buonamici, S., Watkins, D. N., Peacock, C. D., Sage, J. 2011; 17 (11): 1504-U1506

  Abstract

  Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

  View details for DOI 10.1038/nm.2473

  View details for PubMedID 21983857

• PDGF signalling controls age-dependent proliferation in pancreatic beta-cells NATURE Chen, H., Gu, X., Liu, Y., Wang, J., Wirt, S. E., Bottino, R., Schorle, H., Sage, J., Kim, S. K. 2011; 478 (7369): 349-?

  Abstract

  Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islets. With age, declining β-cell Pdgfr levels were accompanied by reductions in β-cell enhancer of zeste homologue 2 (Ezh2) levels and β-cell replication. Conditional inactivation of the Pdgfra gene in β-cells accelerated these changes, preventing mouse neonatal β-cell expansion and adult β-cell regeneration. Targeted human PDGFR-α activation in mouse β-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult β-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated β-cell proliferation. The discovery of a conserved pathway controlling age-dependent β-cell proliferation indicates new strategies for β-cell expansion.

  View details for DOI 10.1038/nature10502

  View details for Web of Science ID 000296021100038

  View details for PubMedID 21993628

  View details for PubMedCentralID PMC3503246

• Discovery and Preclinical Validation of Drug Indications Using Compendia of Public Gene Expression Data (vol 3, 96ra77, 2011) SCIENCE TRANSLATIONAL MEDICINE Sirota, M., DUDLEY, J. T., Kim, J., Chiang, A. P., Morgan, A. A., Sweet-Cordero, A., Sage, J., Butte, A. J. 2011; 3 (102)

  View details for DOI 10.1126/scitranslmed.3003215

  View details for Web of Science ID 000295323200013

• Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway JOURNAL OF EXPERIMENTAL MEDICINE Viatour, P., Ehmer, U., Saddic, L. A., Dorrell, C., Andersen, J. B., Lin, C., Zmoos, A., Mazur, P. K., Schaffer, B. E., Ostermeier, A., Vogel, H., Sylvester, K. G., Thorgeirsson, S. S., Grompe, M., Sage, J. 2011; 208 (10): 1963-1976

  Abstract

  Hepatocellular carcinoma (HCC) is the third cancer killer worldwide with >600,000 deaths every year. Although the major risk factors are known, therapeutic options in patients remain limited in part because of our incomplete understanding of the cellular and molecular mechanisms influencing HCC development. Evidence indicates that the retinoblastoma (RB) pathway is functionally inactivated in most cases of HCC by genetic, epigenetic, and/or viral mechanisms. To investigate the functional relevance of this observation, we inactivated the RB pathway in the liver of adult mice by deleting the three members of the Rb (Rb1) gene family: Rb, p107, and p130. Rb family triple knockout mice develop liver tumors with histopathological features and gene expression profiles similar to human HCC. In this mouse model, cancer initiation is associated with the specific expansion of populations of liver stem/progenitor cells, indicating that the RB pathway may prevent HCC development by maintaining the quiescence of adult liver progenitor cells. In addition, we show that during tumor progression, activation of the Notch pathway via E2F transcription factors serves as a negative feedback mechanism to slow HCC growth. The level of Notch activity is also able to predict survival of HCC patients, suggesting novel means to diagnose and treat HCC.

  View details for DOI 10.1084/jem.20110198

  View details for PubMedID 21875955

• Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models CANCER CELL Taguchi, A., Politi, K., Pitteri, S. J., Lockwood, W. W., Faca, V. M., Kelly-Spratt, K., Wong, C., Zhang, Q., Chin, A., Park, K., Goodman, G., Gazdar, A. F., Sage, J., Dinulescu, D. M., Kucherlapati, R., DePinho, R. A., Kemp, C. J., Varmus, H. E., Hanash, S. M. 2011; 20 (3): 289-299

  Abstract

  We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models.

  View details for DOI 10.1016/j.ccr.2011.08.007

  View details for Web of Science ID 000295205700006

  View details for PubMedID 21907921

  View details for PubMedCentralID PMC3406925

• Newly identified aspects of tumor suppression by RB DISEASE MODELS & MECHANISMS Viatour, P., Sage, J. 2011; 4 (5): 581-585

  Abstract

  The retinoblastoma (RB) tumor suppressor belongs to a cellular pathway that plays a crucial role in restricting the G1-S transition of the cell cycle in response to a large number of extracellular and intracellular cues. Research in the last decade has highlighted the complexity of regulatory networks that ensure proper cell cycle progression, and has also identified multiple cellular functions beyond cell cycle regulation for RB and its two family members, p107 and p130. Here we review some of the recent evidence pointing to a role of RB as a molecular adaptor at the crossroads of multiple pathways, ensuring cellular homeostasis in different contexts. In particular, we discuss the pro- and anti-tumorigenic roles of RB during the early stages of cancer, as well as the importance of the RB pathway in stem cells and cell fate decisions.

  View details for DOI 10.1242/dmm.008060

  View details for Web of Science ID 000294411800005

  View details for PubMedID 21878458

  View details for PubMedCentralID PMC3180221

• Discovery and Preclinical Validation of Drug Indications Using Compendia of Public Gene Expression Data SCIENCE TRANSLATIONAL MEDICINE Sirota, M., Dudley, J. T., Kim, J., Chiang, A. P., Morgan, A. A., Sweet-Cordero, A., Sage, J., Butte, A. J. 2011; 3 (96)

  Abstract

  The application of established drug compounds to new therapeutic indications, known as drug repositioning, offers several advantages over traditional drug development, including reduced development costs and shorter paths to approval. Recent approaches to drug repositioning use high-throughput experimental approaches to assess a compound's potential therapeutic qualities. Here, we present a systematic computational approach to predict novel therapeutic indications on the basis of comprehensive testing of molecular signatures in drug-disease pairs. We integrated gene expression measurements from 100 diseases and gene expression measurements on 164 drug compounds, yielding predicted therapeutic potentials for these drugs. We recovered many known drug and disease relationships using computationally derived therapeutic potentials and also predict many new indications for these 164 drugs. We experimentally validated a prediction for the antiulcer drug cimetidine as a candidate therapeutic in the treatment of lung adenocarcinoma, and demonstrate its efficacy both in vitro and in vivo using mouse xenograft models. This computational method provides a systematic approach for repositioning established drugs to treat a wide range of human diseases.

  View details for DOI 10.1126/scitranslmed.3001318

  View details for PubMedID 21849665

• Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis CANCER CELL McEvoy, J., Flores-Otero, J., Zhang, J., Nemeth, K., Brennan, R., Bradley, C., Krafcik, F., Rodriguez-Galindo, C., Wilson, M., Xiong, S., Lozano, G., Sage, J., Fu, L., Louhibi, L., Trimarchi, J., Pani, A., Smeyne, R., Johnson, D., Dyer, M. A. 2011; 20 (2): 260-275

  Abstract

  It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.

  View details for DOI 10.1016/j.ccr.2011.07.005

  View details for Web of Science ID 000294099700013

  View details for PubMedID 21840489

  View details for PubMedCentralID PMC3551581

• miR than meets the eye GENES & DEVELOPMENT Sage, J., Ventura, A. 2011; 25 (16): 1663-1667

  Abstract

  Retinoblastoma is a rare pediatric cancer that has served as a paradigm to investigate the mechanisms of tumorigenesis. In this issue of Genes & Development, Conkrite and colleagues (pp. 1734-1745) found high levels of the miR-17~92 and miR-106b-25 microRNAs in primary retinoblastomas and show that overexpression of miR-17~92 accelerates retinoblastoma development in mice by promoting proliferation, in part by reducing expression of the cell cycle inhibitor p21. These experiments identify the RB/miR-17~92/p21 axis as a critical regulator of retinoblastoma tumorigenesis and potentially many other cancers.

  View details for DOI 10.1101/gad.17454011

  View details for Web of Science ID 000294003800001

  View details for PubMedID 21852531

  View details for PubMedCentralID PMC3165930

• Characterization of the cell of origin for small cell lung cancer CELL CYCLE Park, K., Liang, M., Raiser, D. M., Zamponi, R., Roach, R. R., Curtis, S. J., Walton, Z., Schaffer, B. E., Roake, C. M., Zmoos, A., Kriegel, C., Wong, K., Sage, J., Kim, C. F. 2011; 10 (16): 2806-2815

  Abstract

  Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.

  View details for DOI 10.4161/cc.10.16.17012

  View details for Web of Science ID 000294155600035

  View details for PubMedID 21822053

  View details for PubMedCentralID PMC3219544

• Functional Interactions between Retinoblastoma and c-MYC in a Mouse Model of Hepatocellular Carcinoma PLOS ONE Saddic, L. A., Wirt, S., Vogel, H., Felsher, D. W., Sage, J. 2011; 6 (5)

  Abstract

  Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes.

  View details for DOI 10.1371/journal.pone.0019758

  View details for PubMedID 21573126

• MicroRNA programs in normal and aberrant stem and progenitor cells GENOME RESEARCH Arnold, C. P., Tan, R., Zhou, B., Yue, S., Schaffert, S., Biggs, J. R., Doyonnas, R., Lo, M., Perry, J. M., Renault, V. M., Sacco, A., Somervaille, T., Viatour, P., Brunet, A., Cleary, M. L., Li, L., Sage, J., Zhang, D., Blau, H. M., Chen, C., Chen, C. 2011; 21 (5): 798-810

  Abstract

  Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

  View details for DOI 10.1101/gr.111385.110

  View details for Web of Science ID 000290088000018

  View details for PubMedID 21451113

  View details for PubMedCentralID PMC3083097

• RB deletion disrupts coordination between DNA replication licensing and mitotic entry in vivo MOLECULAR BIOLOGY OF THE CELL Bourgo, R. J., Ehmer, U., Sage, J., Knudsen, E. S. 2011; 22 (7): 931-939

  Abstract

  The integrity of the retinoblastoma tumor suppressor (RB) pathway is critical for restraining inappropriate proliferation and suppressing tumor development in a plethora of tissues. Here adenovirus-mediated RB deletion in the liver of adult mice led to DNA replication in the absence of productive mitotic condensation. The replication induced by RB loss was E2F-mediated and associated with the induction of DNA damage and a nontranscriptional G2/M checkpoint that targeted the accumulation of Cyclin B1. In the context of RB deletion or E2F activation, there was an increase in hepatocyte ploidy that was accompanied by hyperphysiological assembly of prereplication complexes. In keeping with this dysregulation, initiation of DNA replication was readily observed in hepatocytes that were phenotypically in G2/M. Under such conditions, uncoupling of replication initiation from mitotic progression led to altered genome ploidy in the liver. Interestingly, these findings in hepatocytes were not recapitulated in the basally proliferative tissues of the gastrointestinal tract, where RB deletion, while increasing DNA replication, did not lead to a profound uncoupling from mitosis. Combined, these findings demonstrate the critical role of RB in controlling cell-cycle transitions and underscore the importance of intrinsic tissue environments in resultant phenotypes.

  View details for DOI 10.1091/mbc.E10-11-0895

  View details for Web of Science ID 000288995800005

  View details for PubMedID 21289097

• Methylation of the Retinoblastoma Tumor Suppressor by SMYD2 JOURNAL OF BIOLOGICAL CHEMISTRY Saddic, L. A., West, L. E., Aslanian, A., Yates, J. R., Rubin, S. M., Gozani, O., Sage, J. 2010; 285 (48): 37733-37740

  Abstract

  The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

  View details for DOI 10.1074/jbc.M110.137612

  View details for Web of Science ID 000284424000065

  View details for PubMedID 20870719

  View details for PubMedCentralID PMC2988378

• G1 arrest and differentiation can occur independently of Rb family function JOURNAL OF CELL BIOLOGY Wirt, S. E., Adler, A. S., Gebala, V., Weimann, J. M., Schaffer, B. E., Saddic, L. A., Viatour, P., Vogel, H., Chang, H. Y., Meissner, A., Sage, J. 2010; 191 (4): 809-825

  Abstract

  The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

  View details for DOI 10.1083/jcb.201003048

  View details for Web of Science ID 000284737200014

  View details for PubMedID 21059851

  View details for PubMedCentralID PMC2983066

• Transient Inactivation of Rb and ARF Yields Regenerative Cells from Postmitotic Mammalian Muscle CELL STEM CELL Pajcini, K. V., Corbel, S. Y., Sage, J., Pomerantz, J. H., Blau, H. M. 2010; 7 (2): 198-213

  Abstract

  An outstanding biological question is why tissue regeneration in mammals is limited, whereas urodele amphibians and teleost fish regenerate major structures, largely by cell cycle reentry. Upon inactivation of Rb, proliferation of postmitotic urodele skeletal muscle is induced, whereas in mammalian muscle this mechanism does not exist. We postulated that a tumor suppressor present in mammals but absent in regenerative vertebrates, the Ink4a product ARF (alternative reading frame), is a regeneration suppressor. Concomitant inactivation of Arf and Rb led to mammalian muscle cell cycle reentry, loss of differentiation properties, and upregulation of cytokinetic machinery. Single postmitotic myocytes were isolated by laser micro-dissection-catapulting, and transient suppression of Arf and Rb yielded myoblast colonies that retained the ability to differentiate and fuse into myofibers upon transplantation in vivo. These results show that differentiation of mammalian cells is reversed by inactivation of Arf and Rb and support the hypothesis that Arf evolved at the expense of regeneration.

  View details for DOI 10.1016/j.stem.2010.05.022

  View details for Web of Science ID 000281107400012

  View details for PubMedID 20682446

  View details for PubMedCentralID PMC2919350

• RB's original CIN? GENES & DEVELOPMENT Sage, J., Straight, A. F. 2010; 24 (13): 1329-1333

  Abstract

  The retinoblastoma tumor suppressor RB is the downstream mediator of a cellular pathway that is thought to prevent cancer by controlling the ability of cells to enter or exit the cell cycle in G0/G1. Recently, however, accumulating evidence has suggested that RB, its family members p107 and p130, and their partners, the E2F family of transcription factors, may have important cellular functions beyond the G1/S transition of the cell cycle, including during DNA replication and at the transition into mitosis. In this issue of Genes & Development, three studies demonstrate a critical role for RB in proper chromosome condensation, centromeric function, and chromosome stability in mammalian cells, and link these cellular functions of RB to tumor suppression in mice. Here we discuss how transcriptional and post-transcriptional mechanisms under the control of the RB pathway ensure accurate progression through mitosis, thereby preventing cancer development.

  View details for DOI 10.1101/gad.1948010

  View details for Web of Science ID 000279405000001

  View details for PubMedID 20551167

  View details for PubMedCentralID PMC2895191

• Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions PLOS GENETICS Burkhart, D. L., Wirt, S. E., Zmoos, A., Kareta, M. S., Sage, J. 2010; 6 (6)

  Abstract

  The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells.

  View details for DOI 10.1371/journal.pgen.1001003

  View details for Web of Science ID 000279805200032

  View details for PubMedID 20585628

• Loss of p130 Accelerates Tumor Development in a Mouse Model for Human Small-Cell Lung Carcinoma CANCER RESEARCH Schaffer, B. E., Park, K., Yiu, G., Conklin, J. F., Lin, C., Burkhart, D. L., Karnezis, A. N., Sweet-Cordero, E. A., Sage, J. 2010; 70 (10): 3877-3883

  Abstract

  Small-cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer. Although SCLC patients often initially respond to therapy, tumors nearly always recur, resulting in a 5-year survival rate of less than 10%. A mouse model has been developed based on the fact that the RB and p53 tumor suppressor genes are mutated in more than 90% of human SCLCs. Emerging evidence in patients and mouse models suggests that p130, a gene related to RB, may act as a tumor suppressor in SCLC cells. To test this idea, we used conditional mutant mice to delete p130 in combination with Rb and p53 in adult lung epithelial cells. We found that loss of p130 resulted in increased proliferation and significant acceleration of SCLC development in this triple-knockout mouse model. The histopathologic features of the triple-mutant mouse tumors closely resembled that of human SCLC. Genome-wide expression profiling experiments further showed that Rb/p53/p130-mutant mouse tumors were similar to human SCLC. These findings indicate that p130 plays a key tumor suppressor role in SCLC. Rb/p53/p130-mutant mice provide a novel preclinical mouse model to identify novel therapeutic targets against SCLC.

  View details for DOI 10.1158/0008-5472.CAN-09-4228

  View details for Web of Science ID 000278486300004

  View details for PubMedID 20406986

  View details for PubMedCentralID PMC2873158

• Complex transcriptional regulatory networks around the retinoblastoma tumor suppressor gene CELL CYCLE Burkhart, D. L., Sage, J. 2010; 9 (9): 1663-1664

  View details for Web of Science ID 000277402200002

  View details for PubMedID 20404509

• p107 in the public eye: an Rb understudy and more CELL DIVISION Wirt, S. E., Sage, J. 2010; 5

  Abstract

  p107 and its related family members Rb and p130 are critical regulators of cellular proliferation and tumorigenesis. Due to the extent of functional overlap within the Rb family, it has been difficult to assess which functions are exclusive to individual members and which are shared. Like its family members, p107 can bind a variety of cellular proteins to affect the expression of many target genes during cell cycle progression. Unlike Rb and p130, p107 is most highly expressed during the G1 to S phase transition of the cell cycle in actively dividing cells and accumulating evidence suggests a role for p107 during DNA replication. The specific roles for p107 during differentiation and development are less clear, although emerging studies suggest that it can cooperate with other Rb family members to control differentiation in multiple cell lineages. As a tumor suppressor, p107 is not as potent as Rb, yet studies in knockout mice have revealed some tumor suppressor functions in mice, depending on the context. In this review, we identify the unique and overlapping functions of p107 during the cell cycle, differentiation, and tumorigenesis.

  View details for DOI 10.1186/1747-1028-5-9

  View details for Web of Science ID 000283795500001

  View details for PubMedID 20359370

• Regulation of RB Transcription In Vivo by RB Family Members MOLECULAR AND CELLULAR BIOLOGY Burkhart, D. L., Ngai, L. K., Roake, C. M., Viatour, P., Thangavel, C., Ho, V. M., Knudsen, E. S., Sage, J. 2010; 30 (7): 1729-1745

  Abstract

  In cancer cells, the retinoblastoma tumor suppressor RB is directly inactivated by mutation in the RB gene or functionally inhibited by abnormal activation of cyclin-dependent kinase activity. While variations in RB levels may also provide an important means of controlling RB function in both normal and cancer cells, little is known about the mechanisms regulating RB transcription. Here we show that members of the RB and E2F families bind directly to the RB promoter. To investigate how the RB/E2F pathway may regulate Rb transcription, we generated reporter mice carrying an eGFP transgene inserted into a bacterial artificial chromosome containing most of the Rb gene. Expression of eGFP largely parallels that of Rb in transgenic embryos and adult mice. Using these reporter mice and mutant alleles for Rb, p107, and p130, we found that RB family members modulate Rb transcription in specific cell populations in vivo and in culture. Interestingly, while Rb is a target of the RB/E2F pathway in mouse and human cells, Rb expression does not strictly correlate with the cell cycle status of these cells. These experiments identify novel regulatory feedback mechanisms within the RB pathway in mammalian cells.

  View details for DOI 10.1128/MCB.00952-09

  View details for Web of Science ID 000275302000013

  View details for PubMedID 20100864

  View details for PubMedCentralID PMC2838078

• Keeping an Eye on Retinoblastoma Control of Human Embryonic Stem Cells JOURNAL OF CELLULAR BIOCHEMISTRY Conklin, J. F., Sage, J. 2009; 108 (5): 1023-1030

  Abstract

  Human embryonic stem cells (hESCs) hold great promise in regenerative medicine. However, before the full potential of these cells is achieved, major basic biological questions need to be addressed. In particular, there are still gaps in our knowledge of the molecular mechanisms underlying the derivation of hESCs from blastocysts, the regulation of the undifferentiated, pluripotent state, and the control of differentiation into specific lineages. Furthermore, we still do not fully understand the tumorigenic potential of hESCs, limiting their use in regenerative medicine. The RB pathway is a key signaling module that controls cellular proliferation, cell survival, chromatin structure, and cellular differentiation in mammalian cells. Members of the RB pathway are important regulators of hESC biology and manipulation of the activity of this pathway may provide novel means to control the fate of hESCs. Here we review what is known about the expression and function of members of the RB pathway in hESCs and discuss areas of interest in this field.

  View details for DOI 10.1002/jcb.22342

  View details for Web of Science ID 000272640900001

  View details for PubMedID 19760644

• Novel roles for A-type lamins in telomere biology and the DNA damage response pathway EMBO JOURNAL Gonzalez-Suarez, I., Redwood, A. B., Perkins, S. M., Vermolen, B., Lichtensztejin, D., Grotsky, D. A., Morgado-Palacin, L., Gapud, E. J., Sleckman, B. P., Sullivan, T., Sage, J., Stewart, C. L., Mai, S., Gonzalo, S. 2009; 28 (16): 2414-2427

  Abstract

  A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.

  View details for DOI 10.1038/emboj.2009.196

  View details for Web of Science ID 000269074400010

  View details for PubMedID 19629036

  View details for PubMedCentralID PMC2735177

• The role of the retinoblastoma/E2F1 tumor suppressor pathway in the lesion recognition step of nucleotide excision repair DNA REPAIR Lin, P. S., McPherson, L. A., Chen, A. Y., Sage, J., Ford, J. M. 2009; 8 (7): 795-802

  Abstract

  The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb-/-, p107-/-, p130-/- MEFs repaired both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1-/- MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1-/- cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair.

  View details for DOI 10.1016/j.dnarep.2009.03.003

  View details for PubMedID 19376752

• The retinoblastoma gene Rb and its family member p130 suppress lung adenocarcinoma induced by oncogenic K-Ras ONCOGENE Ho, V. M., Schaffer, B. E., Karnezis, A. N., Park, K. S., Sage, J. 2009; 28 (10): 1393-1399

  Abstract

  Mutations of the retinoblastoma tumor suppressor gene RB are frequently observed in human cancers, but rarely in non-small cell lung carcinomas (NSCLCs). Emerging evidence also suggests that the RB-related gene p130 is inactivated in a subset of human NSCLCs. To directly test the specific tumor suppressor roles of RB and p130 in NSCLC, we crossed Rb and p130 conditional mutant mice to mice carrying a conditional oncogenic K-Ras allele. In this model, controlled oncogenic K-Ras activation leads to the development of adenocarcinoma, a major subtype of NSCLC. We found that loss of p130 accelerated the death of mice, providing direct evidence in vivo that p130 is a tumor suppressor gene, albeit a weak one in this context. Loss of Rb increased the efficiency of lung cancer initiation and resulted in the development of high-grade adenocarcinomas and rapid death. Thus, despite the low frequency of RB mutations in human NSCLCs and reports that K-Ras activation and loss of RB function are rarely found in the same human tumors, loss of Rb clearly cooperates with activation of oncogenic K-Ras in lung adenocarcinoma development in mice.

  View details for DOI 10.1038/onc.2008.491

  View details for Web of Science ID 000264116000012

  View details for PubMedID 19151761

  View details for PubMedCentralID PMC2834234

• Hematopoietic Stem Cell Quiescence Is Maintained by Compound Contributions of the Retinoblastoma Gene Family CELL STEM CELL Viatour, P., Somervaille, T. C., Venkatasubrahmanyam, S., Kogan, S., McLaughlin, M. E., Weissman, I. L., Butte, A. J., Passegue, E., Sage, J. 2008; 3 (4): 416-428

  Abstract

  Individual members of the retinoblastoma (Rb) tumor suppressor gene family serve critical roles in the control of cellular proliferation and differentiation, but the extent of their contributions is masked by redundant and compensatory mechanisms. Here we employed a conditional knockout strategy to simultaneously inactivate all three members, Rb, p107, and p130, in adult hematopoietic stem cells (HSCs). Rb family triple knockout (TKO) mice develop a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors and is accompanied by increased apoptosis in lymphoid progenitor populations. Loss of quiescence in the TKO HSC pool is associated with an expansion of these mutant stem cells but also with an enhanced mobilization and an impaired reconstitution potential upon transplantation. The presence of a single p107 allele is sufficient to largely rescue these defects. Thus, Rb family members collectively maintain HSC quiescence and the balance between lymphoid and myeloid cell fates in the hematopoietic system.

  View details for DOI 10.1016/j.stem.2008.07.009

  View details for Web of Science ID 000260149800012

  View details for PubMedID 18940733

  View details for PubMedCentralID PMC2646421

• Cellular mechanisms of tumour suppression by the retinoblastoma gene NATURE REVIEWS CANCER Burkhart, D. L., Sage, J. 2008; 8 (9): 671-682

  Abstract

  The retinoblastoma (RB) tumour suppressor gene is functionally inactivated in a broad range of paediatric and adult cancers, and a plethora of cellular functions and partners have been identified for the RB protein. Data from human tumours and studies from mouse models indicate that loss of RB function contributes to both cancer initiation and progression. However, we still do not know the identity of the cell types in which RB normally prevents cancer initiation in vivo, and the specific functions of RB that suppress distinct aspects of the tumorigenic process are poorly understood.

  View details for DOI 10.1038/nrc2399

  View details for Web of Science ID 000258662900012

  View details for PubMedID 18650841

• GFP reporter mice for the retinoblastoma-related cell cycle regulator p107 CELL CYCLE Burkhart, D. L., Viatour, P., Ho, V. M., Sage, J. 2008; 7 (16): 2544-2552

  Abstract

  The RB tumor suppressor gene is mutated in a broad range of human cancers, including pediatric retinoblastoma. Strikingly, however, Rb mutant mice develop tumors of the pituitary and thyroid glands, but not retinoblastoma. Mouse genetics experiments have demonstrated that p107, a protein related to pRB, is capable of preventing retinoblastoma, but not pituitary tumors, in Rb-deficient mice. Evidence suggests that the basis for this compensatory function of p107 is increased transcription of the p107 gene in response to Rb inactivation. To begin to address the context-dependency of this compensatory role of p107 and to follow p107 expression in vivo, we have generated transgenic mice carrying an enhanced GFP (eGFP) reporter inserted into a bacterial artificial chromosome (BAC) containing the mouse p107 gene. Expression of the eGFP transgene parallels that of p107 in these transgenic mice and identifies cells with a broad range of expression level for p107, even within particular organs or tissues. We also show that loss of Rb results in the upregulation of p107 transcription in specific cell populations in vivo, including subpopulations of hematopoietic cells. Thus, p107 BAC-eGFP transgenic mice serve as a useful tool to identify distinct cell types in which p107 is expressed and may have key functions in vivo, and to characterize changes in cellular networks accompanying Rb deficiency.

  View details for Web of Science ID 000258829900016

  View details for PubMedID 18719374

• Hope in sight for retinoblastoma NATURE MEDICINE Sage, J. 2007; 13 (1): 30-31

  View details for Web of Science ID 000243301800024

  View details for PubMedID 17206131

• pRB family proteins are required for H3K27 trimethylation and polycomb repression complexes binding to and silencing p16(INK4a) tumor suppressor gene GENES & DEVELOPMENT Kotake, Y., Cao, R., Viatour, P., Sage, J., Zhang, Y., Xiong, Y. 2007; 21 (1): 49-54

  Abstract

  Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16(INK4a) expression. Here, we demonstrate that Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16.

  View details for DOI 10.1101/gad.1499407

  View details for Web of Science ID 000243382700005

  View details for PubMedID 17210787

  View details for PubMedCentralID PMC1759899

• The related retinoblastoma (pRb) and p130 proteins cooperate to regulate homeostasis in the intestinal epithelium JOURNAL OF BIOLOGICAL CHEMISTRY Haigis, K., Sage, J., Glickman, J., Shafer, S., Jacks, T. 2006; 281 (1): 638-647

  Abstract

  pRb, p107, and p130 are related proteins that play a central role in the regulation of cell cycle progression and terminal differentiation in mammalian cells. Nevertheless, it is still largely unclear how these proteins achieve this regulation in vivo. The intestinal epithelium is an ideal in vivo system in which to study the molecular pathways that regulate proliferation and differentiation because it exists in a constant state of development throughout an animal's lifetime. We studied the phenotypic effects on the intestinal epithelium of mutating Rb and p107 or p130. Although mutating these genes singly had little or no effect, loss of pRb and p107 or p130 together produced chronic hyperplasia and dysplasia of the small intestinal and colonic epithelium. In Rb/p130 double mutants this hyperplasia was associated with defects in terminal differentiation of specific cell types and was dependent on the increased proliferation seen in the epithelium of mutant animals. At the molecular level, dysregulation of the Rb pathway led to an increase in the expression of Math1, Cdx1, Cdx2, transcription factors that regulate proliferation and differentiation in the intestinal epithelium. The absence of Cdx1 function in Rb/p130 double mutant mice partially reverted the histologic phenotype by suppressing ectopic mitosis in the epithelium. These studies implicate the Rb pathway as a regulator of epithelial homeostasis in the intestine.

  View details for Web of Science ID 000234307200076

  View details for PubMedID 16258171

• C/EBP beta cooperates with RB : E2F to implement Ras(V12)-induced cellular senescence EMBO JOURNAL Sebastian, T., Malik, R., Thomas, S., Sage, J., Johnson, P. F. 2005; 24 (18): 3301-3312

  Abstract

  In primary cells, overexpression of oncogenes such as Ras(V12) induces premature senescence rather than transformation. Senescence is an irreversible form of G1 arrest that requires the p19ARF/p53 and p16INK4a/pRB pathways and may suppress tumorigenesis in vivo. Here we show that the transcription factor C/EBPbeta is required for Ras(V12)-induced senescence. C/EBPbeta-/- mouse embryo fibroblasts (MEFs) expressing Ras(V12) continued to proliferate despite unimpaired induction of p19ARF and p53, and lacked morphological features of senescent fibroblasts. Enforced C/EBPbeta expression inhibited proliferation of wild-type MEFs and also slowed proliferation of p19Arf-/- and p53-/- cells, indicating that C/EBPbeta acts downstream or independently of p19ARF/p53 to suppress growth. C/EBPbeta was unable to inhibit proliferation of MEFs lacking all three RB family proteins or wild-type cells expressing dominant negative E2F-1 and, instead, stimulated their growth. C/EBPbeta decreased expression of several E2F target genes and was associated with their promoters in chromatin immunoprecipitation assays, suggesting that C/EBPbeta functions by repressing genes required for cell cycle progression. C/EBPbeta is therefore a novel component of the RB:E2F-dependent senescence program activated by oncogenic stress in primary cells.

  View details for DOI 10.1038/sj.emboj.7600789

  View details for Web of Science ID 000232551600014

  View details for PubMedID 16107878

  View details for PubMedCentralID PMC1224679

• Making young tumors old: a new weapon against cancer? Science of aging knowledge environment : SAGE KE Sage, J. 2005; 2005 (33): pe25-?

  Abstract

  As the population of industrial nations ages, the incidence of cancer and cancer mortality is increasing. Intuitively, older organisms may be less able to cope with accumulated damage and thus be more prone to develop cancer. However, so far, the links between aging and cancer have been only partially explored. Strikingly, four recent studies now indicate that premature senescence accompanied by cell cycle arrest occurs in tumors initiated by an oncogenic mutation. Thus, senescence may act as a key tumor suppressor mechanism in young tumors in vivo.

  View details for PubMedID 16107660

• Cell type-specific effects of Rb deletion in the murine retina GENES & DEVELOPMENT Macpherson, D., Sage, J., Kim, T., Ho, D., McLaughlin, M. E., Jacks, T. 2004; 18 (14): 1681-1694

  Abstract

  Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.

  View details for DOI 10.1101/gad.1203304

  View details for Web of Science ID 000222742100005

  View details for PubMedID 15231717

• Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents ONCOGENE Mayhew, C. N., Perkin, L. M., Zhang, X. P., Sage, J., Jacks, T., Knudsen, E. S. 2004; 23 (23): 4107-4120

  Abstract

  The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.

  View details for DOI 10.1038/sj.onc.1207503

  View details for Web of Science ID 000221520200009

  View details for PubMedID 15064736

• Cyclin C makes an entry into the cell cycle DEVELOPMENTAL CELL Sage, J. 2004; 6 (5): 607-608

  Abstract

  From yeast to humans, cell cycle progression is orchestrated by the oscillation of kinase activities associated with cyclins. In an article published recently in Cell, Ren and Rollins investigate mechanisms controlling the G0/G1 transition in quiescent cells and identify new cyclin C/Cdk3 complexes as key regulators of cell cycle reentry in human cells.

  View details for Web of Science ID 000222443100001

  View details for PubMedID 15130482

• RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult NUCLEIC ACIDS RESEARCH Bosco, E. E., Mayhew, C. N., Hennigan, R. F., Sage, J., Jacks, T., Knudsen, E. S. 2004; 32 (1): 25-34

  Abstract

  Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (gammaH2AX) foci, which indicate DNA double-strand breaks. The formation of gammaH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak gammaH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these gammaH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce gammaH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.

  View details for DOI 10.1093/nar/gkg919

  View details for Web of Science ID 000188988700018

  View details for PubMedID 14704340

• Recapitulation of the effects of the human papillomavirus type 16 E7 oncogene on mouse epithelium by somatic Rb deletion and detection of pRb-independent effects of E7 in vivo MOLECULAR AND CELLULAR BIOLOGY Balsitis, S. J., Sage, J., Duensing, S., Munger, K., Jacks, T., Lambert, P. F. 2003; 23 (24): 9094-9103

  Abstract

  Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.

  View details for DOI 10.1128/MBC.23.24.9094-9103.2003

  View details for Web of Science ID 000187040400017

  View details for PubMedID 14645521

• Perp is a mediator of p53-dependent apoptosis in diverse cell types CURRENT BIOLOGY Ihrie, R. A., Reczek, E., Horner, J. S., Khachatrian, L., Sage, J., Jacks, T., Attardi, L. D. 2003; 13 (22): 1985-1990

  Abstract

  The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.

  View details for DOI 10.1016/j.cub.2003.10.055

  View details for Web of Science ID 000186558100026

  View details for PubMedID 14614825

• Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry NATURE Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M., Jacks, T. 2003; 424 (6945): 223-228

  Abstract

  Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumour suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumour suppressor gene in humans, we have produced a conditional allele of the mouse Rb gene. We show here that acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. We also show that acute loss of Rb in senescent cells leads to reversal of the cellular senescence programme. Thus, the use of conditional knockout strategies might refine our understanding of gene function and help to model human cancer more accurately.

  View details for DOI 10.1038/nature01764

  View details for Web of Science ID 000184032700049

  View details for PubMedID 12853964

• Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system MOLECULAR AND CELLULAR BIOLOGY Macpherson, D., Sage, J., Crowley, D., Trumpp, A., Bronson, R. T., Jacks, T. 2003; 23 (3): 1044-1053

  Abstract

  Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.

  View details for DOI 10.1128/MCB.23.3.1044-1053.2003

  View details for Web of Science ID 000180673600026

  View details for PubMedID 12529408

• An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation CELL Klappacher, G. W., Lunyak, V. V., Sykes, D. B., Sawka-Verhelle, D., Sage, J., Brard, G., Ngo, S. D., Gangadharan, D., Jacks, T., Kamps, M. P., Rose, D. W., Rosenfeld, M. G., Glass, C. K. 2002; 109 (2): 169-180

  Abstract

  Defining the molecular mechanisms that coordinately regulate proliferation and differentiation is a central issue in development. Here, we describe a mechanism in which induction of the Ets repressor METS/PE1 links terminal differentiation to cell cycle arrest. Using macrophages as a model, we provide evidence that METS/PE1 blocks Ras-dependent proliferation without inhibiting Ras-dependent expression of cell type-specific genes by selectively replacing Ets activators on the promoters of cell cycle control genes. Antiproliferative effects of METS require its interaction with DP103, a DEAD box-containing protein that assembles a novel corepressor complex. Functional interactions between the METS/DP103 complex and E2F/ pRB family proteins are also necessary for inhibition of cellular proliferation, suggesting a combinatorial code that directs permanent cell cycle exit during terminal differentiation.

  View details for Web of Science ID 000175082600007

  View details for PubMedID 12007404

• Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA de Vries, A., Flores, E. R., Miranda, B., Hsieh, H. M., van Oostrom, C. T., Sage, J., Jacks, T. 2002; 99 (5): 2948-2953

  Abstract

  The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

  View details for DOI 10.1073/pnas.052713099

  View details for Web of Science ID 000174284600065

  View details for PubMedID 11867759

• The RB gene family and control of the cell cycle BULLETIN DU CANCER Sage, J. 2001; 88 (6): 541-543

  View details for Web of Science ID 000169993400001

  View details for PubMedID 11459699

• Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization GENES & DEVELOPMENT Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S. Q., Williams, B., Theodorou, E., Jacks, T. 2000; 14 (23): 3037-3050

  Abstract

  The retinoblastoma protein, pRB, and the closely related proteins p107 and p130 are important regulators of the mammalian cell cycle. Biochemical and genetic studies have demonstrated overlapping as well as distinct functions for the three proteins in cell cycle control and mouse development. However, the role of the pRB family as a whole in the regulation of cell proliferation, cell death, or cell differentiation is not known. We generated embryonic stem (ES) cells and other cell types mutant for all three genes. Triple knock-out mouse embryonic fibroblasts (TKO MEFs) had a shorter cell cycle than wild-type, single, or double knock-out control cells. TKO cells were resistant to G(1) arrest following DNA damage, despite retaining functional p53 activity. They were also insensitive to G(1) arrest signals following contact inhibition or serum starvation. Finally, TKO MEFs did not undergo senescence in culture and do possess some characteristics of transformed cells. Our results confirm the essential role of the Rb family in the control of the G(1)/S transition, place the three Rb family members downstream of multiple cell cycle control pathways, and further the link between loss of cell cycle control and tumorigenesis.

  View details for Web of Science ID 000165788600011

  View details for PubMedID 11114892

• Sex hormone-induced carcinogenesis in Rb-deficient prostate tissue CANCER RESEARCH Wang, Y. H., Hayward, S. W., Donjacour, A. A., Young, P., Jacks, T., Sage, J., Dahiya, R., Cardiff, R. D., Day, M. L., Cunha, G. R. 2000; 60 (21): 6008-6017

  Abstract

  The retinoblastoma (Rb) gene product is a prototypic tumor suppressor. Mice lacking the Rb gene are not viable and die in utero at approximately 13 days of gestation. In this study, we have rescued Rb-/- prostates by grafting pelvic organ rudiments from Rb-/- mouse embryos under the renal capsule of adult male nude mouse hosts. Grafts of embryonic pelvic organs developed into functional prostatic tissue. Some of the prostatic tissue generated was further used to construct chimeric prostatic tissue recombinants by combining wild-type rat urogenital mesenchyme (rUGM) with Rb-/- and Rb+/+ prostatic epithelium (PRE). The tissue recombinants were grown as subcapsular renal grafts and treated from the time of grafting with Silastic capsules containing 25 mg of testosterone plus 2.5 mg of estradiol. During 5-8 weeks of hormone treatment, rUGM+Rb+/+PRE tissue recombinants developed prostatic hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue recombinants developed hyperplasia, atypical hyperplasia, and carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue recombinants, prostatic epithelial cells of the basal lineage disappeared, whereas the luminal cells underwent carcinogenesis. Epithelial E-cadherin almost totally disappeared. In all cases, epithelial PCNA labeling was elevated in tissue recombinants containing Rb-/- versus Rb+/+ epithelium. These epithelial changes were associated with almost total loss of smooth muscle cells in the stroma. In contrast, in untreated hosts rUGM+Rb+/+PRE tissue recombinants developed normally, and rUGM+Rb-/-PRE tissue recombinants developed mild epithelial hyperplasia. The results of this study demonstrate that Rb-/- prostatic tissue can be rescued from embryonic lethal mice and used to test its susceptibility to hormonal carcinogenesis. Deletion of the Rb gene predisposes prostatic epithelium to hyperplasia and increases proliferative activity Susceptibility to hormonal carcinogenesis in response to exogenous testosterone + estradiol is manifested in the progression from atypica hyperplasia to carcinoma. Thus, these findings demonstrate that the absence of the Rb tumor suppressor gene may predispose prostatic epithelial cells to carcinogenesis. Rescue of organs from Rb-/- embryos not only provides an opportunity to analyze the Rb gene pathway in the development and progression of prostate cancer but also provides an opportunity for specifically evaluating the role of the Rb pathway in development and carcinogenesis in other organs, such as the mammary gland and colon. Because rUGM greatly stimulates prostatic epithelial proliferation, the tissue recombinant model is a particularly useful tool for assessing the functional role of other genes in prostatic carcinogenesis through use of the appropriate transgenic or gene knockout mice.

  View details for Web of Science ID 000165230300021

  View details for PubMedID 11085521

• NF-kappa B is developmentally regulated during spermatogenesis in mice DEVELOPMENTAL DYNAMICS Lilienbaum, A., Sage, J., Memet, S., Rassoulzadegan, M., Cuzin, F., Israel, A. 2000; 219 (3): 333-340

  Abstract

  To analyze NF-kappa B activity in the testis, we used murine transgenic lines carrying a LacZ reporter gene under the control of a NF-kappa B-responsive promoter (Schmidt-Ullrich et al. [1996] Dev 122:2117-2128). We constructed three independent lines containing the promoter of the gene encoding p105, the precursor of the p50 subunit. This promoter contains three NF-kappa B-binding sites in its proximal part. Our results show that in adult mice, the beta-galactosidase activity which reflects nuclear NF-kappa B activity, is first detected in spermatocytes at the pachytene stage and remains activated in the following steps of germ cell differentiation and maturation. Using transgenic mice carrying a p105nlslacZ construct with the 3 NF-kappa B sites mutated in the p105 promoter, we found a significant reduction in the transgene activity, confirming the important role of NF-kappa B in the activation of the transgene. To confirm the stage of induction during spermatogenesis, we analysed the beta-galactosidase activity in the testes from prepuberal mice in which cells synchrouneously enter meiosis. We detected the transgene activity at 18 days after birth, corresponding to the pachytene stage in spermatocytes. In nuclear extracts prepared from prepuberal mice, we found a peak of NF-kappa B DNA-binding activity made of p50 and p65 subunits at day 18 after birth, which remains high in the later stages. Further analysis showed that I kappa B alpha and beta, but not epsilon are expressed in the testes. Altogether, these data suggest that NF-kappa B factors are stage specifically controlled and may play a role during the development of sperm cells.

  View details for Web of Science ID 000165122200005

  View details for PubMedID 11066090

• Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21(WAF1/CIP1) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, Y., Griffith, E. C., Sage, J., Jacks, T., Liu, J. O. 2000; 97 (12): 6427-6432

  Abstract

  Angiogenesis has been demonstrated to be essential for tumor growth and metastasis, and inhibition of angiogenesis is emerging as a promising strategy for treating cancer. Among the most potent inhibitors of angiogenesis is the fumagillin family of natural products. An analog of fumagillin, known as TNP-470 or AGM-1470, has been undergoing clinical trials for treating a variety of cancers. TNP-470 has been shown to block endothelial cell cycle progression in the late G(1) phase. Although the direct molecular target for TNP-470 has been identified as the type 2 methionine aminopeptidase (MetAP2), how inhibition of this enzyme leads to cell cycle arrest has remained unclear. We report that treatment of endothelial and other drug-sensitive cell types leads to the activation of the p53 pathway, causing an accumulation of the G(1) cyclin-dependent kinase inhibitor p21(WAF1/CIP1). The requirement of p53 and p21(WAF1/CIP1) for the cell cycle inhibition by TNP-470 is underscored by the observation that cells deficient in p53 and p21(WAF1/CIP1) are resistant to TNP-470. These results shed significant light on the mechanism of cell cycle inhibition by TNP-470 and suggest an alternative method of activating p53 in endothelial cells to halt angiogenesis and tumor progression.

  View details for Web of Science ID 000087526300037

  View details for PubMedID 10841547

• Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad MECHANISMS OF DEVELOPMENT Sage, J., Martin, L., Meuwissen, R., Heyting, C., Cuzin, F., Rassoulzadegan, M. 1999; 80 (1): 29-39

  Abstract

  Transcription controls active at the initial stages of meiosis are clearly key elements in the regulation of germinal differentiation. Transcription of the Sycp1 gene (synaptonemal complex protein 1) starts as early as the leptotene and zygotene stages. Constructs with Sycp1 5' upstream sequences directed the expression of reporter genes to pachytene spermatocytes in transgenic mice. A short fragment encompassing the transcription start (n.t. -54 to +102) was sufficient for stage-specific expression in the adult male and for temporal regulation during development. Upstream enhancer element(s) quantitatively regulating expression were localized in the region between -54 and -260. The gene is normally expressed both in the male and female gonads, but none of the promoter sequences active in the testis allowed the expression of reporter genes during meiosis in the ovary.

  View details for Web of Science ID 000078473700003

  View details for PubMedID 10096061

• Cre expression in primary spermatocytes: A tool for genetic engineering of the germ line MOLECULAR REPRODUCTION AND DEVELOPMENT Vidal, F., Sage, J., Cuzin, F., Rassoulzadegan, M. 1998; 51 (3): 274-280

  Abstract

  Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1-Cre and a second transgene where LoxP sites flank either the beta geo coding region, the Pgk1 promoter, or a tk-neo cassette inserted into the Rxr alpha locus. The LoxP-flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double-transgenic males with normal females showed extensive deletions of the LoxP-flanked sequences. When the males were hemizygous for the Sycp1-Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1-Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process.

  View details for Web of Science ID 000076145500006

  View details for PubMedID 9771647

• Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis DEVELOPMENT Vincent, S., Segretain, D., Nishikawa, S., Nishikawa, S., Sage, J., Cuzin, F., Rassoulzadegan, M. 1998; 125 (22): 4585-4593

  Abstract

  The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.

  View details for Web of Science ID 000077649200022

  View details for PubMedID 9778516

• Stage-specific signals in germ line differentiation: Control of Sertoli cell phagocytic activity by spermatogenic cells DEVELOPMENTAL BIOLOGY Grandjean, V., Sage, J., Ranc, F., Cuzin, F., Rassoulzadegan, M. 1997; 184 (1): 165-174

  Abstract

  Differentiation of male germ cells requires a continuous cross-talk with their somatic support, the Sertoli cell. An in vitro model of Sertoli cells was recently provided by established cell lines which maintain Sertoli-specific characteristics, among which is a regulated phagocytic capacity. In vivo, Sertoli cells take up the residual cytoplasm expelled from the maturing sperm, a process restricted to a limited period of germinal maturation, and they also eliminate abnormally differentiated germ cells in case of hormonal deficiency. Cells of the Sertoli line efficiently take up latex beads, as well as dead cells in the cultures. A semiquantitative assay of phagocytosis was developed, based on the uptake of fluorescent latex beads. 15P-1 cultures were found to contain a minor fraction of active phagocytes. After addition of a defined fraction of germ cells, however, all the cells internalized beads as efficiently as macrophages. The inducing cell was identified as the pachytene spermatocyte, a cell type which, in vivo, is associated with Sertoli cells when they express their phagocytic potential. These inducing meiotic cells were not internalized themselves. Rather, they interacted with Sertoli cells via a surface signal that was resistant to formaldehyde fixation. The whole induction process does not involve changes in Sertoli gene expression, since it occurs even in the presence of high doses of cycloheximide. After the required initial contact, further maintenance of the activity was dependent on factor(s) secreted in the medium of the activated culture. Phagocytosis was, on the other hand, abrogated in the presence of factor(s) secreted by a distinct fraction of germ cells, enriched in the late stages (second division) of meiosis.

  View details for Web of Science ID A1997WV15600015

  View details for PubMedID 9142992

• TRANSMEIOTIC DIFFERENTIATION OF MALE GERM-CELLS IN CULTURE CELL Rassoulzadegan, M., PAQUISFLUCKLINGER, V., Bertino, B., Sage, J., Jasin, M., Miyagawa, K., VANHEYNINGEN, V., Besmer, P., Cuzin, F. 1993; 75 (5): 997-1006

  Abstract

  A cell culture system that supports the differentiation of male germ cells through meiosis is described. It takes advantage of the properties of a cell line, 15P-1, established from testicular cells of transgenic mice that express the large T protein of polyoma virus in the seminiferous epithelium. This line exhibits features characteristics of Sertoli cells, including transcription of the Wilms' tumor (WT1) and Steel genes. Cells of the 15P-1 type support the meiotic and postmeiotic differentiation in cocultures of diploid premeiotic germ cells into haploid spermatids expressing the protamine (Prm-1) gene. When cocultured with 15P-1 cells, testicular cells explanted from immature 9-day-old animals, before the onset of the first meiosis, generated tetrads of haploid cells with the morphology of round spermatids and initiated protamine transcription.

  View details for Web of Science ID A1993MK96600019

  View details for PubMedID 7504588