Monte Winslow

All Publications

• CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors. Nature Skoulidis, F., Araujo, H. A., Do, M. T., Qian, Y., Sun, X., Cobo, A. G., Le, J. T., Montesion, M., Palmer, R., Jahchan, N., Juan, J. M., Min, C., Yu, Y., Pan, X., Arbour, K. C., Vokes, N., Schmidt, S. T., Molkentine, D., Owen, D. H., Memmott, R., Patil, P. D., Marmarelis, M. E., Awad, M. M., Murray, J. C., Hellyer, J. A., Gainor, J. F., Dimou, A., Bestvina, C. M., Shu, C. A., Riess, J. W., Blakely, C. M., Pecot, C. V., Mezquita, L., Tabbó, F., Scheffler, M., Digumarthy, S., Mooradian, M. J., Sacher, A. G., Lau, S. C., Saltos, A. N., Rotow, J., Johnson, R. P., Liu, C., Stewart, T., Goldberg, S. B., Killam, J., Walther, Z., Schalper, K., Davies, K. D., Woodcock, M. G., Anagnostou, V., Marrone, K. A., Forde, P. M., Ricciuti, B., Venkatraman, D., Van Allen, E. M., Cummings, A. L., Goldman, J. W., Shaish, H., Kier, M., Katz, S., Aggarwal, C., Ni, Y., Azok, J. T., Segal, J., Ritterhouse, L., Neal, J. W., Lacroix, L., Elamin, Y. Y., Negrao, M. V., Le, X., Lam, V. K., Lewis, W. E., Kemp, H. N., Carter, B., Roth, J. A., Swisher, S., Lee, R., Zhou, T., Poteete, A., Kong, Y., Takehara, T., Paula, A. G., Parra Cuentas, E. R., Behrens, C., Wistuba, I. I., Zhang, J., Blumenschein, G. R., Gay, C., Byers, L. A., Gibbons, D. L., Tsao, A., Lee, J. J., Bivona, T. G., Camidge, D. R., Gray, J. E., Lieghl, N., Levy, B., Brahmer, J. R., Garassino, M. C., Gandara, D. R., Garon, E. B., Rizvi, N. A., Scagliotti, G. V., Wolf, J., Planchard, D., Besse, B., Herbst, R. S., Wakelee, H. A., Pennell, N. A., Shaw, A. T., Jänne, P. A., Carbone, D. P., Hellmann, M. D., Rudin, C. M., Albacker, L., Mann, H., Zhu, Z., Lai, Z., Stewart, R., Peters, S., Johnson, M. L., Wong, K. K., Huang, A., Winslow, M. M., Rosen, M. J., Winters, I. P., Papadimitrakopoulou, V. A., Cascone, T., Jewsbury, P., Heymach, J. V. 2024

  Abstract

  For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4+ and CD8+ T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

  View details for DOI 10.1038/s41586-024-07943-7

  View details for PubMedID 39385035

  View details for PubMedCentralID 6030433

• Experimental variables determine the outcome of RAS-RAS interactions. The Journal of biological chemistry Zhou, Z., Nguyen, T. L., Li, X., Poujol, C., Berlinska, E., Michelina, S. V., Kapp, J. N., Plückthun, A., Winslow, M. M., Ambrogio, C., Shan, Y., Santamaría, D., Westover, K. D. 2024: 107859

  Abstract

  RAS clustering at the cell membrane is critical to activate signaling in cells, but whether this clustering is mediated exclusively by its c-terminal hypervariable region, receives contributions from the G-domain of RAS, and/or is influenced by secondary effectors has been intensely debated. Reports that G-domain mutations do not modulate RAS-RAS interactions, have led some to question the validity of previous experiments that indicate the G-domain plays a role in RAS clustering/interactions. Here we reconcile these findings by clarifying the impact of experimental variables, such as protein expression levels, cellular context, RAS zygosity, and secondary effector interactions on RAS clustering. Lack of control over these variables impact the results using G-domain mutations across various assay systems and can lead to unsound conclusions.

  View details for DOI 10.1016/j.jbc.2024.107859

  View details for PubMedID 39374781

• A Lung Cancer Mouse Model Database. bioRxiv : the preprint server for biology Cai, L., Gao, Y., DeBerardinis, R., Chen, T., Winslow, M., Xiao, G., Rudin, C., Oliver, T., Minna, J., Xie, Y. 2024

  Abstract

  Lung cancer, the leading cause of cancer mortality, exhibits diverse histological subtypes and genetic complexities. Numerous preclinical models have been developed to study lung cancer, but data from these models are disparate, siloed, and difficult to compare in a centralized fashion. Here we established the Lung Cancer Mouse Model Database (LCMMDB), an extensive repository of 1,354 samples from 77 transcriptomic datasets. Meticulous curation and collaboration with data depositors have produced a robust and comprehensive database, enhancing the fidelity of the genetic landscape it depicts. The LCMMDB aligns 859 tumors from genetically engineered mouse models (GEMMs) with human lung cancer mutations, enabling comparative analysis and revealing a pressing need to broaden the diversity of genetic aberrations modeled in GEMMs. Accompanying this resource, we developed a web application that offers researchers intuitive tools for in-depth gene expression analysis and fostering potential collaborations. With standardized reprocessing of gene expression data, the LCMMDB serves as a powerful platform for cross-study comparison and lays the groundwork for future research, aiming to bridge the gap between mouse models and human lung cancer for improved translational relevance.

  View details for DOI 10.1101/2024.02.28.582577

  View details for PubMedID 38464291

• Oncogenic context shapes the fitness landscape of tumor suppression. Nature communications Blair, L. M., Juan, J. M., Sebastian, L., Tran, V. B., Nie, W., Wall, G. D., Gerceker, M., Lai, I. K., Apilado, E. A., Grenot, G., Amar, D., Foggetti, G., Do Carmo, M., Ugur, Z., Deng, D., Chenchik, A., Paz Zafra, M., Dow, L. E., Politi, K., MacQuitty, J. J., Petrov, D. A., Winslow, M. M., Rosen, M. J., Winters, I. P. 2023; 14 (1): 6422

  Abstract

  Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged-the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context-and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

  View details for DOI 10.1038/s41467-023-42156-y

  View details for PubMedID 37828026

  View details for PubMedCentralID 8412936

• 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

• Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma. Proceedings of the National Academy of Sciences of the United States of America Yousefi, M., Andrejka, L., Szamecz, M., Winslow, M. M., Petrov, D. A., Boross, G. 2023; 120 (38): e2303224120

  Abstract

  Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

  View details for DOI 10.1073/pnas.2303224120

  View details for PubMedID 37695905

• Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression. Nature methods Zengel, J., Wang, Y. X., Seo, J. W., Ning, K., Hamilton, J. N., Wu, B., Raie, M., Holbrook, C., Su, S., Clements, D. R., Pillay, S., Puschnik, A. S., Winslow, M. M., Idoyaga, J., Nagamine, C. M., Sun, Y., Mahajan, V. B., Ferrara, K. W., Blau, H. M., Carette, J. E. 2023

  Abstract

  The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

  View details for DOI 10.1038/s41592-023-01896-x

  View details for PubMedID 37291262

  View details for PubMedCentralID 3337962

• High-Throughput Identification, Modeling, and Analysis of Cancer Driver Genes In Vivo. Cold Spring Harbor perspectives in medicine Tang, Y. J., Shuldiner, E. G., Karmakar, S., Winslow, M. M. 2023

  Abstract

  The vast number of genomic and molecular alterations in cancer pose a substantial challenge to uncovering the mechanisms of tumorigenesis and identifying therapeutic targets. High-throughput functional genomic methods in genetically engineered mouse models allow for rapid and systematic investigation of cancer driver genes. In this review, we discuss the basic concepts and tools for multiplexed investigation of functionally important cancer genes in vivo using autochthonous cancer models. Furthermore, we highlight emerging technical advances in the field, potential opportunities for future investigation, and outline a vision for integrating multiplexed genetic perturbations with detailed molecular analyses to advance our understanding of the genetic and molecular basis of cancer.

  View details for DOI 10.1101/cshperspect.a041382

  View details for PubMedID 37277208

• Phagocytosis increases an oxidative metabolic and immune suppressive signature in tumor macrophages. The Journal of experimental medicine Gonzalez, M. A., Lu, D. R., Yousefi, M., Kroll, A., Lo, C. H., Briseño, C. G., Watson, J. E., Novitskiy, S., Arias, V., Zhou, H., Plata Stapper, A., Tsai, M. K., Ashkin, E. L., Murray, C. W., Li, C. M., Winslow, M. M., Tarbell, K. V. 2023; 220 (6)

  Abstract

  Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.

  View details for DOI 10.1084/jem.20221472

  View details for PubMedID 36995340

• Dissecting metastasis using preclinical models and methods. Nature reviews. Cancer Hebert, J. D., Neal, J. W., Winslow, M. M. 2023

  Abstract

  Metastasis has long been understood to lead to the overwhelming majority of cancer-related deaths. However, our understanding of the metastatic process, and thus our ability to prevent or eliminate metastases, remains frustratingly limited. This is largely due to the complexity of metastasis, which is a multistep process that likely differs across cancer types and is greatly influenced by many aspects of the in vivo microenvironment. In this Review, we discuss the key variables to consider when designing assays to study metastasis: which source of metastatic cancer cells to use and where to introduce them into mice to address different questions of metastasis biology. We also examine methods that are being used to interrogate specific steps of the metastatic cascade in mouse models, as well as emerging techniques that may shed new light on previously inscrutable aspects of metastasis. Finally, we explore approaches for developing and using anti-metastatic therapies, and how mouse models can be used to test them.

  View details for DOI 10.1038/s41568-023-00568-4

  View details for PubMedID 37138029

  View details for PubMedCentralID 5308465

• 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

• Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth. Nature cell biology Tang, R., Shuldiner, E. G., Kelly, M., Murray, C. W., Hebert, J. D., Andrejka, L., Tsai, M. K., Hughes, N. W., Parker, M. I., Cai, H., Li, Y. C., Wahl, G. M., Dunbrack, R. L., Jackson, P. K., Petrov, D. A., Winslow, M. M. 2023

  Abstract

  Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.

  View details for DOI 10.1038/s41556-022-01049-w

  View details for PubMedID 36635501

• A new system for multiplexed mosaic analysis of gene function in the mouse. Cell reports methods Cai, H., Winslow, M. M. 2022; 2 (9): 100295

  Abstract

  In a recent issue of Cell, Liu etal. present an innovative mouse model system in which Cre/lox stochastically turns on transgenic expression of one out of up to 100 sgRNAs in somatic cells, creating genetic mosaicism that enables the multiplexed assessment of gene function invivo.

  View details for DOI 10.1016/j.crmeth.2022.100295

  View details for PubMedID 36160047

• Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles. Molecular cell Hughes, N. W., Qu, Y., Zhang, J., Tang, W., Pierce, J., Wang, C., Agrawal, A., Morri, M., Neff, N., Winslow, M. M., Wang, M., Cong, L. 2022

  Abstract

  The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.

  View details for DOI 10.1016/j.molcel.2022.06.001

  View details for PubMedID 35752172

• Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities. Cancer research Yousefi, M., Boross, G., Weiss, C., Murray, C. W., Hebert, J. D., Cai, H., Ashkin, E. L., Karmakar, S., Andrejka, L., Chen, L., Wang, M., Tsai, M. K., Lin, W., Li, C., Yakhchalian, P., Colon, C. I., Chew, S., Chu, P., Swanton, C., Kunder, C. A., Petrov, D. A., Winslow, M. M. 2022; 82 (8): 1589-1602

  Abstract

  Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma.SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

  View details for DOI 10.1158/0008-5472.CAN-22-0059

  View details for PubMedID 35425962

• LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer. Nature communications Murray, C. W., Brady, J. J., Han, M., Cai, H., Tsai, M. K., Pierce, S. E., Cheng, R., Demeter, J., Feldser, D. M., Jackson, P. K., Shackelford, D. B., Winslow, M. M. 2022; 13 (1): 1090

  Abstract

  LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the cancer state that stems from Lkb1 deficiency can be reverted remains unknown. To identify the processes governed by LKB1 in vivo, we generated an allele which enables Lkb1 inactivation at tumor initiation and subsequent Lkb1 restoration in established tumors. Restoration of Lkb1 in oncogenic KRAS-driven lung tumors suppressed proliferation and led to tumor stasis. Lkb1 restoration activated targets of C/EBP transcription factors and drove neoplastic cells from a progenitor-like state to a less proliferative alveolar type II cell-like state. We show that C/EBP transcription factors govern a subset of genes that are induced by LKB1 and depend upon NKX2-1. We also demonstrate that a defining factor of the alveolar type II lineage, C/EBPalpha, constrains oncogenic KRAS-driven lung tumor growth in vivo. Thus, this key tumor suppressor regulates lineage-specific transcription factors, thereby constraining lung tumor development through enforced differentiation.

  View details for DOI 10.1038/s41467-022-28619-8

  View details for PubMedID 35228570

• Tumor suppressor pathways shape EGFR-driven lung tumor progression and response to treatment. Molecular & cellular oncology Foggetti, G., Li, C., Cai, H., Petrov, D. A., Winslow, M. M., Politi, K. 2022; 9 (1): 1994328

  Abstract

  In vivo modeling combined with CRISPR/Cas9-mediated somatic genome editing has contributed to elucidating the functional importance of specific genetic alterations in human tumors. Our recent work uncovered tumor suppressor pathways that affect EGFR-driven lung tumor growth and sensitivity to tyrosine kinase inhibitors and reflect the mutational landscape and treatment outcomes in the human disease.

  View details for DOI 10.1080/23723556.2021.1994328

  View details for PubMedID 35252550

  View details for PubMedCentralID PMC8890383

• Tumor suppressor pathways shape EGFR-driven lung tumor progression and response to treatment MOLECULAR & CELLULAR ONCOLOGY Foggetti, G., Li, C., Cai, H., Petrov, D. A., Winslow, M. M., Politi, K. 2021

  View details for DOI 10.1080/23723556.2021.1994328

  View details for Web of Science ID 000742922000001

• LKB1 inactivation modulates chromatin accessibility to drive metastatic progression. Nature cell biology Pierce, S. E., Granja, J. M., Corces, M. R., Brady, J. J., Tsai, M. K., Pierce, A. B., Tang, R., Chu, P., Feldser, D. M., Chang, H. Y., Bassik, M. C., Greenleaf, W. J., Winslow, M. M. 2021

  Abstract

  Metastasis is the leading cause of cancer-related deaths and enables cancer cells to compromise organ function by expanding in secondary sites. Since primary tumours and metastases often share the same constellation of driver mutations, the mechanisms that drive their distinct phenotypes are unclear. Here we show that inactivation of the frequently mutated tumour suppressor gene LKB1 (encoding liver kinase B1) has evolving effects throughout the progression of lung cancer, which leads to the differential epigenetic re-programming of early-stage primary tumours compared with late-stage metastases. By integrating genome-scale CRISPR-Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumours. Using an in vivo model of metastatic progression, we further show that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumours. The expression of SOX17 is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution.

  View details for DOI 10.1038/s41556-021-00728-4

  View details for PubMedID 34341533

• miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts. Genes & development Xue, B., Chuang, C., Prosser, H. M., Fuziwara, C. S., Chan, C., Sahasrabudhe, N., Kuhn, M., Wu, Y., Chen, J., Biton, A., Chen, C., Wilkinson, J. E., McManus, M. T., Bradley, A., Winslow, M. M., Su, B., He, L. 2021

  Abstract

  Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the Kras LSL-G12D/+ ; Trp53 flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

  View details for DOI 10.1101/gad.347344.120

  View details for PubMedID 34301766

• Quantitative in vivo analyses reveal a complex pharmacogenomic landscape in lung adenocarcinoma. Cancer research Li, C., Lin, W., Rizvi, H., Cai, H., McFarland, C. D., Rogers, Z. N., Yousefi, M., Winters, I. P., Rudin, C. M., Petrov, D. A., Winslow, M. M. 2021

  Abstract

  The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most critical gaps in enabling the effective use of cancer therapies. Here we couple a multiplexed and quantitative experimental platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. The complex map of genotype-specific treatment responses uncovered that over 20% of possible interactions show significant resistance or sensitivity. Known and novel interactions were identified, and one of these interactions, the resistance of KEAP1 mutant lung tumors to platinum therapy, was validated using a large patient response dataset. These results highlight the broad impact of tumor suppressor genotype on treatment responses and define a strategy to identify the determinants of precision therapies.

  View details for DOI 10.1158/0008-5472.CAN-21-0716

  View details for PubMedID 34215621

• 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

• Genetic determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo. Cancer discovery Foggetti, G., Li, C., Cai, H., Hellyer, J. A., Lin, W., Ayeni, D., Hastings, K., Choi, J., Wurtz, A., Andrejka, L., Maghini, D. G., Rashleigh, N., Levy, S., Homer, R., Gettinger, S. N., Diehn, M., Wakelee, H. A., Petrov, D. A., Winslow, M. M., Politi, K. 2021

  Abstract

  In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations, however the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating ten putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2 - the strongest drivers of growth in a Kras-driven model - reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, Keap1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib and mutations in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differ across oncogenic contexts and that the fitness landscape shifts upon treatment.

  View details for DOI 10.1158/2159-8290.CD-20-1385

  View details for PubMedID 33707235

• Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects. Nucleic acids research Wang, C., Cheng, J. K., Zhang, Q., Hughes, N. W., Xia, Q., Winslow, M. M., Cong, L. 2021

  Abstract

  Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however,engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps.Finally, our REDIT method is applicable across cell types including human stem cells,and is generalizable to different Cas9 enzymes.

  View details for DOI 10.1093/nar/gkaa1264

  View details for PubMedID 33619540

• A functional taxonomy of tumor suppression in oncogenic KRAS-driven lung cancer. Cancer discovery Cai, H. n., Chew, S. K., Li, C. n., Tsai, M. K., Andrejka, L. n., Murray, C. W., Hughes, N. W., Shuldiner, E. G., Ashkin, E. L., Tang, R. n., Hung, K. L., Chen, L. C., Lee, S. Y., Yousefi, M. n., Lin, W. Y., Kunder, C. A., Cong, L. n., McFarland, C. D., Petrov, D. A., Swanton, C. n., Winslow, M. M. 2021

  Abstract

  Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multi-step process, however the importance and specific roles of many of these genes during tumor initiation, growth and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of forty-eight known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, while the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression.

  View details for DOI 10.1158/2159-8290.CD-20-1325

  View details for PubMedID 33608386

• Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis. Cell reports Dorsch, M., Kowalczyk, M., Planque, M., Heilmann, G., Urban, S., Dujardin, P., Forster, J., Ueffing, K., Nothdurft, S., Oeck, S., Paul, A., Liffers, S. T., Kaschani, F., Kaiser, M., Schramm, A., Siveke, J. T., Winslow, M. M., Fendt, S. M., Nalbant, P., Grüner, B. M. 2021; 37 (8): 110056

  Abstract

  Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

  View details for DOI 10.1016/j.celrep.2021.110056

  View details for PubMedID 34818551

• 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

• Altered mitochondria functionality defines a metastatic cell state in lung cancer and creates an exploitable vulnerability. Cancer research Chuang, C., Dorsch, M., Dujardin, P., Silas, S., Ueffing, K., Holken, J. M., Yang, D., Winslow, M. M., Gruner, B. M. 2020

  Abstract

  Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for most cancer deaths but the cellular changes that enable cancer cells to leave the primary tumor and establish inoperable and lethal metastases remain poorly understood. To uncover genes that are specifically required to sustain metastasis survival or growth, we performed a genome-scale pooled lentiviral-shRNA library screen in cells that represent non-metastatic and metastatic states of lung adenocarcinoma. Mitochondrial ribosome and mitochondria-associated genes were identified as top gene sets associated with metastasis-specific lethality. Metastasis-derived cell lines in vitro and metastases analyzed ex vivo from an autochthonous lung cancer mouse model had lower mitochondrial membrane potential and reduced mitochondrial functionality than non-metastatic primary tumors. Electron microscopy of metastases uncovered irregular mitochondria with bridging and loss of normal membrane structure. Consistent with these findings, compounds that inhibit mitochondrial translation or replication had a greater effect on the growth of metastasis-derived cells. Finally, mice with established tumors developed fewer metastases upon treatment with phenformin in vivo. These results suggest that the metastatic cell state in lung adenocarcinoma is associated with a specifically altered mitochondrial functionality that can be therapeutically exploited.

  View details for DOI 10.1158/0008-5472.CAN-20-1865

  View details for PubMedID 33239425

• Zmat3 Is a Key Splicing Regulator in the p53 Tumor Suppression Program. Molecular cell Bieging-Rolett, K. T., Kaiser, A. M., Morgens, D. W., Boutelle, A. M., Seoane, J. A., Van Nostrand, E. L., Zhu, C., Houlihan, S. L., Mello, S. S., Yee, B. A., McClendon, J., Pierce, S. E., Winters, I. P., Wang, M., Connolly, A. J., Lowe, S. W., Curtis, C., Yeo, G. W., Winslow, M. M., Bassik, M. C., Attardi, L. D. 2020; 80 (3): 452

  Abstract

  Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens invivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.

  View details for DOI 10.1016/j.molcel.2020.10.022

  View details for PubMedID 33157015

• A versatile system to record cell-cell interactions. eLife Tang, R., Murray, C. W., Linde, I. L., Kramer, N. J., Lyu, Z., Tsai, M. K., Chen, L. C., Cai, H., Gitler, A. D., Engleman, E., Lee, W., Winslow, M. M. 2020; 9

  Abstract

  Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher-order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

  View details for DOI 10.7554/eLife.61080

  View details for PubMedID 33025906

• CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities NATURE Han, K., Pierce, S. E., Li, A., Spees, K., Anderson, G. R., Seoane, J. A., Lo, Y., Dubreuil, M., Olivas, M., Kamber, R. A., Wainberg, M., Kostyrko, K., Kelly, M. R., Yousefi, M., Simpkins, S. W., Yao, D., Lee, K., Kuo, C. J., Jackson, P. K., Sweet-Cordero, A., Kundaje, A., Gentles, A. J., Curtis, C., Winslow, M. M., Bassik, M. C. 2020

  View details for DOI 10.1038/s41586-020-2099-x

  View details for Web of Science ID 000519162500002

• 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

• An Lkb1-Sik axis suppresses lung tumor growth and controls differentiation. Cancer discovery Murray, C. W., Brady, J. J., Tsai, M. K., Li, C. n., Winters, I. P., Tang, R. n., Andrejka, L. n., Ma, R. K., Kunder, C. A., Chu, P. n., Winslow, M. M. 2019

  Abstract

  The kinase, LKB1, is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of Lkb1 substrates in vivo, we employed CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic Kras-driven lung adenocarcinoma. We demonstrate that members of the salt-inducible kinase (Sik) family are critical for constraining tumor development. Histological and gene expression similarities between Lkb1- and Sik-deficient tumors suggest that Siks and Lkb1 operate within the same axis. Furthermore, a gene expression signature reflecting Sik deficiency is enriched in LKB1 mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key Lkb1-Sik tumor-suppressive axis and underscore the need to redirect the focus of efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression.

  View details for DOI 10.1158/2159-8290.CD-18-1237

  View details for PubMedID 31350327

• Towards quantitative and multiplexed in vivo functional cancer genomics. Nature reviews. Genetics Winters, I. P., Murray, C. W., Winslow, M. M. 2018

  Abstract

  Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.

  View details for PubMedID 30267031

• 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

• Hmga2 is dispensable for pancreatic cancer development, metastasis, and therapy resistance. Scientific reports Chiou, S., Dorsch, M., Kusch, E., Naranjo, S., Kozak, M. M., Koong, A. C., Winslow, M. M., Gruner, B. M. 2018; 8 (1): 14008

  Abstract

  Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence ofdisseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.

  View details for PubMedID 30228296

• Tumor Suppressor Activity of Selenbp1, a Direct Nkx2-1 Target, in Lung Adenocarcinoma. Molecular cancer research : MCR Caswell, D. R., Chuang, C., Ma, R. K., Winters, I. P., Snyder, E. L., Winslow, M. M. 2018

  Abstract

  The Nkx2-1 transcription factor in lung epithelial lineages promotes differentiation and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic deletion of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate Selenbp1 as a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo.IMPLICATIONS: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome.

  View details for PubMedID 30002193

• Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice NATURE GENETICS Rogers, Z. N., McFarland, C. D., Winters, I. P., Seoane, J. A., Brady, J. J., Yoon, S., Curtis, C., Petrov, D. A., Winslow, M. M. 2018; 50 (4): 483-+

  Abstract

  The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.

  View details for PubMedID 29610476

• A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo. Nature methods Rogers, Z. N., McFarland, C. D., Winters, I. P., Naranjo, S., Chuang, C., Petrov, D., Winslow, M. M. 2017

  Abstract

  Cancer growth is a multistage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remain largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor-suppressor genes. Here, we present a method that integrates tumor barcoding with ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes. By combining Tuba-seq with multiplexed CRISPR-Cas9-mediated genome editing, we quantified the effects of 11 tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. Tuba-seq enables the broad quantification of the function of tumor-suppressor genes with unprecedented resolution, parallelization, and precision.

  View details for DOI 10.1038/nmeth.4297

  View details for PubMedID 28530655

• Molecular definition of a metastatic lung cancer state reveals a targetable CD109-Janus kinase-Stat axis. Nature medicine Chuang, C., Greenside, P. G., Rogers, Z. N., Brady, J. J., Yang, D., Ma, R. K., Caswell, D. R., Chiou, S., Winters, A. F., Grüner, B. M., Ramaswami, G., Spencley, A. L., Kopecky, K. E., Sayles, L. C., Sweet-Cordero, E. A., Li, J. B., Kundaje, A., Winslow, M. M. 2017; 23 (3): 291-300

  Abstract

  Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.

  View details for DOI 10.1038/nm.4285

  View details for PubMedID 28191885

• Blimp1 induces transient metastatic heterogeneity in pancreatic cancer. Cancer discovery Chiou, S. H., Risca, V. I., Wang, G. X., Yang, D. n., Grüner, B. M., Kathiria, A. S., Ma, R. K., Vaka, D. n., Chu, P. n., Kozak, M. n., Castellini, L. n., Graves, E. E., Kim, G. E., Mourrain, P. n., Koong, A. C., Giaccia, A. J., Winslow, M. M. 2017

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. Using a novel genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor Blimp1 as a key driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes and hypoxia-mediated induction of Blimp1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which up-regulation of Blimp1 links microenvironmental cues to a metastatic stem cell character.

  View details for PubMedID 28790031

• 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

• An Arntl2-Driven Secretome Enables Lung Adenocarcinoma Metastatic Self-Sufficiency CANCER CELL Brady, J. J., Chuang, C., Greenside, P. G., Rogers, Z. N., Murray, C. W., Caswell, D. R., Hartmann, U., Connolly, A. J., Sweet-Cordero, E. A., Kundaje, A., Winslow, M. M. 2016; 29 (5): 697-710

  Abstract

  The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments.

  View details for DOI 10.1016/j.ccell.2016.03.003

  View details for PubMedID 27150038

• Recombinase-based conditional and reversible gene regulation via XTR alleles NATURE COMMUNICATIONS Robles-Oteiza, C., Taylor, S., Yates, T., Cicchini, M., Lauderback, B., Cashman, C. R., Burds, A. A., Winslow, M. M., Jacks, T., Feldser, D. M. 2015; 6

  Abstract

  Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).

  View details for DOI 10.1038/ncomms9783

  View details for Web of Science ID 000366294800001

  View details for PubMedID 26537451

  View details for PubMedCentralID PMC4635517

• Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery. ACS chemical biology Ofori, L. O., Withana, N. P., Prestwood, T. R., Verdoes, M., Brady, J. J., Winslow, M. M., Sorger, J., Bogyo, M. 2015; 10 (9): 1977-1988

  Abstract

  There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

  View details for DOI 10.1021/acschembio.5b00205

  View details for PubMedID 26039341

  View details for PubMedCentralID PMC4577961

• Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2. PLoS genetics Madison, B. B., Jeganathan, A. N., Mizuno, R., Winslow, M. M., Castells, A., Cuatrecasas, M., Rustgi, A. K. 2015; 11 (8)

  Abstract

  Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

  View details for DOI 10.1371/journal.pgen.1005408

  View details for PubMedID 26244988

  View details for PubMedCentralID PMC4526516

• Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing GENES & DEVELOPMENT Chiou, S., Winters, I. P., Wang, J., Naranjo, S., Dudgeon, C., Tamburini, F. B., Brady, J. J., Yang, D., Gruener, B. M., Chuang, C., Caswell, D. R., Zeng, H., Chu, P., Kim, G. E., Carpizo, D. R., Kim, S. K., Winslow, M. M. 2015; 29 (14): 1576-1585

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

  View details for DOI 10.1101/gad.264861.115

  View details for Web of Science ID 000358596300010

  View details for PubMedCentralID PMC4526740

• Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes & development Chiou, S. H., Winters, I. P., Wang, J., Naranjo, S., Dudgeon, C., Tamburini, F. B., Brady, J. J., Yang, D., Grüner, B. M., Chuang, C. H., Caswell, D. R., Zeng, H., Chu, P., Kim, G. E., Carpizo, D. R., Kim, S. K., Winslow, M. M. 2015; 29 (14): 1576-85

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

  View details for DOI 10.1101/gad.264861.115

  View details for PubMedID 26178787

  View details for PubMedCentralID PMC4526740

• Upregulation of the microRNA cluster at the Dlkl-Dio3 locus in lung adenocarcinoma ONCOGENE Valdmanis, P. N., Roy-Chaudhuri, B., Kim, H. K., Sayles, L. C., Zheng, Y., Chuang, C., Caswell, D. R., Chu, K., Zhang, Y., Winslow, M. M., Sweet-Cordero, E. A., Kay, M. A. 2015; 34 (1): 94-103

  Abstract

  Mice in which lung epithelial cells can be induced to express an oncogenic Kras(G12D) develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of Kras(G12D) tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured Kras(G12D) tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.Oncogene advance online publication, 9 December 2013; doi:10.1038/onc.2013.523.

  View details for DOI 10.1038/onc.2013.523

  View details for Web of Science ID 000349740100009

• An AMPK-Independent Signaling Pathway Downstream of the LKB1 Tumor Suppressor Controls Snail1 and Metastatic Potential. Molecular cell Goodwin, J. M., Svensson, R. U., Lou, H. J., Winslow, M. M., Turk, B. E., Shaw, R. J. 2014; 55 (3): 436-450

  Abstract

  The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.

  View details for DOI 10.1016/j.molcel.2014.06.021

  View details for PubMedID 25042806

• Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis. Proceedings of the National Academy of Sciences of the United States of America Sinkevicius, K. W., Kriegel, C., Bellaria, K. J., Lee, J., Lau, A. N., Leeman, K. T., Zhou, P., Beede, A. M., Fillmore, C. M., Caswell, D., Barrios, J., Wong, K., Sholl, L. M., Schlaeger, T. M., Bronson, R. T., Chirieac, L. R., Winslow, M. M., Haigis, M. C., Kim, C. F. 2014; 111 (28): 10299-10304

  Abstract

  Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.

  View details for DOI 10.1073/pnas.1404399111

  View details for PubMedID 24982195

  View details for PubMedCentralID PMC4104911

• Obligate Progression Precedes Lung Adenocarcinoma Dissemination CANCER DISCOVERY Caswell, D. R., Chuang, C., Yang, D., Chiou, S., Cheemalavagu, S., Kim-Kiselak, C., Connolly, A., Winslow, M. M. 2014; 4 (7): 781-789

  Abstract

  Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis.Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression.

  View details for DOI 10.1158/2159-8290.CD-13-0862

  View details for Web of Science ID 000338708900024

  View details for PubMedCentralID PMC4090265

• Obligate progression precedes lung adenocarcinoma dissemination. Cancer discovery Caswell, D. R., Chuang, C. H., Yang, D., Chiou, S. H., Cheemalavagu, S., Kim-Kiselak, C., Connolly, A., Winslow, M. M. 2014; 4 (7): 781-9

  Abstract

  Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis.Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression.

  View details for DOI 10.1158/2159-8290.CD-13-0862

  View details for PubMedID 24740995

  View details for PubMedCentralID PMC4090265

• A Conditional System to Specifically Link Disruption of Protein-Coding Function with Reporter Expression in Mice CELL REPORTS Chiou, S., Kim-Kiselak, C., Risca, V. I., Heimann, M. K., Chuang, C., Burds, A. A., Greenleaf, W. J., Jacks, T. E., Feldser, D. M., Winslow, M. M. 2014; 7 (6): 2078-2086

  View details for DOI 10.1016/j.celrep.2014.05.031

  View details for Web of Science ID 000338325400031

• A conditional system to specifically link disruption of protein-coding function with reporter expression in mice. Cell reports Chiou, S., Kim-Kiselak, C., Risca, V. I., Heimann, M. K., Chuang, C., Burds, A. A., Greenleaf, W. J., Jacks, T. E., Feldser, D. M., Winslow, M. M. 2014; 7 (6): 2078-2086

  Abstract

  Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

  View details for DOI 10.1016/j.celrep.2014.05.031

  View details for PubMedID 24931605

  View details for PubMedCentralID PMC4113058

• Differential Tks5 isoform expression contributes to metastatic invasion of lung adenocarcinoma GENES & DEVELOPMENT Li, C. M., Chen, G., Dayton, T. L., Kim-Kiselak, C., Hoersch, S., Whittaker, C. A., Bronson, R. T., Beer, D. G., Winslow, M. M., Jacks, T. 2013; 27 (14): 1557-1567

  Abstract

  Metastasis accounts for the vast majority of cancer-related deaths, yet the molecular mechanisms that drive metastatic spread remain poorly understood. Here we report that Tks5, which has been linked to the formation of proteolytic cellular protrusions known as invadopodia, undergoes an isoform switch during metastatic progression in a genetically engineered mouse model of lung adenocarcinoma. Nonmetastatic primary tumor-derived cells predominantly expressed a short isoform, Tks5short, while metastatic primary tumor- and metastasis-derived cells acquired increased expression of the full-length isoform Tks5long. This elevation of Tks5long to Tks5short ratio correlated with a commensurate increase in invadopodia activity in metastatic cells compared with nonmetastatic cells. Further characterization of these isoforms by knockdown and overexpression experiments demonstrated that Tks5long promoted invadopodia in vitro and increased metastasis in transplant models and an autochthonous model of lung adenocarcinoma. Conversely, Tks5short decreased invadopodia stability and proteolysis, acting as a natural dominant-negative inhibitor to Tks5long. Importantly, high Tks5long and low Tks5short expressions in human lung adenocarcinomas correlated with metastatic disease and predicted worse survival of early stage patients. These data indicate that tipping the Tks5 isoform balance to a high Tks5long to Tks5short ratio promotes invadopodia-mediated invasion and metastasis.

  View details for DOI 10.1101/gad.222745.113

  View details for Web of Science ID 000322011500004

  View details for PubMedID 23873940

• MicroRNA-33a Mediates the Regulation of High Mobility Group AT-Hook 2 Gene (HMGA2) by Thyroid Transcription Factor 1 (TTF-1/NKX2-1) JOURNAL OF BIOLOGICAL CHEMISTRY Rice, S. J., Lai, S., Wood, L. W., Helsley, K. R., Runkle, E. A., Winslow, M. M., Mu, D. 2013; 288 (23): 16348-16360

  Abstract

  In lung cancers, TTF-1 displays seemingly paradoxical activities. Although TTF-1 is amplified in primary human lung cancers, it inhibits primary lung tumors from metastasizing in a mouse model system. It was reported that the oncogenic proepithelial mesenchymal transition (EMT) high mobility group AT-hook 2 gene (HMGA2) mediates the antimetastatic function of TTF-1. To gain mechanistic insight into the metastasis-critical signaling axis of TTF-1 to HMGA2, we used both reverse and forward strategies and discovered that microRNA-33a (miR-33a) is under direct positive regulation of TTF-1. By chromatin immunoprecipitation, we determined that TTF-1 binds to the promoter of SREBF2, the host gene of miR-33a. The 3'-untranslated region (UTR) of HMGA2 contains three predicted binding sites of miR-33a. We showed that the first two highly conserved sites are conducive to HMGA2 repression by miR-33a, establishing HMGA2 as a genuine target of miR-33a. Functional studies revealed that enforced expression of miR-33a inhibits the motility of lung cancer cells, and this inhibition can be rescued by overexpression of the form of HMGA2 without the 3'-UTR, suggesting that TTF-1 keeps the prometastasis gene HMGA2 in check via up-regulating miR-33a. This study reports the first miRNAs directly regulated by TTF-1 and clarifies how TTF-1 controls HMGA2 expression. Moreover, the documented importance of SREBF2 and miR-33a in regulating cholesterol metabolism suggests that TTF-1 may be a modulator of cholesterol homeostasis in the lung. Future studies will be dedicated to understanding how miRNAs influence the oncogenic activity of TTF-1 and the role of TTF-1 in cholesterol metabolism.

  View details for DOI 10.1074/jbc.M113.474643

  View details for Web of Science ID 000320378900013

  View details for PubMedID 23625920

  View details for PubMedCentralID PMC3675572

• Characterizing deformability and surface friction of cancer cells. Proceedings of the National Academy of Sciences of the United States of America Byun, S., Son, S., Amodei, D., Cermak, N., Shaw, J., Kang, J. H., Hecht, V. C., Winslow, M. M., Jacks, T., Mallick, P., Manalis, S. R. 2013; 110 (19): 7580-7585

  Abstract

  Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.

  View details for DOI 10.1073/pnas.1218806110

  View details for PubMedID 23610435

  View details for PubMedCentralID PMC3651488

• A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis NATURE COMMUNICATIONS Reticker-Flynn, N. E., Malta, D. F., Winslow, M. M., Lamar, J. M., Xu, M. J., Underhill, G. H., Hynes, R. O., Jacks, T. E., Bhatia, S. N. 2012; 3

  Abstract

  Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.

  View details for DOI 10.1038/ncomms2128

  View details for Web of Science ID 000313514100032

  View details for PubMedID 23047680

• Occludin Is a Direct Target of Thyroid Transcription Factor-1 (TTF-1/NKX2-1) JOURNAL OF BIOLOGICAL CHEMISTRY Runkle, E. A., Rice, S. J., Qi, J., Masser, D., Antonetti, D. A., Winslow, M. M., Mu, D. 2012; 287 (34): 28790-28801

  Abstract

  The thyroid transcription factor 1 gene (TTF-1 or NKX2-1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-β on occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of occludin restored cellular sensitivity to anoikis. Overexpression of occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates occludin, which represents another avenue of TTF-1-mediated metastasis suppression.

  View details for DOI 10.1074/jbc.M112.367987

  View details for Web of Science ID 000308074600052

  View details for PubMedID 22761434

• Treating metastatic cancer with nanotechnology NATURE REVIEWS CANCER Schroeder, A., Heller, D. A., Winslow, M. M., Dahlman, J. E., Pratt, G. W., Langer, R., Jacks, T., Anderson, D. G. 2012; 12 (1): 39-50

  View details for DOI 10.1038/nrc3180

  View details for Web of Science ID 000298369300012

• Response and Resistance to NF-kappa B Inhibitors in Mouse Models of Lung Adenocarcinoma CANCER DISCOVERY Xue, W., Meylan, E., Oliver, T. G., Feldser, D. M., Winslow, M. M., Bronson, R., Jacks, T. 2011; 1 (3): 236-247

  Abstract

  Lung adenocarcinoma is a leading cause of cancer death worldwide. We recently showed that genetic inhibition of the NF-κB pathway affects both the initiation and the maintenance of lung cancer, identifying this pathway as a promising therapeutic target. In this investigation, we tested the efficacy of small-molecule NF-κB inhibitors in mouse models of lung cancer. In murine lung adenocarcinoma cell lines with high NF-κB activity, the proteasome inhibitor bortezomib efficiently reduced nuclear p65, repressed NF-κB target genes, and rapidly induced apoptosis. Bortezomib also induced lung tumor regression and prolonged survival in tumor-bearing Kras(LSL-G12D/wt);p53(flox/flox) mice but not in Kras(LSL-G12D/wt) mice. After repeated treatment, initially sensitive lung tumors became resistant to bortezomib. A second NF-κB inhibitor, Bay-117082, showed similar therapeutic efficacy and acquired resistance in mice. Our results using preclinical mouse models support the NF-κB pathway as a potential therapeutic target for a defined subset of lung adenocarcinoma.Using small-molecule compounds that inhibit NF-κB activity, we provide evidence that NF-κB inhibition has therapeutic efficacy in the treatment of lung cancer. Our results also illustrate the value of mouse models in validating new drug targets in vivo and indicate that acquired chemoresistance may later influence bortezomib treatment in lung cancer.

  View details for DOI 10.1158/2159-8290.CD-11-0073

  View details for Web of Science ID 000295783300021

  View details for PubMedID 21874163

• Nuclear factor I/B is an oncogene in small cell lung cancer GENES & DEVELOPMENT Dooley, A. L., Winslow, M. M., Chiang, D. Y., Banerji, S., Stransky, N., Dayton, T. L., Snyder, E. L., Senna, S., Whittaker, C. A., Bronson, R. T., Crowley, D., Barretina, J., Garraway, L., Meyerson, M., Jacks, T. 2011; 25 (14): 1470-1475

  Abstract

  Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

  View details for DOI 10.1101/gad.2046711

  View details for Web of Science ID 000292758200004

  View details for PubMedID 21764851

• Suppression of lung adenocarcinoma progression by Nkx2-1 NATURE Winslow, M. M., Dayton, T. L., Verhaak, R. G., Kim-Kiselak, C., Snyder, E. L., Feldser, D. M., Hubbard, D. D., DuPage, M. J., Whittaker, C. A., Hoersch, S., Yoon, S., Crowley, D., Bronson, R. T., Chiang, D. Y., Meyerson, M., Jacks, T. 2011; 473 (7345): 101-U120

  Abstract

  Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

  View details for DOI 10.1038/nature09881

  View details for Web of Science ID 000290218300039

  View details for PubMedID 21471965

• Endogenous T Cell Responses to Antigens Expressed in Lung Adenocarcinomas Delay Malignant Tumor Progression CANCER CELL DuPage, M., Cheung, A. F., Mazumdar, C., Winslow, M. M., Bronson, R., Schmidt, L. M., Crowley, D., Chen, J., Jacks, T. 2011; 19 (1): 72-85

  Abstract

  Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here, we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors.

  View details for DOI 10.1016/j.ccr.2010.11.011

  View details for Web of Science ID 000287290300011

  View details for PubMedID 21251614

• Stage-specific sensitivity to p53 restoration during lung cancer progression NATURE Feldser, D. M., Kostova, K. K., Winslow, M. M., Taylor, S. E., Cashman, C., Whittaker, C. A., Sanchez-Rivera, F. J., Resnick, R., Bronson, R., Hemann, M. T., Jacks, T. 2010; 468 (7323): 572-U249

  Abstract

  Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.

  View details for DOI 10.1038/nature09535

  View details for Web of Science ID 000284584200044

  View details for PubMedID 21107428

• Selective role of calcineurin in haematopoiesis and lymphopoiesis EMBO REPORTS Gallo, E. M., Ho, L., Winslow, M. M., Staton, T. L., Crabtree, G. R. 2008; 9 (11): 1141-1148

  Abstract

  The calcineurin/NFAT (nuclear factor of activated T-cells) signalling pathway is essential for many aspects of vertebrate development and is the target of the widely used immunosuppressive drugs FK506 and cyclosporine A. The basis for the therapeutic specificity of these drugs has remained unclear, as calcineurin is expressed ubiquitously. By inactivating calcineurin during haematopoietic development, we found that although this signalling pathway has an important, non-redundant role in the regulation of lymphocyte developmental checkpoints, it is not essential for the development of blood myeloid lineages. These studies have shown that the specificity of calcineurin inhibitors arises from the selective use of calcineurin at distinct developmental stages. The requirement for calcineurin/NFAT in the development of the adaptive but not of the innate immune system is consistent with the idea that the evolutionary appearance of this pathway was involved in the emergence of vertebrates.

  View details for DOI 10.1038/embor.2008.174

  View details for Web of Science ID 000260586800017

  View details for PubMedID 18818667

  View details for PubMedCentralID PMC2581854

• Targeted deletion reveals essential and overlapping functions of the miR-17 similar to 92 family of miRNA clusters CELL Ventura, A., Young, A. G., Winslow, M. M., Lintault, L., Meissner, A., Erkeland, S. J., Newman, J., Bronson, R. T., Crowley, D., Stone, J. R., Jaenisch, R., Sharp, P. A., Jacks, T. 2008; 132 (5): 875-886

  Abstract

  miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

  View details for DOI 10.1016/j.cell.2008.02.019

  View details for Web of Science ID 000253818000023

  View details for PubMedID 18329372

• Calcineurin sets the bandwidth for discrimination of signals during thymocyte development NATURE Gallo, E. M., Winslow, M. M., Cante-Barrett, K., Radermacher, A. N., Ho, L., McGinnis, L., Iritani, B., Neilson, J. R., Crabtree, G. R. 2007; 450 (7170): 731-U11

  Abstract

  At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

  View details for DOI 10.1038/nature06305

  View details for Web of Science ID 000251209700056

  View details for PubMedID 18046413

• Selective role of NFATc3 in positive selection of thymocytes JOURNAL OF IMMUNOLOGY Cante-Barrett, K., Winslow, M. M., Crabtree, G. R. 2007; 179 (1): 103-110

  Abstract

  The four Ca(2+)-dependent NFATc proteins are both signal transducers and transcription factors that reside in the cytoplasm until dephosphorylation by calcineurin. Dephosphorylation exposes nuclear import sequences and sends NFATc proteins into the nucleus where they assemble with nuclear partners into NFAT transcription complexes. Recent genetic studies have indicated that calcineurin-NFAT signaling is a major determinant of vertebrate morphogenesis and development. Mice lacking calcineurin activity show a complete block in positive selection of CD4 and CD8 double-positive thymocytes, yet the role of the NFATc proteins in T cell development has been controversial. In this study, we address the requirement for NFATc3 in T cell development by generating NFATc3 conditional knockout mice. We show that specific deletion of NFATc3 in thymocytes causes a partial block at the double-negative stage 3 and also a partial block in positive selection. Furthermore, the defect does not become more pronounced when NFATc2 is also absent, consistent with the fact that NFATc2-null mice do not have a T cell developmental defect. Expression of a nuclear (and constitutively active) NFATc1 even at subphysiological levels can rescue the transition of double-negative to double-positive thymocytes in RAG-null mice, but is unable to rescue development of CD4 and CD8 single-positive cells. In addition to NFATc3, this suggests a role for NFATc1 in T cell development. Our studies indicate that the signals that direct positive selection likely use both NFATc1 and NFATc3 downstream of calcineurin.

  View details for Web of Science ID 000247497600016

  View details for PubMedID 17579027

• Stringent control of NFATc1 nuclear occupancy is critical for maintaining balanced immune response GENE THERAPY AND MOLECULAR BIOLOGY Pan, M., Winslow, M. M., Keum, J. S., Crabtree, G. R. 2007; 11B: 171-176

  View details for Web of Science ID 000251610000008

• Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function NATURE Heit, J. J., Apelqvist, A. A., Gu, X., Winslow, M. M., Neilson, J. R., Crabtree, G. R., Kim, S. K. 2006; 443 (7109): 345-349

  Abstract

  The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.

  View details for DOI 10.1038/nature05097

  View details for PubMedID 16988714

• Calcineurin/NFAT signaling in osteoblasts regulates bone mass DEVELOPMENTAL CELL Winslow, M. M., Pan, M., Starbuck, M., Gallo, E. M., Deng, L., Karsenty, G., Crabtree, G. R. 2006; 10 (6): 771-782

  Abstract

  Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.

  View details for DOI 10.1016/j.devcel.2006.04.006

  View details for Web of Science ID 000238244700011

  View details for PubMedID 16740479

• NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21 NATURE Arron, J. R., Winslow, M. M., Polleri, A., Chang, C., Wu, H., Gao, X., Neilson, J. R., Chen, L., Heit, J. J., Kim, S. K., Yamasaki, N., Miyakawa, T., Francke, U., Graef, I. A., Crabtree, G. R. 2006; 441 (7093): 595-600

  Abstract

  Trisomy 21 results in Down's syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Down's syndrome. Here we report that two genes, DSCR1 and DYRK1A , lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. We use mathematical modelling to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Our observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a-overexpressing mice, mouse models of Down's syndrome and human trisomy 21 are consistent with these predictions. We suggest that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down's syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease.

  View details for DOI 10.1038/nature04678

  View details for PubMedID 16554754

• CD8(+) recent thymic emigrants home to and efficiently repopulate the small intestine epithelium NATURE IMMUNOLOGY Staton, T. L., Habtezion, A., Winslow, M. M., Sato, T., Love, P. E., Butcher, E. C. 2006; 7 (5): 482-488

  Abstract

  Prevailing knowledge dictates that naive alphabeta T cells require activation in lymphoid tissues before differentiating into effector or memory T cells capable of trafficking to nonlymphoid tissues. Here we demonstrate that CD8(+) recent thymic emigrants (RTEs) migrated directly into the small intestine. CCR9, CCL25 and alpha(4)beta(7) integrin were required for gut entry of CD8(+) RTEs. After T cell receptor stimulation, intestinal CD8(+) RTEs proliferated and acquired a surface phenotype resembling that of intraepithelial lymphocytes. CD8(+) RTEs efficiently populated the gut of lymphotoxin-alpha-deficient mice, which lack lymphoid organs. These studies challenge the present understanding of naive alphabeta T cell trafficking and suggest that RTEs may be involved in maintaining a diverse immune repertoire at mucosal surfaces.

  View details for DOI 10.1038/ni1319

  View details for Web of Science ID 000237008800013

  View details for PubMedID 16582913

• The calcineurin phosphatase complex modulates immunogenic B cell responses IMMUNITY Winslow, M. M., Gallo, E. M., Neilson, J. R., Crabtree, G. R. 2006; 24 (2): 141-152

  Abstract

  A series of signal-directed transitions regulates the development of distinct populations of self-tolerant B cells and ultimately the production of antibody-producing plasma cells. We studied the role of calcineurin/NFAT signaling in B cells by deleting the regulatory b1 subunit of calcineurin specifically in B cells. Follicular (FO) and marginal zone (MZ) B cells develop normally in these mice, but B1 cell numbers are reduced. In vitro, calcineurin b1-deficient B cells have a cell-intrinsic proliferation defect downstream of the B cell receptor. These mice have higher total serum IgM despite the absence of B1 cells and have enhanced T cell-independent-1 responses. Conversely, mice with calcineurin b1-deficient B cells develop larger germinal centers and have reduced plasma cell development and antigen-specific antibody production during T cell-dependent immune responses. By several different criteria, calcineurin is dispensable for B cell tolerance, indicating that this phosphatase complex modulates immunogenic, but not tolerogenic, responses in vivo.

  View details for DOI 10.1016/j.immuni.2005.12.013

  View details for Web of Science ID 000235479000006

  View details for PubMedID 16473827

• Calcineurin B1 is essential for positive but not negative selection during thymocyte development IMMUNITY Neilson, J. R., Winslow, M. M., Hur, E. M., Crabtree, G. R. 2004; 20 (3): 255-266

  Abstract

  During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.

  View details for Web of Science ID 000221442600004

  View details for PubMedID 15030770