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2021-22 Courses


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  • INK4 tumor suppressor proteins mediate resistance to CDK4/6 kinase inhibitors. Cancer discovery Li, Q., Jiang, B., Guo, J., Shao, H., Del Priore, I. S., Chang, Q., Kudo, R., Li, Z., Razavi, P., Liu, B., Boghossian, A. S., Rees, M. G., Ronan, M. M., Roth, J. A., Donovan, K. A., Palafox, M., Reis-Filho, J. S., de Stanchina, E., Fischer, E. S., Rosen, N., Serra, V., Koff, A., Chodera, J. D., Gray, N. S., Chandarlapaty, S. 2021

    Abstract

    Cyclin-dependent kinases 4 and 6 (CDK4/6), represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identify several genetic alterations (e.g. FAT1, PTEN or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g. p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C/D-cyclin/CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance.

    View details for DOI 10.1158/2159-8290.CD-20-1726

    View details for PubMedID 34544752

  • TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma CANCER CELL Seong, B. A., Dharia, N., Lin, S., Donovan, K. A., Chong, S., Robichaud, A., Conway, A., Hamze, A., Ross, L., Alexe, G., Adane, B., Nabet, B., Ferguson, F. M., Stolte, B., Wang, E., Sun, J., Darzacq, X., Piccioni, F., Gray, N. S., Fischer, E. S., Stegmaier, K. 2021; 39 (9): 1262-+

    Abstract

    Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.

    View details for DOI 10.1016/j.ccell.2021.07.003

    View details for Web of Science ID 000695626600016

    View details for PubMedID 34329586

    View details for PubMedCentralID PMC8443273

  • Exploring Ligand-Directed N-Acyl-N-alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development. ACS medicinal chemistry letters Teng, M., Jiang, J., Ficarro, S. B., Seo, H., Bae, J. H., Donovan, K. A., Fischer, E. S., Zhang, T., Dhe-Paganon, S., Marto, J. A., Gray, N. S. 2021; 12 (8): 1302-1307

    Abstract

    Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting N-acyl-N-alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4CRBN E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.

    View details for DOI 10.1021/acsmedchemlett.1c00285

    View details for PubMedID 34413960

  • Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy. Cell Koikawa, K., Kibe, S., Suizu, F., Sekino, N., Kim, N., Manz, T. D., Pinch, B. J., Akshinthala, D., Verma, A., Gaglia, G., Nezu, Y., Ke, S., Qiu, C., Ohuchida, K., Oda, Y., Lee, T. H., Wegiel, B., Clohessy, J. G., London, N., Santagata, S., Wulf, G. M., Hidalgo, M., Muthuswamy, S. K., Nakamura, M., Gray, N. S., Zhou, X. Z., Lu, K. P. 2021

    Abstract

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

    View details for DOI 10.1016/j.cell.2021.07.020

    View details for PubMedID 34388391

  • ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1-mutant lung cancer NATURE CANCER Deng, J., Thennavan, A., Dolgalev, I., Chen, T., Li, J., Marzio, A., Poirier, J. T., Peng, D. H., Bulatovic, M., Mukhopadhyay, S., Silver, H., Papadopoulos, E., Pyon, V., Thakurdin, C., Han, H., Li, F., Li, S., Ding, H., Hu, H., Pan, Y., Weerasekara, V., Jiang, B., Wang, E. S., Ahearn, I., Philips, M., Papagiannakopoulos, T., Tsirigos, A., Rothenberg, E., Gainor, J., Freeman, G. J., Rudin, C. M., Gray, N. S., Hammerman, P. S., Pagano, M., Heymach, J. V., Perou, C. M., Bardeesy, N., Wong, K. 2021
  • Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. Nature chemical biology Dubiella, C., Pinch, B. J., Koikawa, K., Zaidman, D., Poon, E., Manz, T. D., Nabet, B., He, S., Resnick, E., Rogel, A., Langer, E. M., Daniel, C. J., Seo, H., Chen, Y., Adelmant, G., Sharifzadeh, S., Ficarro, S. B., Jamin, Y., Martins da Costa, B., Zimmerman, M. W., Lian, X., Kibe, S., Kozono, S., Doctor, Z. M., Browne, C. M., Yang, A., Stoler-Barak, L., Shah, R. B., Vangos, N. E., Geffken, E. A., Oren, R., Koide, E., Sidi, S., Shulman, Z., Wang, C., Marto, J. A., Dhe-Paganon, S., Look, T., Zhou, X. Z., Lu, K. P., Sears, R. C., Chesler, L., Gray, N. S., London, N. 2021

    Abstract

    The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

    View details for DOI 10.1038/s41589-021-00786-7

    View details for PubMedID 33972797

  • Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2. Angewandte Chemie (International ed. in English) Du, G., Jiang, J., Wu, Q., Henning, N. J., Donovan, K. A., Yue, H., Che, J., Lu, W., Fischer, E. S., Bardeesy, N., Zhang, T., Gray, N. S. 2021

    Abstract

    Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.

    View details for DOI 10.1002/anie.202101328

    View details for PubMedID 33915015

  • PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors. Analytical chemistry Zhu, H., Ficarro, S. B., Alexander, W. M., Fleming, L. E., Adelmant, G., Zhang, T., Willetts, M., Decker, J., Brehmer, S., Krause, M., East, M. P., Gray, N. S., Johnson, G. L., Kruppa, G., Marto, J. A. 2021

    Abstract

    Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

    View details for DOI 10.1021/acs.analchem.1c02349

    View details for PubMedID 34606255

  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition SCIENCE ADVANCES Fassl, A., Brain, C., Abu-Remaileh, M., Stukan, I., Butter, D., Stepien, P., Feit, A. S., Bergholz, J., Michowski, W., Otto, T., Sheng, Q., Loo, A., Michael, W., Tiedt, R., DeAngelis, C., Schiff, R., Jiang, B., Jovanovic, B., Nowak, K., Ericsson, M., Cameron, M., Gray, N., Dillon, D., Zhao, J. J., Sabatini, D. M., Jeselsohn, R., Brown, M., Polyak, K., Sicinski, P. 2020; 6 (25)
  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances Fassl, A. n., Brain, C. n., Abu-Remaileh, M. n., Stukan, I. n., Butter, D. n., Stepien, P. n., Feit, A. S., Bergholz, J. n., Michowski, W. n., Otto, T. n., Sheng, Q. n., Loo, A. n., Michael, W. n., Tiedt, R. n., DeAngelis, C. n., Schiff, R. n., Jiang, B. n., Jovanovic, B. n., Nowak, K. n., Ericsson, M. n., Cameron, M. n., Gray, N. n., Dillon, D. n., Zhao, J. J., Sabatini, D. M., Jeselsohn, R. n., Brown, M. n., Polyak, K. n., Sicinski, P. n. 2020; 6 (25): eabb2210

    Abstract

    Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

    View details for DOI 10.1126/sciadv.abb2210

    View details for PubMedID 32704543

    View details for PubMedCentralID PMC7360435

  • Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances Fassl, A. n., Brain, C. n., Abu-Remaileh, M. n., Stukan, I. n., Butter, D. n., Stepien, P. n., Feit, A. S., Bergholz, J. n., Michowski, W. n., Otto, T. n., Sheng, Q. n., Loo, A. n., Michael, W. n., Tiedt, R. n., DeAngelis, C. n., Schiff, R. n., Jiang, B. n., Jovanovic, B. n., Nowak, K. n., Ericsson, M. n., Cameron, M. n., Gray, N. n., Dillon, D. n., Zhao, J. J., Sabatini, D. M., Jeselsohn, R. n., Brown, M. n., Polyak, K. n., Sicinski, P. n. 2020; 6 (25)

    Abstract

    Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

    View details for DOI 10.1126/sciadv.abb2210

    View details for PubMedID 32937480

  • Coordinating Tissue Regeneration Through Transforming Growth Factor-beta Activated Kinase 1 Inactivation and Reactivation STEM CELLS Hsieh, H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P. H., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019; 37 (6): 766–78

    View details for DOI 10.1002/stem.2991

    View details for Web of Science ID 000474038500008

  • Coordinating Tissue Regeneration through TGF-beta Activated Kinase 1 (TAK1) In-activation and Re-activation. Stem cells (Dayton, Ohio) Sung Hsieh, H. H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S. U., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019

    Abstract

    Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of TGF-beta activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by re-activation to elicit differentiation and extracellular matrix (ECM) production. While the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens are unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and re-activate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated re-activation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. SIGNIFICANCE STATEMENT: We target the TAK1 pathway to reduce heterotopic ossification, a pathologic condition in which bone develops within muscle or soft tissues. We show that Tak1 knockout leads to cellular proliferation; this can be harnessed to increase the number of cells present at the injury site. Using a mouse model, we inactivate and reactivate the Tak1 gene. We show that inactivation and reactivation of Tak1 can improve bony healing through the coordination of increased proliferation (inactivation) followed by differentiation (reactivation). This approach elucidates a new paradigm in regenerative medicine in which coordination between treatment and withdrawal of treatment can augment healing. © AlphaMed Press 2019.

    View details for PubMedID 30786091

  • A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles CELL Subramanian, A., Narayan, R., Corsello, S. M., Peck, D. D., Natoli, T. E., Lu, X., Gould, J., Davis, J. F., Tubelli, A. A., Asiedu, J. K., Lahr, D. L., Hirschman, J. E., Liu, Z., Donahue, M., Julian, B., Khan, M., Wadden, D., Smith, I. C., Lam, D., Liberzon, A., Toder, C., Bagul, M., Orzechowski, M., Enache, O. M., Piccioni, F., Johnson, S. A., Lyons, N. J., Berger, A. H., Shamji, A. F., Brooks, A. N., Vrcic, A., Flynn, C., Rosains, J., Takeda, D. Y., Hu, R., Davison, D., Lamb, J., Ardlie, K., Hogstrom, L., Greenside, P., Gray, N. S., Clemons, P. A., Silver, S., Wu, X., Zhao, W., Read-Button, W., Wu, X., Haggarty, S. J., Ronco, L. V., Boehm, J. S., Schreiber, S. L., Doench, J. G., Bittker, J. A., Root, D. E., Wong, B., Golub, T. R. 2017; 171 (6): 1437-+

    Abstract

    We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

    View details for DOI 10.1016/j.cell.2017.10.049

    View details for Web of Science ID 000417362700023

    View details for PubMedID 29195078

  • SIKs control osteocyte responses to parathyroid hormone NATURE COMMUNICATIONS Wein, M. N., Liang, Y., Goransson, O., Sundberg, T. B., Wang, J., Williams, E. A., O'Meara, M. J., Govea, N., Beqo, B., Nishimori, S., Nagano, K., Brooks, D. J., Martins, J. S., Corbin, B., Anselmo, A., Sadreyev, R., Wu, J. Y., Sakamoto, K., Foretz, M., Xavier, R. J., Baron, R., Bouxsein, M. L., Gardella, T. J., Divieti-Pajevic, P., Gray, N. S., Kronenberg, H. M. 2016; 7

    Abstract

    Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

    View details for DOI 10.1038/ncomms13176

    View details for PubMedID 27759007

  • Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma. Blood Matthews, J. M., Bhatt, S., Patricelli, M. P., Nomanbhoy, T. K., Jiang, X., Natkunam, Y., Gentles, A. J., Martinez, E., Zhu, D., Chapman, J. R., Cortizas, E., Shyam, R., Chinichian, S., Advani, R., Tan, L., Zhang, J., Choi, H. G., Tibshirani, R., Buhrlage, S. J., Gratzinger, D., Verdun, R., Gray, N. S., Lossos, I. S. 2016; 128 (2): 239-248

    Abstract

    Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), yet 40-50% of patients will eventually succumb to their disease demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNA's that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that Germinal Center Kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Since the majority of DLBCL tumors (~80%) exhibit activation of GCK, this therapy may be applicable to most patients.

    View details for DOI 10.1182/blood-2016-02-696856

    View details for PubMedID 27151888

  • Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis. Neuron Lee, S., Shang, Y., Redmond, S. A., Urisman, A., Tang, A. A., Li, K. H., Burlingame, A. L., Pak, R. A., Jovicic, A., Gitler, A. D., Wang, J., Gray, N. S., Seeley, W. W., Siddique, T., Bigio, E. H., Lee, V. M., Trojanowski, J. Q., Chan, J. R., Huang, E. J. 2016; 91 (1): 41-55

    Abstract

    Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.neuron.2016.05.021

    View details for PubMedID 27321923

    View details for PubMedCentralID PMC4938715

  • The immunological evolution of catalysis SCIENCE Patten, P. A., Gray, N. S., Yang, P. L., Marks, C. B., Wedemayer, G. J., Boniface, J. J., Stevens, R. C., Schultz, P. G. 1996; 271 (5252): 1086–91

    Abstract

    The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

    View details for DOI 10.1126/science.271.5252.1086

    View details for Web of Science ID A1996TW70100029

    View details for PubMedID 8599084