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The Discovery of SWI/SNF Chromatin Remodeling Activity As a Novel and Targetable Dependency In Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: The discovery of SWI/SNF chromatin remodeling activity as a novel and targetable dependency in 2 uveal melanoma 3 Authors: Florencia Rago1†, GiNell Elliott1†, Ailing Li1†, Kathleen Sprouffske2†, Grainne Kerr2†, Aurore 4 Desplat2†, Dorothee Abramowski2†, Julie T. Chen1†, Ali Farsidjani1†, Kay X. Xiang1†, Geoffrey Bushold1†, 5 Yun Feng1†, Matthew D. Shirley1†, Anka Bric1†, Anthony Vattay1†, Henrik Mӧbitz2◊, Katsumasa 6 Nakajima1◊, Christopher D. Adair1◊, Simon Mathieu1◊, Rukundo Ntaganda1◊, Troy Smith1◊, Julien P.N. 7 Papillon1◊, Audrey Kauffmann2†, David A. Ruddy1†, Hyo-eun C. Bhang1†, Deborah Castelletti2†, Zainab 8 Jagani1†* 9 † Oncology, ◊ Global Discovery Chemistry 10 1 Novartis Institutes for Biomedical Research, Cambridge, MA, USA 11 2 Novartis Institutes for Biomedical Research, Basel, Switzerland 12 * Corresponding Author: 13 Zainab Jagani 14 Novartis Institutes for Biomedical Research 15 250 Massachusetts Ave 16 600/03/3C-282 17 Cambridge, MA 02139 18 Phone: +16178714276 19 [email protected] 20 21 Running title: SWI/SNF complex is a novel target in uveal melanoma 22 Key words: BRM/SMARCA2, BRG1/SMARCA4, SWI/SNF, Uveal Melanoma, Chromatin Remodeling 23 Conflict of Interest Disclosure Statement: All authors performed the work herein as employees of the 24 Novartis Institutes for BioMedical Research. 25 Figures: 5 main, 6 supplemental 26 Tables: 4 supplemental 27 1 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no 3 approved targeted therapies and very few effective treatments for this cancer. While activating mutations 4 in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, few additional 5 drug targets have been identified. Recently, studies have identified context specific roles for the 6 mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer 7 lineages. Here we find evidence that the SWI/SNF complex is essential through analysis of functional 8 genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools 9 and small molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the 10 SWI/SNF complex and the melanocyte lineage specific transcription factor MITF, suggesting that these 11 two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These 12 studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the 13 treatment of this cancer. 14 15 16 17 18 19 20 21 22 23 2 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 Uveal melanoma is a rare cancer that arises from the melanocytes in of the uvea. Although it originates in 3 melanocytes, the underlying mutations and unique immune environment of the eye distinguish this cancer 4 from the more common cutaneous melanoma. Uveal melanoma is mainly characterized by driver 5 mutations in GNAQ or GNA11 which lead to hyperactivation of the G proteins, resulting in downstream 6 mitogen-activated protein kinase (MAPK) pathway activation (1,2). In addition, chromosome 8q 7 amplification and BAP1 loss of heterozygosity (chromosome 3) or silencing are also commonly observed 8 and correlate with increased aggressiveness and poor prognosis (3). To date, targeted agents, primarily 9 against the MAPK pathway and its effectors, such as PKC, and even immunotherapy, have resulted in 10 limited clinical responses, and surgery and radiation remain the most common treatments, with overall 11 poor prognosis upon detection of metastatic disease (4). Due to the paucity of available treatments, 12 further studies to understand the biology of the disease, as well as elucidate novel therapeutic targets, 13 remain critical. 14 We set out to uncover novel dependencies in uveal melanoma through analysis of previously 15 performed unbiased pooled short hairpin RNA (shRNA) screens, and discovered an unanticipated role of 16 the SWI/SNF chromatin-remodeling complex in survival of uveal melanomas. The SWI/SNF complex 17 represents an important tumor suppressor in cancer, with approximately 20% of tumors harboring 18 mutations in one or more of its subunits (5,6). More recently however, BRG1/SMARCA4, the catalytic 19 subunit of the SWI/SNF complex, has been shown to be essential for cancer survival, such as in AML 20 where it works in concert with leukemic transcription factors to facilitate MYC expression (7,8). 21 Additionally, it has been demonstrated that SWI/SNF drives active enhancer state maintenance in a 22 lineage specific manner, further suggesting its context specific role in tumor maintenance (9-11). By this 23 logic, the SWI/SNF complex represents a targetable node in the complex lineage-specific transcriptional 24 machinery that may drive certain cancers. 25 In this study, we show that uveal melanoma models are dependent on the SWI/SNF catalytic 26 subunits BRG1 and BRM (also known as SMARCA4 and SMARCA2, respectively) by genetic 27 knockdown. We also demonstrate broad activity of small molecule inhibitors of BRM/BRG1 ATPase 3 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 activity (12,13) across a panel of uveal melanoma cell lines. In an effort to dissect the mechanism of 2 SWI/SNF dependence, our work reveals a functional link between SWI/SNF and the transcription factor 3 Microphthalmia associated Transcription Factor (MITF). Together, our data identify SWI/SNF as a novel 4 target in uveal melanoma and reveal the therapeutic potential of applying small molecule inhibition of 5 SWI/SNF for the treatment of uveal melanoma. 6 7 Materials and Methods: 8 Cell lines and reagents 9 BRM011, BRM014 and BRM017 (synthesis described in (12,13)) stocks were dissolved at 10 mM in 10 DMSO. Doxycycline stock solution was made at 100 µg/ml in water. Shield 1 (Clontech) was dissolved at 11 0.5 mM in ethanol. 12 Cell lines were obtained from ATCC (MP41, MM28, MP46, MP65, MP38, and SW13), Sigma (Mel202), 13 Leiden University medical center (92.1 and OMM1), and Lonza (human epidermal melanocytes), and 14 cultured in manufacturer recommended media (92.1 and OMM1 were cultured in RPMI1640 + 10% FBS). 15 All parental lines were SNP profiled using the Fluidigm assay decribed previously (14) and tested 16 negative for Mycoplasma infection by qPCR (tests performed by Idexx Biosciences between March 2016 17 – February 2019). Cell lines used for no more than 1-3 months after thawing depending on doubling time. 18 Cell line engineering 19 shRNA cloning into pLKO based inducible vectors and cell line generation were described previously 20 (15). Cell line details annotated in Supplementary Table S3. 21 DD-BRG1 was assembled by adding Shield destabilization domain (DD) sequence (16) to the N-terminus 22 of BRG1 open reading frame (ORF). ACTL6A cDNA (Invitrogen) and DD-BRG1 were cloned into an in 23 house constitutive expression vector (lentiviral with EF1alpha promoter driving ORF expression). Flag- 24 HA-streptavidin (FHS) tagged MITF-M ORF was cloned into pLNCX-2 (Clontech). 4 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Growth, viability and apoptosis assays 2 To measure cell growth, cell lines were plated in 96 well plates (92.1 5000 cells, OMM1 2500, MP41 2500), 3 then imaged on an IncuCyte (4x objective) and analyzed using Incucyte Zoom 2016B software. 4 For viability assays, cells were plated in 384 well plates (Corning 3765) (92.1 500 cells, OMM1 500, MP41 5 500, Mel202 1500, MP46 1000, MM28 4000, MP38 3000, MP65 1500, SW13 1000, melanocytes 3000). 6 Plates were dosed with an 11 point, 3-fold serial dilution using the Echo550 (Labcyte). After 5 days, Cell 7 Titer Glo (Promega) was added and luminescence measured (PHERAstar, BMG Labtech). Growth 8 inhibition values were calculated as described previously (17). Normalized data were fit using the three 9 parameters nonlinear regression function in GraphPad Prism 7. Absolute AC50s (AAC50) reported as 10 concentrations of compound where curve fit crosses 0.5. 11 For caspase activity, cells were plated and treated as above (92.1 1500 cells, OMM1 1500, MP41 1500, 12 Mel202 3000, MP46 3000, MM28 5000, MP38 5000, MP65 3500, SW13 3000). After 48 h, Caspase 3/7 13 Glo (Promega) was added and luminescence measured. Normalization was performed relative to 14 untreated wells and plotted as fold activity. 15 Single point viability and caspase activity assays were performed in indicated 92.1 shRNA lines treated 16 with 250 nM BRM011, BRM014, BRM017 or 100 ng/ml doxycycline for 3 days and then assayed as 17 above and analyzed by normalizing relative to untreated wells.
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