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The Discovery of SWI/SNF Chromatin Remodeling Activity As a Novel And

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The Discovery of SWI/SNF Chromatin Remodeling Activity As a Novel And Published OnlineFirst August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGY AND TRANSLATIONAL STUDIES The Discovery of SWI/SNF Chromatin Remodeling Activity as a Novel and Targetable Dependency in Uveal Melanoma Florencia Rago1, GiNell Elliott1, Ailing Li1, Kathleen Sprouffske2, Grainne Kerr2, Aurore Desplat2, Dorothee Abramowski2, Julie T. Chen1, Ali Farsidjani1, Kay X. Xiang1, Geoffrey Bushold1, Yun Feng1, Matthew D. Shirley1, Anka Bric1, Anthony Vattay1, Henrik Mobitz€ 2, Katsumasa Nakajima1, Christopher D. Adair1, Simon Mathieu1, Rukundo Ntaganda1, Troy Smith1, Julien P.N. Papillon1, Audrey Kauffmann2, David A. Ruddy1, Hyo-eun C. Bhang1, Deborah Castelletti2, and Zainab Jagani1 ABSTRACT ◥ Uveal melanoma is a rare and aggressive cancer that originates screens and further validation in a panel of uveal melanoma cell in the eye. Currently, there are no approved targeted therapies lines using both genetic tools and small-molecule inhibitors of and very few effective treatments for this cancer. Although SWI/SNF. In addition, we describe a functional relationship activating mutations in the G protein alpha subunits, GNAQ between the SWI/SNF complex and the melanocyte lineage– and GNA11, are key genetic drivers of the disease, few additional specific transcription factor Microphthalmia-associated Tran- drug targets have been identified. Recently, studies have identi- scription Factor, suggesting that these two factors cooperate to fied context-specific roles for the mammalian SWI/SNF chro- drive a transcriptional program essential for uveal melanoma cell matin remodeling complexes (also known as BAF/PBAF) in survival. These studies highlight a critical role for SWI/SNF in various cancer lineages. Here, we find evidence that the SWI/ uveal melanoma, and demonstrate a novel path toward the SNF complex is essential through analysis of functional genomics treatment of this cancer. Introduction We set out to uncover novel dependencies in uveal melanoma through analysis of previously performed unbiased pooled short Uveal melanoma is a rare cancer that arises from the melanocytes of hairpin RNA (shRNA) screens, and discovered an unanticipated role the uvea. Although it originates in melanocytes, the underlying of the SWI/SNF chromatin–remodeling complex in survival of uveal mutations and unique immune environment of the eye distinguish melanomas. The SWI/SNF complex represents an important tumor this cancer from the more common cutaneous melanoma. Uveal suppressor in cancer, with approximately 20% of tumors harboring melanoma is mainly characterized by driver mutations in GNAQ or mutations in one or more of its subunits (5, 6). More recently however, GNA11 which lead to hyperactivation of the G proteins, resulting BRG1/SMARCA4, the catalytic subunit of the SWI/SNF complex, has in downstream MAPK pathway activation (1, 2). In addition, been shown to be essential for cancer survival, such as in acute myeloid chromosome 8q amplification and BAP1 loss of heterozygosity leukemia where it works in concert with leukemic transcription factors (chromosome 3) or silencing are also commonly observed and to facilitate MYC expression (7, 8). In addition, it has been demon- correlate with increased aggressiveness and poor prognosis (3). To strated that SWI/SNF drives active enhancer state maintenance in a date, targeted agents, primarily against the MAPK pathway and its lineage-specific manner, further suggesting its context-specific role in effectors, such as PKC, and even immunotherapy, have resulted in tumor maintenance (9–11). By this logic, the SWI/SNF complex limited clinical responses, and surgery and radiotherapy remain the represents a targetable node in the complex lineage-specific transcrip- most common treatments, with overall poor prognosis upon detection tional machinery that may drive certain cancers. of metastatic disease (4). Due to the paucity of available treatments, In this study, we show that uveal melanoma models are dependent further studies to understand the biology of the disease, as well as on the SWI/SNF catalytic subunits BRG1 and BRM (also known as elucidate novel therapeutic targets, remain critical. SMARCA4 and SMARCA2, respectively) by genetic knockdown. We also demonstrate broad activity of small-molecule inhibitors of BRM/ BRG1 ATPase activity (12, 13) across a panel of uveal melanoma cell lines. In an effort to dissect the mechanism of SWI/SNF dependence, 1Novartis Institutes for Biomedical Research, Cambridge, Massachusetts. 2Novartis Institutes for Biomedical Research, Basel, Switzerland. our work reveals a functional link between SWI/SNF and the tran- scription factor Microphthalmia associated Transcription Factor Note: Supplementary data for this article are available at Molecular Cancer (MITF). Together, our data identify SWI/SNF as a novel target in Therapeutics Online (http://mct.aacrjournals.org/). uveal melanoma and reveal the therapeutic potential of applying small- Corresponding Author: Zainab Jagani, Novartis Institutes for Biomedical molecule inhibition of SWI/SNF for the treatment of uveal melanoma. Research, 250 Massachusetts Avenue, 600/03/3C-282, Cambridge, MA 02139. Phone: 617-8714241; Fax: 617-8714083; E-mail: [email protected] Materials and Methods – Mol Cancer Ther 2020;XX:XX XX Cell lines and reagents doi: 10.1158/1535-7163.MCT-19-1013 BRM011, BRM014, and BRM017 (synthesis described in refs. 12, 13) Ó2020 American Association for Cancer Research. stocks were dissolved at 10 mmol/L in DMSO. Doxycycline stock AACRJournals.org | OF1 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 Rago et al. solution was made at 100 mg/mL in water. Shield 1 (Clontech) was cocktail (Thermo Fisher Scientific), and then diluted in Laemmli dissolved at 0.5 mmol/L in ethanol. sample buffer (Bio-Rad). Proteins were separated by SDS-PAGE and Cell lines were obtained from the ATCC (MP41, MM28, MP46, transferred to 0.2 mmol/L nitrocellulose membrane (Bio-Rad). Anti- MP65, MP38, and SW13), Sigma (Mel202), Leiden University medical body information can be found in Supplementary Table S4. Blots were center (92.1 and OMM1), and Lonza (human epidermal melanocytes), visualized on a Bio-Rad Chemidoc imager by chemiluminescence and cultured in manufacturer-recommended media (92.1 and OMM1 (Pierce ECL, Thermo Fisher Scientific). were cultured in RPMI1640 þ 10% FBS). All parental lines were SNP profiled using the Fluidigm assay described previously (14) and tested RNA sequencing negative for Mycoplasma infection by qPCR (tests performed by Idexx Sample preparation and sequencing Biosciences between March 2016 and February 2019). Cell lines were 92.1 cells were plated in triplicate at 150,000 (72- or 48-hour used for no more than 1 to 3 months after thawing depending on treatments) or 250,000 (24-hour treatment) cells per well in a 6-well doubling time. plate. After treatment, RNA was isolated using Qiagen's RNeasy Plus kit according to the manufacturer's instructions. We assessed RNA Cell line engineering integrity using the Agilent 2100 and Agilent RNA 6000 Nano Kit. shRNA cloning into pLKO-based inducible vectors and cell line Sample libraries were generated as per the manufacturer's specifica- generation were described previously (15). Cell line details are anno- tions on the Hamilton STAR robotics platform using the TruSeq tated in Supplementary Table S3. Stranded mRNA Library Prep Kit, High Throughput (Illumina), and DD-BRG1 was assembled by adding Shield destabilization domain 200 ng input RNA. The PCR-amplified RNA sequencing (RNA-seq) (DD) sequence (16) to the N-terminus of BRG1 open reading frame library products were quanti fied using the Advanced Analytical Frag- (ORF). ACTL6A cDNA (Invitrogen) and DD-BRG1 were cloned into ment Analyzer Standard Sensitivity NGS Fragment Analysis Kit an in-house constitutive expression vector (lentiviral with EF1alpha (Agilent). Samples were diluted to 10 nmol/L in Elution Buffer promoter driving ORF expression). Flag-HA-streptavidin–tagged (Qiagen), denatured, and loaded between 2.5 and 4.0 pmol/L on an MITF-M ORF was cloned into pLNCX-2 (Clontech). Illumina cBOT using the HiSeq 4000 PE Cluster Kit (Illumina). Sequencing was performed on a HiSeq 4000 at 75 base pair paired Growth, viability, and apoptosis assays end with 8 base pair dual indexes using the HiSeq 4000 SBS Kit, 150 To measure cell growth, cell lines were plated in 96-well plates (92.1 cycles (Illumina), and sequence intensity files were generated on 5,000 cells, OMM1 2,500, MP41 2,500), then imaged on an IncuCyte instrument using the Illumina Real Time Analysis software. The (4x objective), and analyzed using IncuCyte Zoom 2016B software. resulting intensity files were demultiplexed with the bcl2fastq2 soft- For viability assays, cells were plated in 384-well plates (Corning ware and aligned to the human transcriptome using PISCES version 3765; 92.1 500 cells, OMM1 500, MP41 500, Mel202 1,500, MP46 2018.04.01. 1,000, MM28 4,000, MP38 3,000, MP65 1,500, SW13 1,000, and melanocytes 3,000). Plates were dosed with an 11-point, 3-fold serial Differential expression and pathway enrichment analysis dilution using the Echo550 (Labcyte). After 5 days, Cell Titer Glo Differential expression was determined using limma from Biocon- (Promega) was added and luminescence measured (PHERAstar, BMG ductor (PMID: 25605792). Genes were called differentially expressed
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