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]

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

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.

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

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.

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).

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.

18 Compound profiling in 92.1

19 Cells were plated in 384 well plates in duplicate at 2000 cells per well. Cells were dosed with an 8 point,

20 3-fold serial dilution (compounds 8-14 Supplementary Table S1) on an Echo 550. Three days after

21 treatment, cell viability was measured using ATPlite 1 step (Perkin Elmer) and luminescence measured.

22 AAC50 for compounds were determined using an in house statistics package (HELIOS).

23 Western blot

24 Cells were harvested in T-PER lysis buffer (Thermo Fisher Scientific), 50 mM DTT, and HALT

25 protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific), then diluted in Laemmli sample buffer

1 (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to 0.2 µM nitrocellulose membrane

2 (Bio-Rad). Antibody information can be found in Supplementary Table S4. Blots were visualized on a Bio-

3 Rad Chemidoc imager by chemiluminescence (Pierce ECL, Thermo Fisher Scientific).

4 RNA-Seq

5 Sample preparation and sequencing

6 92.1 cells were plated in triplicate at 150,000 (72 or 48 h treatments) or 250,000 (24 h treatment) cells per

7 well in a 6-well plate. After treatment, RNA was isolated using Qiagen’s RNeasy Plus kit according to

8 manufacturer’s instructions. RNA integrity we assessed with using the Agilent 2100 and Agilent RNA

9 6000 Nano Kit.

10 Sample libraries were generated per manufacturer’s specifications on the Hamilton STAR

11 robotics platform using the TruSeq Stranded mRNA Library Prep Kit, High Throughput (Illumina) and 200

12 ng input RNA. The PCR amplified RNA-Seq library products were quantified using the Advanced

13 Analytical Fragment Analyzer Standard Sensitivity NGS Fragment Analysis Kit (Agilent). Samples were

14 diluted to 10 nM in Elution Buffer (Qiagen), denatured, and loaded between 2.5 to 4.0 pM on an Illumina

15 cBOT using the HiSeq® 4000 PE Cluster Kit (Illumina). Sequencing was performed on a HiSeq® 4000 at

16 75 base pair paired end with 8 base pair dual indexes using the HiSeq® 4000 SBS Kit, 150 cycles

17 (Illumina), and sequence intensity files were generated on instrument using the Illumina Real Time

18 Analysis software. The resulting intensity files were demultiplexed with the bcl2fastq2 software and

19 aligned to the human transcriptome using PISCES version 2018.04.01.

20 Differential Expression and Pathway Enrichment Analysis

21 Differential expression was determined using limma from Bioconductor (PMID: 25605792). Genes were

22 called differentially expressed if they had an average Log2 expression ≥ 0 (reported as AveExpr in

23 limma), an adjusted p-value ≤ 0.01, and an absolute Log2 fold-change of 0.5 relative to DMSO control.

24 Gene set enrichment analysis for differentially expressed genes was performed using the hypergeometric

25 test with FDR-adjusted p-values for 2 pathway sets downloaded from MSigDB (PMID:16199517; Hallmark

26 and KEGG).

1 ChIP qPCR

2 Assay was designed and performed by Active Motif. Details can be found in Supplemental Methods.

3 ATAC-Seq

4 Sample preparation and sequencing

5 100,000 cells were processed for ATAC-Seq using OMNI-ATAC-Seq protocol (18). After tagmentation,

6 samples were purified using Zymo DNA clean and concentration kit according to manufacturer’s protocol.

7 Purified DNA was used for library amplification using Nextera Index kit (Illumina) and NEBNext High-

8 Fidelity PCR master mix (New England biolabs). Number of amplification cycles (final <11) was

9 determined individually for each sample by qPCR using SYBR green and calculated as number of PCR

10 amplification cycles required to achieve one-third maximum fluorescent intensity. Amplified libraries were

11 purified using AMPure XP beads (Beckman). Final product quality and concentration was determined

12 using the D5000 DNA Tape on the Agilent Tapestation. Samples were diluted to 8 nM in Elution Buffer

13 (Qiagen), denatured, and loaded at 6.4 pM on a Miseq at 75 base pair paired end with 8 base pair dual

14 indexes using the MiSeq Reagent Kit v3, 150 cycles (Illumina). Sequencing data were used to recalculate

15 sample concentration adjusting for sample representation. Using updated concentrations, samples were

16 diluted to 8 nM in Elution Buffer, denatured, and loaded at 6.4 pM on an Illumina cBOT using the HiSeq®

17 4000 PE Cluster Kit (Illumina). Libraries were sequenced on a HiSeq® 4000 at 75 base pair paired end

18 with 8 base pair dual indexes using the HiSeq® 4000 SBS Kit, 150 cycles (Illumina), and sequence

19 intensity files were generated on instrument using the Illumina Real Time Analysis software. Detailed data

20 analysis methods can be found in Supplemental Methods.

21 RT-qPCR

22 Cells were plated at 15,000 cells per well in 96 well plate in biological quadruplicate per treatment

23 condition and treated with BRM011, BRM014 or BRM017 (0, 0,1, 1, 10, 100, 1000 nM) for 24 h. After

24 treatment, cells were lysed using Cells-to-Ct Bulk Lysis reagents (Thermo Fisher Scientific) and cDNA

25 synthesized using Cell-to-Ct Bulk RT reagents (Thermo Fisher Scientific) according to manufacturer’s

26 protocol. RT-qPCR was performed with Taqman Fast Advanced Master Mix (Applied Biosystems) on a

1 Viia 7 real-time PCR system (Applied Biosystems). Probes are annotated in Supplementary Table S4.

2 Relative quantification for each sample was calculated using the 2-ΔΔCt method (TBP normalized and

3 expressed as fold change relative to DMSO treated). Graphing and analyses were performed using

4 GraphPad Prism 7 (Graphpad Software).

5 In vivo efficacy study

6 Mice were maintained and handled in accordance with the Novartis Institutes for BioMedical Research

7 (NIBR) Institutional Animal Care and Use Committee (IACUC) and all studies were approved by the NIBR

8 IACUC. Female athymic nude mice (Charles River) were acclimated in NIBR animal facility (12 hour

9 light/dark cycle) with ad libitum access to food and water for at least 3 days before manipulation. Mice

10 (6–8 wk old) were inoculated subcutaneously in the right dorsal axillary region with the 92.1 cell line

11 (10x106 cells in 50% Matrigel). Tumor volumes and body weights were monitored twice per week and the

12 general health condition of mice was monitored daily. Tumor volume was determined by measurement

13 with calipers and calculated using a modified ellipsoid formula, where tumor volume (TV) (mm3) = [((l ×

14 w2) × 3.14159))/6], where l is the longest axis of the tumor and w is perpendicular to l. When average

15 tumor volume reached approximately 200 mm3, animals were randomly assigned to receive daily dosing

16 of either vehicle or BRM014 20 mg/kg. Compound treatments began 20 days post 92.1 cell implantation

17 and tumor samples were collected for PD analysis post 27 days of daily dosing.

18 RT-qPCR

19 Lysis buffer from Qiagen RNeasy plus mini kit was added to tumors samples, which were then

20 homogenized using Lysing Matrix D beads using a Precellys 24 homogenizer. Samples were further

21 homogenized using Qiashredder columns (Qiagen), and RNA isolated using the Qiagen RNeasy plus

22 mini kit according to manufacturer’s protocol. cDNA synthesis was performed according to manufacturer’s

23 protocol using ABI high capacity cDNA synthesis kit and 1 µg input RNA. RT-qPCR was performed in

24 technical duplicate (2 reactions per tumor) with FastStart Universal Probe master mix with Rox

25 (MilliporeSigma) on a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad). Probes are

26 annotated in Supplementary Table S4. Relative quantification for each sample was calculated using the 2-

1 ΔΔCt method (β-actin normalized and expressed as fold change relative to vehicle control). Graphing and

2 analyses were performed using GraphPad Prism 7 (Graphpad Software).

3 Data Accession

4 RNA-Seq and ATAC-Seq datasets can be accessed under BioProject ID: PRJNA622863

5 (http://www.ncbi.nlm.nih.gov/bioproject/622863).

7 Results

8 SWI/SNF complex is essential for uveal melanoma proliferation

9 Pooled screening efforts using shRNA have uncovered novel dependencies across a variety of cancer

10 types (19,20), but uveal melanoma is underrepresented across these studies due to the general scarcity

11 of models in this field. Due to this, only three cell lines, OMM1 (GNA11 Q209L), 92.1 (GNAQ Q209L) and

12 MEL285 (GNAQ/11 WT), were included in the large scale DRIVE screening effort (19). Notably, in the

13 two GNAQ/11 mutant cell lines, OMM1 and 92.1, various subunits of the SWI/SNF chromatin remodeling

14 complexes, including BRG1/SMARCA4, ACTL6A/BAF53a, SMARCB1, ARID1A and SMARCE1, were

15 among the top sensitizers (Figure 1A).

16 To validate the SWI/SNF subunit sensitizers, we engineered doxycycline-inducible shRNAs

17 against the genes that scored as hits. Interestingly, 92.1 showed a profound dependency on BRG1 alone

18 (Figure 1B and C), but not BRM (Supplementary Fig. S1D-E). However, in two other uveal melanoma

19 lines, OMM1 and MP41, knockdown of neither BRG1 nor BRM alone had an effect on cell growth (Fig.

20 1B, Supplementary Fig. S1A-E). Consistent with the screening data, robust depletion of obligate

21 SWI/SNF subunit ACTL6A (21,22) in OMM1 led to a growth arrest (Fig. 1D, Supplementary Fig. S1G),

22 whereas partial depletion was insufficient (Supplementary Fig. S1F-G). This phenotype was confirmed to

23 be on-target as evidenced by a growth rescue with ectopic expression of shRNA resistant ACTL6A (Fig.

24 1D, Supplementary Fig. S1G). These results suggest that the ACTL6A dependency potentially reflects

25 the importance of losing the combined catalytic activity of BRG1 and BRM, since ACTL6A is present in

1 both BRG1 and BRM containing SWI/SNF complexes. We tested this possibility by simultaneous

2 knockdown of BRM and BRG1 in the same cell lines, which notably led to a robust growth effect (Fig. 2,

3 Supplementary Fig. S2A-B). Together these data reveal that the SWI/SNF complex is essential in uveal

4 melanoma.

5 Next, we wished to determine the specific role of SWI/SNF ATPase activity in the observed

6 dependency. While the K785R mutation has been shown to abolish BRG1 catalytic activity without

7 affecting the protein’s ability to incorporate into the complex (23), it has also been described as a

8 dominant negative allele (24). Due to this, we used a destabilization domain (DD) BRG1 construct to

9 engineer 92.1 (Supplementary Fig. S2C), which repressed expression of the allele in the absence of the

10 stabilizing Shield 1 ligand (Supplementary Fig. S2D) (16). We observed successful rescue of proliferation

11 upon expression of the wild type allele, but did not see any rescue with the ATPase dead DD-BRG1

12 (Supplementary Fig. S2E). These data suggest the importance of the ATPase activity of BRG1, however

13 we cannot rule out other explanations due to the dominant negative behavior of this allele.

14 Uveal melanoma cell lines are exquisitely sensitive to small molecule inhibition of SWI/SNF

15 ATPase activity

16 The discovery of genetic dependency on SWI/SNF provided a clear rationale to test the activity of dual

17 BRG1/BRM ATPase small molecule inhibitors in this lineage. The two most potent inhibitors, BRM011

18 and BRM014, show equivalent inhibition of BRG1 and BRM catalytic activity in vitro, as well as robust

19 inhibition of BRM-dependent gene expression and cancer cell growth (13). The activity of these

20 compounds against BRM and BRG1 in biochemical characterization suggests that they would similarly

21 inhibit both proteins in cells. This was tested and confirmed by measuring concordant anti-proliferative

22 activity with analogs of various biochemical potencies in the 92.1 cell line which shows growth arrest upon

23 BRG1 or dual BRG1/BRM knockdown but not BRM knockdown alone ((13); Supplementary Table S1).

24 Taken together, these results suggest that these compounds are capable of inhibiting both BRG1 and

25 BRM cellular activity. We then treated a panel of uveal melanoma cell lines all expressing key members

26 of the SWI/SNF complex (BRG1, BRM, ARID1A and ACTL6A; Supplementary Fig. S3A), with BRM011

27 and BRM014. Cell viability was measured after treatment with BRM011 or BRM014 and compared to

1 BRM017, a structurally related cell-inactive analog (12). Across the cell line panel, there was a profound

2 dose-dependent sensitivity to both active compounds, whereas the control analog BRM017 showed a

3 growth effect at only the highest concentrations (>100 fold shifted from concentrations in which an

4 equivalent phenotype is seen with BRM011) (Fig. 3A). Additionally, increased caspase 3/7 activity after

5 compound treatment was measured in 92.1, Mel202, MP38 and MP41, indicating that these cells are

6 undergoing apoptosis upon SWI/SNF inhibition (Fig. 3B), while OMM1, MM28, MP46 and MP65 show a

7 stasis phenotype (Fig. 3A, Supplementary Fig. S3B). The increase in caspase activity observed upon

8 compound treatment was similar to that observed in the 92.1 dual BRG1/BRM shRNA lines, suggesting

9 that the apoptotic phenotype is likely the result of SWI/SNF inhibition (Supplementary Fig. S3C). In

10 summary, 7 of the 8 cell lines in our panel were highly sensitive to the dual inhibitor BRM014 with

11 absolute AC50s (AAC50) < 100 nM (Supplementary Table S2). Of note, we found no underlying mutation

12 pattern in the SWI/SNF complex members that would explain the observed sensitivity to inhibition

13 (Supplementary Fig. S3D).

14 To confirm the selectivity of our compounds we treated the BRG1/BRM deficient cell line SW13

15 (25,26). In this cell line we only saw partial activity of BRM011 in the viability assay (Amax = 50%), and no

16 activation of caspase activity (Supplementary Fig. S3E, Supplementary Table S2). In order to understand

17 the potential for differential responses between tumor and normal tissue, we also treated non-transformed

18 melanocytes and observed a partial anti-proliferative effect. This activity, particularly for compound

19 BRM014, was significantly less potent on the melanocytes (BRM011 AAC50 = 46.2 nM, BRM014 AAC50

20 = 454.8 nM; Supplementary Fig. S3F, Supplementary Table S2) than observed for many of the uveal cell

21 lines (more than 500 fold shifted relative to 92.1), suggesting the potential for a window of SWI/SNF

22 inhibition between the malignant uveal and normal melanocyte population.

23 The sensitivity observed with small molecule inhibition of SWI/SNF ATPase activity further

24 validates the SWI/SNF dependency observed with genetic perturbation of the complex, and indicates that

25 the dependency may be more broadly applicable across uveal melanoma models. The activity of

26 BRM011 and BRM014 against both BRG1 and BRM allowed successful interrogation of this dependency

1 across a panel of uveal melanoma lines, which may have otherwise not have shown sensitivity to

2 inhibition of only one catalytic subunit alone, such as in the case of OMM1 and MP41.

3 Genomic profiling reveals a functional link between SWI/SNF and MITF activity

4 We next interrogated the mechanisms underlying SWI/SNF driven growth in uveal melanoma, testing the

5 hypothesis that a functional relationship with an essential transcription factor may direct the complex to

6 important loci where SWI/SNF mediated chromatin remodeling could drive an essential transcriptional

7 program. We identified MITF as another dependency common to both 92.1 and OMM1 in the pooled

8 screening data (Fig. 1A). MITF and BRG1 have been previously described to work together in various

9 reports showing that BRG1 is essential to drive the MITF dependent transcriptional program (27-31). In

10 order to determine whether this is relevant in the uveal lineage, we first knocked down MITF using an

11 inducible shRNA in 92.1, OMM1, and MP41, and observed a growth arrest in all three cell lines (Fig. 4A,

12 Supplementary Fig. S4A-B). This phenotype was confirmed to be on target by rescuing the growth arrest

13 in 92.1 through exogenous overexpression of MITF (Fig. 4A).

14 To identify whether MITF and SWI/SNF share transcriptional targets as previously demonstrated

15 in cutaneous melanoma (28,31), expression analysis by RNA sequencing (RNA-Seq) was performed

16 after SWI/SNF (BRG1/BRM) or MITF knockdown or treatment with BRM011 in 92.1. As expected, robust

17 down-regulation of the shRNA target genes was observed across the knockdown conditions

18 (Supplementary Fig. S4C). In addition, there was a significant overlap in genes affected by BRG1/BRM

19 dual knockdown versus MITF knockdown (p<1E-5) (Fig. 4B), as well as these two genetic perturbations

20 and chemical inhibition of SWI/SNF activity by BRM011 (Supplementary Fig. S4D). Among the genes

21 affected by both MITF and SWI/SNF knockdown, pathways affecting hypoxia, apoptosis, cell cycle and

22 differentiation were enriched (p<1E-4), consistent with the profound growth effect observed upon

23 knockdown (Supplementary Fig. S4E). The pattern of transcriptional changes across the three different

24 perturbations indicates that SWI/SNF and MITF control an overlapping gene set (Fig. 4C). Taken

25 together, these data suggest that SWI/SNF catalytic activity plays an important role in the MITF

26 transcriptional program, and support a functional relationship between SWI/SNF and MITF.

1 To further interrogate the relationship between SWI/SNF and MITF, we performed chromatin

2 immunoprecipitation and quantification by PCR (ChIP-qPCR) to monitor co-occupancy of BRG1 and MITF

3 at discrete genomic loci. Specifically, the DCT and TYR promoters were probed in 92.1 cells because

4 both show modulation by MITF and SWI/SNF knockdown (Supplementary Fig. S4F). Both MITF and

5 BRG1 localized to the promoters of DCT and TYR consistent with previous reports (Fig. 4D) (31),

6 suggesting that SWI/SNF and MITF co-localize to certain loci to modulate gene expression.

7 Next, assay for transposase accessible chromatin (ATAC-Seq) was used to investigate the

8 changes in chromatin structure that occur upon SWI/SNF inhibition. The resulting reads (indicating open

9 regions of chromatin) from 92.1 cells treated with BRM011 or DMSO for 24 hours were annotated relative

10 to their closest gene as residing either downstream, overlapping, or upstream of the gene body or in the

11 promoter (Supplementary Fig. S4F, top). Among these peaks, a large proportion were observed to

12 change in accessibility (~18 %) when cells were treated with BRM011, with the majority decreasing in

13 accessibility (Supplementary Fig. S4F bottom). We chose to focus on changes in promoter associated

14 peaks because SWI/SNF has been previously described to play an important role in nucleosome

15 remodeling at promoters (32,33), and, as expected, we observed a marked decrease in chromatin

16 accessibility near transcriptional start sites (TSS) (Fig. 4E). Of note, chromatin closing was observed at

17 the promoters of DCT and TYR (Fig. 4F, Supplementary Fig. S4F, bottom), the same loci where BRG1

18 occupancy was observed by ChIP-qPCR (Fig. 4D), and which showed decreased expression upon

19 compound treatment by RNA-Seq (Supplementary Fig. S4G). To further probe the relationship between

20 SWI/SNF and MITF, we tested for enrichment of MITF binding sites in the accessible chromatin regions

21 identified by ATAC-Seq. We observed a significantly larger fraction of MITF binding sites in the peaks

22 which were depleted upon BRM011 treatment than those that either did not change or were enriched

23 (Supplementary Fig. S4H), supporting a subset of overlapping activity for SWI/SNF and MITF in the

24 genome.

25 Finally, we wished to confirm the effect of SWI/SNF inhibition on MITF and MITF target gene

26 expression. In order to do this, we treated the panel of uveal melanoma cell lines with increasing doses of

27 BRM011, BRM014 and BRM017 and measured expression of MITF-M, TYR, and DCT by RT-qPCR. We

1 consistently observed across the cell lines more potent repression of the target genes by BRM011 and

2 BRM014 as compared to BRM017 (Supplementary Fig. S5A), and this sensitivity to target gene

3 modulation upon compound treatment was generally consistent with the BRM014 AAC50s of these cell

4 lines.

5 MITF overexpression partially rescues cell death after SWI/SNF inhibition

6 The RNA-Seq data showed modulation of MITF transcript levels upon BRM011 treatment, and a

7 similar phenotype was observed at the protein level (Supplementary Fig. S5B). To determine the extent to

8 which the phenotype upon inhibition of SWI/SNF was dependent on the downregulation of MITF, we

9 overexpressed MITF in 92.1 (Supplementary Fig. S5B), and then treated these cells with BRM011,

10 BRM014 and BRM017. The resulting viability effects were compared to empty vector alone. Upon

11 compound treatment, MITF expression was retained in the overexpression cell line, and there was a large

12 shift in compound sensitivity as compared to empty vector (Fig. 4G, Supplementary Fig. S5C), indicating

13 a critical role for SWI/SNF mediated regulation of MITF in maintaining survival of uveal melanoma cells.

14 However, due to the incomplete rescue observed, additional growth promoting pathways affected by

15 SWI/SNF independently of MITF likely contribute to the phenotype.

16 BRM014 treatment results in the growth arrest of a uveal melanoma tumor xenograft

17 Finally, to characterize the phenotype of SWI/SNF inhibition in vivo, the 92.1 model was grown as a tumor

18 xenograft in SCID mice and treated with BRM014. Once the tumors reached an average of 200 mm3, we

19 orally dosed the animals with either vehicle or BRM014 at 20 mg/kg once daily. Similar to the in vitro

20 sensitivity, treatment with BRM014 led to significant tumor growth inhibition (Day 41 - 10.32 %T/C) (Fig.

21 5A). As observed in independent studies (13), the 20mg/kg daily dosing regimen did not result in any

22 significant changes in body weight (Fig. 5B). We tested the modulation of key genes identified in the

23 RNA-Seq dataset in tumors collected at the end of treatment by RT-qPCR, and saw robust inhibition

24 across a number of these genes (Fig. 5C). In particular, genes involved in cell proliferation such as CDK2,

25 as well as MITF target genes involved in melanocyte differentiation (MLANA, PMEL, and RAB27A), were

26 down-regulated, suggesting successful target engagement in tumors. Of note, we did not observe tumor

1 regression as would be expected from the apoptosis seen in vitro, and in fact saw outgrowth in a subset

2 of the tumors after 2 weeks (Fig. 5D), indicating that SWI/SNF activity may not be fully inhibited in this

3 dosing regimen.

5 Discussion

6 Uveal melanoma is a rare cancer with limited treatment options, especially targeted therapeutics.

7 Recent work has suggested that hyper-activating mutations in GNAQ antagonize PRC2 mediated

8 repression of differentiation markers, maintaining cells in a de-differentiated state, and that inhibition of

9 mutant GNAQ leads to differentiation of these cells, which can be counter-acted by inhibition of PRC2

10 (34). Similar to the epigenetic factor PRC2, SWI/SNF has also been shown to be a key player across

11 cancers of many different genetic backgrounds, and provides an exciting potential therapeutic target (35).

12 Here, we describe a dependency on the SWI/SNF complex in uveal melanoma using both genetic and

13 chemical perturbation of the complex. In contrast to our findings, a recent report identified BRD9, a

14 member of the non-canonical BAF complex (ncBAF) as a tumor suppressor in uveal melanoma (36).

15 Such work suggests there are potential differences in penetrance of perturbing BRD9, which is specific to

16 nc-BAF versus BRG1 and BRM, which are members of all the functional SWI/SNF complexes.

17 Although models for uveal melanoma are limited, the significant anti-proliferative activity of

18 BRM011 and BRM014 across the various uveal melanoma models suggests that this may represent a

19 dependency on SWI/SNF that could broadly impact approaches to disease treatment. Importantly, there

20 are examples of cancers, such as small cell carcinoma of the ovary hypercalcemic type, in which

21 expression of both BRG1 and BRM has been lost (37,38), suggesting that SWI/SNF complex activity is

22 not universally essential in cancers, and further defining the importance of the lineage specific context

23 when defining SWI/SNF dependence. Along those lines, we find that the dependency in this lineage is

24 mediated, at least in part, by the melanocyte lineage specific transcription factor MITF. Importantly, these

25 factors co-localize to certain genetic loci and affect transcription of an overlapping gene set. Furthermore,

26 the data shown here suggest a novel therapeutic strategy for treatment of uveal melanoma via inhibition

1 of the SWI/SNF complex. The extensive investigation of the effects of dual inhibitors in vivo reported in

2 Jagani et al (12), suggests that a major limitation of dual BRM/BRG1 inhibition may be due to on-target

3 toxicity. Interestingly, while 40-70% modulation of downstream transcriptional markers of SWI/SNF

4 activity was sufficient to see robust growth inhibition in 92.1, similar levels of target

5 gene/pharmacodynamic inhibition in BRG1-mutant lung cancer models did not induce a growth arrest

6 (12,13), suggesting that the requirements of SWI/SNF inhibition for efficacy can vary depending on the

7 disease context. It is also important to note that although compound treatment was able to elicit apoptosis

8 in 92.1 in vitro, this was not true in vivo. This could be due to resistance to treatment or indicate that a

9 higher dose is required for a robust growth inhibition, and merits further investigation. Our work provides

10 an important foundation from which future studies to investigate rational combination partners with

11 SWI/SNF inhibition should provide important insights into design of highly efficacious treatments for uveal

12 melanoma. It will also be interesting to determine if these discrepancies between lineages hold true using

13 molecules with alternative mechanisms of action, such as the recently described BRG1/BRM PROTAC

14 (39), as this will provide further mechanistic insight into the structural versus catalytic role of the SWI/SNF

15 catalytic subunits. Together these data provide an exciting new paradigm for treatment of SWI/SNF

16 dependent cancers.

18 Acknowledgements

19 The authors would like to thank Lin Fan and Vera Ruda for sequencing of RNA-Seq samples, Lindsey U.

20 Rodrigues for assistance with sample processing, Emilie Niemezyk, Franklin Chung and Dan Rakiec for

21 technical expertise, Matthew Crowe for sharing non-transformed melanocytes, Joshua Korn for cell line

22 mutational data, and Eusebio Manchado, Andrew Wylie, Joshua Korn, Vesselina Cooke, Serena Silver,

23 E. Rob McDonald, Darrin Stuart, Francesco Hofmann, and Jeffrey Engelman for discussions and

24 feedback.

1 Author Contributions:

2 Conception and design: F. Rago and Z. Jagani

3 Development of methodology: F. Rago, G. Elliott, K. Sprouffske, G. Kerr, H.C. Bhang, Z. Jagani

4 Acquisition of data: F. Rago, A. Li, A. Desplat, D. Abramowski, J. Chen, A. Farsidjani, K.X. Xiang, G.

5 Bushold, Y. Feng, A. Bric, A. Vattay, H. Mobitz, K. Nakajima, C.D. Adair, S. Mathieu, R. Ntaganda, T.

6 Smith

7 Analysis and interpretation of data: F. Rago, G. Elliott, K. Sprouffske, G. Kerr, M. Shirley

8 Writing, review and/or revision of the manuscript: F. Rago and Z. Jagani

9 Study supervision: J.P.N. Papillon, A. Kauffmann, D. Ruddy, H.C. Bhang, D. Castelletti, Z. Jagani

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1 Figure Legends

2 Figure 1: Uveal melanoma cell lines are dependent on SWI/SNF complex members. A) OMM1 and

3 92.1 pooled shRNA screen data from McDonald et al (2017) showing all genes tested ranked by

4 sensitivity score (calculated using ATARiS method). MITF, GNAQ, GNA11 and SWI/SNF subunits that

5 scored in the screen are highlighted. B-D) Top - growth as measured by confluence for indicated cell lines

6 is graphed. Expression of shRNAs was induced by addition of doxycycline. (●) + doxycycline (100

7 ng/mL), ■ untreated. N=3, error bars shown as s.d. Bottom - representative images for indicated time

8 point. Scale bar = 0.5 mm.

9 Figure 2: Uveal melanoma cell lines are dependent on the ATPase activity of SWI/SNF. Top - growth

10 as measured by confluence for indicated cell lines is graphed. Expression of shRNAs was induced by

11 addition of doxycycline. (●) + doxycycline (100 ng/mL), ■ untreated. N=3, error bars shown as s.d.

12 Bottom - representative images at t = 156 h. Scale bar = 0.5 mm.

13 Figure 3: Chemical inhibition of SWI/SNF causes growth arrest in uveal melanoma cell lines. A)

14 Viability after uveal melanoma cell lines were treated with BRM011, BRM014 and BRM017 for 5 days is

15 plotted relative to DMSO treated and baseline (0.0) is set at day 0 viability. Error bars are shown as s.d.,

16 N=4. Data were fit using GraphPad Prism. B) Fold caspase activity in uveal melanoma cell lines treated

17 with BRM011, BRM014 and BRM017 for 48 h is plotted relative to DMSO treated. Error bars are shown

18 as s.d., N=4.

19 Figure 4: SWI/SNF perturbation affects an MITF dependent transcriptional program. A) Top - growth

20 as measured by confluence for indicated cell lines. Expression of shRNAs was induced by addition of

21 doxycycline. (●) + doxycycline (100 ng/mL), ■ untreated. N=3, error bars shown as s.d. Bottom -

22 representative images for indicated time point. Scale bar = 0.5 mm. B) Venn diagram of overlap in

23 transcriptional changes under indicated conditions. Genes were determined to be significantly up or

24 down-regulated if absolute log fold change (logFC) ≥ 0.5, average expression ≥ 1 and p ≤ 0.01. C)

25 Heatmap of genes for which expression changes were measured under indicated conditions (absolute

26 logFC ≥ 0.5, average expression ≥ 1 and p ≤ 0.01) in at least one condition are plotted together with

1 expression across all other conditions. The color scale bar indicates the logFC ranges for the

2 comparisons to untreated samples. D) ChIP-qPCR for MITF and BRG1 at indicated loci. N =3. Error bars

3 shown as s.d. E) Median ATAC-Seq read density (RPKM (reads per kilobase per million mapped reads))

4 is plotted for peaks at TSS. Data is shown for 92.1 treated with either BRM011 (100 nM) or DMSO for 24

5 h. F) ATAC-Seq tracks showing DCT and TYR promoter regions in 92.1 treated with BRM011 or DMSO

6 for 24 h. Average density for 3 replicates is shown. G) Viability is plotted for 92.1 engineered to express

7 exogenous MITF or empty vector, then treated with BRM011 or BRM014 for 5 days. Viability is plotted

8 relative to DMSO treated and baseline (0.0) is set at day 0 viability. Error bars are shown as s.d., N=4.

9 Figure 5: Chemical inhibition of SWI/SNF activity leads to growth arrest of 92.1 in vivo A) Tumor

10 growth as measured by tumor volume for either vehicle or BRM014 treatment. B) Body weight for animals

11 in study shown in 5A. C) Expression of indicated genes in endpoint samples of tumors in study shown in

12 5A. Error bars shown as s.e.m for all graphs. N = 6 for vehicle treatment and 10 for BRM014 treatment. P-

13 values indicated on figure were calculated using student’s t-test (* = p < 0.05; ** = p < 0.01; *** = p <

14 0.001). D) Tumor volume of BRM014 treated tumors are plotted individually. Tumors shown in purple

15 showed stasis and tumors shown in blue demonstrated regrowth at end of treatment.

A C 92.1 shBRG1 2202 92.1 shBRG1 2527 150 150 0 Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 -1 -2 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. -3 100 100 -4 -5 -6 50 50

-7 Confluence % OMM1 Confluence %

MITF 0 0 0 50 100 150 200 250 GNA11 0 50 100 150 200 250 ACTL6A Time (h) Time (h) SMARCA4

1 Untreated + Dox Untreated + Dox 0 -1 Sensitivity Score -2 -3 -4 -5 -6 -7 t = 156 h t = 156 h -8 -9 -10 92.1 D

MITF OMM1 shACTL6A 1632 OMM1 shACTL6A 1632 GNAQ

ARID1A + ex. ACTL6A ACTL6A 150 150 SMARCE1 SMARCB1 SMARCA4

100 100

B 50 50 % Confluence % 92.1 shNTC OMM1 shNTC Confluence %

150 150 0 0 0 50 100 150 200 250 0 50 100150 200 250 300 Time (h) Time (h) 100 100 Untreated + Dox Untreated + Dox

50 50 % Confluence % % Confluence %

0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (h) Time (h) t = 156 h t = 156 h

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 Figure 2 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

92.1 shBRG1 2202/ OMM1 shBRG1 2202/ MP41 shBRG1 2202/ 150 shBRM 5537 150 shBRM 5537 150 shBRM 5537

100 100 100

50 50 50 % Confluence % Confluence % % Confluence %

0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Time (h) Time (h) Time (h) Untreated + Dox Untreated + Dox Untreated + Dox

t = 156 h t = 156 h t = 156 h

Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 A Author manuscripts have been peer reviewed and accepted forB publication but have not yet been edited. 92.1 OMM1 BRM011 92.1 BRM011 1.5 1.5 BRM014 6 BRM014 5 1.0 BRM017 BRM017 1.0 4 0.5 3

0.0 Viability Viability 0.5 2

-0.5 Activity Caspase 1

-1.0 0.0 0 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 [Compound] µM [Compound] µM [Compound] µM

Mel202 MP41 Mel202 1.5 1.5 4

1.0 1.0 3 0.5 0.5 2

Viability 0.0 Viability 0.0 1 -0.5 -0.5 Activity Caspase

-1.0 -1.0 0 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 [Compound] µM [Compound] µM [Compound] µM

MM28 MP38 MP38 1.5 1.5 2.0

1.0 1.0 1.5

0.5 1.0 Viability 0.5 Viability 0.0 0.5 Caspase Activity Caspase 0.0 -0.5 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 0.0 [Compound] µM [Compound] µM 0.0001 0.001 0.01 0.1 1 10 100 [Compound] µM MP46 MP65 MP41 1.5 2.0 3 1.5 1.0

1.0 2 0.5

Viability Viability 0.5 0.0 1 0.0 Caspase Activity Caspase -0.5 -0.5 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 0 [Compound] µM [Compound] µM 0.0001 0.001 0.01 0.1 1 10 100 [Compound] µM

92.1 A Author Manuscript Published OnlineFirstB on August92.1 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013C 92.1 shMITF 10 150 92.1 shMITF 10 + ex. MITF BRM011 100 nM BRM 5537 72 h BRM 5537 24 h 150 BRM011 10 nM

shBRG1/BRM shMITF 10 24 h shMITF 10 48 h

Author manuscripts have been peer reviewed andshMITF accepted for publication but have not yet beenshBRG1 2202/ edited.shBRG1 2202/ shMITF 6 24 h shMITF 6 48 h 24 h 24 h 100 100 24 h 24 h

50 50 % Confluence %

0 0 0 50 100 150 200 250 0 50 100 150 200 250 300 700 592 479 5 Time (h) Time (h)

Untreated + Dox Untreated + Dox 0

−5

t = 156 h t = 156 h

D MITF ChIP BRG1 ChIP 35 6 30 25 10 4

5 2

0 0 5 3 6 1 0 k k k 5 2 k k 3 6 k 1 0 k k k 5 12 3 1k 5 2 9k 8k 8 7k 1 3 2 1 4 1 1 5 2 9k 8 8 7k 1 3 2 1 4 r 5 7 6 6 6 7 - - - 1 tr 82 7 6 6 6 - - - 1 Binding Events Detected / 1000 Cells 1000 / Detected Events Binding - - Cells 1000 / Detected Events Binding - nt + 95 93 + 75 + 6 R R n + 95 93 + + 75 + 67 R U P 6 6 6 T 6 Y Y R U T 6 6 T 6 6 YR YR R A CT CT CT + C T T TYRY L2 + C + + C CT + CT + TY T T R D T + T +D D D T C D T D D D T TY A C C CT + CT + B CT C CT C B D D D D D D D D A G Primer location Primer location

E Fchr13 94,470,000 94,475,000 94,480,000 94,485,000 BRM011 127 17.5 DMSO

92.1 DMSO

15.0 0 127 RPKM

92.1 BRM011

12.5 0

−1000 −500 0 500 1000 DCT Distance to transcription start site (TSS) chr11 89,170,000 89,180,000 89,190,000 89,200,000 89,210,000 127 G 92.1 DMSO 92.1 + MITF+ empty vector 1.5 BRM011 0 1.0 BRM014 127

0.5 92.1 BRM011

Viability 0.0

-0.5 0

-1.0 0.0001 0.001 0.01 0.1 1 10 100 [Compound] µM TYR

Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Figure 5 A B Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-19-1013 1200 Author manuscripts haveVehicle been peer reviewed and accepted for10 publication but have not yet beenVehicle edited. 20 mpk BRM014 QD 20 mpk BRM014 QD 1000 5 ± SEM) 3 800

600 0

400 -5 Tumor volume (mm 200

0 SEM) ± (Mean (%) Change Weight Body -10 22 24 26 28 30 32 34 36 38 40 42 44 46 22 24 26 28 30 32 34 36 38 40 42 44 46 Days Post Implantation Days Post Implantation begin dosing

C D BRM014 Treated Tumors 2.5 Vehicle 800 * BRM014

2.0 600 ± SEM) 3 1.5 400 1.0 * * * *** 200 0.5 *** *** * *** *** Tumor volume (mm

Fold change rel. to Vehicle to rel. change Fold 0.0 0 2 T M A A 0 R 1 22 24 26 28 30 32 34 36 38 40 42 44 46 K C N EL 7 YP P D D F A M 2 S TY C IT L P B OX1 Days Post Implantation M A S TYR M R Gene

The discovery of SWI/SNF chromatin remodeling activity as a novel and targetable dependency in uveal melanoma

Florencia Rago, GiNell Elliott, AILING LI, et al.

Mol Cancer Ther Published OnlineFirst August 3, 2020.

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