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Immunoprecipitation of BRCA2 . List of proteins identified via mass spectrometry.

Single nucleotide polymorphisms (SNP) in Capan1 olaparib resistant cells

Gene Expression of Capan1 olaparib resistant cells

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

DNA methylation is an epigenetic mark that is altered in cancer and aging tissues. The effects of extrinsic factors on DNA methylation remain incompletely understood. Microbial dysbiosis is a hallmark of colorectal cancer, and infections have been linked to aberrant DNA methylation in cancers of the GI tract. To determine the microbiota’s impact on DNA methylation, we studied the methylomes of colorectal mucosa in germ-free (no microbiota) and specific-pathogen-free (controlled microbiota) mice, as well as in Il-10 KO mice (Il10−/−) which are prone to inflammation and tumorigenesis in the presence of microbiota. The presence of microbiota was associated with changes in 5% of the methylome and Il10−/− mice showed alterations in 4.1% of the methylome. These changes were slightly more often hypo than hypermethylation and affected preferentially CpG sites located in gene bodies and intergenic regions. Mice with both Il-10 KO and microbiota showed much more pronounced alterations, affecting 18% of the methylome. When looking specifically at CpG island methylation alterations, a hallmark of aging and cancer, 0.4% were changed by the microbiota, 0.4% were changed by Il10−/−, while 4% were changed by both simultaneously. These effects are comparable to what is typically seen when comparing colon cancer to normal. We next compared these methylation changes to those seen in aging, and after exposure to the colon carcinogen Azoxymethane (AOM). Aging was associated with alterations in 18% of the methylome, and aging changes were accelerated in the Il10−/− /SPF mice. By contrast, AOM induced profound hypomethylation that was distinct from the effects of aging or of the microbiota. CpG sites modified by the microbiota were over-represented among DNA methylation changes in colorectal cancer. Thus, the microbiota affects the DNA methylome of colorectal mucosa in patterns reminiscent of what is observed in aging and in colorectal cancer.

SUMMARY ARID1A , a subunit of the SWI/SNF complex, is among the most frequently mutated genes across cancer types. ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCCs), diseases that have no effective therapy. Here, we show that ARID1A mutation confers sensitivity to pan-HDAC inhibitors such as SAHA in ovarian cancers. This correlated with enhanced growth suppression induced by the inhibition of HDAC2 activity in ARID1A-mutated cells. HDAC2 interacts with EZH2 in an ARID1A status-dependent manner. HDAC2 functions as a co-repressor of EZH2 to suppress the expression of EZH2/ARID1A target tumor suppressor genes such as PIK3IP1 to inhibit proliferation and promote apoptosis. SAHA reduced the growth and ascites of the ARID1A-inactivated OCCCs in both orthotopic and genetic mouse models. This correlated with a significant improvement of survival of mice bearing ARID1A-mutated OCCCs. These findings provided preclinical rationales for repurposing FDA-approved pan-HDAC inhibitors for treating ARID1A-mutated cancers., In Brief Fukumoto et al. show that ARID1A mutation confers sensitivity to pan-HDAC inhibitors such as SAHA in ovarian cancers. This correlated with enhanced growth suppression induced by the inhibition of HDAC2 activity in ARID1A-mutated cells. These findings provided preclinical rationales for repurposing FDA-approved pan-HDAC inhibitors for treating ARID1A-mutated cancers.

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.

Inactivation of the subunits of SWI/SNF complex such as ARID1A is synthetically lethal with inhibition of EZH2 activity. However, mechanisms of de novo resistance to EZH2 inhibitors in cancers with inactivating SWI/SNF mutations are unknown. Here we show that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 drives resistance to EZH2 inhibitors in ARID1A-mutated cells. SMARCA4 loss upregulates anti-apoptotic genes in the EZH2 inhibitor-resistant cells. EZH2 inhibitor-resistant ARID1A-mutated cells are hypersensitive to BCL2 inhibitors such as ABT263. ABT263 is sufficient to overcome resistance to an EZH2 inhibitor. In addition, ABT263 synergizes with an EZH2 inhibitor in vivo in ARID1A-inactivated ovarian tumor mouse models. Together, these data establish that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 underlies the acquired resistance to EZH2 inhibitors. They suggest BCL2 inhibition alone or in combination with EZH2 inhibition represents urgently needed therapeutic strategy for ARID1A-mutated cancers., The mechanism of resistance to EZH2 inhibitors in cancers with inactivating SWI/SNF mutations is unknown. Here, the authors demonstrate that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 drives resistance to EZH2 inhibitors in ARID1A-mutated ovarian cancer cells.

CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in human cancers. However, clinically applicable cancer therapeutic strategies based on CARM1 expression remain to be explored. Here, we report that EZH2 inhibition is effective in CARM1-expressing epithelial ovarian cancer. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppresses the growth of CARM1-expressing, but not CARM1-deficient, ovarian tumors in two xenograft models and improves the survival of mice bearing CARM1-expressing ovarian tumors. The observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes in a CARM1-dependent manner. Mechanistically, CARM1 promotes EZH2-mediated silencing of EZH2/BAF155 target tumor suppressor genes by methylating BAF155, which leads to the displacement of BAF155 by EZH2. Together, these results indicate that pharmacological inhibition of EZH2 represents a novel therapeutic strategy for CARM1-expressing cancers., CARM1 is an arginine methyltransferase often overexpressed in human cancer. Here, the authors show that EZH2 inhibition suppresses growth in CARM1-expressing epithelial ovarian cancer, and examine the mechanism of how CARM1 promotes EZH2-mediated tumor suppressor gene silencing.

The SWI/SNF chromatin remodeling complex is altered in ~20% of human cancers. ARID1A, a component of the SWI/SNF chromatin-remodeling complex, is the most frequently mutated epigenetic regulator in human cancers. Inactivation of the SWI/SNF complex is synthetically lethal with inhibition of EZH2 activity. EZH2 inhibitors are entering clinical trials for specific tumor types with SWI/SNF mutations. However, mechanisms of de novo or acquired resistance to EZH2 inhibitors in cancers with inactivating SWI/SNF mutations are unknown. Here we show that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 drives resistance to EZH2 inhibitors in ARID1A-mutated ovarian cancer cells. SMARCA4 decease dominates over SMARCA2 increase in the switch. SMARCA4 loss leads to suppression of apoptotic pathways through upregulating anti-apoptotic genes such as BCL2 in the EZH2 inhibitor resistant cells. EZH2 inhibitor resistant ARID1A-mutated cells are hypersensitive to BCL2 inhibitors such as ABT263. ABT263 is sufficient to overcome resistance to EZH2 inhibitor and synergistic with EZH2 inhibitor in vivo in ARID1A-inactivted ovarian tumour mouse models. Together, these data establish that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 underlies the acquired resistance to EZH2 inhibitors and BCL2 inhibition alone or in combination with EZH2 inhibition represents a novel strategy to overcome and/or prevent EZH2 inhibitor resistance in ARID1A-mutated cancers. Given that the SWI/SNF subunits are among the most frequently mutated genes in human cancers and EZH2 inhibitors are in the clinical trials for tumor with mutations in the SWI/SNF complex, we expect our findings to have far-reaching implications for developing cancer epigenetic therapeutics. Citation Format: Shuai Wu, Nail Fatkhutdinov, Takeshi Fukumoto, Benjamin G. Bitler, Pyoung Hwa Park, Andrew V. Kossenkov, Marco Trizzino, Alessandro Gardini, David W. Speicher, Rugang Zhang,. CATALYTIC SUBUNITS SWITCH DRIVES RESISTANCE TO EZH2 INHIBITORS IN ARID1A-MUTATED CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-114.

DNA methylation changes are important in cancer development and the etiology of the CpG Island Methylator Phenotype (CIMP), the most extreme form of aberrant promoter DNA methylation in cancer, can only partially be explained by genetic changes. An association between CIMP and the gut microbiota has been implicated in colorectal cancer (CRC) pathogenesis but the exact mechanisms underlying the observations are poorly understood. In order to clarify the link between CIMP and the microbiome in CRC, we studied human primary tumor and adjacent mucosal tissues (normal) from CRC patients (n=46). The patient samples were categorized by tumor sites (23 proximal and 23 distal), sex (28 males and 18 females), and age (69 yrs ±11). Bisulfite pyrosequencing and Digital Restriction Enzyme Analysis of Methylation (DREAM) were performed to determine the methylation of CpG sites in the CRC tumors. The tumor samples were selected based on their CIMP status (10 high, 10 low, and 26 negative) and the tumor sites. 16S rRNA gene sequencing using Illumina-Hi-Seq was performed on those samples. Principle Coordinate analysis of the 16S rRNA sequences showed that the microbiota of CIMP-positive (CIMP-high and -low combined) tumors was significantly different from the microbiota of the CIMP-negative tumors (PERMANOVA, P = 0.013). Linear discriminant analysis effect size (LEfSe) showed an enrichment of specific bacterial taxa in the CIMP-positive microbiota. Fusobacterium (LDA score = 4.71, p=0.033) and Erysipelotrichaceae (LDA score = 3.77, p=0.004) were most enriched in the CIMP-positive tumors along with Bacteroides. The microbiota of different tumor sites also showed significant difference with Fusobacterium (LDA score = 4.32, p=0.047) and Erysipelotrichaceae (LDA score = 3.45, p=0.039) being enriched in the proximal tumors compared to the distal tumors. All these bacterial taxa have previous been found to be associated with CRC and/or metabolic disorders. We validated the 16S rRNA data by qPCR analysis using genus-specific probes. In the same samples, Pan-Fusobacterium and Bacteroides fragilis were enriched in the CIMP-positive tumors. The median number of Pan-Fusobacterium per 100 human cells was 12-fold higher in the CIMP-positive tumors (p=0.0015) and 6-fold higher in the case of B. fragilis (p=0.0228). Thus, our data show broad difference in the microbiota of CIMP-positive CRCs compared to CIMP-negative CRCs. The significantly enriched bacterial taxa in CIMP-positive CRC suggest that these specific taxa could play an important role in aberrant DNA methylation modulation in colorectal cancer, and further studies should elucidate the mechanism underlying the CIMP/microbiota link in the context of CRC. Citation Format: Pyoung Hwa Park, Raad Z. Gharaibeh, Lauren Cole, Ang Sun, Woonbok Chung, Jaroslav Jelinek, Jillian L. Pope, Christian Jobin, Jean-Pierre J. Issa. CIMP is associated with altered microbiota composition in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-141.

PURPOSE OF THE STUDY: ARID1A is mutated ~50% and 30% of clear cell (OCCC) and endometrioid (OEC) ovarian cancers, respectively. Over 90% of the ARID1A mutations observed in ovarian cancer are frame-shift or nonsense mutations that result in loss of ARID1A protein expression. In several cancers including OCCC, ARID1A and p53 mutations are often mutually exclusive. OCCC carries a worse prognosis compared to the other histosubtypes of ovarian cancer. Thus, there is an urgent clinical need for improved therapeutic strategies. EXPERIMENTAL PROCEDURE: To investigate the role of specific HDACs in the context of ARID1A expression, we examined the effect of knocking down HDACs in an isogenic OCCC cell line with and without ARID1A expression. Utilizing a panel of ARID1A-mutated and wildtype OCCC cell lines, we evaluated the effect of HDAC knockdown and inhibition through a variety of 2D and 3D assays. We also examined the consequence of HDAC inhibition on tumor growth, survival, and dissemination in an in vivo mouse model of ovarian cancer. SUMMARY OF THE DATA: We have discovered that ARID1A is involved in the repression of HDACs, and ARID1A-inactivation promotes aberrant transcriptional regulation of HDACs. Importantly, we uncovered a novel HDAC-dependent regulatory mechanism of p53. Several HDAC inhibitors are currently in clinical trials. We found HDAC inhibitors were more selective in ARID1A-mutated OCCC cell lines compared to ARID1A-wildtype cells. We observed that HDAC inhibition led to a significant increase in apoptosis in ARID1A-mutated cells. In an orthotopic intra-bursal xenograft model using ARID1A wildtype and deficient cells, the HDAC inhibitor suppressed primary tumor growth and inhibited tumor cell dissemination only in the ARID1A-mutated tumors. Significantly, HDAC inhibition significantly improved the survival of mice bearing ARID1A-mutated tumors. CONCLUSIONS: These findings imply that HDAC inhibition represents a novel therapeutic strategy for ARID1A-deficient cancers. This study further elucidates the observed mutual exclusivity of ARID1A and p53. Importantly, we have uncovered a novel regulatory mechanism of p53 mediated through ARID1A and HDACs. Clinically, this study describes a precision medicine approach to the treatment of ARID1A-mutated OCCC. Citation Format: Benjamin G. Bitler, Pyoung Hwa Park, Yang Hai, Katherine M. Aird, Yemin Wang, David G. Huntsman, Kathleen R. Cho, David W. Christianson, Rugang Zhang. INHIBITION OF HDAC ACTIVITY SELECTIVELY INHIBITS ARID1A–MUTATED OVARIAN CLEAR CELL CARCINOMA THROUGH A NOVEL P53 REGULATORY MECHANISM [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP25.