Small Cell Carcinoma Of The Ovary, Hypercalcemic Type Cell Research Articles

Establishment and characterization of a novel cell line (SCCOHT-CH-1) and PDX models derived from Chinese patients of small cell ovarian carcinoma of the hypercalcemic type

Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is a rare and aggressive malignancy that poses a significant clinical challenge due to its grim prognosis. Unfortunately, only three SCCOHT cell lines are currently available for scientific research. In this study, we have successfully established a novel SCCOHT cell line from a recurrent lesion of a SCCOHT patient, named SCCOHT-CH-1. We comprehensively characterized the novel cell line by employing techniques such as morphological observation, CCK-8 assay, Transwell assay, clone formation assay, short tandem repeat sequence (STR) analysis, karyotype analysis, immunohistochemical staining, western blot assay, and xenograft tumor formation assay. SCCOHT-CH-1 cells were small circular and had a unique STR profile. The population-doubling time of SCCOHT-CH-1 was 33.02 h. The cell line showed potential migratory and invasive ability. Compared with another SCCOHT cell line COV434, SCCOHT-CH-1 exhibited higher expression of AKT, VIM, and CCND1. At the same time, SCCOHT-CH-1 has the ability of tumorigenesis in vivo. We also successfully constructed three patient-derived xenograft (PDX) models of SCCOHT, which were pathologically diagnosed to be consistent with the primary tumor, accompanied by loss of SAMRCA4 protein expression. The establishment of SCCOHT-CH-1 cell line and PDX models from Chinese people represent a pivotal step toward unraveling the molecular mechanism of SCCOHT and fostering the development of targeted interventions to tackle this challenging malignancy.

Re-assigning the histologic identities of COV434 and TOV-112D ovarian cancer cell lines

ObjectiveThe development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors. MethodsFor COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical follow-up on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells. ResultsThe available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically. ConclusionsCOV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.

Abstract LB-038: Novel functional insights revealed by distinct protein-protein interactions of the residual SWI/SNF complex in SMARCA4-deficient small cell carcinoma of the ovary, hypercalcemic type

Abstract Chromatin remodeling plays a critical role in tumor suppression as demonstrated by 20% of human cancers bearing inactivating mutations in SWI/SNF chromatin remodeling complex members. Mutations in different SWI/SNF subunits drive a variety of adult and pediatric tumor types, including non-small cell lung cancers, rhabdoid tumors, medulloblastomas, and ovarian cancers. Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is an aggressive subtype of ovarian cancer occurring in young women. Nearly all (>98%) SCCOHTs have inactivating mutations in SMARCA4, which encodes 1 of 2 mutually exclusive catalytic subunits of the SWI/SNF complex. Less than half of SCCOHT patients survive 5 years despite aggressive surgery and multimodal chemotherapy. Empirical support for effective SCCOHT treatments is scarce, in part because of the poor understanding of SCCOHT tumorigenesis. To gain insight into the functional consequences of SWI/SNF subunit loss, we defined SWI/SNF composition and its protein-protein interactions (PPIs) by immunoprecipitation and mass spectrometry (IP-MS) of SWI/SNF subunits in 3 SCCOHT cell lines. Comparing these results to a cell line containing a wild-type SWI/SNF complex, the interaction of most canonical core SWI/SNF subunits was observed in all SCCOHT cell lines at a lower abundance. The SCCOHT SWI/SNF also lacked ATPase module subunits and showed a drastic reduction in PBAF-specific subunit interactions. The wild-type and SCCOHT SWI/SNF subunits immunoprecipitated a shared set of 26 proteins, including core SWI/SNF subunits and RNA processing proteins. We observed 131 proteins exclusively interacting with the wild-type SWI/SNF complex including isoform-specific SWI/SNF subunits, members of the NuRD complex, and members of the MLL3/4 complex. We observed 60 PPIs exclusive to the SCCOHT residual SWI/SNF shared in at least 2 of the 3 SCCOHT cell lines, including many proteins involved in RNA processing. Differential interactions with the residual SWI/SNF complex in SCCOHT may further elucidate altered functional consequences of SMARCA4 mutations in these tumors as well as identify synthetic lethal targets that translate to other SWI/SNF-deficient tumors. Citation Format: Elizabeth Raupach, Krystine Garcia-Mansfield, Ritin Sharma, Apurva Hegde, Victoria David-Dirgo, Yemin Wang, Chae Young Shin, Lan Tao, Salvatore Facista, Rayvon Moore, Jessica Lang, Victoria Zismann, Krystal Orlando, Monique Spillman, Anthony Karnezis, Lynda Bennett, David Huntsman, Jeffrey Trent, William Hendricks, Bernard Weissman, Patrick Pirrotte. Novel functional insights revealed by distinct protein-protein interactions of the residual SWI/SNF complex in SMARCA4-deficient small cell carcinoma of the ovary, hypercalcemic type [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-038.

The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer.

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.

Abstract 3869: The reversible LSD1 inhibitor SP-2509 promotes anti-tumor immunity in small cell carcinoma of the ovary-hypercalcemic type (SCCOHT)

Abstract Purpose: Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer of young women characterized by SWI/SNF ATPase SMARCA4 (BRG1) genetic inactivation alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Because the SWI/SNF complex associates with histone modifying complexes (HMCs) containing targetable epigenetic enzymes, the lack of SWI/SNF ATPase activity is hypothesized to interrupt normal function of HMCs that result in altered epigenetic landscape. Lysine-specific histone demethylase 1 (LSD1 or KDM1) regulates the chromatin landscape and gene expression by removing specific methyl groups from methylated proteins including histone H3. It associates with a number of transcriptional regulatory complexes including SWI/SNF and acts in a context-dependent manner. We and others have found that LSD1 is among the most highly expressed histone modifiers in SCCOHT. SCCOHT cell lines are very sensitive to the reversible LSD1 inhibitor SP-2509 compared to other epigenetic agents like EZH2 and bromodomain inhibitors, suggesting that LSD1 dependence is a defining feature of these SWI/SNF-deficient ovarian cancers. Recently it has been shown that LSD1 inhibition stimulates an INF-dependent anti-tumor immunity and overcomes resistance to checkpoint blockade through induction of Endogenous Retroviruses (ERVs) in breast cancer. Interestingly, SCCOHT have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T-cell infiltration (TIL). In this study we investigate the ability of SP-2509 to promote anti-tumor immunity and T-cell infiltration in SCCOHT. Experimental Design: SCCOHT cell line BIN67, COV434 and SCCOHT1 were subjected to RT-PCR to evaluate ERVs induction and INF expression in response to SP-2509 treatment. SCCOHT cells were also co-cultured with peripheral blood mononuclear cells (PBMCs) in a 3D platform to evaluate T-cell infiltration following SP-2509 treatment. Results: SP-2509 promotes ERV-mediated immune response in SCCOHT cell lines. Additionally, SP-2509 stimulates T-cell infiltration in SCCOHT in a spheroid platform. Conclusion: Our data suggest that the reversible LSD1 inhibitor SP-2509 stimulates an INF-dependent anti-tumor immunity in SCCOHT cells in vitro and in 3D platform. Given that LSD-1 is a potential therapeutic target in SWI/SNF mutated SCCOHT, the combination of SP-2509 and PD-1 inhibitor represents a plausible combination to induce or augment immunogenic responses in these tumors. Citation Format: Raffaella Soldi, Alexis Weston, Trason Thode, Rhonda Lewis, Mohan Kaadige, Hariprasad Vankayalapati, William Hendricks, Sunil Sharma. The reversible LSD1 inhibitor SP-2509 promotes anti-tumor immunity in small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) [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 3869.

Abstract LB-058: GB-3103, an epigenetic immunomodulator, shows potent antitumor activity against tumors harboring dual loss of SMARCA4/SMARCA2 ATPases

Abstract Dual loss of SMARCA4/SMARCA2 ATPases of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex has been reported in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) and other tumors. Loss of SMARCA4 is the result of inactivating mutations, and the loss of SMARCA2 results from the absence of mRNA expression. Restoration of either SMARCA4 or SMARCA2 can inhibit the growth of these cancers. We have evaluated the activity of a novel, structurally rigid, and potent, Class I/IIb HDAC inhibitor, GB-3103, against human SCCOHT and other cells lines deficient in SWI/SNF complex. GB-3103 shows potent anti-proliferative activities with low nM IC50 values against human SCCOHT lines BIN67 (51nM), COV434 (35nM) and SCCOHT-1 (293nM), and SWI/SNF-deficient rhabdoid and lung tumor lines A204 (95nM), A427 (174nM), G401 (138 nM), G402 (71nM), H522 (102nM). Treatment of human BIN67 SCCOHT cell line for 72h with GB-3103 revealed potent concentration- and time-dependent induction of SMARCA2 expression at both mRNA and protein levels. Treatment of mice bearing G401 human malignant rhabdoid tumor xenografts with GB-3103 at 5 mg/kg, QD resulted in 70% tumor growth inhibition (TGI) compared to vehicle control (P<0.05). Treatment of mice bearing BIN67 human tumor xenografts with GB-3103 at 5 mg/kg and 10 mg/kg, QD resulted in mean tumor regression of 26% and 33%, respectively, at two weeks post-treatment initiation. RNA-Seq analyses of BIN67 cells treated with GB-3103 revealed that GB-3103 affects DNA replication and mRNA stability as well as inducing the expression of MHC Class II proteins. Given the importance of MHC Class II expression and response to checkpoint inhibitor therapies, we tested the activity of GB-3103 alone and in combination with anti-mPD-1 and anti-mPD-L1 antibodies in a syngeneic CT-26 mouse colon cancer model. GB-3103 induced a 93% TGI as a single agent and caused regression of established CT-26 tumors when combined with either anti-mPD-1 or anti-mPD-L1 demonstrating the potent immunomodulatory activity of GB-3103 in addition to direct anticancer activities. GB-3103 is a novel epigenetic immunomodulator with potent anticancer activity against SWI/SNF-deficient cancers. Clinical development of GB-3103 in these genetically defined rare cancers for which no treatments currently exist provides unique clinical and regulatory opportunity for breakthrough therapy designation where approval could be based on smaller single arm clinical studies. Citation Format: Tong Wang, Paul Gonzales, Kari Kotlarczyk, Myung-Ja Lee, Erin Bossert, Courtney Devore, Haiyong Han, Jessica Lang, William Hendricks, Jeffrey Trent, Stephen Gately. GB-3103, an epigenetic immunomodulator, shows potent antitumor activity against tumors harboring dual loss of SMARCA4/SMARCA2 ATPases [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-058.

CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

Ponatinib Shows Potent Antitumor Activity in Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) through Multikinase Inhibition.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.

Abstract 1238: Targeting the platelet derived growth factor receptor (PDGFR) with the receptor tyrosine kinase inhibitor ponatinib in small cell carcinoma of the ovary, hypercalcemic type

Abstract Subunits of the SWI/SNF chromatin-remodeling complex are tumor suppressors that are inactivated in ~20% of all cancers, yet few targeted treatments have shown selective activity in SWI/SNF-mutant cancers. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by SWI/SNF dysregulation. Given that two-year survival following standard high-dose chemotherapy and radiation in SCCOHT is less than 35%, a great need exists for effective targeted therapies to improve outcomes for these women. We previously demonstrated that SCCOHT tumors are driven by inactivating mutations in SMARCA4, one of two mutually exclusive SWI/SNF ATPases. In addition, we have shown that SCCOHT lacks expression of the alternative SWI/SNF ATPase, SMARCA2. We have now found through integrated genomic and functional analyses in SCCOHT tumors and cell lines that SMARCA4 loss correlates with increased expression of receptor tyrosine kinases (RTKs) including the platelet derived growth factor receptors (PDGFRs). Through integration of high-throughput RNA interference and drug screens in SCCOHT cells we have identified sensitivity to RTK knockdown and RTK inhibitors including the FDA-approved oncology drug, ponatinib. These data corroborate prior studies showing RTK dependence in rhabdoid tumors, rare cancers that are also driven by mutations in the SWI/SNF complex. Of the known ponatinib targets, PDGFR-alpha and FGFR1 were highly expressed in SCCOHT tumors, as confirmed in RNA-Seq data (four tumors) and a SCCOHT tissue microarray (TMA; ten tumors). Furthermore, PDGFR-alpha and -beta phosphorylation and downstream signaling are inhibited by ponatinib in SCCOHT cells, suggesting that these tumors are sensitive to ponatinib due to dependence on signaling through these RTKs. Finally, given ponatinib’s potency in vitro and the proposed mechanism of action, we tested this agent in xenograft models of SCCOHT. In addition to confirming efficacy in a SCCOHT cell line xenograft model, superior efficacy was demonstrated in two patient-derived xenograft (PDX) models of SCCOHT with ponatinib. Thus, ponatinib effectively targets SWI/SNF-mutant SCCOHT tumors through inhibition of PDGFR signaling and may have clinical utility for the treatment of these cancers. Citation Format: Jessica Diane Lang, William Hendricks, Pilar Ramos, Holly Yin, Chris Sereduk, Jeffrey Kiefer, Yemin Wang, Anthony N. Karnezis, Bernard Weissman, David Huntsman, Jeffrey Trent. Targeting the platelet derived growth factor receptor (PDGFR) with the receptor tyrosine kinase inhibitor ponatinib in small cell carcinoma of the ovary, hypercalcemic type [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1238. doi:10.1158/1538-7445.AM2017-1238

Abstract AP32: THE METHYLTRANSFERASE EZH2 IS A VULNERABLE TARGET IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE (SCCOHT)

Abstract PURPOSE: SCCOHT is a rare, poorly differentiated and very deadly ovarian tumor that mainly affects young women with the median age in twenties. Recently, we and others have discovered that the genome of SCCOHT was minimally disturbed compared to common malignancies with the inactivation mutations of SMARCA4 being the only recurrent genetic alternation in SCCOHT genome. SMARCA4 is the ATPase of the SWI/SNF chromatin-remodeling complex, which is the master regulator of gene transcription through controlling the accessibility of chromatin and therefore plays critical roles in many biological processes such as cell cycle control, apoptosis, and differentiation. Recently, a genetic antagonism has been observed between some SWI/SNF complex components and the histone methyltransferase EZH2 of the polycomb repressive complex 2 (PRC2), suggesting that EZH2 may be a vulnerable target in certain SWI/SNF-deficient tumor. Therefore, we aimed to determine the promise of defeating SCCOHT through targeting EZH2 in the present study. METHODS: The EZH2 protein expression was determined by immunohistochemistry on the SCCOHT tissue microarray. Cell viability, cell cycle and apoptosis were measured by crystal violet staining, FACS analysis and live cell imaging with activated caspase-3/7 staining, respectively. The proteomic profiles of SCCOHT cells were determined by Mass Spectrometry using the TMT-labeled peptide fractions. Two SCCOHT xenograft mouse models were established from BIN67 and SCCOHT1 cells, respectively, in immune-deficient mice were used for the evaluation of EPZ-6438 efficacy in vivo. RESULT: All (24/24) SCCOHT expressed abundant EZH2 protein. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to two EZH2 inhibitors (GSK126 and EPZ-6438) and EZH2 shRNA. Furthermore, EPZ-6438 induced cell cycle arrest, apoptosis and cell differentiation in SCCOHT cells, accompanying by the induction of genes involved in cell proliferation, apoptosis and differentiation. Treatment of EPZ-6438 in SCCOHT xenograft-bearing mice delayed tumor progression, improved the survival of mice. Further, re-expression of SMARCA4 suppressed the expression of EZH2 and the global level of histone H3K27 trimethylation, induced the expression of PRC2 targets that are induced by EZH2 inhibitors and triggered differentiation of SCCOHT cells. In addition, we also discovered that SCCOHT cells are hypersensitive to multiple inhibitors of histone deacetylase (HDAC), which is known to cooperate with EZH2 to silence certain target genes. Combined treatment of EPZ-6438 and Quisinostat (an HDAC inhibitor) synergistically suppressed the proliferation of SCCOHT cells. CONCLUSION: Our data suggest that EZH2 is a promising therapeutic target for fighting SCCOHT. Combined pharmacological targeting of both EZH2 and HDAC may provide a novel treatment option for SCCOHT patients. Citation Format: Yemin Wang, Shary Chen, Anthony Karnezis, Nancy Dos Santos, Pilar Ramos, Sarah Maines-Bandiera, Christine Chow, Bernard Weissman, Jeffrey Trent, David Huntsman. THE METHYLTRANSFERASE EZH2 IS A VULNERABLE TARGET IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE (SCCOHT) [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 AP32.

Abstract NTOC-090: TARGETING AND EFFICACY OF THE RECEPTOR TYROSINE KINASE INHIBITOR PONATINIB IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE, WORKS THROUGH INHIBITION OF PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR)

Abstract The work that will be presented strongly suggests the identification of a unique vulnerability of Small Cell Carcinoma of the Ovary-Hypercalcemic Type (SCCOHT) to PDGFR inhibition. SCCOHT is an aggressive subtype of ovarian cancer afflicting young women and children with fewer than 35% of patients surviving two years after diagnosis. Currently, these patients must endure high dose chemotherapy because no effective targeted therapies have been identified. We previously demonstrated that these tumors are driven by inactivating mutations in SMARCA4, one of two ATPases in the SWI/SNF chromatin-remodeling complex. In addition, we have also shown that SCCOHT lacks expression of the alternative SWI/SNF ATPase, SMARCA2. SWI/SNF is a key tumor suppressor complex in diverse cancers and its members are mutationally inactivated in up to 50% of other ovarian cancer subtypes. However, no therapeutic approaches selective for SWI/SNF-dysregulation in cancers are routinely used. We have now found through multiomic (DNA/RNA), pathway analysis, immunohistochemistry and functional studies in SCCOHT tumors and cell lines that SMARCA4 loss correlates with increased expression of receptor tyrosine kinases (RTKs) including the platelet derived growth factor receptors (PDGRs). Through integration of high-throughput RNA interference and drug screens in SCCOHT cell lines we have also identified sensitivity to RTK knockdown and inhibitors such as ponatinib. These data corroborate prior studies showing RTK dependence in rhabdoid tumors, rare cancers that are also driven by mutations in the SWI/SNF complex. As ponatinib is approved for cancer treatment and showed great potency, we tested this agent in two patient-derived xenograft (PDX) models of SCCOHT. Ponatinib significantly reduced tumor growth in both models. Of the known ponatinib targets, PDGFR and FGFR1 were highly expressed in SCCOHT tumors, suggesting that SCCOHTs are sensitive to ponatinib through the expression of these RTKs. Further, PDGFR-alpha phosphorylation and downstream signaling are strongly inhibited by ponatinib in SCCOHT. In summary we believe this finding may allow a way to be exploited therapeutically through the FDA-approved inhibitor ponatinib. Citation Format: Jessica D. Lang, William Hendricks, Pilar Ramos, Holly Yin, Chris Sereduk, Jeffrey Kiefer, Yemin Wang, Anthony N. Karnezis, Bernard Weissman, David Huntsman, Jeffrey Trent. TARGETING AND EFFICACY OF THE RECEPTOR TYROSINE KINASE INHIBITOR PONATINIB IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE, WORKS THROUGH INHIBITION OF PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR) [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 NTOC-090.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850-60. ©2017 AACR.

Abstract B15: Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type.

Abstract Objective: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive subtype of ovarian cancer with limited treatment options. Inactivating SMARCA4 germline and somatic mutations are present in nearly all SCCOHT cases, representing a signature molecular feature of this disease. Concomitant loss of SMARCA2, a mutually exclusive catalytic subunit of the SWI/SNF chromatic remodeling complex, has been attributed to worse overall survival in non-small cell lung carcinoma (NSCLC) when compared to cases with loss of SMARCA4 only. The objective of this study was to evaluate the mutational status and expression of SMARCA2 in SCCOHT. Methods: We evaluated 10 archival cases of SCCOHT and 1 newly established case from a PDX model of recurrent disease. Specialty gynecologic pathologists reviewed all cases to confirm the diagnosis. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumors with at least 50% tumor cell nuclei. Using the MSK-IMPACT assay, samples were deep-sequenced for a panel of 341 known oncogenes and tumor suppressor genes frequently altered in cancer. Identified SMARCA4 mutations were validated using Sanger sequencing. Sequencing of the SMARCA2 coding regions and splice sites was done using AmpliSeq. We performed immunoblotting for SMARCA4 and SMARCA2 on available frozen tumor samples using standard protocol. To confirm loss of protein expression, immunohistochemistry (IHC) for the SMARCA4 and SMARCA2 was performed on FFPE slides of all cases; the absence of tumor cell nuclear staining in the presence of internal positive controls scored as loss-of-expression. For cell-line over-expression studies, SCCOHT BIN67 and NSCLC H1299 cells were transiently transfected with an expression plasmid containing cDNA for SMARCA4 using FuGene reagent (Invitrogen) according to manufacturer's instructions. Immunoblotting for SMARCA2 and SMARCA4 was performed on protein lysates extracted from cell pellets. Results: IHC demonstrated loss of SMARCA2 expression in 9 (90%) of 10 archival SCCOHT cases and the PDX model. Sequencing of the SMARCA2 coding region and splice sites revealed no mutations, suggesting a non-mutational etiology of SMARCA2 protein loss. Previous work revealed biallelic SMARCA4 mutations in the 9 archival cases with SMARCA2 loss. SMARCA4 mutations were confirmed in both the PDX model and the one additional archival case, resulting in universal SMARCA4 mutations. The only case with normal SMARCA2 expression was also the only case with retained SMARCA4 expression. To explore the relationship between SMARCA2 and SMARCA4, we re-introduced SMARCA4 into BIN67 SCCOHT and H1299 NSCLC cell lines that lack expression. Wild-type BIN67 cells have very low levels of SMARCA2 expression, while wild-type H1299 cells express SMARCA2 normally. After transfection with plasmid cDNA and confirmation of SMARCA4 expression by immunoblotting, there was no increase in SMARCA2 expression in either BIN67 or H1299 cells. Conclusions: Concomitant loss of SMARCA2 and SMARCA4 expression was seen in nearly all SCCOHT cases and our PDX model. While loss of SMARCA4 expression is due to biallelic mutations, the absence of SMARCA2 expression is non-mutational and potentially the result of epigenetic or post-translational silencing. Our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of SCCOHT. The reversal of SMARCA2 loss may serve as a potential novel therapeutic approach for SCCOHT. Citation Format: Jill H. Tseng, Petar Jelinic, Brooke A. Schlappe, Niamh Conlon, Narciso Olvera, Fanny Dao, Jennifer J. Mueller, Yaser Hussein, Robert A. Soslow, Douglas A. Levine. Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B15.

Abstract A1-27: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) beyond SMARCA4 mutations: A comprehensive genomic analysis

Abstract Background: Small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) is an extremely rare and aggressive tumor that affects mainly young women (median age = 24 years). Prognosis is poor as most patients die within 2 years of diagnosis. Until recently, the literature describing the genomic profile of SCCOHT was scarce. In the last few months, four groups have now confirmed that SMARCA4 mutations are a frequent event in SCCOHT. Methods: We performed an integrated genomic analysis using WES, RNA-seq and CGH approaches to identify other recurrent genomic alteration and potential therapeutic targets. Candidate SNVs identified by WES were validated by Sanger and RNA-Seq. 8 frozen tumors were available, including 6 with matching normal control DNA. In addition 33 tumor samples were available as FFPE. Results: Despite their high grade and aggressive clinical course, SCCOHT tumors display genomic stability, low mutation load (0.53mut.Mb) and few SCNAs. However when present, genomic alterations were recurrent. Integrated WES and CGH analysis revealed a frequent and unusual 19p CN LOH (in 5 of 6 tumors). Although overall mutation rate was low, the mutations that did occur were novel and recurrent in 50%-80% of SCCOHT. In addition to SMARCA4 mutation (M+), we validated 10 novel recurrent M+ (including ABCA7, ANKRD24, CACTIN, EMR1, FBN3, FUT5, GRIN3B, KANK3, KRI1 and PLK5) in 50%-80% of tumors at high allelic frequencies (mean=0.87). Similarly, when present, amplifications were recurrent, common to a third of tumors: such as the mitochondrial redox enzymes (SHMT2, NDUFA4L2) or the transmembrane transporter LRP1 were amplified (Log2>2.3). No common fusion transcripts were identified. Since SCCOHT often display initial chemosensitivity, but rapid progressions, we investigated the differential genomic profiles of treatment naive versus chemotherapy treated tumors to uncover candidate resistance genes. The only alteration significantly enriched in post treatment tumors were M+ in the putative efflux pump, ABCA7. These M+ could not be identified even at low allele fractions in treatment naive tumors, but were identified in all 3 treated tumors with a mean allelic frequency of 0.83. In terms of therapeutic implications, modulating mitochondrial metabolism or targeting the membrane transporter LRP1 or the ABCA7 efflux pump could provide therapeutic venues in the future. However the most promising approaches for now may be targeting cell cycle checkpoint regulators or chromatin remodelling. M+ in the tumor suppressor PLK5 were confirmed in 79% of a cohort of 33 SCCOHT and many were predicted as damaging. Inactivation of PLK5 is associated with a loss of G2/M checkpoint regulation and cell harbouring PLK5 mutations were sensitive to PLK1 inhibitors. More interestingly, SMARCA4-M+ SCCOHT demonstrated complete loss of SMARCA4 expression as well as loss of the only other SWI/SNF catalytic subunit SMARCA2 suggesting that double SMARCA4/SMARCA2 negative SCCOHTs are characterized by a catalytically inactive SWI/SNF complex. This hypothesis was supported by changes in RNA expression levels in the main transcriptional targets for SWI/SNF (RHOA, RB and the E2F family). Importantly, the double SMARCA2/4 negative BIN-67 SCCOHT cell line displaying both a SMARCA4 M+ and loss of SMARCA2 expression was exquisitely sensitive to HDAC and MT inhibitors, while cell lines with either a SMARCB1-M+ or SMARCA4-M+ with retained SMARCA2 expression were not. These data make double SMARCA2/4 negative by IHC a potential predictive biomarker for histone modifying inhibitors in SCCOHT or potentially in other double SMARCA2/4 negative malignancies. Conclusions: We present the 1st integrated genomic analysis of SCCOHT. SCCOHT demonstrate a remarkably homogeneous genomic profile and potentially actionable alterations. In particular, dual null SMARCA2/SMARCA4 SCCOHTs could be selected for clinical trials with EZH2, HDAC or MT inhibitors. Citation Format: Aurélie Auguste, Marc Deloger, Joanna Cyrta, Audrey Le Formal, Catherine Richon, Nathalie Droin, Beatrice Brunn, Olivier Caron, Mojgan Devouassoux, Catherine Lhomme, Patricia Pautier, Catherine Genestie, Alexandra Leary. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) beyond SMARCA4 mutations: A comprehensive genomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-27.

Abstract AS01: Molecular genetic approaches to understanding ovarian cancer

Abstract Ovarian cancer remains a challenging condition to prevent, diagnose and treat. We have conducted several different studies that have attempted to advance knowledge in one or more of these areas. These projects are briefly summarized below. 1) Sequencing of samples from a BRCA1 mutation carrier with high grade serous carcinoma We performed whole exome sequencing (WES) on the blood, primary tumor, omental metastasis and recurrence following therapy with carboplatin and paclitaxel, from a patient carrying a BRCA1 p.S1841R mutation. We observed loss of heterozygosity in the BRCA1 mutation in the primary and subsequent tumors, and somatic mutations in TP53 and NF1 were identified, suggesting their role along with BRCA1 driving the tumor development. We were able to detect large chromosomal rearrangements using WES. A large deletion was present in the three tumors in the regions containing BRCA1, TP53, and NF1 mutations, and an amplification in the regions containing MYC. We did not observe the emergence of new mutations among tumors from diagnosis to relapse after chemotherapy. Mutations present in the primary tumor were the main drivers of metastases and chemotherapy resistance. 2) Identification of DICER1 mutations in non -epithelial ovarian cancer We used Sanger sequencing to sequence the RNase IIIa and IIIb domains of DICER1 in 154 gonadal tumours from 135 females and 19 males, and 43 extra-gonadal germ cell tumors (GCTs) from 26 females and 17 males. We identified heterozygous non-synonymous mutations in the RNase IIIb domain of DICER1 in 14/197 non-epithelial tumours (7.1%). Mutations were found in 9/28 SCSTs (32%), 5/118 gonadal GCTs (4.2%), 0/43 extra-gonadal GCTs and 0/8 miscellaneous tumours. The 14 mutations affected only five residues: E1705, D1709, E1788, D1810 and E1813. In all five patients where matched and constitutional DNA was available, the mutations were somatic. There were no mutations found in the RNase IIIa domain. More than half (8/15) of Sertoli-Leydig cell tumours (SLCTs) harbour DICER1 mutations in the RNase IIIb domain, while mutations are rarely found in GCTs. 3) Characterization of small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches. Citation Format: Leora Witkowski, Jian Carrot-Zhang, Jacek Majewski, William D. Foulkes. Molecular genetic approaches to understanding ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS01.

Abstract 5381: Therapeutic potential of HDAC inhibitors in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)

Abstract SCCOHT is a rare but deadly type of ovarian cancer. It mainly affects young women with the median age about 28 years. Although often diagnosed at an early stage, the prognosis of SCCOHT is nonetheless dismal with a 2-year survival less than 35% due to lack of effective treatments. Unlike common malignancies, the genome of SCCOHT is minimally disturbed. Recently, we and others have discovered inactivating mutations of SMARCA4, the ATPase of the SWI/SNF chromatin remodeling complex, in the majority of SCCOHT along with loss of SMARCA4 protein. Interestingly, SMARCA2, the alternative ATPase of the SWI/SNF complex is also inactivated in SCCOHT without apparent mutations. Re-expression of either SMARCA4 or SMARCA2 robustly inhibited the growth of SCCOHT cells. Therefore, the dual deficiency of SMARCA4 and SMARCA2 may be the primary driver in SCCOHT tumorigenesis by creating distinct epigenetic features that promote oncogenic transformation and can serve as promising therapeutic targets. In an attempt to identify epigenetic drugable targets, we performed the drug screening using an epigenetic drug library (Cayman Chemical) in two SCCOHT cell lines and four other ovarian cancer cell lines with intact SMARCA4 and SMARCA2. We identified several HDAC inhibitors that selectively inhibited the viability of SCCOHT cells (BIN67 and SCCOHT1) compared to that of other ovarian cancer cell lines. We confirmed that in comparison to other ovarian cancer cell lines, SCCOHT cells were significantly more sensitive to the treatment of several pan-HDAC inhibitors including SAHA, an FDA-approved HDAC inhibitor for treatment of cutaneous T cell lymphoma, and two selective HDAC6 inhibitors (CAY10603 and Nexturastat A), but not to Romidepsin, a selective HDAC1/2 inhibitor. Furthermore, the expression of HDAC6 was significantly higher in SCCOHT cells compared with other ovarian cancer cell lines and re-expression of SMARCA4 suppressed the expression of HDAC6. Interestingly, SCCOHT cells were also more sensitive to Pracinostat, a broad HDAC inhibitor with minimum effect on HDAC6. Using Agilent gene expression array, we identified a subset of genes whose expression was upregulated by both SMARCA4 re-expression and SAHA treatment in BIN67 cells. Taken together, our data suggest that the SMARCA4/SMARCA2 dual deficiency may promote hypersensitivity to HDAC inhibitors through both HDAC6-dependent and independent pathways. Ongoing studies will address the detailed mechanisms and evaluate the efficacy of using HDAC inhibitors for the treatment of SCCOHT cell line-derived and patient-derived mouse xenografts to provide requisite evidence for initiating a clinical trial for defeating this notorious disease. Citation Format: Yemin Wang, Pilar Ramos, Anthony N. Karnezis, Jeffrey M. Trent, David G. Huntsman. Therapeutic potential of HDAC inhibitors in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5381. doi:10.1158/1538-7445.AM2015-5381

Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.