Loss Of SMARCA4 Research Articles

Morphologic and molecular analysis of early-onset gastroesophageal adenocarcinomas.

4547 Background: The incidence of early-onset gastroesophageal adenocarcinomas (EO-GEA) is increasing, and these tumors now constitute > 30% of all gastroesophageal cancers. Besides hereditary gastric cancer syndromes, which form ~3% of EO-GEA, the morphologic and molecular spectrum of these tumors is not well-studied. Methods: Next-generation sequencing (NGS) data obtained from routine clinical care from patients with EO-GEA, defined as age ≤50 years, from 3 tertiary care centers was evaluated and compared with tumor profiles of 2,081 patients with GEA from cBioPortal for Cancer Genomics. Available histologic slides were reviewed, and the tumors were classified into Lauren and WHO subtypes. Tumor-detected pathogenic variants of potential germline origin were identified from the NGS data. Results: The study cohort was formed by 79 patients with gastroesophageal (42%) and gastric (58%) adenocarcinoma. The most commonly mutated genes included TP53 (28.5%), CDH1 (10%), ARID1A (5%), KRAS (3.9%) and PIK3CA (3.9%). EO-GEA were less likely to harbor TP53 (28.5% vs. 57.5%, p 0.003) and ARID1A (5% vs. 20.6%, p 0.002) mutations when compared with cBioPortal data. Based on the Lauren scheme, the tumors were classified into intestinal (40%), diffuse (24%), mixed (12%), and indeterminate (15%) subtypes. Driver mutations in CDH1, TP53, FBXW7, BAP1 genes were seen in diffuse/mixed subtype, and TP53, ARID1A, KRAS, PIK3CA, APC, ATM, NBN, MUTYH genes in intestinal subtype. The indeterminate subtype showed TP53 mutations and additional alterations, including SMARCB1/ SMARCA4 loss leading to rhabdoid/undifferentiated morphology. ERBB2 amplification was more likely to be present in intestinal and indeterminate subtypes (p = 0.003). CD274 amplification/PD-L1 expression was more likely to be present in indeterminate subtype (p < 0.0001). Potential germline variants included mutations in gastric cancer susceptibility genes such as CDH1 (2.5%) and APC (1%), and other cancer susceptibility genes such as ATM (4%), NBN (1%), MUTYH (1%) and POLD1 (1%). Conclusions: The molecular profile of EO-GEA is distinct from traditional gastric cancers. Histologic subtypes of EO-GEA correlate with distinct genomic alterations. Our findings also support multigene germline panel testing in parallel for patients with EO-GEA.

Loss of switch/sucrose non-fermenting complex protein expression in undifferentiated gastrointestinal and pancreatic carcinomas.

Undifferentiated carcinoma refers to an epithelial malignancy that lacks morphological evidence of differentiation. Recent studies have implicated the loss of constitutively expressed switch/sucrose non-fermenting (SWI/SNF) complex subunits in undifferentiated carcinomas of the gastrointestinal tract and other sites. In this study we examine the expression of SWI/SNF and mismatch repair (MMR) proteins in a series of undifferentiated carcinomas from the gastrointestinal tract and the pancreas. We searched pathology databases from four Canadian health centres for primary undifferentiated carcinoma from gastrointestinal and pancreatic resection specimens. Upon review of 31 cases, 19 were confirmed to be undifferentiated carcinomas (eight colonic, six gastric, three pancreatic, one appendiceal and one duodenal). Immunohistochemical analysis of SMARCA4, SMARCA2, SMARCB1, ARID1A, ARID1B, MSH2, MSH6, MLH1 and PMS2 was performed on whole sections. Five of 19 (26%) showed loss of core SWI/SNF proteins (two loss of SMARCA4, one loss of SMARCB1 and two concurrent loss of ARID1A and ARID1B). SMARCA4, SMARCB1, or ARID1A/ARID1B-deficient undifferentiated carcinoma consistently exhibited sheet-like growth pattern, with cellular discohesion and rhabdoid morphology. Nine of 17 undifferentiated carcinomas tested were MMR-deficient by immunohistochemistry. In comparison, none of the 12 poorly differentiated carcinomas that were originally diagnosed as undifferentiated carcinomas showed loss of SMARCA4, SMARCA2, SMARCB1 or ARID1B. Undifferentiated gastrointestinal/pancreatic carcinomas show frequent loss of expression of SWI/SNF complex proteins. The loss of these core components of SWI/SNF complex may contribute to the arrest of cellular differentiation, resulting in the undifferentiated histology and aggressive clinical behaviour.

SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.

The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.

SMARCA family of genes

The SMARCA subgroup of genes belong to the SWI1/SNF1 family that are responsible chromatin remodelling and repair. Inactivating mutations in the main SMARCA genes A2 and A4 lead to loss...

SMARCA4-deficient Sinonasal Carcinoma: A Series of 10 Cases Expanding the Genetic Spectrum of SWI/SNF-driven Sinonasal Malignancies.

The molecular pathogenesis of poorly differentiated sinonasal carcinoma received significant attention in recent years. As a consequence, several unclassified carcinomas in the morphologic spectrum of sinonasal undifferentiated carcinoma have been reclassified as distinctive genetically defined variants or entities. Among the latter are NUT-rearranged carcinoma and SMARCB1-deficient carcinomas. In this study, we further characterize a rare variant of sinonasal undifferentiated carcinoma-like tumors characterized by inactivation of the SWItch/Sucrose Nonfermentable chromatin remodeler SMARCA4 (BRG1) detectable by immunohistochemistry. Patients were 7 males and 3 females aged 20 to 67 years (median, 44). Tumors originated in the nasal cavity (6), nose and sinuses (2), or at unspecified site (2). Six tumors were initially misdiagnosed as small cell neuroendocrine carcinoma (SCNEC) or large cell neuroendocrine carcinoma (LCNEC). Histologically, the tumors were composed of small basaloid (3 cases) or large epithelioid (7) cells disposed into nests and solid sheets with extensive areas of necrosis. No glands or other differentiating features were noted. Abortive rosettes were seen in 1 case. Immunohistochemistry showed consistent expression of pankeratin and absence of CK5, p63, p16, and NUT in all tumors tested. Other tested markers were variably positive: CK7 (2/6), synaptophysin (9/10; mostly focal and weak), chromogranin-A (4/10; focal), and CD56 (3/5; focal). All tumors showed total loss of SMARCA4 and retained expression of SMARCB1/INI1. Co-loss of SMARCA2 was seen in 1 of 8 cases. Limited data were available on treatment and follow-up. Two patients received surgery (1 also radiotherapy) and 3 received chemotherapy. Metastases (cervical nodes, liver, bone, and lung/mediastinal) were detected in 3 patients; 2 were alive under palliative chemotherapy at 8 and 9 months while 1 died of progressive lung disease at 7 months. Three patients (1 with brain invasion) died soon after diagnosis (1 to 3 mo). In total, 4 of 6 patients (66%) with follow-up died of disease (median, 3 mo). This series characterizes SMARCA4-deficient sinonasal carcinoma as a genetically distinct aggressive entity in the spectrum of undifferentiated sinonasal carcinomas. These variants add to the spectrum of SWItch/Sucrose Nonfermentable-deficient sinonasal carcinomas, at the same time expanding the topographic distribution of SMARCA4-related malignancies.

Abstract NT-114: CATALYTIC SUBUNITS SWITCH DRIVES RESISTANCE TO EZH2 INHIBITORS IN ARID1A-MUTATED CELLS

Abstract 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.

SMARCA2-deficiency confers sensitivity to targeted inhibition of SMARCA4 in esophageal squamous cell carcinoma cell lines

SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.

Abstract 531: The cGAS-STING pathway as a driver of the immune-reactive microenvironment in small cell carcinoma of the ovary, hypercalcemic type

Abstract Introduction: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive malignancy affecting young women. The disease is characterized by a monogenic mutation of SMARCA4, a key member of the SWI/SNF complex. The treatment options are limited, and survival rates are poor. We have previously reported clinical responses in patients undergoing treatment with anti-PD1 immunotherapy, despite a low mutational burden. The responses were attributed to elevated PD-L1 expression and T-cell infiltration in SCCOHT. To better understand the mechanisms of the immune reactive microenvironment we sought to determine the role of the innate immune response driven by the STING pathway after radiotherapy. Methods: The cGAS-STING expression of cell lines with SMARCA4 loss (H1299), SMARCA4 knockdown (293T SMARCA4) and SMARCA4 wild type (CAOV3, OVCAR4) were analyzed following 10 Gy of irradiation. We evaluated the expression of the cGAS-STING cytosolic DNA sensing pathway by measuring time-dependent expression of cGAS dependent interferon stimulated genes CCL5 and CXCL10. Finally, we analyzed the number of micronuclei formed in SCCOHT model cell lines BIN67 and SCCOHT-1 following irradiation using multi-parametric immunofluorescence. Results: In H1299 (SMARCA4 loss), there was an 1847-fold increase (p=0.02) in CCL5 and 3954-fold increase in CXCL10 (p=0.09) at 6 days post-irradiation in comparison to non-irradiated controls. In 293T cells with stable knockdown of SMARCA4, there was a 2.8-fold increase in expression of CCL5 (p < 0.01) and a 1.6-fold increase CXCL10 (p=0.26), 6 days after irradiation compared to irradiated wild type cells. In the SMARCA4 wild type cell lines CAOV3 and OVCAR4, there was no statistically significant upregulation of CCL5 and CXCL10. There were more micronucleated cells per high power field after irradiation in BIN67 (21.2% vs 9.30%, p=0.1) and SCCOHT-1 (9.4% vs 0.0%, p=0.03) compared to non-irradiated controls. Conclusion: SCCOHT is a low mutational burden cancer with demonstrated response to immune checkpoint blockade. Our research shows that following irradiation, the innate immune response triggered by cGAS-STING may be driving the immune reactive microenvironment of SCCOHT. The loss of SMARCA4 may play a role in activating the innate immune response. Better characterization of the association between the SWI/SNF complex and immune reactivity should lead to a broader use for immune checkpoint inhibitors. Citation Format: Elke Van Oudenhove, Jay R. Patibandla, Selim Misirlioglu, Ernesto Arostegui Fernandez, Petar Jelinic, Douglas A. Levine. The cGAS-STING pathway as a driver of the immune-reactive microenvironment in small cell carcinoma of the ovary, hypercalcemic type [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 531.

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.

Abstract 397: SMARCA4 is a selective vulnerability in SMARCA2-deficient esophageal squamous cell carcinoma cell lines

Abstract Esophageal squamous cell carcinoma (ESCC) is a highly prevalent tumor type with poor prognosis and limited treatment options. In this study, we aimed to discover potential novel drug targets using a pooled epigenome sgRNA viability screen in a panel of ESCC cell models. We identify the BAF chromatin remodeling helicase subunit SMARCA4/BRG1 as a novel dependency in a subset of ESCC cell lines. In line with the established synthetic lethal interaction of SMARCA4 and SMARCA2/BRM, the two mutual exclusive catalytic subunits of the BAF complex, SMARCA4-dependent cell lines display low or absent expression of SMARCA2. In rescue studies using SMARCA4 variants, we demonstrate that SMARCA4-dependency is linked to its ATPase/helicase activity, but not to bromodomain function. Similarly, ectopic expression of wild-type and bromodomain-mutant SMARCA2, but not a helicase-dead variant, rescues from loss of SMARCA4 in SMARCA2-low ESCC cells. SMARCA2-proficient ESCC cell models are rendered SMARCA4-dependent upon CRISPR/Cas9-mediated knock-out of SMARCA2. We further identify SMARCA4-dependent cell models from additional tumor types (colon, ovarian and pancreas carcinoma) with low or absent SMARCA2 expression. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and indicate pharmacological inhibition of SMARCA4 as a novel opportunity for targeted therapy of SMARCA2-deficient cancers. Citation Format: Katharina Ehrenhöfer-Wölfer, Teresa Puchner, Silvia Blaha-Ostermann, Alexandra Hörmann, Wolfgang Sommergruber, Norbert Schweifer, Thomas Zichner, Andreas Schlattl, Ralph A. Neumüller, Manfred Koegl, Mark P. Petronczki, Mark Pearson, Simon Wöhrle. SMARCA4 is a selective vulnerability in SMARCA2-deficient esophageal squamous cell carcinoma cell lines [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 397.

Identification of targetable molecular alterations in the NCI-COG Pediatric MATCH trial.

10011 Background: The screening protocol for the NCI-Children’s Oncology Group (COG) Pediatric Molecular Analysis for Therapy Choice (MATCH) trial detects tumor alterations that are used to assign patients with treatment-refractory or recurrent cancers to phase 2 treatment arms of molecularly-targeted therapies. Methods: Patients age 1 to 21 years old with treatment-refractory or recurrent solid tumors, non-Hodgkin lymphomas, or histiocytic disorders treated at U.S. based COG sites are eligible. DNA and RNA extracted from FFPE tumor samples are sequenced using an Oncomine cancer gene panel for detection of mutations, amplifications, and fusions. Loss of SMARCB1, SMARCA4, and PTEN expression is detected by immunohistochemistry. Lists of actionable mutations (aMOIs) based upon available clinical and pre-clinical data are used a priori to determine eligibility for treatment arms. Results: Between 7/24/17 and 12/31/18, 422 patients with a median age of 13 years (range 1-21) were enrolled from 93 COG sites. Solid tumors comprised 71% (n = 300) of diagnoses, CNS tumors 24% (n = 101) and lymphomas/histiocytoses 5% (n = 21). A tumor sample was submitted for 390 patients, sequencing was attempted for 370 (95%), and results were confirmed for 357 (92%). Median turn-around time was 15 days. An aMOI for at least one of the 10 current treatment arms was identified in 112 patients (29%, 95% CI 24%-33%); 95 patients (24%, 95% CI 20%-29%) were assigned to a treatment arm with 39 patients (10%, 95% CI 7%-13%) enrolled to date. The aMOI rate was similar in patients less than 12 years of age (35%) compared to patients 12 years and older (25%). Actionable MAPK pathway alterations were found in 11% of patients (n = 41), most often HRAS/ KRAS/ NRAS mutations (n = 16), BRAF mutations or fusions (n = 14), or NF1 mutations (n = 11). Other genes with recurrent aMOIs included SMARCB1 (n = 14), ALK (n = 8), CDK4 (n = 8), PIK3CA (n = 7), PTEN (n = 7), FGFR1 (n = 5), and BRCA1/BRCA2 (n = 5). Conclusions: Approximately one-quarter of patients with tumor submitted for Pediatric MATCH screening have been assigned to an investigational therapy, facilitating the evaluation of molecularly-targeted agents in biomarker-positive pediatric cohorts through a collaborative nationwide study. Clinical trial information: NCT03155620.

SMARCA4-deficient Thoracic Sarcomas: Clinicopathologic Study of 30 Cases With an Emphasis on Their Nosology and Differential Diagnoses.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity with an aggressive clinical course and specific genetic alterations of the BAF chromatin remodeling complex. In the present study, we reviewed the clinical and pathologic features of 30 cases of SMARCA4-DTS, discussed its main differential diagnoses and the challenging diagnostic scenarios that the average pathologist may face. In addition, we tested the specificity of the "SMARCA4-DTS immunohistochemical signature" (co-loss of SMARCA4 and SMARCA2 with overexpression of SOX2) in a large cohort of intrathoracic malignancies. Patients ranged from 28 to 90 years of age (median: 48 y), with a marked male predominance (male:female=9:1) and they were usually smokers. Tumors were generally large compressive masses located in the mediastinum (n=13), pleura (n=5), lung (n=2) or in 2 or more of these topographies (n=10). Treatment strategies were varied, including 1 case treated with EZH2 inhibitors. Median overall survival was 6 months. Histologically, tumors were poorly differentiated frequently showing rhabdoid features. A subset of cases showed a focal myxoid stroma (7%, n=2/30) and rare cases displayed a previously unreported pattern simulating desmoplastic small round cell tumors (7%, n=2/30). Making a diagnosis was challenging when dealing with biopsy material from massively necrotic tumors and in this setting the expression of SOX2, CD34, and SALL4 proved useful. All tested cases displayed concomitant loss of SMARCA4 and SMARCA2 and most tumors expressed epithelial markers (Pan-keratin or EMA) (n=29/30), SOX2 (n=26/27), and CD34 (n=17/27). SMARCB1 expression was retained in all cases (23/23). SALL4 and Claudin-4 were expressed in a subset of cases (n=7/21 and 2/19, respectively). TTF-1 and P63 were focally expressed in 1 case each. P40 and NUT were not expressed (0/23 and 0/20, respectively) The SMARCA4-DTS immunohistochemical signature was both sensitive and specific, with only a subset of small cell carcinoma of the ovary hypercalcemic type showing overlapping phenotypes. Our study confirms and expands the specific features of SMARCA4-DTS, emphasizing the fact that they can be straightforwardly identified by pathologists.

SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer

Tumor suppressor SMARCA4 (BRG1), a key SWI/SNF chromatin remodeling gene, is frequently inactivated in cancers and is not directly druggable. We recently uncovered that SMARCA4 loss in an ovarian cancer subtype causes cyclin D1 deficiency leading to susceptibility to CDK4/6 inhibition. Here, we show that this vulnerability is conserved in non-small cell lung cancer (NSCLC), where SMARCA4 loss also results in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. In addition, SMARCA2, another SWI/SNF subunit lost in a subset of NSCLCs, also regulates cyclin D1 and drug response when SMARCA4 is absent. Mechanistically, SMARCA4/2 loss reduces cyclin D1 expression by a combination of restricting CCND1 chromatin accessibility and suppressing c-Jun, a transcription activator of CCND1. Furthermore, SMARCA4 loss is synthetic lethal with CDK4/6 inhibition both in vitro and in vivo, suggesting that FDA-approved CDK4/6 inhibitors could be effective to treat this significant subgroup of NSCLCs.

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.

Histone Deacetylase Inhibitors Synergize with Catalytic Inhibitors of EZH2 to Exhibit Antitumor Activity in Small Cell Carcinoma of the Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation in vitro and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.

SWI/SNF catalytic subunits\u2019 switch drives resistance to EZH2 inhibitors in ARID1A-mutated cells

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.

SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus): a clinicopathologic entity distinct from undifferentiated carcinoma

Small cell carcinoma of the ovary, hypercalcemic type is a rare, aggressive malignancy which usually occurs in young women and is characterized by mutations in SMARCA4, with few other alterations. We recently encountered uterine tumors with morphologic, immunohistochemical, and genetic similarities to small cell carcinoma of the ovary, hypercalcemic type. Herein we report the clinicopathologic and molecular features (using a targeted massively parallel sequencing [MPS] assay) of these tumors. The cases were diagnosed on cervical and endometrial biopsies (n = 2, 34, and 29 years) or hysterectomy and bilateral salpingo-oophorectomy (n = 3, 25, 33, and 58 years). The tumors were composed of sheets of large atypical epithelioid cells with prominent rhabdoid morphology, indistinguishable from the “large cell” variant of small cell carcinoma of the ovary, hypercalcemic type. In three cases, the ovaries were pathologically examined to exclude a primary ovarian malignancy. Immunohistochemically, four of four cases showed SMARCA4 loss, and were negative or only focally positive for keratins, EMA, and claudin-4. One of three cases was positive for WT-1. Targeted MPS was successfully performed on 4 of 5 tumors, and showed recurrent mutations in SMARCA4, with few other alterations. Of the cases diagnosed on hysterectomy, all had extensive lymphovascular invasion, extra-uterine spread, and marked infiltrative growth. These tumors were uniformly aggressive; all patients died of disease (median survival 7 months, range 1–43 months). We propose this entity be called “SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus)”, a term which describes both the tumor's underlying molecular abnormality and its morphology. Its unique clinicopathologic and molecular features differentiate it from other related malignancies, including undifferentiated endometrial carcinoma, small cell carcinoma of the ovary (hypercalcemic type), and epithelioid sarcoma. We review and discuss previously reported “rhabdoid tumors of the uterus;” while they are a heterogenous group of tumors, some of them are likely examples of this entity. Correctly identifying cases of SMARCA4-deficient uterine sarcoma from histologic mimics is important as it may have prognostic, predictive, and germline implications.

Abstract IA21: Small cell carcinomas of the ovary: Strengths and weaknesses

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and very aggressive malignancy that occurs mostly in young women. We and others previously described SCCOHT as a SMARCA4-mutated monogenic disease and unraveled other important molecular features, including apparent universal loss of SMARCA2 through epigenetic silencing. Typically, SMARCA4 and SMARCA2 are redundant catalytic subunits of the SWI/SNF complex and loss of either will result in the overexpression of the other. However, SCCOHT is unique in having concomitant loss of both subunits with restoration of either leading to tumor suppression. Nonetheless, despite these advances in understanding the biology of SCCOHT, there are few effective treatment options and survival rates remain poor. In this presentation we will discuss the role of SMARCA4 and SMARCA2 on tumor growth. We will also present therapeutic options to manipulate SWI/SNF complex dependencies. We will present case histories of patients who have had positive responses to targeted therapies. We will also discuss responses to anti-PD1 immunotherapy and characterize the immune landscape of SCCOHT tumors using quantitative immunofluorescence and gene expression profiling. Unexpectedly for a low mutation burden cancer, the majority of the tumors demonstrated PD-L1 expression with strong associated T-cell infiltration. PD-L1 expression can be detected in both tumor and stromal cells, with macrophages being the most abundant PD-L1 positive cells in some tumors. These findings suggest that although SCCOHT are low-mutational-burden tumors, their immunogenic microenvironment that resembles the landscape of tumors that respond well to treatment with PD-1/PD-L1 blockade. Citation Format: Douglas A. Levine. Small cell carcinomas of the ovary: Strengths and weaknesses. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA21.

SMARCB1-deficient Tumors of Childhood: A Practical Guide.

The SMARCB1 gene ( INI1, BAF47) is a member of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, involved in the epigenetic regulation of gene transcription. SMARCB1 acts as a tumor suppressor gene, and loss of function of both alleles gives rise to SMARCB1-deficient tumors. The prototypical SMARCB1-deficient tumor is the malignant rhabdoid tumor (MRT) which was first described in the kidney but also occurs in soft tissue, viscera, and the brain (where it is referred to as atypical teratoid rhabdoid tumor or AT/RT). These are overwhelmingly tumors of the very young, and most follow an aggressive and ultimately lethal course. Morphologically, most but not all contain a population of "rhabdoid" cells, which are large cells with abundant cytoplasm, perinuclear spherical inclusions, and eccentric vesicular nuclei with large inclusion-like nucleoli. MRT immunohistochemistry reveals complete loss of SMARCB1 nuclear expression, and molecular analysis confirms biallelic SMARCB1 inactivation in the vast majority. Rare AT/RTs have loss of SMARCA4, another SWI/SNF member, rather than SMARCB1. With the widespread adoption of SMARCB1 immunohistochemistry, an increasing number of SMARCB1-deficient tumors outside of the MRT-AT/RT spectrum have been described. In addition to MRT and AT/RT, pediatric tumors with complete loss of SMARCB1 expression include cribriform neuroepithelial tumor, renal medullary carcinoma, and epithelioid sarcoma. Tumors with variable loss of SMARCB1 expression include subsets of epithelioid malignant peripheral nerve sheath tumor, schwannomas arising in schwannomatosis, subsets of chordomas, myoepithelial carcinomas, and sinonasal carcinomas. Variable and reduced expression of SMARCB1 is characteristic of synovial sarcoma. In this review, the historical background, clinical characteristics, morphology, immunohistochemical features, and molecular genetics most germane to these tumors are summarized. In addition, familial occurrence of these tumors (the rhabdoid tumor predisposition syndrome) is discussed. It is hoped that this review may provide practical guidance to pathologists encountering tumors that have altered expression of SMARCB1.

Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

Alveolar epithelial cells (AECs) injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF). Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII) cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO)7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ) model, as well as a new SMARCA4-deleted alveolar type II (ATII)-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.