Krystal Ann Orlando

Re-expression of SMARCA4/BRG1 in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) promotes an epithelial-like gene signature through an AP-1-dependent mechanism

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive form of ovarian cancer. SCCOHT tumors have inactivating mutations in SMARCA4 (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. To address the role that BRG1 loss plays in SCCOHT tumorigenesis, we performed integrative multi-omic analyses in SCCOHT cell lines +/- BRG1 reexpression. BRG1 reexpression induced a gene and protein signature similar to an epithelial cell and gained chromatin accessibility sites correlated with other epithelial originating TCGA tumors. Gained chromatin accessibility and BRG1 recruited sites were strongly enriched for transcription-factor-binding motifs of AP-1 family members. Furthermore, AP-1 motifs were enriched at the promoters of highly upregulated epithelial genes. Using a dominant-negative AP-1 cell line, we found that both AP-1 DNA-binding activity and BRG1 reexpression are necessary for the gene and protein expression of epithelial genes. Our study demonstrates that BRG1 reexpression drives an epithelial-like gene and protein signature in SCCOHT cells that depends upon by AP-1 activity.

Loss of SMARCA4 Leads to Intron Retention and Generation of Tumor-Associated Antigens in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare, deadly form of ovarian cancer that uniformly harbors mutations in SMARCA4, a member of the SWI/SNF chromatin remodeling complex. SWI/SNF impacts RNA splicing, and dysregulation of splicing can generate immunogenic tumor antigens. In this study, we explored the relationship between SMARCA4 loss and RNA splicing dysregulation. SCCOHT primary tumors harbored tumor-associated outlier splicing events compared with normal tissues. Many of the tumor events were retained introns encoding novel peptides predicted to bind to MHC-I complexes. Immune cells were observed in primary SCCOHT tumors, suggesting a potentially immune-reactive tumor microenvironment. Mutations in several switch/sucrose nonfermenting (SWI/SNF) subunits were associated with higher rates of outlier retained introns across tumor types in The Cancer Genome Atlas data. Interestingly, RNA sequencing of isogenic SCCOHT cell lines demonstrated a role for SMARCA4 in intron retention (IR). Distinct protein–protein interactions between splicing factors identified in SCCOHT cell lines supported a role for SMARCA4 in splicing regulation. Furthermore, SWI/SNF localized to genes, which were differentially spliced. Mass spectrometry analyses confirmed expression of some of these novel peptides, and a subset of these are predicted to bind to MHC-I complexes. A pool of these novel peptides derived from retained introns in SCCOHT triggered proliferation and expression of TNFα and INFγ in primary human T cells. Together, these data suggest that SMARCA4 loss in SCCOHT leads to IR. Furthermore, T-cell activation by novel peptides encoded by these tumor-specific splicing events suggests IR could be a source of tumor-associated antigens in SCCOHT. Significance: SCCOHT, a rare ovarian cancer, features splicing dysregulation due to SMARCA4 loss that generates immunostimulatory peptides linked to potential immune responses and therapeutic avenues, challenging traditional views of the role of SMARCA4.