Abstract 405: PBRM1 re-introduction in PBRM1-mutant kidney cancer cell lines drives an Interferon-γ expression signature

Abstract

Abstract Introduction Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer and is driven by loss of chromosome 3p. The 3p tumor suppressor PBRM1 is mutated in about 1/3 of tumors. VHL loss is well-known to drive the hypoxic response, but the role of PBRM1 loss is less well understood. PBRM1 is part of the SWI-SNF complex, an ancient chromatin modifying machine, so we hypothesized that its loss would alter chromatin profiles and subsequently expression of SWI-SNF target genes, and would result therapeutic susceptibility to histone H3-K27 methyltransferase inhibitors. Therefore we sought to reintroduce PBRM1 back into ccRCC cell lines which carry PBRM1 truncations in conjunction with 3p LOH and to characterize these cells in detail. Methods All cell lines harbored chromosome 3p loss with truncation of PBRM1 in the remaining allele. Tet-inducible PBRM1 or GFP was introduced into RCC-ER (D251fs), RCC-FG2 (Q445*), and RCC-MF (N496fs). Western blot and immunohistochemistry were used to confirm BAF180 (the protein encoded by PBRM1) and p21. CellTiter Glo was used to measure cell viability to drug treatments. RNA seq was used followed by gene-set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) for gene expression profiling. Results BAF180 and p21 (a BAF180 target gene) were re-expressed in a time- and dose-dependent manner in all 3 cell lines. BAF180 was localized to the nucleus. EZH2 inhibitor UNC1999 completely ablated clonogenic formation in all 3 cell lines but there was not differential sensitivity between PBRM1-induced and uninduced cells. All cell lines were resistant to EPZ11011989, a different EZH2 inhibitor. We also characterized sensitivity to everolimus, an MTOR inhibitor, and sunitinib, an antiangiogenic multi-tyrosine kinase inhibitor. BAF180 expression did not alter sensitivity to these drugs either. Therefore, we performed RNA sequencing to evaluate the numerous signaling or epigenetic pathways which may have been altered in cells re-expressing BAF180. Using IPA, we saw that several immune signaling pathways were increased in cells upon reintroduction of PBRM1 including Interferon-γ signaling (RCC-ER and RCC-FG2), JAK/STAT signaling, and IL6 pathway. GSEA confirmed the finding that Interferon-γ pathway is activated in each cell line after 7 days of BAF180 induction. These findings were confirmed using Western blot for STAT1/2/3 and their phosphoepitopes, JAK1 and phospo-JAK1, IRF1 and IRF9. Conclusions Our hypothesis that PBRM1 loss would lead to a targeted vulnerability with EZH2 inhibitors was not supported. However, our finding that Interferon-γ signaling is increased when PBRM1 is reintroduced is significant. Given the prominence of Interferon signaling in the response to PD1 and PDL1 inhibitors, somatic PBRM1 inactivation in ccRCC may have significant biological and clinical implications for triaging patients into immune checkpoint therapy. Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. PBRM1 re-introduction in PBRM1-mutant kidney cancer cell lines drives an Interferon-γ expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 405.