Abstract A24: PBRM1 alteration in clear cell renal cell carcinoma increases tumorigenicity through ALDH1A1 upregulation

In this study, we investigated the mechanisms by which PBRM1 functions as a tumor suppressor in clear cell renal cell carcinoma (ccRCC). PBRM1, also known as BAF180 or Polybromo, is a member of the PBAF SWI/SNF chromatin remodeling complex. Cancer sequencing studies have revealed that SWI/SNF components are widely mutated in cancer. PBRM1 mutations in particular are found in ~40% of ccRCC tumors, making it the second most highly mutated gene in ccRCC (behind VHL). Although many recent studies have looked at how other SWI/SNF components function in cancer control, relatively little is known about the tumor suppressive mechanism of PBRM1 in ccRCC. To investigate genes under PBRM1 control, we first generated mouse embryonic fibroblasts in which Pbrm1 was floxed and could be conditionally deleted. Using human ccRCC cell lines, we also stably knocked-down PBRM1 in the wild-type setting, or stably re-expressed it in the mutant setting. We then used gene expression profiling on these lines to look for genes that were consistently altered with PBRM1 loss. Ten genes were significantly altered across all lines, although only six (IGFBP3, ALDH1A1, EFNA3, WBP5, ESCO2, and PBRM1) changed reliably in the same direction. Further expression analysis using two different human primary tumor datasets revealed that only one of these genes was consistently altered in PBRM1-mutant tumors: ALDH1A1. ALDH1A1, or aldehyde dehydrogenase 1, is involved in the retinoic acid metabolic pathway and irreversibly converts retinaldehyde to retinoic acid. In addition to known roles in hematopoietic stem cell development, white vs. brown fat programming, and insulin signaling, ALDH1A1 is marker of tumor-initiating cells in various cancers. We confirmed that stable knockdown of PBRM1 in ccRCC cell lines resulted in higher expression and activity of ALDH1A1. Alternatively, re-expression of wild-type, but not cancer-associated mutant, PBRM1 lowered ALDH1A1 expression and activity in a PBRM1-mutant line. As ALDH1A1 is a tumor-initiating cell marker, we next investigated PBRM19s effects on tumorigenic potential. In xenograft studies, PBRM1 knockdown cells formed significantly larger tumors. We then used the tumorsphere formation assay as a surrogate measure of tumor-initiating capacity. We confirmed that PBRM1 knockdown cells can form more free-floating tumorspheres. These cells also formed more colonies in soft-agar. This increased tumorigenic potential was at least partly dependent upon ALDH1A1, as revealed by chemical inhibition or siRNA knockdown of ALDH1A1. Additionally, we found that another PBAF-specific component, ARID2, is required for the pro-tumorigenic changes observed as a result of PBRM1 depletion. Knockdown of PBRM1 resulted in increased levels of ARID2, while re-expression of wild-type, but not mutant, PBRM1 lowered ARID2 levels. The primary tumor datasets confirmed increased ARID2 expression in PBRM1-mutant tumors. Knockdown of ARID2 in the context of PBRM1 loss reversed the increase in ALDH1A1 levels and ability to form tumorspheres described above. Interestingly, other SWI/SNF components, including SNF5, BRG1, and BRM, were dispensable for these changes. These findings suggest that one key tumor suppressor function of PBRM1 in ccRCC is to repress ALDH1A1 levels, and that mutation of PBRM1 increases a cell9s tumorigenic potential through upregulated ALDH1A1. Citation Format: David Schoenfeld, William Su, Sakellarios Zairis, Deepti Mathur, Raul Rabadan, Ramon Parsons. PBRM1 alteration in clear cell renal cell carcinoma increases tumorigenicity through ALDH1A1 upregulation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A24.