LAUSR

Background: ARID1A mutations (ARID1Amut) occur in up to 30% of metastatic bladder cancers (mBC) and have been associated with poorer response to standard treatments and shorter overall survival. We previously identified bromodomain and extraterminal (BET) protein inhibition as a potential treatment paradigm for mBC, with particular potency of BET inhibitors (BETi) in cells lines containing ARID1A inactivating mutations (ARID1Amut). BETi prevents BET protein bromodomains from interacting with acetylated histone lysine tails and promoting transcription of multiple oncogenes. Our previous data shows that treatment with the pan-BETi OTX-015 reduces ARID1B expression, and significantly reduces both gene and protein expression of RAD51/RAD51 in ARID1Amut cells compared to ARID1A wild type (ARID1AWT), suggesting impaired DNA damage repair function. Here we evaluate the phenotypic effects of BETi on proliferation and migration in preclinical models of BC. Methods: To evaluate OTX-015-induced changes to gene expression, 5637 (ARID1AWT) and HT1197 (ARID1Amut) cells were treated with 1 μM OTX-015 for 48-h. RNA was extracted and sent to Novogene for bulk RNA-seq. Differential expression was quantified using DESeq2, and gene enrichment was analyzed using GSEA. 5637 and HT1197 cells were treated with 1 or 5 μM OTX-015 for 48-h, and colony forming potential was assessed by crystal violet staining after 10 days. Cells were treated with the same treatment schema as above, and a wound-healing assay evaluated wound closure over 48-h. Differences in the percent area of wells covered with colonies and relative wound closure were assessed by FIJI v.2.9.1. Results: BET inhibition of ARID1Amut HT1197 cells lead to a significant decrease in the pathway enrichment for G2/M checkpoint (NES=-2.78, q<0.0001) and E2F target (NES-2.61, q<0.0001) genes. Conversely, BET inhibition of ARID1AWT 5637 cells, lead to more muted response (G2/M: NES=-1.31; q=0.08, and E2F: NES=-1.53; q=0.02). After 1 μM OTX-015, cell cycle pathway targets were significantly impacted in ARID1Amut HT1197 cells (P=0.0002), but not in ARID1AWT 5637 cells (P=0.47). Results from our in vitro phenotypic data confirmed that OTX-015 significantly reduced proliferation and migration in HT1197 cells. After 1 μM OTX-015, 5637 colonies covered only 2% less area than 0.1% DMSO treated control colonies (n=3, P=0.99), and wounds were fully healed at 48-h (n=3, P=0.99). Conversely, HT1197 treated cells covered 96% less area (n=3, P<0.0001), and maintained a 38% larger wound size at 48-h (n=3, P=0.008), when compared to 0.1% DMSO treated colonies. Conclusions: These preliminary data highlight how OTX-015 potently inhibits proliferation and migration in ARID1Amut cells. These data will be confirmed in isogenic cell lines harboring inactivating ARID1A mutations, and support future mechanistic exploration (e.g., BETi-mediated cell cycle dysregulation) in ARID1Amut bladder cancer. Citation Format: Ryan M. Kemper, Manfred Meng, Jeffrey S. Damrauer, William Y. Kim, Daniel J. Crona. OTX-015 inhibits proliferation and migration in ARID1A-mutated bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6281.