Abstract 4350: Characterization of PUF60 splicing activity that is essential for the survival of triple negative breast cancer

Abstract Alternative splicing driven by RNA-binding proteins (RBPs) plays a significant role in TNBC, however, pharmacological modulators of core spliceosomal proteins can disrupt early stages of spliceosome assembly and lead to toxic effects in normal tissues, limiting their utility as safe therapeutics. Here, we used in vivo CRISPR-Cas9 screening to evaluate RBP dependencies in TNBC and identified 17 splicing regulatory candidates that are essential in TNBC but dispensable for normal cell growth and survival. Within this cohort, the alternative splicing factor, PUF60, was the most significantly upregulated candidate in patient TNBC tumors. We found that depletion of PUF60 induced apoptosis in human TNBC cell lines and impeded xenografted tumor growth in vivo while upregulation of PUF60 expression increased TNBC cellular proliferation. Although PUF60 has been shown to also bind DNA and influence transcription, TNBC cells constructed with a previously characterized mutation (L140P) required for 3’ splice site recognition displayed significantly decreased cellular proliferation compared to cells containing wild type PUF60. Integrated eCLIP and KD RNA-sequencing revealed that PUF60 selectively regulates the splicing of mRNAs in TNBC that contribute to essential processes including DNA repair, replication and cell cycle progression. RNA-seq following L140P-mediated inhibition of 3’ ss recognition revealed a cohort of splicing events within genes enriched for similar processes following knockdown of endogenous PUF60 including cell cycle and histone modification. Altogether, our study highlights a critical role for PUF60-mediated splicing in the maintenance of genomic integrity in TNBC as well as the therapeutic potential of identifying and targeting splicing effector RBPs only responsible for TNBC survival. Citation Format: Alexandra Tankka, Trent Gomberg, Cathy Zhou, Vivian Pham, Gene Yeo. Characterization of PUF60 splicing activity that is essential for the survival of triple negative breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4350.