Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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(6_Suppl):Abstract nr 138.