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Abstract Nonbiased mutational screening to identify essential genes and pathways in cancer is a powerful approach for understanding underlying biology as well as the identification of potential therapeutic targets. Epigenetic (EPI) and transcriptional factors (TF) play an important role in the change of phenotype and lineage plasticity from adenocarcinoma to castration resistant prostate cancer (CRPC), double negative (DN) or treatment-induced neuroendocrine prostate cancer (NEPC). When RB1 loss occurs in combination with TP53 alterations, stemness and lineage plasticity increase with one consequence being differentiation to a neuroendocrine (NE) lineage. Here, we are identifying and testing the functional role of epigenetic and transcription factors required for the viability of CRPC, DN and NEPC relative to a matrix of genotypic and phenotypic characteristics. We applied CRISPR-CAS9 dropout screens in prostate organoids and cell lines to identify gene/s associated with CRPC, DN and NEPC viability. We successfully CRISPR-engineered LNCaP clones of single (LNCaP-TP53-SKO, LNCaP-RB1-SKO) and double (LNCaP-TP53-RB1-DKO) gene knockouts, which were subsequently characterized for immuno-phenotype and transcriptome. Our dropout screens, targeting all TFs and EPIs, identified 117 TF and 40 EPI genes responsible for the viability of DN, NEPC and LNCaP models. We found known genes such as FOXA1 and ASCL1, as well as novel genes including PROX1 and KLF3, which were validated through guide RNA competition assays. Our results showed PROX1 is essential for cell viability in a DN organoid model, and its expression is associated with increased aggressiveness, indicating PROX1 has role in stemness/lineage plasticity in treatment resistant adenocarcinoma. RNA-seq analysis following overexpression of PROX1 in the LNCaP-TP53-RB1 null adenocarcinoma model, with and without enzalutamide, identified pathways enriched for cell proliferation, differentiation, neuronal development, and prostate gland development. In summary, our study has suggested an important role for PROX1 in the growth and differentiation of poorly differentiated prostate cancer, which may lead to the identification of potential therapeutic targets. Citation Format: Sonam Raj, Brian Capaldo, Joel Bowman, Margaret White, Xavier Moore, Berkley Gryder, Aian N. Alilin, David Takeda, Kathleen Kelly. Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation 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 554.
Raj et al. (Fri,) studied this question.