Abstract Our group has previously demonstrated that the Trp53 R270H mutation can drive prostate cancer (CaP) initiation in a genetically engineered mouse model, and that the human equivalent, TP53 R273H, can promote development of castration resistant growth of LNCaP cells as well as resistance to commonly used therapeutic agents. The primary objective of the current study was to identify genes that may contribute to the development of these gain-of-function phenotypes. Wildtype mice and mice that were heterozygous or homozygous for the Trp53 R270H mutation (referred to as Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H, respectively) that were ~3 months old were exposed to 5 Gy radiation to activate and stabilize p53, consequently increasing its expression. Mouse prostates were harvested 6 hours post-irradiation and either processed for subsequent histological/immunohistochemistry (IHC) analysis or snap-frozen for subsequent RNA extraction and transcriptome profiling with RNA-Sequencing (RNA-Seq) analysis. P53 expression was determined by IHC. RNA-Seq data were processed to quantify transcript levels and to assess differential gene expression between the 3 groups. PIN lesions were observed in 3-month-old Trp53 R270H/R270H mice prostates, but not in Trp53 +/+ or Trp53 +/ R270H mice prostates. IHC analysis demonstrated that p53 was stabilized in the majority of prostate cells from Trp53 +/+, Trp53 +/ R270H, or Trp53 R270H/R270H mice 6 hours post-irradiation. RNA-Seq analysis of RNA isolated from irradiated mice prostates identified 1,444 genes that were differentially expressed in Trp53 +/+ versus Trp53 R270H/R270H mice prostate cells, and 796 genes that were differentially expressed in Trp53 +/+ versus Trp53 +/ R270H mice. Statistically significant differences in gene expression between the 3 groups were observed for 1,378 genes, including a number of p53 target genes, such as Cdkn1a, Bax, Bcl2, Kras, Mdm2, and Id4. Our data identify multiple genes that may contribute to prostate cancer initiation and/or progression through p53 gain-of-function and loss-of-function mechanisms. It is possible that further analysis of these genes may lead to the development of new therapies and/or biomarkers for prostate cancer patients as well as guide the usage of currently available therapies in men at risk of developing CaP and CaP patients who harbor TP53 mutations. Citation Format: Ruth L. Vinall, Qian Chen, George Talbott, Neil Hubbard, Clifford Tepper, Alexander Borowsky. Use of a genetically engineered mouse model and RNA sequencing to identify genes that are aberrantly regulated by mutant p53 in prostate cells following irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2017-2820