Abstract 5359: Targeted engineering mRNA 3′UTR length enhances immunotherapy response in prostate cancer

Abstract

Abstract Background: Global mRNA 3′untranslated region (3′UTR) shortening through alternative polyadenylation (APA) has been widely observed in most cancers; however, it has not been demonstrated whether targeted interference of specific oncogenic mRNA 3′UTR lengths can inhibit tumor growth and potentiate immunotherapy response in prostate cancer. Methods: DaPars algorithm was applied for de novo identification of dynamic alternative polyadenylation (APA) during prostate cancer progression to the lethal phase; 3′UTR polyadenylation site (PAS) locations and usages were identified by Poly(A)-ClickSeq (PAC-seq) and 3′RACE; APA transcripts were quantified by RHAPA assay; a 3′UTR CRISPR/dCas13 Engineering System (3′UTRCES) was developed to manipulate the length of desired 3′UTRs; RIP-qPCR and PAR-CLIP-qPCR assays were used to determine the mechanistic basis of 3′UTRCES in APA editing; RNA-seq was utilized to evaluate the off-target effects; TT3 Lipid-like nanoparticles (LLN) were applied for intratumoral delivery of 3′UTRCES RNA molecules for targeted 3′UTR therapy. Quantitative proteomics and immunoprecipitation assays were conducted to investigate how major histocompatibility complex class I (MHC-I) substrates were recognized by SPSB1-containing ubiquitin ligase complex for degradation; Flow cytometry and T cell cytotoxicity assays were performed to evaluate MHC-I level and killing of tumor cells by antigen-stimulated CD8+ T cells. Results: 3′UTR globally shortens during prostate cancer progression to castration-resistance. Through blocking the proximal PASs, 3′UTRCES efficiently and specifically reverses the 3′UTR shortening of novel APA-linked, clinically-relevant prostate cancer oncogenic mRNAs, such as SPSB1, leading to reduced SPSB1 mRNA translation and prostate cancer cell proliferation. Intratumoral injection of TT3 LLN encapsulating 3′UTRCES RNA molecules effectively and safely inhibits prostate tumor growth in engrafted and transgenic mouse models. Notably, downregulation of SPSB1 protein by 3′UTRCES disrupts the interactions of SPSB1-containing ubiquitin ligase complex, leading to compromised ubiquitination-mediated MHC-I degradation and increased stability and abundance of MHC-I protein. Consistently, 3′UTRCES enhances MHC-I-regulated antigen presentation and thereby augments CD8+ T cell-mediated cytotoxicity, which suggest the potentials of 3′UTRCES to sensitize prostate cancer to immune checkpoint therapies. Importantly, 3′UTR shortening of SPSB1 mRNA is significantly associated with decreased MHC-I expression, reduced cytotoxic T-cell infiltration and activation in castration resistant prostate cancer patients. Conclusions: Our results establish the concept of “3′UTR targeted therapy” for treatment of prostate cancer with broad applications to other cancers and other 3′UTR-related diseases. Citation Format: Furong Huang, Fuwen Yuan, Ya Cui, Lei Li, Kexin Li, Zhifen Cui, Jingyue Yan, Qiang Chen, Christopher Nicchitta, Wenbin Ye, Yuebao Zhang, William Hankey, Jeffrey Everitt, Ming Chen, Jiaoti Huang, Hongyan Wang, Xin Lu, Eric Wagner, Yizhou Dong, Wei Li, Qianben Wang. Targeted engineering mRNA 3′UTR length enhances immunotherapy response in prostate 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(6_Suppl):Abstract nr 5359.