Abstract 5650: RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in cancer

Abstract

Abstract Background: Ribosomes are a complex ensemble of ribosomal RNA and proteins. As the site where genetic information is translated to protein, they have a key role in cell survival, growth and proliferation. Ribosome biogenesis (RiBi), the process of creating ribosomes, is a complex function governed by precise checkpoints and surveillance mechanisms which may become dysregulated in cancer leading to tumor growth and therapeutic resistance. Ribosome-targeted therapy could provide a promising treatment for cancer. NUC-7738 (3'-deoxyadenosine [3’-dA] which is phosphorylated and protected with a phosphoramidate moiety attached at the 5’-position) generates sustained intracellular levels of 3’-dATP, a molecule that profoundly influences RNA regulatory processes, including poly(A) tail length, leading to alternative polyadenylation (APA) and splicing, resulting in impaired cellular responses. NUC-7738 is currently being investigated in combination with pembrolizumab in patients with advanced melanoma in the Phase 2 part of the clinical study (NCT03829254). Here, we investigate the impact of 3’-dATP on RNA regulation and RiBi utilizing a novel bioinformatic pipeline. Material and Methods: RNA was extracted from melanoma and renal carcinoma cell lines treated with NUC-7738 for 24h and paired biopsies (pre and post treatment) from patients treated with NUC-7738 + pembrolizumab. Sequencing libraries used for long-read PCR-cDNA sequencing were generated. Gene-level expression was quantified using Salmon DeSeq2. In addition to gene expression, poly(A) tail length, APA and isoform switch were analyzed. Protein expression was determined using JESS Western analysis. Multiplexed analysis of protein localization and expression in tissue was carried out using an in-house immunofluorescence protocol. Results: NUC-7738 caused a global reduction in poly(A) tail length in cell lines and patient biopsies that was most pronounced in the poly(A) tail exosome targeting (PAXT) long non-coding RNA (lncRNA) transcripts, including SNHG19, 5’TOP and YAE1, all of which play a role in RiBi. PAXT lncRNAs such as SNHG3 and SNHG19 increased in abundance. Ribosomal subunit proteins such as RPS3, RPS6, RPL17 decreased in expression. Expression of YAE1, a regulator of RiBi, also decreased. Distinct APA usage of various transcripts was observed in cell lines and patient biopsies. Conclusion: Through the development of a novel bioinformatic pipeline we have been able to demonstrate that NUC-7738 disrupts RNA regulation, leading to perturbed RiBi. Targeting RiBi provides a promising approach to treating cancers Citation Format: Mustafa Elshani, Ying Zhang, In Hwa Um, Ruth Plummer, Sarah P. Blagden, Stefan N. Symeonides, Natalie Cook, T.R. Jeffry Evans, Alison L. Dickson, David J. Harrison. RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in 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 5650.