Abstract 277: NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform

Abstract

Abstract Background: Metabolic dysregulation, a hallmark of cancer, allows tumor cells to sustain high rates of growth and proliferation in unfavorable conditions, including hypoxia. Many cancers, including clear cell renal cell carcinoma (ccRCC), demonstrate the Warburg effect with high rates of glycolysis and some are dependent on glutamine. These cancers express high levels of glutaminase (GLS), the enzyme responsible for conversion of glutamine to glutamate. GLS exists as two distinct isoforms, GAC and KGA, which are generated by alternative polyadenylation. The presence of GAC favors more metabolically active cell growth, and the GAC:KGA ratio negatively correlates with survival in ccRCC. Selectively inhibiting the GAC isoform may be a target for therapy. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), has shown encouraging efficacy signals in several solid tumor types in a Phase ½ clinical study (NuTide:701 NCT03829254). It generates 3’-dATP which profoundly affects transcription by altering polyadenylation and has been shown to alter expression of mitochondrial electron transport chain genes. The aim of this study was to investigate the impact of NUC-7738 on GLS alternative polyadenylation and the ratio of GAC:KGA isoforms. Material and Methods: ccRCC (Caki-1 [VHL mutant];786-O [wildtype]) and pancreatic (PANC1; MIAPACA) cancer cell lines were treated with NUC-7738 at IC50 doses in hypoxic and normoxic conditions. NUC-7738 and 3’-dATP were measured by LC-MS. mRNA levels of glutaminase isoforms were determined by RT-qPCR using primers targeting exon 14-15 junction for GAC and exon 16-18 junction for KGA. GAC and KGA protein expression determined by JESS Western analysis. Results: NUC-7738 was converted into 3’-dATP within 6 hours of treatment with an average concentration of 8 pmol/106 cells, which was maintained for ~24 hours and decreased by 50% by 48 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. In ccRCC cells (VHL wildtype and mutant), NUC-7738 reduced transcript expression of GAC isoform, with no change in KGA, indicating a change in polyadenylation site usage. This was reflected in altered protein levels, with decreased GAC in ccRCC and pancreatic cell lines in hypoxic and normoxic conditions after NUC-7738 treatment. Conclusion: NUC-7738 generates sustained levels of 3’-dATP in cells, which is associated with alternative polyadenylation site usage changes. In a variety of cell types, mRNA and protein levels of the more metabolically active GAC isoform were reduced, with an increase in the relative amount of KGA isoform. Increased glutamate production and GAC:KGA ratio are key for survival of some tumors. As alternative polyadenylation is implicated in the control of expression of many genes in cancer, the ability to influence it may lead to new strategies for developing anti-cancer treatments interfering with cellular metabolism. Citation Format: Mustafa Elshani, Ying Zhang, Tia Hawkins, Mary Kudsy, In Hwa Um, Greice Zickuhr, Alison L. Dickson, David J. Harrison. NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 277.