Abstract 6666: Translocation-dependent regulation of nucleotide metabolism in response to drug-induced nucleotide stress

Abstract Nucleotide metabolism plays a significant role in cancer development and progression. Cancer cells have an increased demand for nucleotides, which are the building blocks of DNA and RNA, due to their uncontrolled proliferation. Targeting nucleotide metabolism is an important strategy for cancer therapy. However, existing therapies have a small therapeutic window as they induce toxicity in cancer and normal cells alike. Little is known about the mechanisms of nucleotide stress-induced toxicity in cells. In prokaryotes, the PyrR protein senses nucleotide levels by binding UMP. This regulates the ability of PyrR to bind the attenuator region of the pyr operon transcript, thus altering transcription of downstream nucleotide metabolism genes. PyrR therefore acts as a nucleotide sensor in prokaryotes. The paralog of this protein in mammals is yet to be discovered. It is unknown if similar nucleotide sensing mechanisms occur in mammalian cells. Our study aims focused to identify new nucleotide binding proteins that regulate nucleotide metabolism in humans. Here, we identified human proteins that had conserved homology to regions of the prokaryotic PyrR protein. We found similar amino acid sequences to the UMP binding site of PyrR in some candidate eukaryotic proteins. We analyzed the effect of nucleotide stress induction, via treatment with two clinical nucleotide biosynthesis inhibitors, on candidate protein P. P is normally localized in the cytoplasm but upon inhibitor treatment, P translocates into the nucleus. Knockdown of P resulted in decreased, cell viability, which could be rescued by nucleotide addition. Computational simulations identified potential nucleotide binding sites on P. We mutated the potential binding site aminos acids of P and expressed P mutant protein in cells. Unlike wildtype P, P mutant did not translocate into the nucleus upon nucleotide stress. Chromatin immunoprecipitation with high-throughput sequencing (ChIP)-seq revealed that P binds to the promoter regions of nucleotide metabolic genes. Through these studies, we have identified a novel mammalian protein sensor that transcriptionally regulates nucleotide metabolism through nuclear localization. These findings suggest an additional pathway by which nucleotide metabolism is directly regulated in response to the availability of end-product nucleotides. Knowledge of this pathway could propose ways to improve the therapeutic window of nucleotide biosynthesis-based chemotherapy. Citation Format: Zaffar Iqbal Bhat, Iqra Ishrat, XiaoMi Lu, Justin L. Tan. Translocation-dependent regulation of nucleotide metabolism in response to drug-induced nucleotide stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6666.