Abstract PO-031: Aldolase A (ALDOA) is required for efficient DNA double-strand break (DSB) repair

Abstract

Abstract Introduction: Metabolic reprogramming, known as the Warburg effect, is one of the universal differences between cancer cells and non-cancerous cells. Glucose metabolism and DNA repair are frequently dysregulated in cancer. Metabolic pathways provide cells with nucleic acids and energy required to repair DNA. However, the underlying mechanisms that promote crosstalk between these processes are unknown. ALDOA is a glycolytic enzyme that catalyses the conversion of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA is overexpressed in several types of cancer. In this study, we demonstrate a novel mechanism through which ALDOA directly regulates DSB repair. Methods: ALDOA was depleted from cells using siRNA and single-shot quantitative proteomics were performed. Immunofluorescence was utilized to determine the localization of ALDOA. DSB repair reporter assays were used to measure DSB break repair. Gene expression was quantified by western blot and qPCR. Immunoprecipitations were used to detect protein:protein interactions. Statistical analysis: The experiments were at least n=3, and data are presented as the means ± SEM. Statistical significance was evaluated using Student’s t-test or one-way ANOVA. Results: In order to identify ALDOA-dependent pathways, we performed quantitative mass spectrometry on ALDOA depleted cells. In addition to the expected decrease in glycolysis pathways, we also observed a significant downregulation of DNA repair proteins in ALDOA depleted cells. Further analysis showed that the ALDOA protein responds to DNA damage (IR) and migrates from the cytosol to the nucleus, suggesting that it could be directly involved in DNA damage repair. Slower clearance of γ-H2AX foci (a DSB marker), and decreased clonogenicity following irradiation (IR) treatment were also observed, indicating dysfunctional DNA repair processes. Repair of DSBs is primarily though the NHEJ (non-homologous end-joining) or HR (homologous recombination) -mediated DNA repair pathways. Silencing ALDOA led to a decrease in both NHEJ- and HR-mediated DSB repair efficiency. This disruption was likely due to the significant reduction of both the mRNA and protein of the DNA repair effector kinases, DNA-Dependent Kinase (DNA-PK) and Ataxia and Telangiectasia Mutated (ATM) in ALDOA-depleted cells. In addition to regulating the expression of DNAPK and ATM we also found that ALDOA directly interacted with both kinases, suggesting that it may have a direct role in regulating their function. Here, we define a role for ALDOA in the repair of DNA DSBs, through the regulation of DNA repair effector kinase expression and function. Conclusion: These results identify crosstalk between metabolic and DNA repair pathways and have implications for cancer treatment and tumorigenesis. The role of ALDOA in DNA repair could promote therapeutic resistance in tumors and may be a future therapeutic target to sensitize tumors to DNA-damaging agents such as radiation. Citation Format: Amila Suraweera, Kenneth O’Byrne, Derek Richard, Emma Bolderson, Thais Sobanski. Aldolase A (ALDOA) is required for efficient DNA double-strand break (DSB) repair [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-031.