Project Summary Repair of alkylation damage to the genome is critical, because such damage is cytotoxic and potentially mutagenic. The oxidative demethylase ALKBH3 is a central component of a DNA alkylation damage repair (DAR) pathway. Along with the activating signal co-integrator complex (ASCC), ALKBH3 demethylates adenine methylated on the N1 position (1meA) and cytosine methylated on the N3 position (3meC) in single-strand DNA to maintain genomic integrity. Although much is known about the biochemical mechanisms of DAR, we know very little about the regulation of DAR pathways. DNA-alkylating drugs have been widely used in the treatment of different cancers. Improved understanding of DAR will overcome the challenge to using these drugs more effectively and designing additional personalized treatments for cancer. By studying prostate cancer cells, we found that ASCC3, a subunit of ASCC, is tyrosine phosphorylated at Y1909 by fibroblast growth factor receptor 1 (FGFR1). This phosphorylation event primed ASCC3 for His phosphorylation by the histidine kinase NME2. NME2-mediated ASCC3 His phosphorylation promoted ALKBH3 recruitment to DNA alkylation damage sites and DAR. We knocked out NME2 in neuroendocrine prostate cancer (NEPC) cells and found that 3meC and 1meA accumulated in DNA and that these cells were particularly sensitive to DNA-alkylating agents. The translational relevance of these findings is supported by the amplification of NME2 and ASCC3 in ~10% of NEPC and increased expression of NME2 correlates with poor survival in NEPC patients. Based on these preliminary findings, we hypothesize that NME2 governs genome stability by promoting ASCC3/ALKBH3 function in DAR and that targeting this pathway sensitizes cancer cells to DNA-alkylating agents. To test this hypothesis, in this application we propose to dissect the molecular mechanism by which NME2 promotes DAR, determine how NME2-mediated ASCC3 phosphorylation is modulated by DNA damage signals, and decipher the role of NME2 in promoting resistance to alkylating chemotherapy in vivo. Our studies will characterize a previously unknown role of His phosphorylation in DAR and genome stability. Built upon multidisciplinary expertise, compelling scientific premises, and rigorous research strategy, our studies promise to provide new insights into tumor responses to alkylating chemotherapy and may reveal targets for inhibition by small molecules that will sensitize NEPC and other NME- and ASCC3-dependent cancers to chemotherapy with DNA-alkylating agents.