The role of histidine phosphorylation in the DNA alkylation damage response

项目摘要 修复基因组的烷基化损伤至关重要，因为这种损伤具有细胞毒性，并且可能 致突变的氧化脱甲基酶ALKBH 3是DNA烷基化损伤修复（DAR）的核心组分。 通路沿着激活信号共整合复合物（ASCC），ALKBH 3使腺嘌呤去甲基化 单链DNA中N1位甲基化（1 meA）和N3位胞嘧啶甲基化（3 meC） 来维持基因组的完整性虽然我们对DAR的生化机制了解很多，但我们知道， 关于DAR通路的调节知之甚少DNA烷基化药物已被广泛用于治疗 不同的癌症。提高对DAR的理解将克服更多使用这些药物的挑战 设计更多的个性化癌症治疗方法。通过研究前列腺癌细胞， 发现ASCC的一个亚基ASCC 3在Y1909处被成纤维细胞生长因子受体酪氨酸磷酸化 1（FGFR 1）。该磷酸化事件引发ASCC 3通过组氨酸激酶NME 2进行His磷酸化。 NME 2介导的ASCC 3 His磷酸化促进ALKBH 3募集至DNA烷基化损伤位点， 亲爱的。我们敲除神经内分泌前列腺癌（NEPC）细胞中的NME 2，发现3 meC和1 meA 这些细胞在DNA中积累，并且这些细胞对DNA烷化剂特别敏感。平移 这些发现的相关性得到了~10% NEPC中NME 2和ASCC 3扩增的支持， NME 2表达增加与NEPC患者的生存率低相关。基于这些初步 研究结果，我们假设NME 2通过促进DAR中ASCC 3/ALKBH 3功能来控制基因组稳定性。 并且靶向该途径使癌细胞对DNA烷化剂敏感。为了验证这一假设，在 应用，我们建议解剖NME 2促进DAR的分子机制，确定如何 NME 2介导的ASCC 3磷酸化受DNA损伤信号调节，并破译NME 2的作用 在体内促进对烷基化化疗的抗性。我们的研究将描述一种以前未知的 His磷酸化在DAR和基因组稳定性中的作用。基于多学科专业知识， 科学的前提和严谨的研究策略，我们的研究有望为肿瘤提供新的见解 对烷化剂化疗的反应，并可能揭示小分子抑制的靶点， NEPC和其他NME和ASCC 3依赖性癌症与DNA烷化剂化疗的关系。

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.