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是至关重要的，因为这种损伤是细胞毒性的，并有可能 致突变性。氧化去甲基酶ALKBH3是DNA烷基化损伤修复(DAR)的核心成分 路径。与激活信号共整合复合体(ASCC)一起，ALKBH3去甲基化腺嘌呤 单链DNA中N1位甲基化(1meA)和N3位胞嘧啶甲基化(3meC) 以保持基因组的完整性。虽然对DAR的生化机制了解很多，但我们知道 关于DAR通路的调节很少。DNA烷基化药物已被广泛应用于治疗 由不同的癌症引起。提高对DAR的了解将克服更多使用这些药物的挑战 有效地为癌症设计更多的个性化治疗方法。通过研究前列腺癌细胞，我们 发现ASCC3是ASCC的一个亚基，在Y1909被成纤维细胞生长因子受体磷酸化 1(FGFR1)。这一磷酸化事件使ASCC3被组氨酸激酶NME2磷酸化。 NME2介导的ASCC3His磷酸化促进ALKBH3募集到DNA烷基化损伤部位和 达尔。我们在神经内分泌前列腺癌(NEPC)细胞中敲除了NME2，发现3meC和1meA 在DNA中积累，这些细胞对DNA烷化剂特别敏感。翻译型 NME2和ASCC3在~10%的NEPC和ASCC3中的扩增支持了这些发现的相关性 NME2的高表达与NEPC患者的生存不良有关。基于这些初步的 结果，我们假设NME2通过促进DAR的ASCC3/ALKBH3功能来控制基因组的稳定性 而以此为靶点的途径会使癌细胞对DNA烷化剂敏感。为了检验这一假设，在这个 应用我们建议剖析NME2促进DAR的分子机制，确定如何 NME2介导的ASCC3磷酸化受DNA损伤信号的调控，并破译NME2的作用 在体内促进对烷基化化疗的耐药性。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。以多学科专业知识为基础，令人信服 科学的前提和严谨的研究策略，我们的研究承诺为肿瘤提供新的见解 对烷化化疗的反应，并可能揭示致敏小分子的抑制靶点 NEPC和其他依赖NME和ASCC3的癌症使用DNA烷化剂进行化疗。

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.