MECHANISM AND REGULATION OF THE DNA ALKYLATION DAMAGE RESPONSE

 描述（由申请方提供）：对基因组的烷基化损伤的修复在所有细胞中均至关重要，因为此类损伤具有细胞毒性和潜在致突变性。这些修复途径抵消内源性和环境烷化剂。烷基化化疗是许多肿瘤的主要治疗方式，强调了这些途径在癌症中的重要性。存在许多不同的烷基化修复途径，包括碱基切除和核苷酸切除修复、甲基鸟嘌呤甲基转移酶（MGMT）的直接逆转和AlkB蛋白家族的脱烷基化。我们最近证明，人类AlkB同源物之一，ALKBH 3，在特定的肿瘤细胞中的烷基化损伤修复中起着重要作用，但在非恶性细胞中的这种功能是不确定的。这表明烷基化修复酶可能在不同的细胞环境中受到不同的调节。虽然我们对介导DNA烷基化修复的生化机制了解很多，但我们对这些途径如何调节知之甚少，特别是在后生动物中。我们最近发现了去泛素化酶OTUD 4在调节人类细胞中的MGMT和AlkB家族酶中的关键作用。我们的数据表明，OTUD 4作为具有多种差异调节的去泛素化酶活性的复合物的组分起作用，以控制烷基化修复，以及影响DNA损伤信号传导。总之，我们的工作暗示泛素化作为烷基化损伤修复的关键调节剂，这是我们在本提案中寻求理解的一个未探索的领域。具体而言，我们将测试OTUD 4作为脱泛素酶复合物支架的假设，其在体外和小鼠肿瘤模型中促进烷基化化疗抗性（Aim 1）。我们还将确定这种去泛素化酶的催化特异性如何通过翻译后修饰来调节DNA损伤反应信号传导（目的2）。最后，我们将检验非蛋白酶体泛素化在ALKBH 3通路激活中发挥作用的假设（目的3）。除了对我们理解DNA修复途径的影响外，这项工作还将为肿瘤对烷基化化疗的反应提供新的见解，并可能揭示化疗增敏的新小分子靶点。

 DESCRIPTION (provided by applicant): The repair of alkylation damage to the genome is critical in all cells, because such damage is cytotoxic and potentially mutagenic. These repair pathways counteract endogenous and environmental alkylating agents. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of these pathways in cancer. A number of different pathways exist for alkylation repair, which include base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB protein family. We recently demonstrated that one of the human AlkB homologues, ALKBH3, plays an important role in alkylation damage repair in specific tumor cells, but is dispensable for this function in nonmalignant cells. This suggests that alkylation repair enzymes may be differentially regulated in various cellular contexts. While much is known about the biochemical mechanisms that mediate DNA alkylation repair, we know very little about how these pathways are modulated, particularly in metazoans. We have recently discovered a critical role for the deubiquitinase OTUD4 in the regulation of MGMT and the AlkB family of enzymes in human cells. Our data suggests that OTUD4 functions as a component of a complex with multiple, differentially regulated deubiquitinase activities to control alkylation repair, as well as impacting DNA damage signaling. Together, our work implicates ubiquitination as a critical regulator of alkylation damage repair, an unexplored area which we seek to understand in this proposal. Specifically, we will test the hypothesis that OTUD4 serves as a deubiquitinase complex scaffold that promotes alkylation chemoresistance, both in vitro and in a mouse tumor model (Aim 1). We will also determine how the catalytic specificity of this deubiquitinase may be regulated by post- translational modification to modulate DNA damage response signaling (Aim 2). Finally, we will test the hypothesis that non-proteasomal ubiquitination plays a role in the activation of the ALKBH3 pathway (Aim 3). Beyond its implications on our understanding of DNA repair pathways, this work will provide new insights into tumor responses to alkylation chemotherapy, and may reveal novel small molecule targets for chemo sensitization.