Investigating DNA repair vulnerabilities in oncometabolite producing cancers

项目摘要 三羧酸循环基因中的癌症相关突变诱导2-羟基戊二酸的产生， 富马酸盐或琥珀酸盐。这些肿瘤抑制同源重组（HR）DNA修复 通路非同源末端连接（NHEJ）是双链断裂（DSB）修复的另一个主要途径， 进一步细分为经典（cNHEJ）和高度致突变的替代末端连接（altNHEJ） 途径。我们小组的初步数据表明，oncometamine诱导NHEJ修复的上调， 然而，这一观察结果的机制基础尚待阐明。多种抑制剂已经 开发了这些NHEJ修复途径中的靶蛋白，包括DNA-PK和pol θ抑制剂， 这表明NHEJ是临床相关的靶点。我假设特异性肿瘤 并动态调节altNHEJ和cNHEJ，这可以靶向治疗增益， 肿瘤细胞 我将研究oncometamine如何改变DSB修复，并评估NHEJ途径作为治疗 通过两个目标。我的第一个目标将阐明NHEJ途径之间的动态平衡， 癌代谢产物生成细胞。我将使用表达HR、总NHEJ和总NHEJ特异性报告基因的U2 OS细胞。 altNHEJ，以确定外源性或由突变产生的内源性致癌物质如何改变 NHEJ频率为了确定cNHEJ和altNHEJ蛋白募集如何在DNA断裂位点改变， 不同的染色质状态，我将使用免疫荧光和染色质免疫沉淀测序 在细胞系中，内源性双链断裂可以在细胞中的数百个位点处被诱导。 基因组将使用ChIP-seq研究cNHEJ或altNHEJ的蛋白募集时间的变化 通过免疫荧光鉴定的蛋白质。这将建立在何种程度上oncometalone改变各种 NHEJ的各个阶段，如DNA末端加工或连接。我的第二个目标是研究 cNHEJ和altNHEJ对产生癌代谢物的癌症的抑制。我会瞄准cNHEJ和altNHEJ 分别用DNA-PK和pol θ抑制剂，通过在 产生癌代谢物的细胞系。这将决定是否癌代谢产物产生的癌症更多 与亲本细胞系相比，对DNA-PK或pol θ抑制剂作为单一药剂敏感。而且这些 细胞系对PARP抑制剂显示出极高的敏感性。我将评估潜在的治疗组合， 测试这些细胞系对DNA-PK抑制剂和pol θ抑制剂与PARP组合的敏感性 抑制剂的这将确定靶向cNHEJ或altNHEJ和PARP是否比靶向cNHEJ或altNHEJ和PARP更有效。 单独行动总的来说，这一建议将导致更完整的理解如何oncometabolism 影响DSB修复并鉴定用于治疗产生癌代谢物的癌症的新的治疗策略。

PROJECT SUMMARY Cancer-associated mutations in tricarboxylic acid cycle genes induce production of 2-hydroxyglutarate , fumarate, or succinate. These oncometabolites suppress the homologous recombination (HR) DNA repair pathway. Nonhomologous end joining (NHEJ) is the other major pathway for double strand break (DSB) repair, which is further sub-divided into classical (cNHEJ) and highly mutagenic alternative end joining (altNHEJ) pathways. Our group’s preliminary data suggests that oncometabolites induce upregulation of NHEJ repair, however, the mechanistic basis for this observation has yet to be elucidated. Multiple inhibitors have been developed that target proteins within these NHEJ repair pathways, including DNA-PK and pol theta inhibitors, which suggests that NHEJ is a clinically relevant target. I hypothesize specific oncometabolites uniquely and dynamically regulate altNHEJ and cNHEJ, which can be targeted for a therapeutic gain against tumor cells. I will investigate how oncometabolites alter DSB repair and evaluate NHEJ pathways as therapeutic targets through two aims. My first aim will elucidate the dynamic balance between NHEJ pathways in oncometabolite producing cells. I will use U2OS cells that express reporters specific for HR, total NHEJ, and altNHEJ to determine how oncometabolites, added exogenously or intrinsically produced by mutations, alter NHEJ frequency. To determine how cNHEJ and altNHEJ protein recruitment is altered at DNA break sites with diverse chromatin states, I will use both immunofluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) in a cell line in which endogenous double strand breaks can be induced at hundreds of sites in the genome. Changes in the timing of protein recruitment will be studied using ChIP-seq for cNHEJ or altNHEJ proteins identified by immunofluorescence. This will establish the extent to which oncometabolites alter various stages of NHEJ, such as DNA end processing or ligation. My second aim will investigate the effect of cNHEJ and altNHEJ inhibition on oncometabolite producing cancers. I will target cNHEJ and altNHEJ with DNA-PK and pol theta inhibitors, respectively, by performing short-term cell viability assays in oncometabolite producing cell lines. This will determine whether oncometabolite producing cancers are more sensitive to DNA-PK or pol theta inhibitors as single agents compared to parental cell lines. Furthermore, these cell lines show exquisite sensitivity to PARP inhibitors. I will evaluate potential therapeutic combinations by testing the sensitivity of these cell lines to DNA-PK inhibitors and pol theta inhibitors in combination with PARP inhibitors. This will determine whether targeting both cNHEJ or altNHEJ and PARP is more effective than single agents alone. Overall, this proposal will lead to a more complete understanding of how oncometabolites affect DSB repair and identify novel therapeutic strategies for treatment of oncometabolite producing cancers.