Investigating DNA repair vulnerabilities in oncometabolite producing cancers

项目总结 三羧酸循环基因的癌症相关突变会诱导2-羟基戊二酸的产生， 富马酸根，或琥珀酸根。这些肿瘤代谢物抑制同源重组(HR)DNA修复 路径。非同源末端连接(NHEJ)是双链断裂(DSB)修复的另一条主要途径， 它又分为经典(CNHEJ)和高度诱变的替代末端连接(AltNHEJ) 小路。我们小组的初步数据表明，肿瘤代谢物诱导NHEJ修复上调， 然而，这一观察的机制基础尚未阐明。多种抑制剂已经被 开发了这些NHEJ修复途径中的靶蛋白，包括DNA-PK和poltheta抑制剂， 这表明NHEJ是一个临床相关的靶点。我假设特定的肿瘤代谢物是唯一的 并动态调节altNHEJ和cNHEJ，这可以作为治疗的靶点 肿瘤细胞。 我将研究肿瘤代谢如何改变DSB修复，并评估NHEJ通路是否具有治疗作用 通过两个目标实现目标。我的第一个目标是阐明NHEJ通路之间的动态平衡 产生肿瘤性代谢的细胞。我将使用U2OS细胞来表达HR、TOTAL NHEJ和 AltNHEJ以确定外源或内在地由突变产生的肿瘤代谢产物如何改变 NHEJ频率。要确定cNHEJ和altNHEJ蛋白募集是如何在DNA断裂位点上改变的，请使用 不同的染色质状态，我将同时使用免疫荧光和染色质免疫沉淀测序 (芯片序列)在细胞系中，内源性双链断裂可以在数百个位置被诱导 基因组。对于cNHEJ或altNHEJ，将使用CHIP-SEQ来研究蛋白质募集时间的变化 免疫荧光法鉴定的蛋白质。这将确定肿瘤代谢在多大程度上改变 NHEJ的各个阶段，如DNA末端加工或连接。我的第二个目标是调查 CNHEJ和altNHEJ对产生肿瘤的肿瘤的抑制。我会瞄准cNHEJ和altNHEJ 分别使用DNA-PK和Poltheta抑制剂，通过在 代谢物产生细胞系。这将决定肿瘤代谢物是否会导致更多的癌症 与亲本细胞系相比，DNA-PK或poltheta抑制剂作为单一药物敏感。此外，这些 细胞系对PARP抑制剂表现出极高的敏感性。我将通过以下方式评估潜在的治疗组合 检测这些细胞系对DNA-PK抑制剂和poltheta抑制剂与PARP的联合敏感性 抑制剂。这将决定同时以cNHEJ或altNHEJ和PARP为目标比 单独一名特工。总体而言，这一建议将导致对肿瘤如何代谢的更全面的理解 影响DSB修复并确定治疗产生肿瘤代谢物的新的治疗策略。

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.