Investigating DNA repair vulnerabilities in oncometabolite producing cancers

研究产生肿瘤代谢物的癌症中的 DNA 修复漏洞

• 批准号: 10672173
• 负责人: Katelyn Noronha
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-17 至 2024-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672173
• 关键词: Affect; Biological Assay; Cancer Patient; Cell Line; Cell Survival; Cells; ChIP-seq; Chromatin; Citric Acid Cycle; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA-dependent protein kinase; Data; Dependence; Development; Disease Progression; Double Strand Break Repair; Enzymes; Equilibrium; Event; Flow Cytometry; Frequencies; Fumarate Hydratase; Fumarates; Future; Genes; Genome; Glioma; Immunofluorescence Immunologic; Isocitrate Dehydrogenase; Knowledge; Lead; Ligands; Ligation; Malignant Neoplasms; Molecular Target; Monitor; Mutation; Nonhomologous DNA End Joining; Paraganglioma; Pathway interactions; Pheochromocytoma; Poly(ADP-ribose) Polymerase Inhibitor; Poly(ADP-ribose) Polymerases; Process; Production; Proteins; Renal Cell Carcinoma; Reporter; Reporting; Research; Role; Signal Transduction; Site; Succinate Dehydrogenase; Succinates; Testing; Therapeutic; Up-Regulation; cancer health disparity; cancer therapy; clinical development; clinically relevant; cytotoxic; efficacy evaluation; homologous recombination; inhibitor; insight; mutant; neoplastic cell; novel therapeutic intervention; novel therapeutics; recruit; repaired; response; small molecule inhibitor; synergism; therapeutic target; therapeutically effective; translational approach; treatment strategy

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.

项目摘要 三核酸循环基因中与癌症相关的突变诱导2-羟基氯丁烷的产生 富马酸或琥珀酸酯。这些oncometabolites抑制同源重组（HR）DNA修复 路径。非动力学末端连接（NHEJ）是双链断裂（DSB）修复的另一个主要途径， 将进一步细分为经典（CNHEJ）和高度诱变的替代端（Altnhej） 途径。我们小组的初步数据表明，oncometabolites诱导了NHEJ修复的上调， 但是，该观察结果的机械基础尚未阐明。多种抑制剂已经 在这些NHEJ修复途径中开发了靶向蛋白，包括DNA-PK和POL THETA抑制剂， 这表明NHEJ是临床上相关的目标。我假设特定的oncometabolites独特 并动态调节Altnhej和cnhej，可以将其瞄准用于治疗的收益 肿瘤细胞。 我将研究oncometabolites如何改变DSB修复并评估NHEJ途径作为治疗 通过两个目标进行目标。我的第一个目标将阐明NHEJ途径之间的动态平衡 oncometabolite产生细胞。我将使用对HR，Total NHEJ和 altnhej确定如何外源或本质上产生的oncometabolites如何改变 NHEJ频率。确定CNHEJ和Altnhej蛋白在DNA断裂部位的改变 多种染色质状态，我将使用免疫荧光和染色质免疫沉淀测序 （chip-seq）在细胞系中，可以在该细胞系中诱导内源性双链断裂的数百个位点。 基因组。使用chip-seq进行cnhej或altnhej的蛋白质募集时间的变化将进行研究 通过免疫荧光鉴定的蛋白质。这将确定oncometabolites在多大程度上改变了各种 NHEJ的阶段，例如DNA末端处理或结扎。我的第二个目标将调查 CNHEJ和Altnhej抑制oncometabolite生产癌症。我将以CNHEJ和Altnhej为目标 分别使用DNA-PK和POL THETA抑制剂，通过对短期细胞活力分析进行 oncometabolite产生细胞系。这将确定oncometabolite产生癌症是否更多 与亲本细胞系相比，对DNA-PK或POL THETA抑制剂敏感。此外，这些 细胞系显示对PARP抑制剂的独家敏感性。我将通过 测试这些细胞系对DNA-PK抑制剂和pol theta抑制剂的敏感性与PARP结合 抑制剂。这将确定针对CNHEJ或Altnhej和Parp是否比 单独的代理。总体而言，该建议将使人们对oncometebolites的方式有更全面的了解 影响DSB修复并确定用于治疗oncometabolite生产癌症的新型治疗策略。

项目成果

Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions.

• DOI: 10.1093/nar/gkq379
• 发表时间: 2010-09
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Yu AM;McVey M
• 通讯作者: McVey M

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

• DOI: 10.1038/s41467-021-23463-8
• 发表时间: 2021-06-17
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Zatreanu D;Robinson HMR;Alkhatib O;Boursier M;Finch H;Geo L;Grande D;Grinkevich V;Heald RA;Langdon S;Majithiya J;McWhirter C;Martin NMB;Moore S;Neves J;Rajendra E;Ranzani M;Schaedler T;Stockley M;Wiggins K;Brough R;Sridhar S;Gulati A;Shao N;Badder LM;Novo D;Knight EG;Marlow R;Haider S;Callen E;Hewitt G;Schimmel J;Prevo R;Alli C;Ferdinand A;Bell C;Blencowe P;Bot C;Calder M;Charles M;Curry J;Ekwuru T;Ewings K;Krajewski W;MacDonald E;McCarron H;Pang L;Pedder C;Rigoreau L;Swarbrick M;Wheatley E;Willis S;Wong AC;Nussenzweig A;Tijsterman M;Tutt A;Boulton SJ;Higgins GS;Pettitt SJ;Smith GCM;Lord CJ
• 通讯作者: Lord CJ

Analysis of drug combinations: current methodological landscape.

• DOI: 10.1002/prp2.149
• 发表时间: 2015-06
• 期刊: PHARMACOLOGY RESEARCH & PERSPECTIVES
• 影响因子: 2.6
• 作者: Foucquier, Julie;Guedj, Mickael
• 通讯作者: Guedj, Mickael

Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

• DOI: 10.1038/nsmb.2796
• 发表时间: 2014-04
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8
• 作者: Aymard F;Bugler B;Schmidt CK;Guillou E;Caron P;Briois S;Iacovoni JS;Daburon V;Miller KM;Jackson SP;Legube G
• 通讯作者: Legube G

Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

• DOI: 10.1158/1535-7163.mct-14-0765
• 发表时间: 2015-02
• 期刊: Molecular cancer therapeutics
• 影响因子: 5.7
• 作者: Goglia AG;Delsite R;Luz AN;Shahbazian D;Salem AF;Sundaram RK;Chiaravalli J;Hendrikx PJ;Wilshire JA;Jasin M;Kluger HM;Glickman JF;Powell SN;Bindra RS
• 通讯作者: Bindra RS