Post-translational modifications control JARID enzyme activity during DNA damage

翻译后修饰控制 DNA 损伤期间 JARID 酶的活性

• 批准号: 10817495
• 负责人: ELISABETH D MARTINEZ
• 金额: $ 7.1万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10817495
• 关键词: Alanine; Aspartic Acid; Cancer Patient; Cell Death; Cell Line; Cell Survival; Cells; Chromatin; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; Enzymes; Epigenetic Process; Foundations; Genetic Transcription; Genomics; Glutamic Acid; Goals; Growth; Histones; In Vitro; Ionizing radiation; KDM5B gene; Knock-out; Local Therapy; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Mass Spectrum Analysis; Measures; Mediating; Modernization; Molecular; Mutate; Non-Small-Cell Lung Carcinoma; Nonhomologous DNA End Joining; Normal tissue morphology; Oncogenic; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Predisposition; Radiation; Radiation therapy; Resistance; Role; Signal Transduction; Site; Stress; Therapeutic; Treatment Protocols; Tumor Tissue; ataxia telangiectasia mutated protein; cancer cell; demethylation; enzyme activity; enzyme substrate; gain of function; histone demethylase; homologous recombination; inhibitor; loss of function; mutant; novel; novel strategies; overexpression; palliative; pharmacologic; radiation resistance; recruit; repaired; response; small molecule inhibitor; tumor

Close to 75% of all non-small cell lung cancer (NSCLC) patients receive radiation therapy at some point in their treatment regimen whether with curative or palliative intent. Radiation, which is currently given in a highly localized fashion, causes massive DNA damage leading to DSBs and cancer cell death, yet on its own is not yet curative. Indeed, while some cancers are intrinsically resistant to IR, others acquire resistance by upregulating DNA repair pathways. Given modern advances in the targeted administration of radiotherapy, a paradigm shift leading to curing NSCLC or other tumors could occur if tumor tissue could be globally sensitized to radiation therapy. The epigenetic susceptibilities we propose to investigate here, may be the key. The oncogenic JARID/KDM5 histone demethylase subfamily of Jumonji enzymes, which are overexpressed in multiple malignancies, have recently defined roles in the DNA damage pathway: they mediate DNA repair by erasing trimethyl marks on active chromatin harboring H3K4me3 marks, thus stopping transcription and facilitating the recruitment of both homologous recombination and non-homologous end joining repair factors. The novel concept we propose here is that JARID enzymes must be modified post-translationally upon DNA damage, likely by radiation-activated ATM or ATR kinases, to enhance their histone demethylating activity on active chromatin, thus providing a mechanism to stop transcription and recruit repair factors to these sites, for cancer-cell survival. If true, this novel oncogenic activity of JARID enzymes would have significant implications for developing new approaches to sensitize lung tumors to ionizing radiation (IR), by selectively inhibiting the enhanced demethylase activity of JARID enzymes on chromatin. Our specific aims are to: 1.Determine if JARID enzymes are substrates of ATM kinases during the DNA damage response to IR: we will determine by mass spectrometry if JARID1B is phosphorylated by ATM/ATR kinases during the DNA damage response. We will map the site of phosphorylation and will mutate it to alanine (loss of function) or glutamic or aspartic acid (gain of function) to determine the impact on DNA repair dynamics, using isogenic cells lines expressing endogenous wt JARID1B (as controls) or null for JARID1B (knock out cells available). 2.Define how oncogenic JARID enzyme activity is modulated by post-translational modifications: we will measure the histone demethylase activity of unphosphorylated vs phosphorylated JARID1B enzyme in vitro and in cells. We will also determine the genomic sites JARID1B associates with in control vs. in cells undergoing DNA damage, and define if JARID1B recruitment to DSBs is dependent on phosphorylation. In this supplement, we will expand the above goals to include a pharmacological approach. We will evaluate if small molecule inhibitors have greater or lesser potency on the loss of function and gain of function JARID1B mutants compared to the wt enzyme in vitro and in cells alone, and in combination with DNA damage. Our study will thus have wide impact to lung cancer patients by providing the molecular and mechanistic foundation for hypersensitizing tumors to radiation by using Jumonji inhibitors to curtail DNA repair through blocking the histone signals that trigger this repair.

近75%的所有非小细胞肺癌（NSCLC）患者在其生命的某个阶段接受放射治疗。 治疗方案，无论是治愈性的还是姑息性的。辐射，目前是在一个高度 局部化的方式，导致大量的DNA损伤，导致DSB和癌细胞死亡，但其本身还没有 有疗效的事实上，虽然一些癌症本质上对IR具有抗性，但其他癌症通过上调IR而获得抗性。 DNA修复途径鉴于放射治疗靶向管理的现代进步， 如果肿瘤组织可以对辐射全面敏感， 疗法我们在这里要研究的表观遗传易感性可能是关键。 Jumonji酶的致癌JARID/KDM 5组蛋白去甲基化酶亚家族，其在大肠杆菌中过表达。 多种恶性肿瘤，最近已确定的作用，在DNA损伤途径：他们介导的DNA修复， 擦除携带H3 K4 me 3标记的活性染色质上的三甲基标记，从而停止转录， 促进同源重组和非同源末端连接修复因子的募集。 我们提出的新概念是，JARID酶必须在DNA上进行后修饰 损伤，可能是由辐射激活的ATM或ATR激酶，以增强其组蛋白去甲基化活性， 活性染色质，从而提供了一种停止转录并将修复因子募集到这些位点的机制， 癌细胞存活率如果这是真的，JARID酶的这种新的致癌活性将具有重要意义。 用于开发使肺肿瘤对电离辐射（IR）敏感的新方法，通过选择性抑制 增强的JARID酶对染色质的脱甲基酶活性。我们的具体目标是： 1.确定JARID酶是否是IR DNA损伤反应期间ATM激酶的底物：我们 将通过质谱法确定JARID 1B在DNA扩增过程中是否被ATM/ATR激酶磷酸化 损伤响应我们将绘制磷酸化位点，并将其突变为丙氨酸（功能丧失）或丙氨酸（功能丧失）。 谷氨酸或天冬氨酸（功能获得），以确定对DNA修复动力学的影响，使用同基因 表达内源性wt JARID 1B（作为对照）或不表达JARID 1B（可获得敲除细胞）的细胞系。 2.定义致癌JARID酶活性如何通过翻译后修饰调节：我们将 在体外测量未磷酸化与磷酸化的JARID 1B酶的组蛋白脱甲基酶活性， 在细胞中。我们还将确定JARID 1B与对照细胞和经历过 DNA损伤，并确定JARID 1B募集到DSB是否依赖于磷酸化。 在本补充中，我们将扩大上述目标，包括药理学方法。我们将评估是否 小分子抑制剂对JARID 1B功能丧失和功能获得具有或大或小的效力 突变体相比，野生型酶在体外和细胞中单独，并与DNA损伤的组合。 因此，我们的研究将通过提供分子和机制对肺癌患者产生广泛的影响。 通过使用Jumonji抑制剂减少DNA修复， 阻断了触发修复的组蛋白信号