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% 的非小细胞肺癌 (NSCLC) 患者在其生命中的某个时刻接受过放射治疗 治疗方案，无论是治愈性的还是姑息性的。辐射，目前给予高度 局部时尚，会造成大量 DNA 损伤，导致 DSB 和癌细胞死亡，但其本身尚不存在 有疗效。事实上，虽然有些癌症本质上对 IR 有抵抗力，但其他癌症则通过上调而获得抵抗力。 DNA 修复途径。鉴于放疗靶向管理的现代进步，范式转变 如果肿瘤组织整体对辐射敏感，那么就可能治愈非小细胞肺癌或其他肿瘤 治疗。我们建议在这里研究的表观遗传敏感性可能是关键。 Jumonji 酶的致癌 JARID/KDM5 组蛋白去甲基化酶亚家族，在 最近确定了多种恶性肿瘤在 DNA 损伤途径中的作用：它们通过以下方式介导 DNA 修复： 擦除含有 H3K4me3 标记的活性染色质上的三甲基标记，从而停止转录并 促进同源重组和非同源末端连接修复因子的招募。 我们在这里提出的新概念是 JARID 酶必须在 DNA 上进行翻译后修饰 损伤，可能是由辐射激活的 ATM 或 ATR 激酶引起的，以增强其组蛋白去甲基化活性 活性染色质，从而提供一种机制来停止转录并将修复因子招募到这些位点， 癌细胞的存活。如果属实，JARID 酶的这种新型致癌活性将产生重大影响 开发新方法，通过选择性抑制肺部肿瘤对电离辐射（IR）的敏感性 增强 JARID 酶对染色质的去甲基化酶活性。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 JARID1B 在 DNA 过程中是否被 ATM/ATR 激酶磷酸化 损害反应。我们将绘制磷酸化位点并将其突变为丙氨酸（功能丧失）或 谷氨酸或天冬氨酸（功能获得），使用等基因确定对 DNA 修复动态的影响 表达内源性 wt JARID1B（作为对照）的细胞系或表达 JARID1B 的空细胞系（可用的敲除细胞）。 2.定义致癌 JARID 酶活性如何通过翻译后修饰调节：我们将 体外测量非磷酸化与磷酸化 JARID1B 酶的组蛋白去甲基化酶活性， 在细胞中。我们还将确定 JARID1B 与对照细胞和经历细胞的基因组位点相关的基因组位点。 DNA 损伤，并确定 JARID1B 招募到 DSB 是否依赖于磷酸化。 在本补充材料中，我们将扩展上述目标以包括药理学方法。我们将评估是否 小分子抑制剂对功能丧失和功能获得具有或多或少的效力 JARID1B 突变体与野生型酶在体外和单独在细胞中以及与DNA损伤相结合的比较。 因此，我们的研究将通过提供分子和机制来对肺癌患者产生广泛的影响。 通过使用 Jumonji 抑制剂来减少 DNA 修复，从而为肿瘤对辐射过敏奠定了基础 阻断触发这种修复的组蛋白信号。