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.

在所有非小细胞肺癌（NSCLC）患者中，近75％的人在其某个时候接受放射治疗 治疗方案是具有治疗性或姑息性意图的。辐射，目前以高度给出 本地化时尚会造成巨大的DNA损害，导致DSB和癌细胞死亡，但尚未 治愈性。确实，尽管有些癌症对IR具有本质上的抵抗力，而另一些癌症则通过上调获得抵抗 DNA修复途径。鉴于放射疗法的靶向给药现代进步，范式转移 如果肿瘤组织可以在全球范围内对辐射敏感，则会导致固化NSCLC或其他肿瘤 治疗。我们建议在这里进行调查的表观遗传敏感性可能是关键。 Jumonji酶的致癌jarID/KDM5组蛋白脱甲基酶亚科，该酶过表达 多种恶性肿瘤最近在DNA损伤途径中定义了作用：它们通过 在含有H3K4me3标记的活性染色质上擦除三甲基标记，从而停止转录和 促进同源重组和非同源终端连接修复因子的募集。 我们在这里提出的新颖概念是，在DNA上必须在翻译后修改JARID酶 受辐射激活的ATM或ATR激酶的损害，以增强其组蛋白脱甲基活性 活性染色质，因此提供了一种机制，可以停止转录并募集这些位点的修复因子，以便 癌症生存。如果是真的，这种新型的Jarid酶的致癌活性将具有显着的影响 为了开发新方法，通过选择性抑制肺部肿瘤对电离辐射（IR）的敏感性 JARID酶在染色质上增强的去甲基酶活性。我们的具体目的是： 1.确定如果Jarid酶是DNA损伤对IR的损伤反应期间ATM激酶的底物：我们 将通过质谱法确定JARID1b在DNA期间用ATM/ATR激酶磷酸化 伤害响应。我们将绘制磷酸化位点，并将其突变为丙氨酸（功能丧失）或 使用等源性的谷氨酸或天冬氨酸（功能增益）以确定对DNA修复动力学的影响 用于表达内源性WT JARID1B（作为对照）的细胞线或用于JARID1b的null（可敲出可用的单元）。 2.定义如何通过翻译后修饰调节致癌性jarid酶的活性：我们将 在体外测量未磷酸化与磷酸化JARID1B酶的组蛋白脱甲基酶活性 在细胞中。我们还将确定基因组位点JARID1b在对照中与正在进行的细胞中 DNA损伤，并定义JARID1b募集到DSB的依赖于磷酸化。 在此补充中，我们将扩大上述目标，以包括药理方法。我们将评估是否 小分子抑制剂在功能丧失和功能增益jarid1b上具有较高的效力 突变体与仅在体外和细胞中的WT酶相比，并与DNA损伤结合使用。 因此，我们的研究将通过提供分子和机械性来对肺癌患者产生广泛的影响 通过使用Jumonji抑制剂来减少通过Jumonji抑制剂减少DNA修复 阻止触发此修复的组蛋白信号。