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 治疗方案，无论是出于治疗还是姑息意图。辐射，这是目前在一个高度 本地化时尚，导致大量DNA损伤，导致DSB和癌细胞死亡，但其本身还不是 治愈了。事实上，虽然一些癌症对IR具有内在抵抗力，但另一些癌症则通过上调表达而获得抵抗力 DNA修复途径。鉴于靶向放射治疗方面的现代进展，范式的转变 如果肿瘤组织可以对辐射全局敏感，就可能导致治愈非小细胞肺癌或其他肿瘤 心理治疗。我们建议在这里研究的表观遗传易感性，可能是关键。 Jumonji酶的致癌JARID/KDM5组蛋白去甲基酶亚家族，在 多种恶性肿瘤，最近确定了在DNA损伤途径中的作用：它们通过以下方式介导DNA修复 清除含有H3K4me3标记的活性染色质上的三甲基标记，从而停止转录和 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贾里德酶必须在DNA上进行翻译后修饰 损伤，可能是通过辐射激活的ATM或ATR激酶来增强它们的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招募到这些部位的机制， 癌细胞存活。如果这是真的，茉莉酶的这种新的致癌活性将具有重大意义。 为了开发新的方法来使肺肿瘤对电离辐射(IR)敏感，通过选择性地抑制 提高茉莉花酶对染色质的脱甲基酶活性。我们的具体目标是： 1.在对IR的DNA损伤反应中，确定茉莉酸酶是否为ATM激酶的底物：WE 将通过质谱学确定JARID1B是否被DNA过程中的ATM/ATR激酶磷酸化 损坏反应。我们将绘制磷酸化的位置图，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)确定对DNA修复动力学的影响，使用等基因 表达内源性wt JARID1B的细胞系(作为对照)或表达JARID1B的空的细胞系(可获得敲除细胞)。 2.定义致癌的JARID酶活性如何受到翻译后修饰的调节：我们将 体外测定未磷酸化和磷酸化JARID1B酶的组蛋白去甲基酶活性 在牢房里。我们还将确定与JARID1B相关的基因组位置在对照组和正在经历 DNA损伤，并确定JARID1B募集到DSB是否依赖于磷酸化。 在本附录中，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JARID1B的功能丧失和功能获得具有或多或少的效力 与wt酶相比，突变体在体外和细胞内单独存在，并结合DNA损伤。 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 Jumonji抑制剂通过抑制DNA修复增强肿瘤辐射敏感性的基础 阻断触发这种修复的组蛋白信号。