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Targeting pancreatic cancer's metabolic addiction to HuR

针对胰腺癌对 HuR 的代谢成瘾

基本信息

批准号：
10611147
负责人：
Jordan M Winter
金额：
$ 43.17万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10611147
关键词：
3&apos Untranslated Regions Acute Antioxidants Binding Binding Proteins Binding Sites Biological Assay Biology CRISPR/Cas technology Cancer Etiology Cell Culture Techniques Cell Line Cell Nucleus Cell Survival Cells Cellular biology Cessation of life Chemistry Chemoresistance Clinical Data Clustered Regularly Interspaced Short Palindromic Repeats Computational Biology Culture Media Cytoplasm Dependence Diabetic mouse Elements Engraftment Enzymes Exhibits Fractionation Genes Genetic Glucose Glutamine Homeostasis Hour Hyperglycemia Hyperglycemic Mice Impairment Isocitrates Knock-out Knockout Mice Lead Maintenance Malignant Neoplasms Malignant neoplasm of pancreas Mediator Medical Messenger RNA Metabolic Metabolic stress Metabolism Mitochondria Modeling Molecular Molecular Biology Techniques Mus Mutation NADP Normal tissue morphology Nude Mice Nutrient Oxidation-Reduction Oxygen Consumption Pancreatic Ductal Adenocarcinoma Pathology Performance Pharmaceutical Chemistry Pharmaceutical Preparations Predisposition Property Proteins Publishing RNA Binding RNA Interference RNA Stability RNA-Binding Proteins Reactive Oxygen Species Refractory Regulation Research Resistance Role Streptozocin Stress Structure Testing Therapeutic Transcript Transfection Translations United States Withdrawal Work Xenograft procedure addiction alpha ketoglutarate alternative treatment antioxidant enzyme arm cancer cell chemotherapy cytotoxic drug testing gemcitabine improved in vivo inhibitor insight lead optimization mRNA Expression mortality mouse model mutant new therapeutic target novel novel therapeutics overexpression pancreatic cancer cells pancreatic ductal adenocarcinoma cell pancreatic ductal adenocarcinoma model posttranscriptional programs protein expression response statistics therapeutic target transcriptome transcriptome sequencing tumor tumor microenvironment tumorigenesis

项目摘要

Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer death in the U.S. and is generally refractory to chemotherapy. We discovered that the harsh PDA microenvironment primes cancer cells against additional cytotoxic insults (e.g., chemotherapy) and promotes PDA aggressiveness. A better understanding of the molecular underpinnings behind this adaptive program would expose PDAs metabolic vulnerabilities. We identified the RNA binding protein, HuR (ELAVL1), as a major player in the acute pro-survival response. Upon stress, HuR translocates from the nucleus to the cytoplasm with key survival transcripts, like IDH1 (an NADPH generating enzyme). HuR enhances RNA stability and protein translation of target mRNAs, to rapidly adjust the transcriptome in response to stress. Our research highlights two metabolic processes in the HuR adaptive program: a) antioxidant defense and b) mitochondrial performance. HuR silencing in PDA cells produced excessive ROS and NADPH depletion under low glucose or chemotherapy stress. An unbiased RNA seq analysis of antioxidant genes in HuR deficient cells identified IDH1 as the leading antioxidant enzyme under HuR control. RNA binding and RNA stability assays showed that HuR regulates IDH1 post-transcriptionally, and HuR deficient PDA cells had markedly reduced IDH1 mRNA and protein expression. HuR-deficient cells failed to engraft in nude mice, while IDH1 overexpression rescued PDA engraftment. HuR-deficient cells also had dysfunctional mitochondria, reflected by reduced oxygen consumption, ATP, and mitochondrial abundance. Based on this body of work, we hypothesize that PDAs reliance on HuR under low nutrient conditions exposes new therapeutic opportunities. In Aim 1, we establish the survival impact of the HuR- IDH1 axis, by editing out HuR binding sites (CRISPR) in the IDH1 3’UTR. We generated a conditional IDH1 knockout mouse, and will cross it with an established PDA model to validate IDH1 as a therapeutic target. We will test an allosteric modulator of mutant IDH1 (GSK-321) as a novel wild type IDH1 inhibitor in PDA, and launch hit-to-lead optimization to improve drug properties. In Aim 2, we identify specific aspects of mitochondrial biology under HuR control through studies of mitochondrial structure and function in HuR- deficient PDA cells. The importance of the HuR-IDH1 axis on mitochondrial ROS levels will be demonstrated. Additional transcripts will impact HuR’s regulation of mitochondrial performance will be identified. A novel mitochondrial inhibitor, CPI-613, will be combined with HuR or IDH1 inhibition as a new synthetic lethal approach against PDAs adaptive metabolic program. In Aim 3, we will use a diabetic mouse model to show that a hyperglycemic state suppresses the HuR pro-survival network, and sensitizes PDA to chemotherapy. Successful engraftment of HuR-deficient cells in hyperglycemic mice would suggest that HuR is less important under these conditions. Our studies of HuR biology will improve understanding of PDA metabolic tendencies, and reveal therapeutic opportunities relevant to PDAs nutrient deprived microenvironment.

胰腺导管腺癌（PDA）是美国癌症死亡的第三大原因，并且通常是化疗无效我们发现，恶劣的PDA微环境使癌细胞对另外的细胞毒性损伤（例如，化疗）并促进PDA侵袭性。更好地了解这种适应性程序背后的分子基础将暴露PDA代谢的脆弱性。我们确定了RNA结合蛋白HuR（ELAVL 1）作为急性促生存反应的主要参与者。后应激时，HuR从细胞核易位到细胞质中，具有关键的存活转录物，如IDH 1（NADPH 产生酶）。HuR增强靶mRNA的RNA稳定性和蛋白质翻译，以快速调节靶mRNA的表达。转录组对压力的反应。我们的研究强调了HuR适应性中的两个代谢过程程序：a）抗氧化防御和B）线粒体性能。产生的PDA细胞中的HuR沉默低葡萄糖或化疗应激下活性氧和NADPH过度消耗。无偏RNA测序 HuR缺陷细胞中抗氧化基因的分析确定IDH 1是在细胞内的主要抗氧化酶。 HuR控制。RNA结合和RNA稳定性测定显示HuR在转录后调节IDH 1， HuR缺陷型PDA细胞IDH 1 mRNA和蛋白表达明显降低。HuR缺陷细胞未能在裸鼠中植入，而IDH 1过表达挽救了PDA植入。HuR缺陷细胞还线粒体功能障碍，表现为耗氧量、ATP和线粒体丰饶。基于这些工作，我们假设PDA在低营养下依赖于HuR 新的治疗方法带来了新的机会。在目标1中，我们建立了HuR的生存影响- 通过编辑IDH 1 3 'UTR中的HuR结合位点（CRISPR），可以在IDH 1轴上进行修饰。我们生成了一个条件IDH 1 敲除小鼠，并将其与已建立的PDA模型杂交以验证IDH 1作为治疗靶标。我们将测试突变IDH 1的变构调节剂（GSK-321）作为PDA中的新型野生型IDH 1抑制剂，并且启动点击到铅优化，以改善药物性能。在目标2中，我们确定了线粒体生物学在HuR的控制下，通过研究HuR中的线粒体结构和功能，缺陷型PDA细胞。将证明HuR-IDH 1轴对线粒体ROS水平的重要性。将鉴定将影响HuR对线粒体性能的调节的其他转录物。一种新型线粒体抑制剂CPI-613将与HuR或IDH 1抑制剂组合，作为新的合成致死性药物。针对PDA的适应性代谢程序的方法。在目标3中，我们将使用糖尿病小鼠模型来显示高血糖状态抑制了HuR促生存网络，并使PDA对化疗敏感。 HuR缺陷细胞在高血糖小鼠中的成功移植表明HuR不太重要在这种情况下。我们对HuR生物学的研究将提高对PDA代谢趋势的理解，并揭示了与PDA营养缺乏微环境相关的治疗机会。