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主要原因，通常是对化学疗法的难治性。我们发现Harmsh PDA微环境素癌细胞反对其他细胞毒性感染（例如化学疗法）并促进PDA侵袭性。更好地理解该自适应程序背后的分子基础将暴露PDA代谢脆弱性。我们将RNA结合蛋白HUR（Elavl1）鉴定为急性促生物活性反应的主要参与者。之上压力，HUR从细胞核易位到细胞质，并具有关键的存活记录，例如IDH1（NADPH）生成酶）。 HUR增强了靶mRNA的RNA稳定性和蛋白质翻译，以快速调整响应压力的转录组。我们的研究强调了HUR自适应中的两个代谢过程程序：a）抗氧化剂防御和b）线粒体性能。在PDA细胞中产生的HUR沉默在低葡萄糖或化学疗法胁迫下的ROS和NADPH耗竭过多。无偏的RNA SEQ 在刺激性缺陷细胞中抗氧化基因的分析，将IDH1鉴定为领先的抗氧化剂酶。 HUR控制。 RNA结合和RNA稳定性测定表明HUR在转录后调节IDH1，缺乏HUR的PDA细胞显着降低了IDH1 mRNA和蛋白质表达。缺陷细胞未能在裸鼠中植入，而IDH1过表达挽救了PDA植入。缺乏hur的细胞线粒体功能障碍，通过减少氧气，ATP和线粒体反映抽象。基于这项工作，我们假设PDA在低营养中对HUR的依赖条件暴露了新的治疗机会。在AIM 1中，我们建立了HUR的生存影响 IDH1轴，通过编辑IDH1 3'Utr中的HUR绑定站点（CRISPR）。我们生成了有条件的IDH1 敲除小鼠，并将使用已建立的PDA模型跨越它，以验证IDH1作为治疗靶标。我们将测试突变体IDH1（GSK-321）的变构调节剂，作为PDA中的一种新型野生型IDH1抑制剂，并且启动命中率优化以改善药物特性。在AIM 2中，我们确定了线粒体生物学通过HUR控制的线粒体结构和功能 PDA细胞不足。将证明HUR-IDH1轴对线粒体ROS水平的重要性。其他成绩单将影响HUR对线粒体性能的监管。小说线粒体抑制剂CPI-613将与HUR或IDH1抑制作用，作为一种新的合成致死反对PDA自适应代谢计划的方法。在AIM 3中，我们将使用糖尿病鼠标模型显示高血糖状态抑制了HUR亲生网络，并将PDA感知到化学疗法。成功植入高血糖小鼠中的HUR缺陷细胞会表明HUR不太重要在这些条件下。我们对HUR生物学的研究将改善对PDA代谢趋势的理解，并揭示了与PDAS营养剥夺的微环境有关的治疗机会。