Targeting pancreatic cancer's metabolic addiction to HuR

胰腺导管腺癌(PDA)是美国癌症死亡的第三大原因，通常是 对化疗无效。我们发现，恶劣的PDA微环境启动了癌细胞对 额外的细胞毒性侮辱(例如，化疗)并促进PDA的侵袭性。更好地理解 这一适应性计划背后的分子基础将暴露PDA代谢的脆弱性。我们 发现RNA结合蛋白Hur(ELAVL1)是急性支持生存反应的主要参与者。vt.在.的基础上 在压力下，HUR通过关键的生存转录本从细胞核转移到细胞质，如IDH1(一种NADPH 产生酶)。HUR增强了RNA的稳定性和靶mRNAs的蛋白质翻译，以快速调整 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 计划：a)抗氧化防御和b)线粒体性能。PDA细胞中产生的HUR沉默 在低糖或化疗压力下，ROS和NADPH过度耗竭。一种无偏的RNA序列 对HUR缺陷细胞的抗氧化基因进行分析，确定IDH1是HUR缺陷细胞中的主要抗氧化酶 HUR控制中心。RNA结合和RNA稳定性分析表明，Hur在转录后调节IDH1， HUR基因缺失的PDA细胞IDH1mRNA和蛋白表达显著降低。HUR缺陷细胞 裸鼠移植失败，IDH1过表达挽救了PDA植入。HUR缺陷细胞也 线粒体功能障碍，表现为氧耗、三磷酸腺苷和线粒体减少 富足。基于这项工作，我们假设PDA在低营养条件下依赖于HUR 疾病暴露出新的治疗机会。在目标1中，我们确定了越族人的生存影响- IDH1轴，编辑掉IDH1 3‘非编码区中的HUR结合位点(CRISPR)。我们生成了一个条件IDH1 基因敲除小鼠，并将其与已建立的PDA模型交叉，以验证IDH1作为治疗靶点。我们 将在PDA中测试突变IDH1的变构调节剂(GSK-321)作为一种新的野生型IDH1抑制剂，以及 开展Hit-to-Lead优化，提高药物性能。在目标2中，我们确定了 HUR控制下的线粒体生物学--线粒体结构和功能的研究 缺乏PDA细胞。Hur-IDH1轴在线粒体ROS水平上的重要性将被证明。 将确定额外的转录本将影响HUR对线粒体性能的调节。一本小说 线粒体抑制剂CPI-613将与HUR或IDH1抑制剂联合作为一种新的合成致死物质 针对PDA适应性代谢计划的方法。在目标3中，我们将使用糖尿病小鼠模型来展示 高血糖状态抑制了HUR支持生存的网络，并使PDA对化疗敏感。 HUR缺陷细胞在高血糖小鼠中的成功植入表明HUR不那么重要 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， 揭示了与PDA营养剥夺微环境相关的治疗机会。

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.