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 对化疗耐药。我们发现恶劣的 PDA 微环境会促使癌细胞抵抗 额外的细胞毒性损伤（例如化疗）并促进 PDA 侵袭性。更好地理解 这种适应性程序背后的分子基础将暴露 PDA 的代谢脆弱性。我们 确定 RNA 结合蛋白 HuR (ELAVL1) 是急性促生存反应的主要参与者。之上 应激时，HuR 会从细胞核转移到细胞质，并携带关键的生存转录本，如 IDH1（一种 NADPH） 产生酶）。 HuR 增强目标 mRNA 的 RNA 稳定性和蛋白质翻译，以快速调节 应激反应的转录组。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 HuR 沉默 excessive ROS and NADPH depletion under low glucose or chemotherapy stress.无偏见的 RNA 测序 对 HuR 缺陷细胞中抗氧化基因的分析确定 IDH1 是主要的抗氧化酶 胡尔控制。 RNA 结合和 RNA 稳定性测定表明 HuR 在转录后调节 IDH1， HuR缺陷的PDA细胞的IDH1 mRNA和蛋白表达显着降低。 HuR缺陷细胞 failed to engraft in nude mice, while IDH1 overexpression rescued PDA engraftment. HuR 缺陷细胞也 had dysfunctional mitochondria, reflected by reduced oxygen consumption, ATP, and mitochondrial 丰富。基于这项工作，我们假设 PDA 在低营养条件下依赖 HuR conditions exposes new therapeutic opportunities.在目标 1 中，我们确定了 HuR- 的生存影响 IDH1 axis, by editing out HuR binding sites (CRISPR) in the IDH1 3'UTR.我们生成了一个条件 IDH1 敲除小鼠，并将其与已建立的 PDA 模型进行杂交，以验证 IDH1 作为治疗靶点。我们 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.在目标 2 中，我们确定了以下具体方面 mitochondrial biology under HuR control through studies of mitochondrial structure and function in HuR- PDA 细胞缺陷。 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.一本小说 mitochondrial inhibitor, CPI-613, will be combined with HuR or IDH1 inhibition as a new synthetic lethal 针对 PDA 适应性代谢程序的方法。 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 在这些条件下。 Our studies of HuR biology will improve understanding of PDA metabolic tendencies, and reveal therapeutic opportunities relevant to PDAs nutrient deprived microenvironment.

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.