Differential function and tumor vulnerabilities revealed by RAS membrane trafficking

RAS 膜运输揭示的差异功能和肿瘤脆弱性

• 批准号: 10468873
• 负责人: MARK Reid PHILIPS
• 金额: $ 99.67万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468873
• 关键词: Address; Biochemistry; Biological Assay; Biology; CRISPR screen; Cell membrane; Cells; Cellular Membrane; Clinic; Development; Enzymes; Farnesyl Transferase Inhibitor; Funding; Genes; Genetic Engineering; Genome; Glycolysis; Human; Image; KRAS2 gene; Laboratories; Malignant Neoplasms; Mediating; Membrane; Methylation; Microscopy; Modification; Monomeric GTP-Binding Proteins; Mutate; Nature; Oncogenes; Oncogenic; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Isoforms; Protein Kinase; Proteins; Proteolysis; RAS genes; RNA Splicing; RNA interference screen; Regulation; Resolution; Rodent; Signal Transduction; Therapeutic; Variant; Work; cancer genomics; cancer therapy; cell transformation; farnesylation; genome-wide; hexokinase; innovation; insight; melanoma; metabolomics; multidisciplinary; mutant; novel; novel therapeutics; palmitoylation; ras Proteins; recruit; structural biology; trafficking; transcriptomics; tumor; tumor metabolism

Mutant RAS genes drive cancer more frequently than any other oncogene. Oncogenic RAS proteins transform cells only when associated with cellular membranes. Membrane association is mediated by post-translational modifications, including farnesylation, aaX proteolysis, carboxyl methylation, and palmitoylation. For more than two decades my laboratory has focused on the post-translational modification and membrane targeting of RAS and related small GTPases. We have made paradigm-shifting contributions to the field including the discovery that RAS traffics upon and signals from endomembranes as well as the plasma membrane (PM). These observations established the field of compartmentalized signaling of RAS. Early attempts to treat cancer with farnesyltransferase inhibitors (FTIs) failed in the clinic not because membrane association is dispensable for RAS function but rather because FTIs did not block membrane association. We have since sought more effective means of limiting membrane association of RAS. In recent work we have focused on KRAS and NRAS, the isoforms most often mutant in tumors. We have established phosphorylation of KRAS4B as a means of modulating membrane association and function, characterized the differential membrane trafficking of KRAS4A and KRAS4B, the two splice variants of the KRAS locus, developed quantitative assays for KRAS4B membrane association that were applied to genome-wide RNAi and CRISPR screens, and discovered differential effects of the two splice variants on tumor metabolism. Perhaps most remarkable is our recent discovery that hexokinase 1 (HK1), the enzyme that catalyzes the first committed step in glycolysis, is an effector of KRAS that is specific to the KRAS4A splice variant by virtue of its unique subcellular trafficking (in press in Nature). We have also discovered that NRAS is uniquely sensitive to inhibition of isoprenylcysteine carboxylmethytransferase (ICMT), the CaaX modifying enzyme we first identified. Over the seven years of funding that we seek through the R35 mechanism we propose to build on these discoveries. The overarching scientific question to be addressed is whether the differential modification and membrane trafficking of RAS proteins can reveal new therapeutic vulnerabilities. Specifically, we will a) characterize HK1 as an effector of KRAS4A and explore more broadly the differential effects on tumor metabolism driven by the two splice variants of the KRAS locus, b) pursue hits from a recent, innovative screen that revealed previously unappreciated genes, including several druggable protein kinases, that are required for efficient membrane association of KRAS4B, and c) determine if ICMT inhibition is viable approach to treating NRAS-driven melanoma. Our approach will be innovative, multidisciplinary, and collaborative. We have recruited experts to serve as collaborators in kinase biochemistry, super-resolution microscopy, structural biology, genome regulation, metabolomics, cancer genomics, single-cell transcriptomics, and rodent genetic engineering and imaging. We expect that the work proposed will lead to new insights into basic RAS biology and reveal vulnerabilities that can be exploited therapeutically.

突变的RAS基因比任何其他癌基因更频繁地驱动癌症。致癌Ras蛋白转化 仅当与细胞膜相关时。膜协会是由翻译后介导的 修饰，包括法尼化，AAX蛋白水解，羧基甲基化和棕榈酰化。更多 超过二十年，我的实验室专注于翻译后修饰和膜的靶向 RAS和相关的小GTPase。我们已经为该领域做出了范式转移的贡献 RAS运输的发现以及内膜和质膜的信号（PM）。这些 观察结果确定了RAS的分室信号传导领域。早期尝试治疗癌症 诊所中的Farnesylsylsylansferase抑制剂（FTI）失败，不是因为膜关联对于RAS是可分配的 功能，而是因为FTI没有阻止膜关联。从那以后，我们寻求更有效 限制RAS膜关联的手段。在最近的工作中，我们专注于Kras和Nras 同工型最常见于肿瘤中的突变体。我们已经建立了KRAS4B的磷酸化作为 调节膜关联和功能，表征了KRAS4A的差异膜运输 KRAS4B是KRAS基因座的两个剪接变体，开发了KRAS4B膜的定量测定 应用于全基因组RNAi和CRISPR屏幕的关联，发现 关于肿瘤代谢的两个剪接变体。也许最引人注目的是我们最近发现的己糖酶 1（hk1），催化糖酵解的第一步的酶是克拉斯的效应子，是特定的 凭借其独特的亚细胞贩运（在自然界中）。我们也有 发现NRA对抑制异on-半胱氨酸羧基递转企业（ICMT），唯一敏感 我们首先确定的CAAX修饰酶。在我们通过R35寻求的七年资金中 我们建议以这些发现为基础的机制。要解决的总体科学问题是 RAS蛋白的差异修饰和膜运输是否可以揭示新的治疗性 漏洞。具体来说，我们将a）将HK1描述为Kras4a的效应子，并更广泛地探索 KRAS基因座的两个剪接变体驱动的肿瘤代谢的差异作用，b）追求命中 从最近的创新屏幕中揭示了先前未欣赏的基因，其中包括几种可吸毒的基因 蛋白激酶是KRAS4B有效膜缔合所需的蛋白激酶，c）确定ICMT是否是否 抑制是治疗NRAS驱动的黑色素瘤的可行方法。我们的方法将是创新的， 多学科和协作。我们已招募专家担任激酶生物化学的合作者， 超分辨率显微镜，结构生物学，基因组调节，代谢组学，癌症基因组学，单细胞 转录组学以及啮齿动物的基因工程和成像。我们希望提出的工作将导致 对基本RAS生物学的新见解，并揭示了可以通过治疗剥削的漏洞。