Differential function and tumor vulnerabilities revealed by RAS membrane trafficking

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 细胞只有在与细胞膜结合时才能生长。膜结合是由翻译后 这些修饰包括法尼基化、aaX蛋白水解、羧基甲基化和棕榈酰化。有关详细 二十多年来，我的实验室一直专注于翻译后修饰和膜靶向， RAS和相关的小GTP酶。我们对该领域做出了范式转变的贡献，包括 发现RAS在内膜以及质膜（PM）上运输并从内膜和质膜发出信号。这些 这些观察建立了RAS的区室化信号传导领域。早期的癌症治疗尝试 法尼基转移酶抑制剂（FTIs）在临床上失败，不是因为膜结合对RAS不利 功能，而是因为FTIs不阻断膜缔合。从那时起，我们一直在寻求更有效的方法， 限制RAS膜结合的手段。在最近的工作中，我们集中在KRAS和NRAS上， 同种型在肿瘤中最常突变。我们已经建立了KRAS 4B的磷酸化作为一种手段， 调节膜缔合和功能，表征KRAS 4A的差异膜运输 和KRAS 4B，KRAS基因座的两个剪接变体，开发了KRAS 4B膜的定量测定法， 应用于全基因组RNAi和CRISPR筛选，并发现了 两种剪接变异体对肿瘤代谢的影响也许最值得注意的是我们最近发现己糖激酶 1（HK1），催化糖酵解中第一个关键步骤的酶，是KRAS的效应子， KRAS4A剪接变体凭借其独特的亚细胞运输（在自然界出版）。我们还 发现NRAS对异戊二烯基半胱氨酸羧基甲基转移酶（ICMT）的抑制是唯一敏感的， 我们首次发现的CaaX修饰酶在我们通过R35寻求的七年资金中， 我们建议建立在这些发现的基础上的机制。要解决的首要科学问题是 RAS蛋白的差异修饰和膜运输是否可以揭示新的治疗方法， 漏洞具体而言，我们将a）将HK1表征为KRAS 4A的效应子，并更广泛地探索 由KRAS基因座的两种剪接变体驱动的对肿瘤代谢的不同影响，B）追求命中 从最近的一项创新性筛选中发现了以前不受重视的基因， 蛋白激酶，其是KRAS 4B的有效膜结合所需的，和c）确定ICMT是否 抑制是治疗NRAS驱动黑色素瘤的可行方法。我们的方法将是创新的， 多学科，协作。我们已经招募了激酶生物化学方面的专家作为合作者， 超分辨率显微镜，结构生物学，基因组调控，代谢组学，癌症基因组学，单细胞 转录组学和啮齿动物基因工程和成像。我们期望拟议的工作将导致 新的见解基本RAS生物学和揭示漏洞，可以利用治疗。

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.