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容易导致癌症。致癌RAS蛋白转化 只有在与细胞膜相关的情况下才有细胞。翻译后介导的膜联结 修饰，包括法尼化、AAX蛋白降解、羧甲基化和棕榈酰化。了解更多 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。我们为该领域做出了改变范式的贡献，包括 发现RAS在膜内和质膜(PM)上运输和发出信号。这些 观察结果确定了RAS信号的区隔领域。治疗癌症的早期尝试 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来说是必不可少的 这不仅是因为FTIs没有阻止膜结合，而是因为FTIs没有阻止膜结合。自那以后，我们一直在寻求更有效的 限制RAS膜结合的方法。在最近的工作中，我们重点研究了KRAS和NRAS， 异构体在肿瘤中最常发生突变。我们已经建立了KRAS4B的磷酸化作为一种手段 调节膜结合和功能，表征KRAS4A的差异性膜转运 和KRAS4B，KRAS基因座的两个剪接变体，建立了KRAS4B膜的定量检测方法 关联应用于全基因组RNAi和CRISPR筛查，并发现 这两个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)是催化糖酵解第一步的酶，是KRAS的特异效应物 KRAS4A剪接变异体凭借其独特的亚细胞贩运(在自然界中)。我们还有 发现NRAS对异丙基半胱氨酸羧甲基转移酶(ICMT)的抑制唯一敏感， 我们首次鉴定的CAAX修饰酶。在我们通过R35寻求的七年资金中 我们建议在这些发现的基础上建立一种机制。需要解决的首要科学问题是 RAS蛋白的差异化修饰和膜转运能否揭示新的治疗方法 漏洞。具体地说，我们将a)将HK1描述为KRAS4A的效应者，并进行更广泛的探索 KRAS基因两个剪接变异体对肿瘤代谢的不同影响，b)追求HITS 来自最近的一次创新筛查，揭示了以前未被欣赏的基因，包括几种可用药 KRAS4B的有效膜结合所需的蛋白激酶，以及c)决定ICMT 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、协作性。我们已经招募了专家作为激酶生物化学的合作者， 超分辨显微镜、结构生物学、基因组调控、代谢组学、癌症基因组学、单细胞 转录学、啮齿动物基因工程和成像。我们期望拟议的工作将导致 对基本RAS生物学的新见解，并揭示可用于治疗的漏洞。

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.