Differential function and tumor vulnerabilities revealed by RAS membrane trafficking

突变的 RAS 基因比任何其他致癌基因更容易引发癌症。致癌 RAS 蛋白转化 仅当与细胞膜结合时才对细胞起作用。膜结合是由翻译后介导的 修饰，包括法呢基化、aaX 蛋白水解、羧基甲基化和棕榈酰化。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。我们对该领域做出了范式转变的贡献，包括 发现 RAS 在内膜和质膜 (PM) 上传输并发出信号。这些 观察结果确立了 RAS 信号传导领域。治疗癌症的早期尝试 法呢基转移酶抑制剂 (FTI) 在临床上失败并不是因为膜结合对于 RAS 来说是可有可无的 功能而是因为 FTI 不会阻断膜缔合。此后我们寻求更有效的 限制 RAS 膜结合的方法。在最近的工作中，我们重点关注 KRAS 和 NRAS， 同种型在肿瘤中最常发生突变。我们已经建立了 KRAS4B 磷酸化作为一种手段 调节膜关联和功能，表征 KRAS4A 的差异膜运输 和 KRAS4B，KRAS 基因座的两个剪接变体，开发了 KRAS4B 膜的定量分析方法 协会被应用于全基因组 RNAi 和 CRISPR 筛选，并发现了不同的影响 两种剪接变异对肿瘤代谢的影响。也许最引人注目的是我们最近发现己糖激酶 1 (HK1) 是催化糖酵解第一个关键步骤的酶，是 KRAS 的特异性效应子 凭借其独特的亚细胞运输功能，将其转化为 KRAS4A 剪接变体（正在《自然》杂志上发表）。我们还有 发现 NRAS 对异戊二烯半胱氨酸羧甲基转移酶 (ICMT) 的抑制具有独特的敏感性， 我们首先鉴定出的 CaaX 修饰酶。七年来，我们通过 R35 寻求资金 我们建议建立在这些发现基础上的机制。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。具体来说，我们将 a) 将 HK1 描述为 KRAS4A 的效应子并进行更广泛的探索 KRAS 基因座的两个剪接变体驱动的对肿瘤代谢的不同影响，b) 追踪命中 最近的一项创新筛选揭示了以前未被重视的基因，其中包括一些可药物化的基因 蛋白激酶，这是 KRAS4B 有效膜结合所必需的，并且 c) 确定 ICMT 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、协作性。我们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、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了可用于治疗的漏洞。

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.