Isoprenylcysteine Carboxyl Methyltransferase (ICMT) as a Target in NRAS Driven Melanoma - Resubmission - 1

项目总结 哺乳动物基因组包含三个RAS基因，HRAS、KRAS和NRAS，它们在癌症中突变更多 比其他任何致癌基因都要频繁。RAS蛋白是一种小的GTP酶，其功能是作为二进制开关 调控一系列广泛的信号通路。RAS蛋白仅在与以下物质结合时才传播信号 细胞膜是C-末端CAAX序列翻译后修饰的结果。 法尼基转移酶(FTase)将聚异戊二烯类脂添加到CAAX半胱氨酸中。RAS转换酶1(Rce1) 去除AAX氨基酸。然后，将新的C-末端的法尼基半胱氨酸甲基化 异丙基半胱氨酸羧甲基转移酶(ICMT)。三种CAAX加工酶中的每一种都是 抗RAS药物发现的潜在靶点。19年前，我们克隆了哺乳动物ICMT，鉴定了 酶及其在RAS生物学中的作用和开发的抑制剂。在这笔赠款的最后一个周期中，我们阐明了 ICMT基因消融矛盾地加重KRAS驱动的小鼠模型的机制 胰腺肿瘤，并表明这一现象与缺口信号有关，并高度 ICMT抑制抑制了其他肿瘤的生长，这一点具有上下文依赖性。我们还发现， 在四种RAS蛋白中，NRAS是唯一对ICMT缺乏敏感的蛋白。不仅是NRAS 完全被阻止在质膜上，但NRAS蛋白的细胞水平显著 减少了。突变的NRAS会导致血液系统恶性肿瘤和黑色素瘤。基于我们激动人心的 初步数据我们假设ICMT抑制可能是NRAS的一种有吸引力的治疗方式 突变型黑色素瘤，一个重要的未得到满足的临床需求。在这笔赠款的下一个周期中，我们将利用 CRISPR/Cas9基因编辑以及最近可获得的一种特定的纳米分子ICMT抑制剂来测试这一点 有三个具体目标的假设。目的1.ICMT在NRAS运输和信号传递中的作用。我们会 研究ICMT缺乏对NRAS翻译后修饰、膜转运、 包括短期培养的黑色素瘤细胞中的相互作用组和信号。目标2.ICMT在NRAS中的作用 表情。我们将确定ICMT缺陷导致表达减少的机制 NRAS。目的3.ICMT在NRAS诱导的黑色素瘤生长和存活中的作用。我们会研究 黑色素瘤细胞(包括短期培养)在2D和3D培养中的增殖和存活 软琼脂和异种移植中，我们将有条件的ICMTfl/fl小鼠与NRAS驱动的模型杂交 以确定ICMT抑制在体内是否具有保护性，我们将确定ICMT缺陷是否在 淋巴细胞改变黑色素瘤的免疫原性。成功起诉这些目标将定义 NRAS突变黑色素瘤细胞依赖ICMT的程度和机制 提供ICMT抑制剂临床试验所需的初步数据。

PROJECT SUMMARY Mammalian genomes harbor three RAS genes, HRAS, KRAS and NRAS, which are mutated in cancer more frequently than any other oncogene. RAS proteins are small GTPases that function as binary switches to regulate a wide array of signaling pathways. RAS proteins propagate signals only when associated with cellular membranes as a consequence of post-translational modification of a C-terminal CAAX sequence. Farnesyltransferase (FTase) adds a polyisoprene lipid to the CAAX cysteine. RAS converting enzyme 1 (RCE1) removes the AAX amino acids. The newly C-terminal farnesylcysteine is then methylesterified by isoprenylcysteine carboxyl methyltransferase (ICMT). Each of the three CAAX processing enzymes is a potential target for anti-RAS drug discovery. We cloned mammalian ICMT 19 years ago, characterized the enzyme and its role in RAS biology and developed inhibitors. In the last cycle of this grant we elucidated the mechanism whereby genetic ablation of ICMT paradoxically exacerbated a mouse model of KRAS-driven pancreatic neoplasia and showed that this phenomenon was related to NOTCH signaling and was highly context-dependent in that ICMT inhibition inhibited the growth of other tumors. We also discovered that among the four RAS proteins, NRAS is uniquely sensitive to ICMT deficiency. Not only was NRAS completely blocked from the plasma membrane, but the cellular levels of the NRAS proteins were significantly decreased. Mutant NRAS drives hematopoietic malignancies and melanoma. Based on our exciting preliminary data we hypothesize that ICMT inhibition may be an attractive therapeutic modality for NRAS mutant melanoma, an important unmet clinical need. In the next cycle of this grant we will take advantage of CRISPR/Cas9 gene editing and the recent availability of a specific, nanomolar ICMT inhibitor to test this hypothesis with three specific aims. Aim 1. Role of ICMT in NRAS trafficking and signaling. We will study the effect of ICMT deficiency on NRAS post-translational modification, membrane trafficking, interactome and signaling in melanoma cells including short-term cultures. Aim 2. Role of ICMT in NRAS expression. We will determine the mechanism whereby ICMT deficiency leads to decreased expression of NRAS. Aim 3. Role of ICMT in growth and survival of NRAS-driven melanoma. We will study the proliferation and survival of melanoma cells (including short-term cultures) ±ICMT in 2D and 3D culture, in soft agar and in xenografts, we will cross our conditional ICMTfl/fl mouse with a model of NRAS-driven melanoma to determine if ICMT inhibition is protective in vivo, and we will determine if ICMT deficiency in lymphocytes alters immunogenicity of melanomas. Successful prosecution of these aims will define the degree and mechanisms whereby NRAS-mutant melanoma cells are dependent on ICMT and thereby provide the preliminary data required for clinical trials of ICMT inhibitors.