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

异戊二烯半胱氨酸羧甲基转移酶 (ICMT) 作为 NRAS 驱动的黑色素瘤的靶标 - 重新提交 - 1

• 批准号: 9891956
• 负责人: MARK Reid PHILIPS
• 金额: $ 42.32万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-16 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891956
• 关键词: 3-Dimensional; Ablation; Address; Affect; Agar; Amino Acids; Animal Model; Antineoplastic Agents; Autophagocytosis; BRAF gene; Biology; C-terminal; CRISPR/Cas technology; Cancer Center; Carcinoma; Catabolism; Cell Fractionation; Cell Line; Cell membrane; Cells; Cellular Membrane; Chemicals; Clinical; Clinical Trials; Codon Nucleotides; Cysteine; Data; Development; Enzyme Inhibitor Drugs; Enzymes; Estrogen receptor positive; Face; GTP Binding; Genes; Genetic; Genetic Transcription; Golgi Apparatus; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Hematopoietic Neoplasms; Human; Human Cloning; Immunotherapy; In Vitro; KRAS2 gene; Lipids; Lymphocyte; Malignant Neoplasms; Malignant neoplasm of pancreas; Maps; Measures; Mediating; Melanoma Cell; Membrane; Methylation; Modality; Modeling; Molecular Chaperones; Monomeric GTP-Binding Proteins; Mus; Mutate; Mutation; Nitrogen; Oncogenes; Oncogenic; Patients; Phosphorylation; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; RAS genes; Raf Kinase Inhibitor; Resistance; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Testing; Therapeutic; Therapeutic Agents; Xenograft procedure; anti-melanoma immunity; base; checkpoint therapy; drug discovery; immunogenicity; in vivo; inhibitor/antagonist; innovation; mammalian genome; melanoma; mouse model; multicatalytic endopeptidase complex; mutant; nanomolar; novel; palmitoylation; pancreatic neoplasm; programs; protein-S-isoprenylcysteine O-methyltransferase; ras Proteins; three dimensional cell culture; tool; trafficking; tumor

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.

项目摘要 哺乳动物基因组中含有三种RAS基因，HRAS，KRAS和NRAS，它们在癌症中突变更多 比任何其他致癌基因都要频繁。RAS蛋白是小GTP酶，其作为二元开关起作用， 调节一系列信号通路。RAS蛋白只有在与 细胞膜的C-末端CAAX序列的翻译后修饰的结果。 法尼基转移酶（FTase）将聚异戊二烯脂质添加到CAAX半胱氨酸。RAS转换酶1（RCE 1） 去除AAX氨基酸。新的C-末端法尼基半胱氨酸然后通过 异戊二烯基半胱氨酸羧基甲基转移酶（ICMT）。三种CAAX加工酶中的每一种都是一种 抗RAS药物发现的潜在靶点。19年前，我们克隆了哺乳动物的ICMT， 酶及其在RAS生物学中的作用和开发的抑制剂。在这批赠款的最后一个周期，我们阐明了 ICMT的遗传消融矛盾地加剧了KRAS驱动的小鼠模型的机制 胰腺肿瘤，并表明这种现象与NOTCH信号有关， ICMT抑制作用抑制了其他肿瘤的生长。我们还发现 在这四种RAS蛋白中，NRAS对ICMT缺陷是唯一敏感的。不仅是NRAS 完全阻断质膜，但NRAS蛋白的细胞水平显著降低。 降低突变的NRAS驱动造血系统恶性肿瘤和黑色素瘤。基于我们令人兴奋的 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，一个重要的未满足的临床需求。在下一轮拨款中，我们将利用 CRISPR/Cas9基因编辑和最近可用的特异性纳摩尔ICMT抑制剂来测试这一点 有三个具体目标的假设。目标1。ICMT在NRAS运输和信号传导中的作用。我们将 研究ICMT缺陷对NRAS翻译后修饰，膜运输， 包括短期培养物在内的黑色素瘤细胞中的相互作用组和信号传导。目标二。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。目标3。ICMT在NRAS驱动的黑色素瘤生长和生存中的作用我们会研究 2D和3D培养中黑色素瘤细胞（包括短期培养物）±ICMT的增殖和存活， 在软琼脂和异种移植物中，我们将我们的条件性ICMTfl/fl小鼠与NRAS驱动的 黑色素瘤，以确定是否ICMT抑制是保护在体内，我们将确定是否ICMT缺陷， 淋巴细胞改变黑色素瘤的免疫原性。成功实现这些目标将决定 NRAS突变型黑色素瘤细胞依赖于ICMT的程度和机制， 提供ICMT抑制剂临床试验所需的初步数据。