Interrogating the role of GTP metabolism in Rac1-driven phenotypes in melanoma

探究 GTP 代谢在 Rac1 驱动的黑色素瘤表型中的作用

• 批准号: 10707950
• 负责人: David W Wolff
• 金额: $ 11.26万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10707950
• 关键词: Actins; Adopted; Affect; Area; Binding; Biosensor; Cells; Clinical; Collaborations; Communication; Data; Dependence; Disease; Dissociation; Dose; Drug resistance; Enzymes; Feedback; Fluorescence Resonance Energy Transfer; GTP Binding; Gene Expression; Genetic; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; High Pressure Liquid Chromatography; Human; Hydrolysis; Immune response; Immunosuppression; Inosine Monophosphate; Invaded; Laboratories; Malignant Neoplasms; Measures; Mediating; Melanoma Cell; Mentors; Metabolism; Metastatic Melanoma; Methods; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Modeling; Movement; Mus; Mutation; Nature; Neoplasm Metastasis; Normal Cell; Nucleotides; Oncogenic; Oncoproteins; Oxidoreductase; Patients; Phenotype; Production; Prognosis; Proteinase 3; Proteins; Publishing; Regulation; Reporting; Research; Resistance; Ribavirin; Role; Series; Serum Response Factor; Structure; Sun Exposure; Technology; Testing; Therapeutic; Time; Transcriptional Regulation; Tumor Promotion; Visualization; Work; Xenograft procedure; cancer cell; clinically relevant; clinically significant; cofactor; efficacy evaluation; enzyme biosynthesis; factor A; gain of function; gain of function mutation; gene induction; imaging study; inhibitor; insight; kinase inhibitor; melanoma; meter; mouse model; multiplexed imaging; myocardin; novel; pharmacologic; pre-clinical; rho GTP-Binding Proteins; spatiotemporal; standard of care; tool; transcription factor; tumor progression

Project Summary Malignant melanoma is a very aggressive form of cancer in humans. Due to its capacity for metastasis and resistance to standard therapeutics, it is extremely difficult to cure. Thus, the median survival of patients with metastatic melanoma is only 8.5 months. Gain of cellular invasive capability occurs in primary melanomas, is prerequisite for metastasis, and is thought to be a critical step in melanoma progression. The Rho GTPase Rac1 is a critical oncoprotein in melanoma which drives tumor progression, cell invasion, and metastasis. A gain-of- function mutation of Rac1 (P29S) is reported to be the third most frequent mutation in sun-exposed melanoma, and is associated with increased disease aggressiveness and resistance to standard-of-care therapeutics. Our laboratory previously uncovered a fundamental connection between GTP metabolism enzymes (GMEs) and Rac1 activity, wherein a noncytotoxic ~25% reduction in cellular GTP levels strongly suppressed Rac1 and invasion in melanoma. Recently, we elucidated the underlying mechanism by demonstrating a dependence of Rac1 activity on local GTP production by key rate-limiting GME inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 directly interacts with Rac1, and disrupting this interaction suppresses Rac1 activity and cell invasion. Moreover, our preliminary data demonstrates that IMPDH inhibition significantly affects melanoma xenograft growth in mice. Importantly, Rac1P29S achieves gain-of-function (higher GTP versus GDP occupancy) relative to Rac1WT through faster displacement of GDP and thus faster GDP/GTP nucleotide exchange. Accordingly, our published data suggest that Rac1P29S is more sensitive to IMPDH inhibition than Rac1WT. Intriguingly, our preliminary data uncovered a potential feed-forward mechanism whereby the activity of Rac1P29S (which is regulated by IMPDH2) also promotes IMPDH2 expression. Therefore, in Specific Aim 1, we will evaluate the efficacy of pharmacological suppression of IMPDH and targeting this feedback mechanism in preclinical Rac1P29S models. In Specific Aim 2, we will investigate this feedback loop by defining the mechanism of Rac1P29S-driven IMPDH2 expression, and characterizing the phenotypic consequences. We previously developed genetically-encoded GTP biosensors (GEVALs) which for the first time visualized free GTP in living cells. By combining this tool with Rac1 activity biosensors, we recently described a correlation between areas of the cell with high GTP and high Rac1 activity. Therefore, in Specific Aim 3, we will characterize a newly generated Rac1P29S biosensor compatible for multiplexing with GEVALs, and directly compare how dependence of Rac1WT versus Rac1P29S activities on local GTP in real time.

项目概要 恶性黑色素瘤是人类一种非常具有侵袭性的癌症。由于其转移能力 并且对标准疗法有抵抗力，治愈起来极其困难。因此，患有以下疾病的患者的中位生存期 转移性黑色素瘤仅8.5个月。原发性黑色素瘤获得细胞侵袭能力，是 转移的先决条件，被认为是黑色素瘤进展的关键步骤。 Rho GTP 酶 Rac1 是黑色素瘤中的一种关键癌蛋白，可驱动肿瘤进展、细胞侵袭和转移。增益- 据报道，Rac1 (P29S) 的功能突变是阳光照射黑色素瘤中第三常见的突变， 并且与疾病侵袭性和对标准治疗的抵抗力增加有关。 我们的实验室之前发现了 GTP 代谢酶之间的基本联系 (GME) 和 Rac1 活性，其中细胞 GTP 水平约 25% 的非细胞毒性降低受到强烈抑制 Rac1 和黑色素瘤的侵袭。最近，我们通过演示阐明了基本机制 Rac1 活性对关键限速 GME 肌苷单磷酸局部 GTP 产生的依赖性 脱氢酶 2 (IMPDH2)。 IMPDH2 直接与 Rac1 相互作用，破坏这种相互作用会抑制 Rac1 活性和细胞侵袭。此外，我们的初步数据表明 IMPDH 抑制显着 影响小鼠黑色素瘤异种移植物的生长。 重要的是，相对于 Rac1WT，Rac1P29S 实现了功能增益（GTP 相对 GDP 占用率更高） 通过更快的 GDP 置换，从而更快的 GDP/GTP 核苷酸交换。据此，我们发布了 数据表明，Rac1P29S 比 Rac1WT 对 IMPDH 抑制更敏感。有趣的是，我们的初步数据 发现了一种潜在的前馈机制，即 Rac1P29S 的活性（受 IMPDH2 调节） 还促进 IMPDH2 表达。因此，在具体目标1中，我们将评估药理功效 抑制 IMPDH 并在临床前 Rac1P29S 模型中针对这种反馈机制。在具体目标 2 中， 我们将通过定义 Rac1P29S 驱动的 IMPDH2 表达机制来研究这个反馈循环，并且 表征表型后果。 我们之前开发了基因编码 GTP 生物传感器 (GEVAL)，这是首次 活细胞中游离 GTP 的可视化。通过将该工具与 Rac1 活性生物传感器相结合，我们最近描述了一种 具有高 GTP 和高 Rac1 活性的细胞区域之间的相关性。因此，在具体目标 3 中，我们将 表征新生成的 Rac1P29S 生物传感器，与 GEVAL 兼容，并直接 实时比较 Rac1WT 与 Rac1P29S 活性对本地 GTP 的依赖性。