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: 10525488
• 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/10525488
• 关键词: Actins; Adopted; Affect; Area; 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; Immune response; Inosine Monophosphate; 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; Pharmaceutical Preparations; Pharmacology; Phenotype; Production; Prognosis; Proteins; Publishing; Regulation; Reporting; Research; Resistance; Ribavirin; Role; Series; Serum Response Factor; Structure; Sun Exposure; Technology; Testing; Therapeutic; Time; Transcriptional Regulation; Work; Xenograft procedure; base; 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; melanoma; mouse model; multiplexed imaging; myocardin; novel; pre-clinical; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; spatiotemporal; standard of care; tool; transcription factor; tumor; 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.

项目摘要 恶性黑色素瘤是一种非常具有侵袭性的人类癌症。由于其转移能力 并且对标准疗法有抵抗力，因此极难治愈。因此，患者的中位生存期 转移性黑色素瘤只有8.5个月。原发性黑色素瘤细胞侵袭能力的获得， 黑色素瘤是转移的先决条件，并且被认为是黑色素瘤进展的关键步骤。The Rho GTQRac 1 是黑色素瘤中的一种关键癌蛋白，其驱动肿瘤进展、细胞侵袭和转移。一个增益- 据报道Rac 1的功能突变（P29 S）是暴露于阳光的黑素瘤中第三常见的突变， 并且与增加的疾病侵袭性和对标准治疗剂的抗性有关。 我们的实验室以前发现了GTP代谢酶之间的基本联系 （GMEs）和Rac 1活性，其中细胞GTP水平的非细胞毒性~25%降低强烈抑制 Rac 1与黑色素瘤侵袭最近，我们阐明了潜在的机制，通过展示一个 Rac 1活性依赖于关键限速GME肌苷单磷酸的局部GTP产生 脱氢酶2（IMPDH 2）。IMPDH 2直接与Rac 1相互作用，破坏这种相互作用抑制 Rac 1活性和细胞侵袭。此外，我们的初步数据表明，IMPDH抑制显着 影响小鼠黑素瘤异种移植物的生长。 重要的是，Rac 1 P29 S相对于Rac 1 WT实现了功能增益（更高的GTP对GDP占用率） 通过更快的GDP置换，从而更快的GDP/GTP核苷酸交换。因此，我们出版的 数据表明Rac 1 P29 S比Rac 1 WT对IMPDH抑制更敏感。有趣的是，我们的初步数据 揭示了一种潜在的前馈机制，即Rac 1 P29 S的活性（由IMPDH 2调节） 还促进IMPDH 2表达。因此，在具体目标1中，我们将评估药理学治疗的有效性。 在临床前Rac 1 P29 S模型中抑制IMPDH并靶向这种反馈机制。在具体目标2中， 我们将通过定义Rac 1 P29 S驱动的IMPDH 2表达的机制来研究这种反馈回路， 表征表型后果。 我们以前开发了基因编码的GTP生物传感器（GEVALs）， 活细胞中的游离GTP。通过将该工具与Rac 1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Rac 1活性的细胞区域之间的相关性。因此，在具体目标3中，我们 表征新产生的与GEVALs多路复用兼容的Rac 1 P29 S生物传感器，并直接 比较Rac 1 WT与Rac 1 P29 S活性如何在真实的时间内依赖于局部GTP。