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

项目摘要 恶性黑色素瘤是人类一种侵袭性很强的癌症。由于它的转移能力 以及对标准疗法的抗药性，这是极其难以治愈的。因此，慢性前列腺炎患者的中位生存期 转移性黑色素瘤只有8.5个月。细胞侵袭能力的获得发生在原发性黑色素瘤，IS 是黑色素瘤转移的先决条件，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，它推动肿瘤的进展、细胞的侵袭和转移。一种收益- Rac1的功能突变(P29S)被报道为在阳光照射的黑色素瘤中第三常见的突变， 并与疾病侵袭性增加和对标准护理疗法的抵抗力有关。 我们的实验室之前发现了GTP代谢酶之间的基本联系 (GMES)和rac1活性，其中非细胞毒性~25%的细胞GTP水平下降强烈抑制 Rac1与黑色素瘤侵袭的关系。最近，我们通过演示一种 关键限速GME肌苷一磷酸对rac1活性与局部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，我们的初步数据显示，IMPDH抑制显著 影响小鼠黑色素瘤异种移植瘤生长。 重要的是，与rac1WT相比，rac1P29S实现了功能增益(GTP比GDP占有率更高) 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 结果表明，Rac1P29S比Rac1WT对IMPDH抑制更为敏感。有趣的是，我们的初步数据 揭示了一种潜在的前馈机制，通过该机制，rac1P29S的活性(受IMPDH2调节) 也促进IMPDH2的表达。因此，在具体目标1中，我们将评估药理作用 在临床前的rac1P29S模型中抑制IMPDH和靶向这种反馈机制。在具体目标2中， 我们将通过定义rac1P29S驱动的IMPDH2表达的机制来研究这种反馈循环，以及 来描述表型后果。 我们之前开发了基因编码的GTP生物传感器(GEVAL)，这是第一次 显示活细胞中的游离GTP。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 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 表征一种新一代的、与GEVALs兼容的、可与GEVALs多路传输的、直接 比较rac1WT和rac1P29S活动对本地GTP的实时依赖性。

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.