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

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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.