The role of regulation and subcellular localization of GTP biosynthesis in melanoma invasion and metastasis

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Metastatic melanoma is the most aggressive type of skin cancer with no treatment. Acquisition of invasive phenotypes is a critical event driving progression from primary to metastatic melanoma which is most detrimental for patient survival in THE case of melanoma brain metastases (MBMs). Invasion of melanoma cells depends on activation of several RHO-GTPases which regulate the biogenesis of cell protrusions. Among RHO-GTPases, RAC1 plays the most prominent role in melanoma progression. Indeed, P29S activating mutation of RAC1 (detected predominately in melanoma) is the 3rd most common mutation in sun-exposed melanomas. In addition, RAC1 and other RHO-GTPases function as effectors and/or regulators of signal transduction programs activated in MBMs, including MAPK, PI3K/AKT or STAT3 pathways. However, mechanisms regulating RHO-GTPase activity in melanoma in general or in MBMs are understudied. Physiological changes of intracellular GTP have not been thought to regulate RHO-GTPase activity because total GTP levels in the cell were considered to be much higher than GTP dissociation constant of RHO proteins (KdRHO•GTP). In our papers (Bianchi-Smiraglia et al. Nature Methods 2017 & Nature Communications 2021; Wolff et al, Cell Chemical Biology 2022), we demonstrated that not only free GTP levels comparable to KdRHO•GTP existed in the cell, but also that RAC1 activation depended on availability of local free GTP. Furthermore, RAC1 interacted with inosine monophosphate dehydrogenase 2 (IMPDH2, a rate limiting enzyme in GTP biosynthesis) and, through this interaction, with other GTP metabolism enzymes (GMEs). Accordingly, IMPDH2 and GMEs were enriched together with RAC1 in cell protrusions of invading cells. Disruption of IMPDH2-RAC1 interaction via sequestration of IMPDH2 away from the plasma membrane did not alter total GTP pools, but decreased local GTP levels in cell protrusions, RAC1 activity, and cell invasion. IMPDH2 and other GTP metabolism enzymes do not contain membrane localization sequences, however, do associate with the plasma membrane. We also demonstrated that high IMPDH2 levels correlate with poor prognosis in melanoma patients. Thus, in the 1st Specific Aim we will establish the mechanisms that recruit IMPDH2 to plasma membrane and cell protrusions. In the 2nd Specific Aim, we will identify and characterize mechanisms maintaining high IMPDH2 levels in metastatic melanoma cells. In the 3rd Specific Aim, we will interrogate the inhibition of IMPDH2 as a novel strategy for treatment of MBMs.