Many cytoplasmic metabolic enzymes are found in the nucleus. The nuclear functions of these enzymes are independent of their catalytic activity and include epigenetic regulation or DNA repair. Our lab has recently shown the critical role of GTP-producing cytosolic IMPDH2 in melanoma progression. However, a subset of melanoma cells displays both cytosolic and nuclear IMPDH2 expression, and we also detected a striking shift from nuclear to cytoplasmic IMPDH2 during melanoma progression. We thus investigated potential nuclear functions of IMPDH2 in melanoma cells.
Melanoma cells with spontaneously occurring high nuclear-to-cytoplasmic (N/C) IMPDH2 ratios formed smaller colonies. Consistent with this, forced overexpression of nuclear IMPDH2 reduced melanoma cell tumorigenicity and increased senescence, supporting a tumour suppressive role for nuclear IMPDH2 in melanoma cells. IMPDH2 ChIPseq and RNAseq analyses demonstrated that nuclear IMPDH2 acts as a senescence inducer by downregulating the E2F target genes and inducing CDKN1A, that are critical regulators of cell cycle arrest and senescence.
We next explored upstream regulators of IMPDH2’s nuclear localisation in melanoma cells. We showed that IMPDH2 underwent cell-cycle- and genotoxic/ metabolic stress-dependent nuclear translocation. Nutlin-3, a p53 activator, also increased nuclear IMPDH2 in a p53-dependent manner, implicating p53 in this relocalisation. To interrogate this further, we performed LC-MS on fractionated nuclear and cytoplasmic IMPDH2, finding that nuclear IMPDH2 is dephosphorylated at serine 122 (S122), which lies within a conserved CDK motif. Treatment with the CDK2 inhibitor INX315 reduced S122 phosphorylation, promoted nuclear translocation of IMPDH2, suppressed clonogenicity and induced senescence. Cells expressing a phospho-mimetic S122D mutant were resistant to these effects, while non-phosphorylatable S122A mutants mirrored INX315’s impact.
These data indicate IMPDH2 has a dual and cell location-dependent role in melanoma. In the cytoplasm, IMPDH2 acts canonically as a metabolic oncoprotein. In contrast, cell-cycle dependent or genotoxic/metabolic stress induced CDK2 inactivation leads to S122 dephosphorylation and nuclear translocation of IMPDH2, where it acts as a tumour suppressor by repressing E2F target genes, inducing CDKN1A, leading to cell-cycle arrest and senescence. Increasing IMPDH2’s N/C ratio in melanoma cells by targeting its S122 phosphorylation is a promising therapeutic strategy.