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 that GTP-producing cytosolic IMPDH2 in melanoma progression. However, a subset of melanoma cells displays both cytosolic and nuclear IMPDH2 expression. We thus investigated potential nuclear functions of IMPDH2 in melanoma cells.
In vitro, 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/ metastasis and increased pigmentation and senescence. IMPDH2 ChIP-seq and RNAseq analyses demonstrated that nuclear IMPDH2 acts as a transcriptional repressor for the E2F target genes and activator for CDK inhibitors that are critical regulators of cell cycle progression.
P53 activation by CX5461, H2O2 or Nutlin-3 led to nuclear translocation of IMPDH2, but this effect was fully abrogated by p53 CRISPR-KO. Based on IMPDH2 LC-MS data, Nutlin-3-treated melanoma cells showed a sharp decrease in serine 122 phosphorylation on IMPDH2. CDK2 inhibition by INX315 or CDK2 silencing dramatically reduced S122 phosphorylation and induced nuclear translocation of IMPDH2. In vitro kinase assay verified that CDK2 phosphorylates IMPDH2 on S122. Nuclear translocation was abrogated in S122D phospho-mimetic form of IMPDH2 upon Nutlin-3 treatment, while S122A nonphospho form of IMPDH2 showed higher nuclear translocation irrespective of the treatment.
These data indicate IMPDH2 has a dual and cell location-dependent role in melanoma. In the cytoplasm, IMPDH2 acts canonically as a metabolic oncoprotein to regulate cellular GTP and promote disease progression. In contrast, nuclear IMPDH2 acts as a tumour suppressor to repress transcription of E2F target genes and induce CDK inhibitor genes 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.