Cellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms are not well understood that regulate distinct cellular states within tumours. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma, and compared protein and RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and invasion, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or DZNep reduced total EZH2 protein and conferred significant inhibition of cell growth and invasion in LPCs. Notably, the EZH2 knockdown phenotype was rescued by both WT-EZH2 as well as methyltransferase-deficient H689A mutant EZH2 overexpression. This indicates a methyltransferase independent role of EZH2 in melanoma cell tumorigenicity and invasion.
To identify potential methyltransferase-independent mechanisms of EZH2 function, we performed LC/MS on EZH2 immunoprecipitates and identified an interacting protein complex with Kinesin-1, the major anterograde motor protein complex for intracellular transport along microtubules, and inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo GTP synthesis, a purine nucleoside that mediates essential requirements of cancer cells such as ribosome biogenesis/ protein synthesis and G-protein coupled receptor signalling via RAS/RAC1 oncogenes. Biochemical studies showed that cytosolic EZH2 interacts with KIF5B through the KLC1-TRP domain in a methylation-independent manner, that EZH2 silencing reduces KLC1 and IMPDH2 interactions, and that KLC1/2 silencing localizes EZH2 and IMPDH2 to nuclei. EZH2 silencing reduces IMPDH2 tetramerization and cellular GTP levels. Guanosine, which replenishes GTP, stabilized ribosomal function and promoted invasive and clonogenic cell states even in siEZH2-treated cells. This indicates methyltransferase-independent and GTP-dependent non-canonical mechanisms of EZH2 regulation of the malignant cell state.