The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation
Although conventional EZH2 enzymatic inhibitors are effective in various tumors, we demonstrated that B-Raf mutant melanoma cells do not respond effectively to both GSK126 and EPZ6438 when used in physiological levels in vitro. In addition, the EZH2 knockdown phenotype (lowered tumorigenesis and metastasis) was rescued by both wild-type EZH2 and methyl-transferase-deficient H689A mutant and cytosolic nuclear localization signal (NLS) deletion-mutant EZH2 overexpression in vitro and in vivo. This clearly indicates a methyl-transferase-independent role of cytosolic EZH2 in melanoma cell tumorigenicity and metastasis. To identify potential methyltransferase-independent mechanisms of EZH2 in melanoma, we performed Liquid Chromatography-Mass Spectrometry (LC-MS) on EZH2 immunoprecipitates from multiple melanoma cell lines and human PDXs. We identified an interacting protein called inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo GTP synthesis. Biochemical studies showed that N-terminal EED-binding domain of cytosolic EZH2 interacts with CBS domain of IMPDH2 in a PRC2- and methylation-independent manner. EZH2 silencing reduces cellular GTP levels by impeding IMPDH2 tetramerization, stability and its cytosolic localization. On the other hand, guanosine, which replenishes GTP, stabilized ribosomal biogenesis and actomyosin contractility and thereby, promoted invasive and clonogenic cell states even in EZH2 silenced cells. In human melanoma clinical samples, high cytosolic EZH2 and IMPDH2 expressions are directly correlated with the nucleolar enlargement in the metastatic melanomas. In addition, IMPDH2 silencing reduces EZH2 overexpression induced proliferation and invasion phenotype that is reversed later by guanosine addition. In addition, EZH2-IMPDH2 complex was also validated across a range of cancers. These results point to a methyltransferase-independent but GTP-dependent non-canonical mechanism of EZH2 regulation in various cancers. Sappanone A (SA), that is shown to inhibit IMPDH2/EZH2 interaction and thereby IMPDH2 tetrametization, is anti-tumorigenic across a range of cancers including melanoma, but not in normal melanocytes or bone marrow progenitor cells. In summary, EZH2 contributes to melanoma tumorigenicity and invasion by upregulating ribosomal biogenesis and actomyosin contractility via IMPDH2-induced GTP synthesis.