EZH2 Inhibition and the Combination with Proteasome Inhibition Are Novel Potential Strategies for the Treatment of Multiple Myeloma

PRC2 catalytic component EZH2 is a methyltransferase which induces trimethylation of histone H3 at lysine 27 (H3K27me3) to repress the transcription of target genes and promotes tumor growth in some cancers. Previous reports demonstrated that EZH2 is overexpressed in multiple myeloma (MM) cells and its expression level correlates with the progression of the disease, suggesting that EZH2 acts as an oncogene in MM. However, the detailed function of EZH2 in MM has not yet been elucidated. Therefore in this study, we examined the impact of EZH2 inhibition on MM cells and evaluated the preclinical efficacy of EZH2 inhibitors in the treatment of MM. We first examined the biologic impact of EZH2 knockdown using lentiviral shRNA and observed significant growth inhibition in knockdown cells. We next examined the effect of EZH2 inhibition using a novel selective EZH2 inhibitor UNC1999 (ACS Chem Biol. 2013). UNC1999 induced significant growth inhibition in MM cell lines such as MM.1S, H929, RPMI8226, and DOX40. We confirmed that UNC1999 reduced the levels of H3K27me3 in dose- and time-dependent manners in western blotting. Importantly, UNC1999 in combination with bortezomib demonstrated synergistic cytotoxicity analyzed by MTS assay in these cell lines. Even in the presence of conditioned media derived from bone marrow stromal cells, UNC1999 enhanced bortezomib-induced cytotoxicity in MM.1S cells. Similar synergistic effects were also observed in the combination of another EZH2 inhibitor GSK126 with bortezomib or in the combination of UNC1999 with another proteasome inhibitor carfilzomib. Moreover, apoptosis induced by bortezomib was enhanced by UNC1999 as evidenced by increased annexin V-positive cells in flow cytometric analysis. Apoptosis was further confirmed by cleavage of caspase-8, -9, -3 and PARP in western blotting. To assess the biologic impact of UNC1999 alone or in combination with bortezomib on MM cells, we performed gene set enrichment analysis using our microarray data. We found significantly positive enrichment of polycomb target genes in both UNC1999- and combination- treated MM.1S cells, and also in bortezomib-treated cells. Of interest, we observed that bortezomib alone downregulated EZH2 mRNA and EZH2 protein, which was enhanced by UNC1999, suggesting one mechanism of the synergistic effects of this combination. In order to examine the anti-MM activities of UNC1999, we used the MM.1S subcutaneous xenograft model in NOG mice. Intraperitoneal dosing of UNC1999 (25mg/kg) twice a week for three weeks significantly reduced the MM tumor growth in comparison to the control group (at day 22, p=0.04). In conclusion, these data indicate that selective EZH2 inhibition and the combination with proteasome inhibition are potential novel therapeutic options in MM.