PHF19 Promotes Drug Resistance through EZH2 Inactivation in Multiple Myeloma

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by mass growth of clonal plasma cells in the bone marrow, the secretion of abnormal monoclonal immunoglobulins and lytic bone lesions. Although current therapies have improved the outcome of patients, MM remains an incurable disease with a high rate of relapse and refractory. The survival of MM patients ranged from less than one month to more than 10 years. MM cells survival are more dependent on bone marrow microenvironment which induced drug-resistance of anti-MM agents. Cytogenetic aberrations of MM cell, such as chromosomal translocation, deletion and amplification induced oncogenes activation and tumor suppressor genes inactivation. Furthermore, epigenetic changes, such as histone methylation, have been suggested as a mediation of chemotherapy resistance in several cancer types, including MM. PcG (Polycomb group) proteins are conserved transcriptional repressors and essential to regulate cell fate.There were two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methytransferase E (Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (polycomb-like) protein. Three PCL paralogues have been identified: PHF1, MTF2 and PHF19. In this study, we found out that PHF19 is overexpressed and was related with relapse and drug resistance in multiple myeloma.

Materials and methods: Gene Expression Profile Assay (GEP) was conducted in sequential patient samples including newly diagnosed, post-treatment and relapsed. 51 newly diagnosed and relapsed paired samples, 19 newly diagnosed and post-treatment paired specimens, 9 patients have newly diagnosed, post-treatment and relapsed samples at different time points were elucidated in this study. MM relapsed and drug-resistance highly correlated genes were screened and the prognosis and survival analysis were conducted. Real time quantitative PCR (RQ-PCR) and Western blotting were used to analysis the expression of the gene in MM cell lines and MM patients. The candidate gene was overexpressed in MM cells by lentiviral infection. The cell proliferation and drug sensitivity of MM cells and proteins in correlated signaling pathways were detected.

Results: GEP assay showed that 56 genes expression in MM resistant clones after treatment and relapse have significantly increased, with 20 genes closely associated with the poor prognosis. Among them, PHF19, a component of the Polycomb repressive complex 2 (PRC2) family,significantly increased in relapsed and refactory patients and myeloma cell lines.The progression-free survival (PFS) and overall survival (OS) were significantly shortened in MM patients with overexpressed PHF19. PHF19 overexpression (OE) promoted the proliferation and inhibited apoptosis of MM cells. The sensitivity to doxorubicin and vincristine was significantly reduced in the PHF19 OE cells. Western blotting showed that the phosphorylation of EZH2 was significantly increased in PHF19 OE cells, while H3K27me3 level was significantly down-regulated. Overexpressed PHF19 through activating NF-κB signaling pathway induced persistent expression of EZH2 and their downstream anti-apoptotic gene, such as IGF1, BCL2 and HIF1α, which induced cell proliferation and drug resistance. Thus targeting PHF19, inhibiting the phosphorylation of EZH2 and sustaining histone H3K27 methylation level may be a potential therapeutic target in relapsed or refractory myeloma patients.

Conclusions: PHF19 expression was obviously increased in MM relapse and drug resistance patients with poor prognosis. Inhibition PHF19 and counteraction EZH2 phosphorylation should be combined to improve chemotherapy induced hypermethylation of H3K27, which may be a new therapeutic strategies in relapsed or refractory myeloma.