Epigenetic Modifiers MMSET and EZH2 Physically Interact and Cooperate to Support Multiple Myeloma Pathophysiology

Multiple myeloma (MM) is a malignant B cell dyscrasia characterized by the accumulation of clonal plasma cells (PC) within the bone marrow (1). Genome sequencing studies have revealed great heterogeneity and genomic instability, a complex mutational landscape and a branching pattern of clonal evolution (2-4). Epigenetic factors are also involved in MM initiation, progression, and occurrence of chemoresistance (5). Among them, EZH2 and MMSET are associated with poor prognosis values and contribute to MM disease. EZH2 is the main catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and deposits H3K27me3 on chromatin (6), whereas MMSET, coded by the gene NSD2, which is the target oncogene of t(4;14) primary translocation, is a histone methyltransferase that methylates H3K36me2 (7). Using proteomics analysis of EZH2-immune precipitates, we identified a constitutive interaction between EZH2 and MMSET independently of t(4;14) translocation and mutational status of EZH2 in MM cells. Furthermore, MAK-683, an allosteric inhibitor of EZH2, disrupted this interaction, but not EPZ-6438, a catalytic inhibitor of EZH2, suggesting that MMSET-EZH2 interaction may be mediated by other PRC2 factors, but is independent of the PRC2 activity. Analysis of EZH2 expression levels and t(4;14) translocation status in 3 independent cohorts of MM patients (CoMMPass (N=674), UAMS-TT2 (N=345), Mtp-Daratumumab (N=51)) identified a worse overall survival of patients with high EZH2 expression combined with t(4;14) chromosomal translocation than patients with only one of these genetic events. We identified 355 differentially expressed genes in this “ EZH2High t(4;14) +” subgroup compared to other patients. GSEA analysis of up-regulated genes in this subgroup identified a significant enrichment for genes involved in cell cycle, KRAS signaling, Cell-Cell signaling, Hypoxia and Epithelial-Mesenchymal transition. These results indicate that EZH2-MMSET interaction is of clinical interest in t(4;14) MM patients. To better understand the molecular relationship between EZH2 and MMSET, we generated MMSET-depleted clones by CRISPR-Cas9. MMSET depletion did not significantly alter cell proliferation and survival, but reduced the clonogenic capacity of MM cells compared to non-depleted cells. Moreover, after IL-6 or serum starvation, IL-6 and IGF-1 sensitivity was significantly affected in MMSET-depleted clones compared to the parental cell line, indicating that MMSET overexpression confers a cell growth advantage to MM cells under stress conditions and an independence of growth factors produced by bone marrow microenvironment Additionally, MMSET depletion induced changes in PRC2 nuclear localization in MM cells, a decrease of H3K36me3 and a concomitant increase in H3K27me3 levels, which altered the transcriptional program of cells showing, among others, a specific enrichment in genes up-regulated in t(4;14) MM subgroup of patients. Finally, we demonstrated that disrupting EZH2-MMSET interaction with MAK-683 may be of therapeutic interest, since MAK-683 combination with conventional drugs used in MM treatment, including melphalan and Panobinostat or BRD4 inhibitors, synergized to kill MM cells. Taken together, our data identified an “ EZH2High t(4;14) +” signature in MM patients with a poor outcome who may benefit from combination therapy of MAK-683 with melphalan or epidrugs. References: (1) van de Donk NWCJ et al. Multiple myeloma. The Lancet. 2021 Jan;397(10272):410-27. (2) Bolli N et al. Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun. 2014;5:2997. (3) Lohr JG et al. Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy. Cancer Cell. 2014 Jan 13;25(1):91-101. (4) Brioli A et al. The impact of intra-clonal heterogeneity on the treatment of multiple myeloma. British Journal of Haematology. 2014;165(4):441-54. (5) Schavgoulidze A et al. Multiple Myeloma: Heterogeneous in Every Way. Cancers (Basel). 2021 Mar 13;13(6):1285. (6) Schuettengruber B et al. Genome Regulation by Polycomb and Trithorax: 70 Years and Counting. Cell. 2017 Sep 21;171(1):34-57. (7) Kuo AJ et al. NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming. Mol Cell. 2011 Nov 18;44(4):609-20.