Targeting DNMT3B impairs multiple myeloma cell proliferation and clonogenic capacity and enhances sensitivity to standard of care agents.

Introduction/Background
In about half of the MM patients, genetic defects and/or abnormal expression are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this further increases at relapse, indicating an important role for epiplayers in MM cell drug resistance (DR). However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR has been formally established. Using RNASeq data from the MMRF CoMMpass study, we found that the epiplayer DNMT3B is significantly increased in the relapsed setting, suggesting a role in MM relapse. Here, we explored the role of DNMT3B in MM cell biology and drug response.

Methods
Both the DNMT3B specific inhibitor Nanaomycin A (NA) and genetic knockdown using a doxycycline inducible shRNA against DNMT3B (shDNMT3B) were used to specifically target DNMT3B. RNA sequencing was performed upon genetic knockdown in AMO-1 (high DNMT3B levels) and XG-2 (intermediate DNMT3B levels) human myeloma cell lines (HMCL). Viability and apoptosis were assessed using a CellTiter-Glo assay and AnnexinV/7AAD stainings. Cell proliferation was measured by BrdU incorporation and cell cycle analysis, while the clonogenic capacity was evaluated by a colony formation assay.

Results
Here, we report that high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. Targeting DNMT3B in HMCL using either shDNMT3B or NA resulted in a strong and significant increase in cell death. NA treatment also strongly reduced viability of primary human MM cells, while human BMSC were much less affected. RNA sequencing identified 794 genes differentially expressed upon DNMT3B depletion, with 394 genes upregulated and 400 genes downregulated in DNMT3B-depleted cells compared to non-depleted cells (Fold change > 1.5; FDR ≤ 0.05). Gene set enrichment analysis (GSEA) of the upregulated genes identified significant enrichment of genes involved in histone methylation and stem cells, whereas downregulated genes are mainly involved in cell cycle, apoptosis, stem cells and MM proliferating molecular subgroup. In line, cell cycle analysis upon DNMT3B silencing and NA treatment revealed impaired cell proliferation due to an arrest in the G1 phase. In addition, a strong and significant reduction in the number of colonies was observed upon DNMT3B targeting, thus supporting a role for DNMT3B in MM cell clonogenicity and thus potentially also in MM stemness. Importantly, combining NA with either bortezomib (Bz) or Melphalan (Mel) resulted in a synergistic inhibition of MM cell viability and further increase in the anti-clonogenic activity. Furthermore, NA was also able to resensitize both Mel and Bz resistant XG-2 cells.

Conclusion
Together, our findings provide for the first time evidence that DNMT3B could be a novel promising epigenetic target to overcome or delay relapse in MM.