The de novo DNA methyltransferase DNMT3B: A new promising target in multiple myeloma

Objectives
Multiple myeloma (MM) is an incurable plasma cell cancer with patients relapsing after every round of therapy due to the development of drug resistance (DR). Recent studies support the involvement of the epiplayers MMSET, EZH2 and G9a in MM cell DR development. However, the role of most epiplayers in MM cell DR remains unknown. To identify new epiplayers involved in DR, we compared the RNA-seq data between newly diagnosed and relapsed patients from the MMRF CoMMpass study and found that DNMT3B is significantly upregulated in relapsed MM patients. This suggests that DNMT3B can be involved in MM relapse. In this study, we examined the role of DNMT3B in MM cell biology and drug response.

Method
The effect of the specific DNMT3B inhibitor Nanaomycin A was evaluated on viability and apoptosis by using a CellTiter-Glo assay and AnnexinV/7AAD staining respectively. In addition, the effect on proliferation was examined using a BrdU incorporation assay and cell cycle analysis. Finally, a colony-forming assay was performed to evaluate the effect of Nanaomycin A alone or in combination with Bortezomib (Bz) in eradicating cells with clonogenic capacities. After 14 days, the number of colonies was assessed with the EVOS M7000.

Results
Short-term Nanaomycin A treatment (up to 72 hours) significantly and dose-dependently reduced viability and increased apoptosis in the human myeloma cell lines AMO-1, XG-2 and XG-7. In addition, low-dose Nanaomycin A treatment (25 to 400 nM) reduced proliferation as evidenced by a clear decrease in BrdU incorporation. Moreover, cell cycle analysis revealed an arrest in the G1 phase for the AMO-1 and XG-2 cells and an arrest in the G2 phase for the XG-2 and XG-7 cells. Importantly, we also observed a clear and dose-dependent decrease of the protein DNMT3B levels upon Nanaomycin A treatment, thus validating its on-target effect.
Since DNMT3B has been suggested to play a significant role in cancer cell stemness, we next performed colony-forming assays with AMO-1 cells treated with Nanaomycin A (100, 200 and 800 nM) and/or Bz (4 nM). We found that at relatively low concentrations (100 and 200 nM), Nanaomycin A was capable of significantly and dose-dependently reducing the number of colonies when added on day 0, but not when added on day 7. In contrast, treatment with a high Nanaomycin A concentration (800 nM) either on day 0 or on day 7, both significantly reduced the number of colonies, confirming that high Nanaomycin A concentrations are indeed cytotoxic. Combining Nanaomycin A (100 nM) with Bz (4 nM), with the Bz added either on day 0 or day 7, resulted in a significant further decrease in colony formation compared to both single agents.

Conclusions
Together, these results indicate that DNMT3B is a novel promising target for MM therapy. Low concentrations of the specific DNMT3B inhibitor Nanaomycin A have an impact on MM cell proliferation and clonogenicity, while high Nanaomycin A concentrations are cytotoxic. These promising results will be further validated using genetic inhibition and in vivo using the 5TMM murine MM models.