The De Novo DNA Methyltransferase DNMT3B Plays an Important Role in MM Cell Growth, Clonogenicity and Drug Response

Objectives
Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse.
Aim
The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response.
Methods
Publicly available gene expression profiling data of two independent cohorts of newly diagnosed and relapsed MM patients (GSE4581 and GSE36133), and BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by applying the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition was achieved using a doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Fourteen days after plating the MM cells, the number of colonies were assessed with the EVOS M7000.
Results
We found that DNMT3B mRNA levels increase during disease progression and 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.
In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. Since DNMT3B might play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to both AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM added either on day 0 or day 7) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone.

Conclusion/summary
Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse. Low Nanaomycin A concentrations impair MM cell proliferation and clonogenicity and sensitize them to the proteasome inhibitor bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.