Contributions
Abstract: PB2126
Type: Publication Only
Background
A significant progress has been made in the treatment strategies for multiple myeloma (MM) over the last decade. However, the genetic complexity is hindering the cure of the disease. Epigenetic therapies have shown clinical efficacy in several hematological malignancies. The most investigated drugs modulating DNA methylation are 5-azacitidine and decitabine, which are approved by FDA for treatment of myelodysplastic syndromes. Although both agents have been investigated in MM, their therapeutic benefit is rather marginal. Other compounds like zebularine, which is considered a second generation DNA methylation inhibitor, has not been well explored in MM. In contrast to 5-azacitidine and decitabine, zebularine is stable, less toxic and inhibits not only DNA methylation but also cytidine deaminase (CDA), an enzyme responsible for a resistance to nucleoside analogs.
Aims
To investigate the cytotoxic effect of zebularine on MM and to elucidate it potential mechanisms of action.
Methods
The KMS12BM, KMS12PE, MM1s/r, OPM2, H929, RPMI and U266 human MM cell lines were obtained from ATCC, and JJN3 was obtained from DSMZ. Zebularine was bought from Santa Cruz Biotech and decitabine was obtained from Sigma Aldrich. The following antibodies were purchased and used as recommended by the manufacturer: GAPDH and cMyc (Abcam), DNMT3a, DNMT3b, E2F1 and PSME1 (Santa Cruz Biotech), pH2Ax was bought from (Novius). Total DNA methylation was estimated by ELISA kit and Anexin/PI kit was used to analyze apoptosis by FacsScalibur.
Results
DNA methylation evaluated by ELISA showed that zebularine treatment induced DNA demethylation in a panel of 6 MM cell lines and this effect was similar to demethylation induced by decitabine. DNA demethylation was associated with DNMT3a/b decrease and was accompanied by the reduction in cell viability tested by MTT and Anexin V experiments in 8 out of 9 MM cell lines. Interestingly, zebularine reduced cell viability more than decitabine in H929, KMS12BM, MM1s and RPMI cells. The cytotoxic effect of zebularine was associated with the induction of DNA damage and the decrease of cMyc protein in 4 cell lines, regardless of TP53 mutation status. Moreover, zebularine potentiated the effect of bortezomib by decreasing PSME1 protein involved in bortezomib resistance. Surprisingly, overexpression of CDA enzyme that can be inhibited by zebularine, reduced cytotoxic effect of the compound and abrogated DNA damage and cMyc protein decrease.
Conclusion
Zebularine induces cytotoxicity in MM cell lines resistant to decitabine and sensitizes myeloma cells to bortezomib treatment. The anti-myeloma activity of zebularine is associated with the decrease of cMyc protein.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Methylation, MYC, Myeloma
Abstract: PB2126
Type: Publication Only
Background
A significant progress has been made in the treatment strategies for multiple myeloma (MM) over the last decade. However, the genetic complexity is hindering the cure of the disease. Epigenetic therapies have shown clinical efficacy in several hematological malignancies. The most investigated drugs modulating DNA methylation are 5-azacitidine and decitabine, which are approved by FDA for treatment of myelodysplastic syndromes. Although both agents have been investigated in MM, their therapeutic benefit is rather marginal. Other compounds like zebularine, which is considered a second generation DNA methylation inhibitor, has not been well explored in MM. In contrast to 5-azacitidine and decitabine, zebularine is stable, less toxic and inhibits not only DNA methylation but also cytidine deaminase (CDA), an enzyme responsible for a resistance to nucleoside analogs.
Aims
To investigate the cytotoxic effect of zebularine on MM and to elucidate it potential mechanisms of action.
Methods
The KMS12BM, KMS12PE, MM1s/r, OPM2, H929, RPMI and U266 human MM cell lines were obtained from ATCC, and JJN3 was obtained from DSMZ. Zebularine was bought from Santa Cruz Biotech and decitabine was obtained from Sigma Aldrich. The following antibodies were purchased and used as recommended by the manufacturer: GAPDH and cMyc (Abcam), DNMT3a, DNMT3b, E2F1 and PSME1 (Santa Cruz Biotech), pH2Ax was bought from (Novius). Total DNA methylation was estimated by ELISA kit and Anexin/PI kit was used to analyze apoptosis by FacsScalibur.
Results
DNA methylation evaluated by ELISA showed that zebularine treatment induced DNA demethylation in a panel of 6 MM cell lines and this effect was similar to demethylation induced by decitabine. DNA demethylation was associated with DNMT3a/b decrease and was accompanied by the reduction in cell viability tested by MTT and Anexin V experiments in 8 out of 9 MM cell lines. Interestingly, zebularine reduced cell viability more than decitabine in H929, KMS12BM, MM1s and RPMI cells. The cytotoxic effect of zebularine was associated with the induction of DNA damage and the decrease of cMyc protein in 4 cell lines, regardless of TP53 mutation status. Moreover, zebularine potentiated the effect of bortezomib by decreasing PSME1 protein involved in bortezomib resistance. Surprisingly, overexpression of CDA enzyme that can be inhibited by zebularine, reduced cytotoxic effect of the compound and abrogated DNA damage and cMyc protein decrease.
Conclusion
Zebularine induces cytotoxicity in MM cell lines resistant to decitabine and sensitizes myeloma cells to bortezomib treatment. The anti-myeloma activity of zebularine is associated with the decrease of cMyc protein.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Methylation, MYC, Myeloma