The DNA Methyltransferase Inhibitor Decitabine Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Multiple Myeloma

Abstract 1833

Multiple myeloma (MM) is still an incurable plasma cell malignancy, thus highlighting the need for alternative treatment options. Currently, strategies for therapy are being developed targeting epigenetic modification using epigenetic modulating agents like histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi). 5-aza-2'-deoxycitidine or decitabine (DAC) is a DNMTi and is FDA approved for treatment of myelodysplastic syndrome and has beneficial clinical effects against leukemia. The anti-tumor effects are ascribed to two non-mutual exclusive modes of action. Relative low doses are thought to lead to passive CpG demethylation resulting in re-expression of genes silence by DNA methylation and apoptosis, while relative high doses are cytotoxic by inducing a DNA damage response together with cell cycle arrest and apoptosis. In multiple myeloma (MM), preclinical data regarding the effects of DAC is, however, limited.

Therefore, we investigated the cytotoxic effects of DAC in MM both in vitro and in vivo. In addition, we evaluated the combination of DAC with the pan-HDAC inhibitor JNJ-26481585. First, we assessed the effects of DAC on cell cycle progression and apoptosis on a panel of MM cell lines. We used one murine (5T33MMvt) and 5 human (OPM-2, RPMI 8226, LP-1, KMS-11 and NCI-H929) MM cell lines. In general, DAC could affect cell cycle progression by inducing either a G0/G1-phase arrest or a G2/M-phase arrest. The 5T33MMvt and LP-1 cells were arrested in the G2/M-phase, while OPM-2 and NCI-H929 cells underwent a G0/G1-phase arrest. Subsequently, apoptosis occurred in all cell lines. Interestingly, the 5T33MMvt cells were relatively sensitive, as nM doses of DAC were sufficient to induce massive apoptosis in a relative short incubation time (2 days). The human cell lines were less sensitive since higher doses (μM range) and longer incubation time (3–5 days) were necessary to induce apoptosis, with the OPM-2 cells being the least sensitive. To determine the potential mechanisms more in detail, we focused on the 5T33MMvt and OPM-2 cells. In both cell lines, DAC-mediated apoptosis was associated with caspase activation and PARP cleavage, Bim upregulation and posttranslational changes in Mcl-1 expression. The G2/M-phase arrest in the 5T33MMvt cells was accompanied by phosphorylation of CDK-1 and an increase in cyclinB1 expression. In both cell lines, p27 protein expression was increased, what may contribute to the cell cycle arrest. Furthermore, in the 5T33MMvt cells, a DNA damage response was activated as evidenced by a clear induction of ATM and H2AX phosphorylation. This was not the case for the OPM-2 cells, in which we observed no ATM activation and only a modest H2AX phosphorylation upon DAC treatment. In addition, the tumor suppressor p53 was phosphorylated on ser15 upon DAC treatment in both cell lines, indicating a potential role of p53. However, a p53 inhibitor, pifithrin-α, could not abrogate DAC-induced apoptosis indicating that p53 transactivation is not essential in this process. Next, we used the syngeneic 5T33 murine MM model (5T33MM) to investigate the in vivo effects of DAC. 5T33MM mice were daily treated with 0.1, 0.2 and 0.5 mg/kg DAC. We observed a significant decrease in serum M-protein, bone marrow plasmacytosis and spleno- and hepatomegaly compared to vehicle treated mice. These effects led to a significant increase in survival probability of DAC treated mice (p≤0.001). Lastly, we evaluated the possibility of combining DAC with a pan-HDAC inhibitor JNJ-26481585 (JNJ-585). DAC and JNJ-585 synergistically induced cell death in RPMI-8226, OPM-2 and 5T33MMvt cells. We further demonstrated the combinatory effects of DAC and JNJ-585 in the 5T33MM murine model. Here, we observed enhanced effects of DAC and JNJ-585 on serum M-protein, BM tumor load and survival (p≤0.001) compared to either agent alone.

In conclusion, DAC shows potent anti-MM effects both in vitro and in vivo. Mechanistically, we observed induction of a DNA damage response and/or cell cycle arrest. Apoptosis was caspase-mediated but independent of the transactivation of p53. DAC was also efficient in the murine 5T33MM model in which DAC treatment led to a survival benefit. In addition, DAC showed useful in a combination with the HDAC inhibitor JNJ-585.