Inhibition of NF-κB Activation Augments the Effect of 5-Azacytidine On Acute Myeloid Leukemia Cell Line

Abstract 1369

Dehydroxymethylepoxyquinomycin (DHMEQ) is a unique NF-kB inhibitor that is a 5-dehydroxymethyl derivative of novel compound epoxyquinomicin C and is currently under development for clinical use. We have shown that DHMEQ specifically binds to NF-kB having transactivation domain and inhibits NF-kB activation at the level of the nuclear translocation and the DNA binding. Constitutive activation of NF-kB has turned out to be a hallmark of various types of cancers originated from hematopoietic and other organs. We showed the effectiveness of DHMEQ in hematological malignancies with constitutive activation of NF-kB including multiple myeloma, chronic lymphocytic leukemia, adult T-cell leukemia, pleural effusion lymphoma and Hodgkin lmphoma. However, significance of NF-kB as a molecular target in acute myeloid leukemia (AML) is not well understood.

Recently 5-azacytidine is widely used for the treatment of myelodysplastic syndrome and other cancers. 5-Azacytidine is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. Therefore it is possible that 5-azacytidine induces molecules required for NF-kB activation. However, the effect of 5-azacytidine on NF-kB induction, which may antagonize the anti-cancer effect is poorly understood. In this study we examined the constitutive NF-kB activation and the effectiveness of DHMEQ on AML cell lines, HL-60, ML-2, HEL, K562, CMK and Meg0-1. We also examined the induction of NF-kB by 5-azacytidine and evaluated the combined treatment of AML cell lines by 5-azacytidine and DHMEQ.

AML cell lines except for K562 showed constitutive activation of NF-κB consisting of RelA and p50 subcomponent. The NF-κB binding activity observed in AML cell lines were relatively modest compared to that observed in Jurkat cells treated with tumor necrosis factor (TNF). Treatment of AML cell lines with constitutive activation of NF-κB by DHMEQ inhibited NF-κB and significantly reduced their viability. Treatment with 5-azacytidine also significantly reduced the viability of AML cell lines, however K562 showed resistance against the treatment. Addition of DHMEQ enhanced the effect of 5-azacytidine on cell viability of HL-60 and CMK cells. The treatment with 5-azacytidine enhanced NF-kB activation although the induction was modest and DHMEQ abolished both constitutive and induced NF-kB activation in HL-60 and CMK cells.

The results indicate that NF-κB is a molecular target of AML cells and DHMEQ is a potential compound for the treatment of AML. Although the NF-κB activity is relatively low compared to that in lymphoid malignancies, the constitutive activation of NF-κB in AML cell lines appears to play a significant role for their survival. The results also indicate that 5-azacytidine can reduce viability of AML cells and combined use of an NF-κB inhibitor augments the effect of 5-azacytidine by inhibiting constitutive and induced NF-κB. Since NF-κB cooperates with various transcription factors and supports the survival of cells, interruption of the crosstalk between NF-kB and molecules induced by 5-azacytidine might also be responsible for the combined effect of DHMEQ and 5-azacytidine.