Comparison of the Cytotoxicity of Cladribine and Clofarabine When Combined with Fludarabine and Busulfan in AML Cells: Enhancement of Cytotoxicity with Epigenetic Modulators

            Clinical trials have shown the efficacy of clofarabine (Clo), fludarabine (Flu) and busulfan (Bu) combinations in hematopoietic stem cell transplantation (HSCT) for myeloid leukemia. In the present study, we determined if the much more affordable cladribine (Clad) can supplant Clo with or without epigenetic modulators, using panobinostat (Pano) and 5-aza-2′-deoxycytidine (DAC) as prototype representatives of such agents. Both [Clad+Flu+Bu] and [Clo+Flu+Bu] combinations showed synergistic cytotoxicity in KBM3/Bu250⁶, HL60 and OCI-AML3 cell lines with 60%-80% inhibition of proliferation. Drug exposure resulted in activation of the ATM pathway, increase in histone modifications with corresponding decreased levels of HDAC3, HDAC4, HDAC5 and SirT7, decrease in mitochondrial membrane potential, activation of apoptosis and stress signaling pathways, and  down-regulation of the pro-survival AKT pathway.  Similarly, these drug combinations activated DNA-damage response and apoptosis in primary cell samples from AML patients. At lower concentrations of Clad/Clo, Flu and Bu, inclusion of Pano and DAC yielded a significant increase in histone modifications and DNA demethylations, resulting in further enhanced cell killing.  Inclusion of Pano decreased histone deacetylases and demethylases. The presence of DAC decreased the level of DNA methyl transferase 1 (DNMT1), which consequently increased the levels of cell-cycle checkpoint proteins such as P16/INK4a, P15/INK4b and P21/Waf1/Cip1. We also demonstrated the DNA demethylating activity of Clad and Clo, which may be augmented by DAC activity and caused degradation of β-catenin in cells exposed to [Clad/Clo+Flu+Bu+DAC+Pano].  Increased demethylations of the gene promoters for SFRP1, DKK3 and WIF1 were observed with concomitant up-regulation of their expressions; the encoded proteins are known to antagonize the pro-survival Wnt/β-catenin pathway. The overlapping activities of [Clad/Clo+Flu+Bu], Pano and DAC in DNA-damage formation, histone modifications, DNA demethylation and apoptosis may explain their synergism. A proposed model for the observed drug synergism in AML cells is shown below. Our results provide a basis for supplanting Clo with Clad and for including epigenetic modifiers in the pre-HSCT conditioning regimen for myeloid leukemia patients.