The synergistic cytotoxicity of clofarabine, fludarabine and busulfan in AML cells involves ATM pathway activation and chromatin remodeling

Abstract

DNA alkylating agents alone or with ionizing radiation have been the preferred conditioning treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In search of less toxic alternatives, we hypothesized that combination of busulfan (Bu), fludarabine (Flu) and clofarabine (Clo) would provide superior efficacy. At low concentrations, these drugs show synergistic cytotoxicity in Bu-resistant AML KBM3/Bu250 6 cells. Similar molecular responses were observed in other AML cell lines and in primary explanted AML cells. The [Clo + Flu + Bu] combination activates an intense DNA damage response through the ATM pathway, leading to cell cycle checkpoint activation and apoptosis. Phosphorylations of SMC1 and SMC3, and methylations of histones 3 and 4, are much more pronounced in cells exposed to [Clo + Flu + Bu] than [Clo + Flu], suggesting their relevance in the efficacy of the triple-drug combination. A possible mechanism for these observed synergistic effects involves the capability of [Clo + Flu] to induce histone methylations and subsequent chromatin remodeling, which may render the genomic DNA more accessible to Bu alkylation. The Bu-mediated DNA cross-linking may provide a feedback loop which perpetuates the DNA damage response initiated by [Clo + Flu] and commits the cells to apoptosis. Our results provide a conceptual mechanistic basis for exploring this triple-drug combination in pretransplant conditioning therapy for allo-HSCT.