Upregulation of XAF1 by 5-Aza-2′-Deoxycytidine Sensitizes Busulfan-Resistant Human CML Cells; Implications for Pretransplant Conditioning Therapy in Myeloid Leukemia

The DNA-alkylating drug busulfan (Bu) is commonly used in myeloablative pretransplant conditioning therapy in patients with chronic myelogenous leukemia (CML). A major obstacle to successful treatment is inherent or acquired cellular Bu-resistance. We hypothesized that cellular Bu-resistance can be reversed by epigenetic upregulation of pro-apoptotic genes. We established a Bu-resistant CML cell line B5/Bu250⁶ which is 4-fold more resistant to Bu than the parental B5 cells but 4-fold collaterally more sensitive to DNA demethylating agent 5-aza-2′-deoxycytidine (DAC). Exposure to DAC synergistically increased Bu-mediated cytotoxicity in B5/Bu250⁶ cells as evaluated by the MTT assay and analyzed by the median-effect method (combination index CI < 0.5). The DAC-induced sensitivity to Bu of B5/Bu250⁶ cells was associated with PARP1 cleavage, phosphorylation of histone 2AX and activation of caspase 8. Real-time PCR and immunostaining analyses of the expressions of various pro-apoptotic genes, which are known to be epigenetically regulated, showed a highly significant increase (approximately 40-fold) in the expression of XAF1 (X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1) in B5/Bu250⁶ cells that were exposed to 0.5 microM DAC for 48 hrs. Analysis of the XAF1 promoter by methylation-specific PCR showed a significant decrease in its methylation status which correlates with the alteration in its gene expression in the presence of DAC. These findings will be discussed relative to our analysis of XAF1 gene methylation status in leukemia cell samples obtained from patients who underwent allogeneic stem cell transplantation after high dose Bu-based conditioning therapy and either remained in remission for more than one year or who experienced rapidly progressive leukemia. Our results suggest that DNA methylation status (eg. XAF1 gene) may be used to identify leukemia patients who could benefit from Bu-DAC combinations to achieve improved leukemic cytoreduction with the conditioning program.