Anti-Apoptotic Proteins, HSP90 and Activated STAT3 Contribute to Busulfan Resistance of Myeloid Leukemia Cells.

Busulfan(Bu)-based chemotherapy is a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) of patients with acute and chronic myelogenous leukemia (AML, CML). A major obstacle to successful HSCT is Bu resistance, which might be attributed to individual differences in drug pharmacokinetics and metabolism, or inherent resistance of cancer cells. We hypothesize that gene expression profiling of leukemia cells can be used to dissect the factors that contribute to their Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed by microarray and real-time RT-PCR techniques to identify differentially expressed genes. The CML B5/Bu250⁶ cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu250⁶ cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with constitutively up-regulated anti-apoptotic genes (BCL-X_L, BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR).). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by ∼ 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. Inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu250⁶ and KBM3/Bu250⁶ more Bu-sensitive. Analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy had alterations in gene expression that were analogous to those observed in the in-vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance. Our results suggest the important roles of apoptotic signaling mechanism, HSP90 and STAT3 and should be considered in the classification of AML patients who will likely benefit from busulfan-based pretransplant conditioning therapy and those who should be offered alternative treatment modalities.