BCR/ABL-kinase mutations frequently mediate clinical resistance to the selective tyrosine kinase inhibitor Imatinib mesylate (IM, Gleevec®). However, mechanisms that promote survival of BCR/ABL-positive cells before clinically overt IM resistance occurs have poorly been defined so far. Here, we demonstrate that IM-treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTor)-pathway in BCR/ABL-positive LAMA-cells and primary leukemia cells in vitro, as well as in a chronic phase CML patient in vivo. In fact, PI3K/Akt-activation critically mediated survival during the early phase of IM resistance development before manifestation of BCR/ABL-dependent strong IM resistance such as through a kinase mutation. Accordingly, inhibition of IM-induced Akt activation using mTor inhibitors and Akt-specific siRNA effectively antagonized development of incipient IM-resistance in vitro. In contrast, IM-resistant chronic myeloid leukemia (CML) patients with BCR/ABL kinase mutations (n=15), and IM-refractory BCR/ABL-positive acute lymphatic leukemia patients (n=2) displayed inconsistent and kinase mutation-independent autonomous patterns of Akt-pathway activation, and mTor-inhibition overcame IM resistance only if Akt was strongly activated. Together, an IM-induced compensatory Akt/mTor activation may represent a novel mechanism for the persistence of BCR/ABL-positive cells in IM-treated patients. Treatment with mTor inhibitors may thus be particularly effective in IM-sensitive patients, whereas Akt-pathway activation variably contributes to clinically overt IM resistance.

BCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFN gamma-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors.

Chronic myeloid leukemia (CML) is a clonal disease of hematopoietic stem cells caused by a reciprocal translocation of the long arms of chromosomes 9 and 22. In human leukocyte antigen A*0201(+) (HLA-A*0201(+)) individuals, response after interferon-alpha (IFN-alpha) was shown to be associated with the emergence of CML-specific cytotoxic T cells that recognize PR-1, a myeloblastin (MBN)-derived nonapeptide. In contrast, imatinib potently induces remissions from CML by specific inhibition of the ABL tyrosine kinase. Here, we explored molecular regulations associated with CML responses under different treatment forms using cDNA-array. Expression of MBN was found to be down-regulated in remission under imatinib therapy (0 of 7 MBN(+) patients). In contrast, MBN transcription was readily detectable in the peripheral blood in 8 of 8 tested IFN-alpha patients in complete remission (P =.0002). IFN-alpha-dependent MBN transcription was confirmed in vitro by stimulation of peripheral blood mononuclear cells (PBMCs) with IFN-alpha and by IFN-alpha-mediated activation of the MBN promoter in reporter gene assays. Finally, with the use of HLA-A*0201-restricted, MBN-specific tetrameric complexes, it was demonstrated that all of 4 IFN-alpha-treated patients (100%), but only 2 of 11 imatinib patients (19%), in complete hematological or cytogenetic remission developed MBN-specific cytotoxic T cells (P =.011). Together, the induction of MBN expression by IFN-alpha, but not imatinib, may contribute to the specific ability of IFN-alpha to induce an MBN-specific T-cell response in CML patients. This also implies that the character of remissions achieved with either drug may not be equivalent and therefore a therapy modality combining IFN-alpha and imatinib may be most effective.