Bcr-Abl-Independent Activation of Src Kinases Associated with Development of Dasatinib Resistance in a CML Patient.

Activation of Src family of kinases (SFK) is associated with chronic myeloid leukemia (CML) disease progression and resistance to imatinib (IM) therapy. Activation of SFK can be either Bcr-Abl-dependent or Bcr-Abl-independent in IM-resistant CML patients and clinical importance of this phenomenon is not completely understood. Second generation of tyrosine kinase inhibitors (TKIs) such as dasatinib, targeting both Bcr-Abl kinase as well as SFK, could represent a logical alternative for treatment of patients with acquired IM resistance due to SFK activation. Recent studies raised question whether overexpression of SFK could lead to an acquired resistance to dasatinib. Sensitivity of individual patients’ leukemia cells to TKIs can be carried out by assessment of inhibition of phosphorylation of Crkl and SFK after incubation of patient’s leukocytes with the drug in vitro. We used 10 μM IM or 250 nM dasatinib in vitro and detection with western blot analysis with anti-Crkl 32H4 monoclonal antibody (Cell SignalingTechnology, Beverly, MA) and Phospho-Src Family (Tyr416) Antibody (Cell Signaling Technology Inc., Danvers, MA). By this functional approach we identified several IM-resistant CML cases, where IM failed to inhibit phospho-Crkl and phospho-SFK in vitro; these patients were either in blast crisis or carried a BCR-ABL mutation. In most cases, however, phospho-SFK were inhibited after incubation of the patients’ leukocytes with dasatinib. The exception was a patient with hematological resistance to IM and acquired resistance to dasatinib therapy due to Bcr-Abl-independent activation of SFK. This patient was a 56-year-old female diagnosed with CML in chronic phase. The cytogenetic examination at the time of diagnosis revealed Ph chromosome and an additional aberration involving derivative chromosomes 2, 3 and 5. After 10 months of IM therapy the BCR-ABL-positive clone was eradicated, but the clinical response to the treatment was unsatisfactory. At the same time the number of cells with the complex additional aberration remained high (>70%). Western blot analysis of the patient’s leukocytes revealed overexpression of phospho-SFK suggesting activation of an alternative signaling pathway not inhibited by IM and independent of Bcr-Abl. In vitro sensitivity to dasatinib provided a rationale for switch to dasatinib therapy. Therefore, dasatinib was administered to the patient (2×70 mg daily). However, a rapid development of resistance to dasatinib followed, which corresponded with a loss of inhibition of SFK in in vitro phosphorylation assay. Interestingly, the phenotype of the patient’s disease resembles a mouse model of v-src gene induced myeloproliferative disease, characterized by splenomegaly, anemia, and increased numbers of immature erythroid cells (