Abstract
Background: The Bcr-Abl tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, in some patients cells are selected with point mutations in the Bcr-Abl kinase domain that decrease drug sensitivity. A deletion mutant lacking amino acids 248–274 was recently reported in 2 patients with the L248V mutation (Gruber et al, Leukemia, 2006). In addition, missplicing of BCR to ABL exon 4 or 5 has been reported to be common in CML patients (Volpe et al, Cancer Research, 2007). However, the incidence and functional relevance of deletion mutants involving ABL exons including the kinase domain are unknown. Wild type Bcr-Abl coexpression with kinase dead mutants that retain the coiled-coil domain of Bcr may result in hetero-dimer and -tetramer formation. In turn, this could produce a dominant-negative effect through decreased Bcr-Abl transphosphorylation.
Methods: Bone marrow and peripheral blood samples were obtained following informed consent from randomly selected CML patients followed in the OHSU outpatient clinic. Nested RT-PCR for BCR-ABL was performed using 2 sets of primers from the breakpoint to the kinase domain. Sequencing covered the Cap, SH3, SH2, linker and kinase domains. Deletions involving the P-loop of the kinase domain were further characterized. To test activity, the mutations introduced into Abl were expressed in HEK293 cells and immunoprecipitated for kinase assays. To test for dominant-negative effects, we generated Ba/F3 lines coexpressing low (L), medium (M) and high (H) levels of deletion mutant Bcr-Abl together with native Bcr-Abl. These cells were tested for IL-3 independence, autophosphorylation and imatinib sensitivity.
Results: We found 14 of 101 (14%) patients to have a deletion mutation. Five patients were found to have a deletion of amino acids 184–274 (Δ184–274), corresponding to loss of ABL exon 4. Other mutants were Δ27–183 (out of frame, stop at 296) (N=3), Δ248–274 with the L248V mutant (N=2) and Δ342–383 (N=2). Also, one patient each had Δ362–444 (loss of ABL exon 7, out of frame, stop at 458) and Δ151–183. Immunoprecipitated Abl proteins with the P-loop deletion mutations were kinase inactive (Figure 1A). P-loop deletion mutants were transformation defective, while coexpression of deletion mutant and native Bcr-Abl conferred growth factor independence and Bcr-Abl signaling was unimpaired. Unexpectedly, imatinib sensitivity was consistently increased in cells coexpressing native and deletion mutant Bcr-Abl (Figure 1B).
Conclusions:
Deletion mutants are relatively common in patients on imatinib therapy (14% in our series) and most frequently involve missplicing.
P-loop deletion mutants are kinase inactive.
Coexpression of deletion mutants with wild type Bcr-Abl confers increased sensitivity to imatinib and thus may improve response to the drug.
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