Imatinib and Leptomycin B Are Effective in Overcoming Imatinib-Resistance Due to Bcr-Abl Amplification or Clonal Evolution but Not Due to Bcr-Abl Kinase Domain Mutation.

Constitutive activity of the Bcr-Abl kinase has been shown to be the causative event in the molecular pathogenesis of CML and Ph+ ALL. Even though Imatinib, a small molecule inhibitor of the Abl kinase, is very effective in treating the disease, development of resistance to this drug is a common phenomenon in advanced stage disease. Using Leptomycin B (LMB), a nuclear export blocker, Vigneri et al. have shown that Bcr-Abl kinase activity trapped in the nucleus induces cell death, thus being a new potential treatment option for Bcr-Abl expressing diseases. To test whether this approach might be effective in the case of imatinib resistant leukaemia several Bcr-Abl positive, imatinib-resistant Ba/F3 clones were established and characterized: 1. Ba/F3 cells resistant due to a point mutation in the Abl kinase domain at position T315I. 2. Ba/F3 cells resistant due to amplification of the Bcr-Abl kinase. 3. Ba/F3 cell clones resistant to imatinib with clear features of clonal evolution. All four cell lines were tested for the efficacy of a combined treatment approach with imatinib and leptomycin B. This combination was very effective in inducing apoptosis in imatinib resistant Ba/F3 cells displaying Bcr-Abl amplification. One out of two cell lines with signs of clonal evolution also responded to this combination treatment. However, imatinib resistant cells expressing the Bcr-AblT315I mutant kinase were completely resistant to this approach. Thus, these results are in agreement with the proposed hypothesis of Vigneri and wang that the combined effect of leptomycin B and imatinib requires Bcr-Abl kinase inhibition (which is not achieved in cells expressing Bcr-Abl T315I) by imatinib and nuclear entrapment by LMB To check the feasibility of this combined treatment for purging purposes, in vitro colony forming assays were performed with mixed cultures of Bcr-Abl positive BaF3 cells and primary murine bone marrow. All colonies formed after combined treatment with imatinib and LMB were derived from BM cells, indicating selective toxicity towards Bcr-Abl positive cells while sparing primary murine bone marrow cells. We are currently evaluating the effectivity of the combined LMB/imatinib treatment as purging strategy in a murine bone marrow transplantation model.