Previous investigations showed that the combination of the transcriptional inhibitor flavopiridol and/or the translational inhibitor homoharringtonine were synergistic with the Abl kinase inhibitor imatinib, in the Bcr-Abl-dependent chronic myelogenous leukemia cell line, K562. This result served as a proof of concept of the sequential blockade strategy, which postulates that reducing the production of the oncogenic kinase by inhibition of transcription and translation would augment the effect of the specific kinase inhibitor. To investigate whether this combination would overcome resistance to imatinib, we studied a series of murine lymphoid Ba/F3 cell lines that were transfected to express either the wild-type Bcr-Abl, or Bcr-Abl with the Abl kinase domain mutations E255K or T315I. Immunoblotting analysis showed that flavopiridol or homoharringtonine decreased the total protein level of Bcr-Abl in all cell line, and hence its kinase activity, as measured by its auto-phosphorylation. The Ba/F3-p210 cells, which express the wild-type Bcr-Abl, were sensitive to imatinib with an IC50 of 0.4 μM in the inhibition of proliferation. The Ba/F3-E255K cells were moderately resistant to imatinib (IC50 = 3.9 μM), whereas the Ba/F3-T315I cells were highly resistant (IC50 = 18.3 μM). Nevertheless, all these cells lines were equally sensitive to flavopiridol (IC50 = 0.2 μM) and homoharringtonine (IC50 = 13.5-17.0 nM), indicating lack of cross-resistance. Clonogenic assays demonstrated similar results: the IC50s for imatinib were 3.1 μM, 10.7 μM and 55.7 μM for Ba/F3-p210, Ba/F3-E255K and Ba/F3-T315I cells, respectively; the IC50s for flavopiridol were within the range of 0.2-0.5 μM, and for homoharringtonine were 127-232 nM for all cell lines. To test for sequence dependence, flavopiridol or homoharringtonine were added prior to, at the same time or after imatinib, and their inhibition of growth was measured by cell counting. There was no apparent difference when the drugs were added either in sequence or simultaneously, suggesting neither sequence advantage, nor adverse drug interaction. Further, the combination of the all three drugs together was additive or mildly synergic in growth inhibition. Clonogenic assays also demonstrated an additive effect as evaluated by the median-effect method. In conclusion, flavopiridol and homoharringtonine reduced the expression of Bcr-Abl protein. They were effective in imatinib-resistant cells and sensitized these cells to imatinib. Clinical application of this sequential blockade strategy could help overcome resistance to imatinib due to the Abl kinase domain mutations.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]