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Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against unmutated or mutant Bcr-Abl-expressing human leukemia cells.

6592 Background: AMN107 has potent in vitro and in vivo activity against the un-mutated and most mutant forms of Bcr-Abl tyrosine kinase (TK). We have previously demonstrated that in CML cells the pan-histone deacetylase inhibitor LBH589 depletes both unmutated and mutated Bcr-Abl, as well as attenuates p-AKT and Raf-1 levels. In the present studies we determined the effects of AMN107 and/or LBH589 in unmutated or mutated Bcr-Abl-expressing mouse BaF3 cells, or human CML K562 and LAMA-84 cells, as well as in primary CML cells procured from patients with imatinib mesylate (IM)-refractory CML.


METHODS
Cultured and primary CML cells were exposed to 20 to 100 nM AMN107 and/or LBH589 for 24 to 48 hours. Following this, the % of apoptotic cells was determined, as well as immunoblot analyses were performed to determine Bcr-Abl, p-STAT5, p-AKT, p-CrkL, Bim, Bcl-xL and p27 levels. Synergy between the apoptotic effects was determined by the median dose-effect isobologram analysis.


RESULTS
AMN107 was 20-fold more potent than IM, and was more potent in inhibiting Bcr-Abl TK activity and attenuating p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in K562 and LAMA-84 cells. Co-treatment with LBH589 and AMN107 exerted synergistic apoptotic effects against human CML (but not the normal bone marrow progenitor cells), with more attenuation of p-STAT5, p-ERK1/2, c-Myc and Bcl-xL and increased p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of unmutated Bcr-Abl or the IM-resistant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis in IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared to either agent alone, co-treatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells.


CONCLUSIONS
These findings support in vivo testing of the combination of LBH589 and AMN107 against IM naïve or resistant CML, with the goal to eradicate IM sensitive or resistant CML. [Table: see text].