Cytoprotective Autophagy Induction by Imatinib Mesylate In Non-BCR-ABL Expressing Cells

Abstract 4937

Autophagy is an intracellular degradation system that delivers cytoplasmic constitutions to the lysosome. Intracellular proteins and organelle are sequestered by the autophagosomes, then delivered to the lysosome and degraded. It has been reported that combining imatinib mesylate (IM) with an autophagy inhibitor such as chloroquine resulted in enhanced elimination of CML clones in vitro and in vivo (Bellodi C et al. J. Clin. Invest. 2009). We here report that IM induces the cytoprotective effect in non-BCR-ABL expressing cells along with autophagosome formation.

Treatment with IM resulted in an increased viable cell number of non-BCR-ABL expressing leukemia cell lines by inhibiting spontaneous apoptosis. IM did not show any effects on cell-cycle progression. When HL-60 cells were cultured in the presence of 3–30 μM of IM for 24–96 hr, up to a 1.6-fold increase in the viable cell number was observed in a dose-dependent manner. Furthermore, when HL-60 cells were cultured either under serum depletion or at lower concentrations of FBS, the cytoprotection by IM became more pronounced as compared with the cells cultured with 10% FBS. As well as in BCR-ABL expressing CML cell lines, electron microscopy revealed an increased autophagosomes after IM treatment in non-BCR-ABL expressing cells. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). IM attenuated the cytotoxic effect of cytosine arabinoside and bortezomib in leukemia cells.

Inhibition of autophagy by knockdown of LC3 gene using shRNA in HL-60 as well as complete knockout of atg5 in the Tet-off atg5^-/- MEF system attenuated the cytoprotective effect of IM. This indicates that the effect is at least in part dependent on autophagy. However, the cytoprotection by IM was not medicated through supression of ROS production via “mitophagy” or reduction of ER stress via “ribophagy”. The cytoprotection with autophagy was still detectable in the HL-60 cells after knockdown of c-abl gene and in an ABL deficient-HL-60 cell line. This indicates that the proapoptotic function of ABL kinase is not involved. In addition, this effect was still observed in the presence of cycloheximide, suggesting that the effect is not mediated through translation process.

Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of targeting tyrosine kinase(s) for induction of cytoprotective autophagy.