ABT-737 Cooperates in a Strong Synergism with Tyrosine Kinase Inhibitors to Induce Apoptosis of Chronic Myeloid Leukemia Cells.

BCR-ABL confers to several cell lines an apoptosis-resistant phenotype that is critically dependent on its kinase activity. It has been shown that BCR-ABL can block the mitochondrial step of apoptosis and that tyrosine kinase inhibition induces apoptosis in BCR-ABL expressing cells. Several pro- and anti-apoptotic proteins of the Bcl-2 family modulate the mitochondrial apoptotic signal. Amongst them, anti-apoptotic Bcl-x_L and Mcl-1 are induced by BCR-ABL. Several studies underlined the probable role of Bim expression during tyrosine kinase inhibition-induced apoptosis and we previously confirmed that cells which were depleted in Bim were unable to undergo apoptosis when treated by two tyrosine kinase inhibitors (TKI) : imatinib (IMA) and nilotinib (NIL). Altogether, these results strongly support the prominent role of the balance between pro- and anti-apoptotic proteins of the Bcl-2 family in the apoptotic response of CML cells after TKI treatment. It has been shown that Bim actually is an indirect activator of apoptosis through its anti-Bcl-x_L, -Mcl-1 and -Bcl-2 effects. Recently, ABT-737, a small molecule which binds Bcl-2 and Bcl-x_L but not Mcl-1, was shown to induce apoptosis in several tumour cell types. In this study, we verify if ABT-737 could cooperate efficiently with the Bim stabilizing TKI.

K562 cells were incubated with increasing concentrations of either imatinib or nilotinib, alone or in combination with ABT-737, in a constant ratio. Both TKI and ABT-737 induced a dose dependant apoptosis and the combination of IMA or NIL with ABT-737 resulted in a synergistic cooperation to induce apoptosis. The combination index (CI) calculated using the Calcusyn software was 0.05 for ABT-737 and either IMA or NIL at ED90. Similar experiments were performed on CD34 expressing cells from 12 CML patients, and the strong synergism of ABT-737 with both IMA and NIL was confirmed with a mean CI of 0.25. To elucidate the mechanisms underlying this cooperation, we analyzed the Bcl-2 family proteins expression and confirmed the increase of Bim and the decrease of Bcl-x_L due to TKI treatment while ABT-737 was without effect on the expression of these proteins. Pull-down experiments on K562 cells using anti-Bcl-2 and anti-Bcl-x_L antibodies followed by analysis of Bim showed that ABT-737 decreases the interaction between BIM and Bcl-2 or Bcl-x_L in cell free system as in intact cells. Moreover, Bim was co-immunoprecipitated more efficiently by anti-MCL-1 antibodies when it was displaced from Bcl-2 and Bcl-x_L by ABT-737. This brings some mechanistic support to the complementary effects of TKI (stabilizing Bim) and ABT-737. The expression of several anti-apoptotic proteins was also analyzed. Surprisingly, the combination of TKI with ABT-737 induced a dramatic decrease in XIAP content while each inhibitor alone was without effect on this protein. The decrease in XIAP was accompanied by an increase in caspase 3 cleavage. Cell sorting experiments showed that the decrease in XIAP preceded the drop in mitochondrial membrane potential and the activation of caspase 3 and it was only partly inhibited by a pan-caspase inhibitor and not at all by proteasome or cathepsin B inhibitors.

ABT-737 cooperates with TKI to induce apoptosis of CML cells. This cooperation can be explained in part by the increase in Bim activity induced by TKI associated to the inactivation of Bcl-2, -x_L by ABT-737. However, a side effect at the level of the caspase inhibitor XIAP also seems to participate, resulting in a strong synergism. Thus, the association between TKI and Bcl-2 inhibitors could be a facilitating strategy to induce apoptosis in some BCR-ABL expressing cells resistant to TKI alone.

Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.