Combined inhibition of the Bcl-2 and mTOR pathways: in vitro pro-apoptotic effects in Acute Lymphoyd Leukemia.

Deregulation of the apoptotic machinery and aberrant activation of the mTOR signalling pathway are frequently described in acute lymphoid leukaemia (ALL) cells, playing a pathogenetic role in the processes of aberrant proliferation and chemoresistance. In this study, we thus evaluated the effects of the combined inhibition of both Bcl-2 and mTOR signalling, by using the BH3 mimetic inhibitor ABT-737 (kindly provided by Abbott Lab.) and the mTOR inhibitor CCI-779, respectively. ABT-737 induced cytotoxic effects on the MOLT4 cells characterized by a low Mcl-1 expression (IC50=198nM). In contrast, CEM-S, CEM-R, and JURKAT proved resistant (IC50>5.4uM); interestingly, they all displaying Mcl-1 overexpression. When the effect of CCI-779 was explored on these cell lines, only a minor cytotoxic effect was observed (IC-50 between 0.5µM-28.2µM). We then investigated the drug combination activity on the resistant cell line phenotype (JURKAT cells); a significant (p<0.01) increase in the level of of apoptosis (51% ± 5.5 in the presence of CCI-779 1000nM + ABT-737 1000nM) compared to the single agents (22.9% ± 3.5 and 7.7% ± 3.4 in the presence of ABT-737 and CCI- 779, respectively) was recorded. A similar activity was observed on the CEMR cell line. WB (Western blot) analysis revealed that in both cell lines exposure to CCI-779, particularly in combination with ABT-737, induced a decrease in Mcl-1. However, the observed Mcl-1 down-regulation was not found in the other resistant phenotype (CEM-S). Moreover, interfering Mcl-1 expression by RNAi we did not revert the resistant phenotype in the CEM-S cell line. Primary ALL blasts, obtained from 18 patients were in the majority of the cases highly sensitive to ABT-737 (50nM); in 9 samples, the combination with CCI-779 (5000nM) showed additive or synergistic effects on apoptosis induction. Resistance to ABT-737 was found in one sample; interestingly, the exposure of this resistant sample to the ABT-737 and CCI-779 combination, induced Mcl-1 down-regulation and induction of apoptosis (57.3% in the presence of ABT-737 50nM + CCI-779 5000nM, compared to 14.1% and 18.8% in the presence of ABT-737 and CCI-779, respectively). In summary, ABT-737 is a highly pro-apoptotic agent in ALL cells; the combined use of the mTOR inhibitor CCI-779 may revert some ABT-737 resistant phenotypes and these effects are associated with Mcl-1 down-regulation.