mTOR and Bcl-2 Combined Inhibition: Synergistic Effects in Acute Lymphoblastic Leukemia Cells on Proliferation and Apoptosis, Through Mcl-1 Down-regulation.

ALL cells are frequently characterized by the constitutive activation of the mTOR signaling cascades. Thus, molecular therapies targeting mTOR have been proposed and    mTOR inhibitors proved effective on reducing cell proliferation with marginal activity on apoptosis. Therefore, a combined treatment with mTOR inhibitors and Bcl‐2 inhibitors has been reported. In addition, since the Mcl‐1 overexpression is reported as the major resistance factors to the Bcl‐2 inhibitor, ABT‐737, the combined use with mTOR inhibitors which control trough the AKT/mTOR pathway, Mcl‐1 levels, is conceivable. The aim of this study was to evaluate the combined effects of mTOR (CCI‐779) and Bcl‐2/Bcl‐XL (ABT‐737) inhibition in ALL cell lines and primary samples. In MOLT‐4 cell line exposure to CCI‐779 induced a flat dose‐response curve (35‐55% growth inhibition) at concentrations ranging between 1 and 5000 nM and apoptosis induction was not seen until 5000 nM. In  CEM‐S, CEM‐R and JURKAT cell lines, only minor cytostatic effects were observed until 20000 nM. In MOLT‐4 cells, ABT‐737 induced dose and time‐dependent growth inhibition (IC‐50= 198nM)    followed at higher concentrations (250‐500nM) by induction of apoptosis. In contrast, the CEM‐S, CEM‐R and JURKAT cells, proved resistant (IC‐50 >5 µM), displaying   Mcl‐1 overexpression. Therefore, we investigated the combined activity of ABT‐737 and CCI‐779, on the resistant phenotypes. The JURKAT cell line showed a significantly higher (p< 0.01) induction of    apoptosis following exposure to ABT‐737 and CCI‐779 (both at 1000nM), as compared to the single agents. A similar activity was observed on the CEM‐R cell line. In both cell lines, CCI‐779 exposure down‐regulate Mcl‐1 protein levels by proteasome degradation. This effect was associated with AKT S473 de‐phosphorylation, following prolonged CCI‐779 exposure. However, Mcl‐1 down regulation was not uniformly induced in the resistant phenotypes, as demonstrated in the CEM‐S cell line. Furthermore, in this case,    RNA interference of Mcl‐1, did not revert the resistant phenotype. In primary ALL blasts, CCI‐779 exposure (5000 nM) induced in 4/21 samples only weakly apoptosis (sub‐G1 peak< 20%) while ABT‐737 (50nM) induced higher levels (> 40%) of apoptosis in the majority (15/21) of them. Among the remaining six samples, resistant to ABT‐737, the combined CCI‐779 and ABT‐737 treatment overcame resistance, inducing Mcl‐1 down‐regulation and apoptosis (in 2/6). In summary, the Bcl‐2/Bcl‐XL (ABT‐737) inhibition is an active proapoptotic treatment of ALL, in addition the combined use of mTOR (CCI‐779) inhibition may revert ABT‐737 resistant phenotypes in a proportion of ALL, via AKT inactivation and Mcl‐1 degradation. However, different resistant mechanisms involved in ALL cells needs further investigation.