COMBINED BCL-2 AND MTOR INHIBITION IN ACUTE LYMPHOBLASTIC LEUKEMIA CELLS
S Iacovelli,1 MR Ricciardi,1 S Decandia,1 P Bergamo,2 A Miele,1
A Vitale,1 AM Testi,1 MT Petrucci,1 M Milella,2 R Foà,1 A Tafuri1
Background. Acute lymphoblastic leukemia (ALL) is a malignant disorder of lymphoid progenitor cells. Leukemic cells resistant to induction chemotherapy lead to persistence of disease or relapse and ultimately to patients’ death. We have previously observed that ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, exerts potent cell growth inhibition and apoptosis induction in ALL cell lines and primary samples. It has also been reported that deregulation of mTOR signalling is frequently found in ALL and mTOR inhibition by CCI-779 is associated with anti-leukemic effects in pre-clinical models. Aim. Aim of the study was to investigate in ALL cell lines and primary leukemic blasts the effect on cell proliferation and apoptosis of ABT-737 and CCI-779 alone or in combination Results. In MOLT-4 cells, ABT-737 induced a dose and time-dependent growth inhibition (IC-50= 198 nM) followed, at higher concentrations (250-500 nM), by apoptosis induction. In contrast, CEM-S (IC-50=12.1 μM), JURKAT (IC-50=66 μM), DAUDI (IC-50=67.8 μM) and RAJI (IC-50=1.6¥1020 μM) proved resistant. Western Blot (WB) analysis revealed that all of them share Mcl-1 overexpression, already reported as a resistant factor to ABT-737. When we explored the effects of CCI-779 on the aforementioned cell lines, only minor cytotoxic effects were found at higher concentrations (IC-50 ranging between 0.5 μM to 28.2 μM), as demonstrated in the MOLT-4 cells which showed a biphasic dose response with a flat curve (35-55% growth inhibition) at concentrations ranging between 1 nM and 5000 nM (IC50=9,87 μM). Apoptosis induction, as measured by Annexin-V positivity, was not observed until 72h at 10000 nM. Instead, cell cycle effects were noticed, as shown by a S-phase decrease, from 38.03%±3.7 (vehicle) to 27.7±7.8% at 5000nM (P=0.03). Aiming at investigating the activity of ABT-737 plus CCI-779 on the resistant phenotypes, we exposed JURKAT cells to the above combination (each of them at 1000nM). A significant (P=0.04) induction of apoptosis was found, as measured by an increase of the sub-G1 peak, to 47.7±5.9% (CCI-779+ABT-737) compared to the effects of the single agents (17.4±1.5% and 4.2±1.5% in the presence of ABT-737 and CCI-779, respectively). WB analysis revealed in the presence of CCI-779 and, particularly, its combination with ABT-737, a decrease of Mcl-1 level. Results obtained on primary cells from 8 ALL patients treated in vitro with of ABT-737 (ranging from 50 to 100 nM) and CCI-779 (ranging from 5000-10000 nM) alone or in combination, showed an increase of the sub-G1 peak in 5/8 and in 3/8 samples exposed to ABT-737 and to CCI-779, respectively, while synergistic effects on apoptosis induction were found in 2/8 primary samples. Summary/Conclusions. We observed that CCI-779 can potentiate growth inhibition and apoptosis induction of ABT-737 on ALL cells. Interestingly, the combined use of both inhibitors exerts synergistic cytotoxic effects on the JURKAT cells, mediated by the down-regulation of Mcl-1. Pre-clinical studies on an initial series of primary ALL samples, support the investigation of the active oral ABT-737 compound, ABT-263, as a novel therapeutic approach in ALL, aimed at overcoming selected resistance mechanisms.