Preclinical Study to Sensitize Acute Lymphoblastic Leukemia Primary Cells by Combined mTOR and BCL-2 Inhibition with CCI-779 and ABT-737.
Authors: Ricciardi Maria Rosaria , Decandia Samantha , Bergamo Paola , Benedictis Cinzia De , Vitale Antonella , Testi Anna Maria , Fei Joanna , Petrucci Maria Teresa , Milella Michele , Foà Robin , Tafuri Agostino 
Published: October 2019

Abstract
Abstract 985
Poster Board I-7
Acute lymphoblastic leukemia (ALL) is characterized by a high relapse rate, especially in adult patients, with the majority developing eventually chemoresistance. Therapeutic strategies based on targeted therapy have improved survival especially in Philadelphia-chromosome positive patients. We have previously demonstrated 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 (Blood, 2007; 110:53a). However, a resistant phenotype, mostly characterized by Mcl-1 overexpression, has been identified for this drug. In this study we have explored the possibility of extending our previous observations by modulating in addition to apoptosis, also aberrant proliferative signals. Since Temsirolimus (CCI-779), a PI3/AKT/mTOR inhibitor, has been reported to induce growth arrest and apoptosis in preclinical models of ALL, we have investigated in ALL cell lines and primary human ALL cells the effect on cell proliferation and apoptosis of CCI-779 alone and in combination with ABT-737. Exploring CCI-779 activity in ALL cells as a single agent, a biphasic dose response was observed in MOLT-4 cells (IC50: 9865 nM), with a flat curve at concentrations (35-55% of inhibition) ranging between 1 nM and 5000 nM, and a more pronounced growth inhibition at concentrations ≥10000 nM, as demonstrated by the MTT based-assay. At these concentrations, induction of apoptosis was not seen up to 5000 nM, while at 24h the S-phase decreased from 37.6% ± 3.2 (vehicle) to 26.1% ±7.8 (p=0.04). Conversely, ABT-737 induced dose- and time-dependent growth inhibition in MOLT-4 cells (IC-50=198nM). Apoptosis induction, as measured by Annexin-V positivity, was not seen at lower concentrations (50 nM), but required higher concentrations of ABT-737 (controls from 11.9% ± 2, ABT-737 at 500 nM 62.7% ± 18.2). Using concentrations that only induce minimal apoptosis (1.3 and 1.4 fold increase with CCI-779 at 5000 nM and 50 nM of ABT-737, respectively), the combined treatment induced a 3.2 fold increase (p= 0.05) after 48 hours. These synergistic effects on induction of apoptosis were not seen on cell cycle modulation. Exploring then the CCI-779 activity on the T-ALL Jurkat cells, a scalar effect on cell growth inhibition was seen at increasing concentrations (from a 34%, to 46% and to 81% at 10, 1.000 and 15.000 nM, respectively). Jurkat cells were resistant to ABT-737 (IC-50=66 uM). This resistant phenotype was however converted to sensitivity by the combined use of ABT-737 and CCI-779 (1000 nM) which increased Annexin-V positive cells from 6.3% ± 1.1 (vehicle) to 10.5 ± 1.5 (CCI-779), 23.7 ± 1.5 (ABT-737) and 54 ± 5.9 (CCI-779 + ABT-737) (p=0.04). Thus, a synergist effect on apoptosis was found in T-ALL by combining CCI-779 and ABT-737. We also confirmed in this resistance model the lack of enhanced cell cycle effects. We then evaluated primary ALL cells obtained from six Philadelphia-chromosome negative patients with B-lineage ALL (5) and T-ALL (1) by treating enriched lymphoblasts with CCI-779 (ranging from 5.000 to 10.000 nM) and ABT-737 (ranging from 50 to 100 nM) alone and in combination. Although ABT-737 was generally effective in inducing apoptosis, an increase above 75% of the sub-G1 peak was seen in 2/6 and in 3/6 samples exposed to CCI-779 and to ABT-737, respectively, while the combination of both inhibitors was effective in 4/6 primary cases. In fact, the combination with CCI-779 increased apoptosis from 14.5% (vehicle), to 28.6% (50 nM of ABT-737), 33.3% (5000 nM of CCI-779), 71.8% (ABT-737 + CCI-779). In conclusion, we demonstrated that CCI-779 can potentiate the effect of ABT-737 in ALL cells.

Disclosures:
Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria.