Multiple Strategies Based on MEK, mTOR and BCL-2 Signal Transduction Inhibition in Acute Lymphoblastic Leukemia (ALL)

Abstract

In spite of improvements in the last decade, relapses still occurs in the majority of ALL patients, with a long-term survival rate of only 30–40%. Recently, small-molecule inhibitors have been developed for targeting deregulated signal transduction pathways involved in proliferation and apoptosis. Current evidence identifies the Raf/MEK/ERK, the PI3K/AKT/mTOR, and the Bcl-2 pathways as potentially relevant targets for therapeutic intervention. We have previously demonstrated that constitutive ERK phosphorylation is an independent predictor of failure to achieve complete remission in adult ALL (Blood 2007; 109:5473) and thus we evaluated the in vitro activity of MEK-inhibitors. However, neither PD98059 nor PD0325901 affected cell growth, cell cycle distribution, and/or apoptosis in ALL cell lines (IC50 >1 μM for PD0325901). These results were confirmed in primary ALL samples, in which PD98059 significantly (P=0.012) inhibited ERK phosphorylation in 8/12 samples, without inducing cell cycle changes or apoptosis. We next investigated the activity of the mTOR inhibitor Temsirolimus. Temsirolimus displayed a biphasic dose-response in a panel of different ALL cell lines, with a flat curve (35–55% of inhibition) at concentrations ranging between 1 and 5,000 nM and more pronounced growth inhibition at concentrations ≥ 10 μM. The CEM cell line was the most sensitive (IC50: 7 nM), Jurkat showed intermediate sensitivity (IC50: 200 nM), while MOLT-4 were resistant (IC50 > 20,000 nM). Cell growth inhibition was associated with inhibition of cell cycle progression, while apoptosis induction was observed in less than 15% of the cells even at the highest concentration of Temsirolimus (20 μM). We then investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories to A.T.), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, in ALL cell lines and in 10 primary samples. ABT-737 showed a potent dose- and time-dependent growth-inhibitory activity in MOLT-4 (IC50: 198 nM), associated with loss of mitochondrial membrane potential, caspase activation, Bcl-2 cleavage, and ultimately apoptosis induction. Conversely, CEM cells proved resistant (IC50: 12,000 nM). We also found that ABT-737 was highly effective in primary adult and childhood ALL, independent of their chromosomal abnormalities, with a significant decrease in viability (p=0.008) and a remarkable induction of apoptosis (from a mean baseline value of 16.8±8.8% to 43.6±22.8%, p=0.04) at 10 nM ABT-737. A dose-dependent down-regulation of Bcl-2 and Bcl-xL expression was observed in sensitive samples, but not in the only resistant one. In summary, our study shows that ALL cells, do not undergo apoptosis in response to single-pathway inhibition, suggesting the presence of multiple, redundant pathways that preserve leukemic cell survival. The only notable exception was the Bcl-2/Bcl-xL inhibitor ABT-737. Studies are ongoing to identify mechanism-driven combinations of agents that would disrupt multiple signal transduction pathways, resulting in synergistic killing and, ultimately, in novel therapeutic strategies for ALL.