Inhibition of Bcl-2 Signaling at Nanomolar Concentrations by Small Molecule BH3 Inhibitor, ABT-737 as a Novel Therapeutic Strategy in Acute Myeloid Leukemia (AML).

In this study, we investigated the anti-leukemic activity of a novel small molecule BH3 inhibitor (ABT-737) that binds with high affinity to Bcl-2, Bcl-xL and Bcl-w (Ki < 1nM). ABT-737 inhibited 50% of cell growth of HL-60, NB4 and KG-1 cells at IC_50’s below 100nM, while its less active enantiomer did not affect cell growth at concentrations of 1μM. The dose-response curve was shifted to the right in cells over-expressing the drug targets, Bcl-2 or Bcl-X_L (IC₅₀ for HL-60/neo - 1μM, HL-60/BclX_L - 4μM and HL-60/Bcl-2 - 5μM). ABT-737 inhibited Bcl-2/Bax heterodimerization starting at 1 hour as evidenced by IP/Western blot analysis. Furthermore, Bax cleavage (at 3 hours) resulted in pro-apoptotic Bax-p18 and later a change to “death conformation” of Bax. This resulted in mitochondrial depolarization and cleavage of caspases-9, -3 and -8. Bax-knockout Hct116 cells were largely protected from cytolytic activity of ABT-737 demonstrating the critical role of Bax in apoptosis induction. In primary AML samples (n=9), 100 nM ABT-737 induced apoptosis in 70±7% of CD34+ progenitor cells (DMSO-control, 28±4%, p<0.001) at 24 hrs. ABT-737 inhibited clonogenic growth of AML with IC₅₀ of 25nM (n=3) but spared normal progenitors n=3 (no inhibition at 100nM). To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC₅₀>500nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC_50s of 50 and 25nM). This suggests that phosphorylation-dependent conformational change of Bcl-2 affects the ability of BH3 inhibitors to disrupt Bcl-2/Bax dimers and induce apoptosis. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.09 to 0.56. The hypothesis that phosphorylation-dependent conformational change of Bcl-2 affects the ability of BH3 inhibitors to disrupt Bcl-2/Bax dimers and induce apoptosis is presently under investigation. Inhibition of MEK signaling may therefore enhance the efficacy of BH3 inhibitors in AML and possibly other malignancies with phosphorylated Bcl-2. In conclusion, small molecule BH3 inhibitors are potent inducers of apoptosis in myeloid leukemias via disruption of Bcl-2/Bax dimerization and activation of the downstream apoptotic cascade. Differential inhibition of AML but not normal progenitor cells at low nanomolar concentrations advocates further development of BH3 inhibitors as a novel therapeutic strategy for AML.