We recently demonstrated that the combined treatment with PD184352, a highly selective inhibitor of MEK1 phosphorylation and activation (kindly provided to us by Dr J. S. Sebolt-Leopold, Cancer Molecular Sciences, Pfizer Global Research & Development, Ann Arbor, MI) and arsenic trioxide (ATO) strikingly potentiates apoptotic cell death induced by ATO in K562 and NB4 leukemic cells through the up-regulation of the proapoptotic p53AIP1 protein (p53-regulated Apoptosis-Inducing Protein 1), via p73 activation (
). Moreover, we showed that MEK1 inhibition sensitizes both parental and arsenic resistant NB4 cell lines to ATO-induced apoptosis through the MEK1 inhibition-mediated dephosphorylation of Bad, increasing its capacity to heterodimerize with Bcl-xL and Bcl-2 thereby blocking theirs antiapoptotic functions (ASH 2003, Abstract 61). p53AIP1 interacts with Bcl-2, affecting p53AIP1-mediated apoptosis through regulation of the mitochondrial transmembrane potential, thus supporting a model in which both pathways (p73-p53AIP1 and Bad phosphorylation) contribute to ATO induced apoptosis and its enhancement by MEK1 inhibitor co-treatment. Collectively, our results prompted us to investigate whether the combination of PD184352 and ATO was effective in inducing apoptosis of primary acute myeloid leukemia (AML) blasts. Twenty primary AML samples with different FAB classification were analyzed at diagnosis. Immunological and genetic characteristics were defined. Primary AML samples were cultured in presence of escalating doses of PD184352 (0.1–20 μM), ATO (0.125–10 μM) or combinations of the 2 agents at a 1:1 ratio (0.25/0.25, 0.5/0.5, 1/1, 1.5/1.5, 2/2). After 48 h of treatment the cells were harvested for mitochondrial transmembrane potential, annexin V and sub-G1 DNA content detection. Combination Index (CI) plots were then generated using the Chou-Talalay method and Calcusyn software (Biosoft, Ferguson, MO). CI less than 1.0 indicates synergism; CI = 1.0 indicates additive effect; CI more than 1.0 indicates antagonistic effect. Interestingly, MEK1 inhibition strikingly increased the apoptosis induced by ATO in sixteen of twenty primary AML samples analyzed. Isobologram analysis confirmed the additive or synergistic nature of this interaction. Five responder patients showed additive effect (one M0, two M2, two M4), eleven synergism (one M1, three M2, three M3, two M4, two M5). In the responsive AML samples the combined treatment with PD184352 and ATO strikingly potentiated loss of mitochondrial transmembrane potential induced by ATO. Treatment with MEK1 inhibitor down-modulated the steady level of ERK and Bad phosphorylation and prevented theirs phosphorylation in rensponse to ATO treatment. Moreover, p53AIP1 expression was enhanced after PD184352 plus ATO treatment compared to ATO alone. In conclusion these results demonstrated a cooperation between these two agents in this setting, and could provide an experimental basis for combined or sequential MEK1 inhibition plus ATO therapy in AML.
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