Abstract
In hematologic malignancies, constitutive activation of the Raf/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Indeed, we have recently demonstrated that selective MEK-I potently inhibit the growth of AML cell lines and ex vivo-cultured primary AML blasts (
Blood 2006, 108:254
). However, these effects are mostly related to the inhibition of cell cycle progression, while apoptosis induction requires higher concentrations of the inhibitors and longer times of exposure. Thus, we investigated MEK-I-induced changes in phospho-protein expression and gene expression profiles, in order to identify relevant downstream targets and to design rational MEK-I-based combination strategies. Analysis of phosphorylation levels of 18 different target proteins performed in OCI-AML3 cells indicated that MEK blockade induces, among other effects, an over-activation of RAF and MEK, suggesting the interruption of a negative feedback loop. Moreover, gene expression profiling indicated that, in the same cellular model, MEK-I induced upregulation of the Flt-3 receptor. Based on these observations, as well as on recent evidence indicating that the Raf inhibitor sorafenib directly inhibits signaling through Flt-3 (JNCI 2008, 100:184
), experiments were performed in OCI-AML3 and MOLM-13 (which harbors a Flt3 ITD) cells to test the activity of MEK-I in combination with sorafenib. Simultaneous inhibition of Flt3/Raf and MEK resulted in the synergistic inhibition of cell growth, as measured by isobologram analysis (Chou–Talalay method) in both model systems, with combination indexes (CI) of 0.12 and 0.48 for OCI-AML3 and MOLM-13 cells, respectively. Neither sorafenib nor MEK-I induced apoptosis in either cell line when used alone; however, apoptosis was observed in up to 50% of the cells with the combined treatment. Based on our previous experience, as well as on the ability of MEK-I to modulate the expression, among others, of genes controlling mitochondrial homeostasis (e.g. PPIF, GRPEL1), we next investigated the impact of simultaneous inhibition of the MEK and Bcl-2 pathways in AML cells. Exposure of OCI-AML3 and MOLM-13 cells to a combination of MEK-I and the Bcl-2/Bcl-xL inhibitor, ABT-737 (kindly provided by Abbott Laboratories) synergistically inhibited cell growth, with CI ranging from 0.45 to 0.04 in OCI-AML3 and from 0.75 to 0.14 in MOLM-13, respectively. In both cellular models, ABT-737dose-dependently induced apoptosis, while MEK-I, at the concentrations used in combination experiments, did not appreciably increase apoptotic cell death; however, simultaneous Bcl- 2/Bcl-xL inhibition and MEK blockade resulted in the massive induction of apoptosis (up to 85% and 67% net apoptosis induction in OCI-AML3 and MOLM-13 cells, respectively). Such pro-apoptotic interaction was highly synergistic with CI of 0.18 and 0.16 in OCIAML3 and MOLM-13 cells, respectively. In contrast, combination with MEK-I did not appreciably sensitize the MEK-I-resistant cell line U937 to either sorafenib- or ABT-737- induced growth inhibitory and pro-apoptotic effects. Overall these results support the role of the Raf/MEK/ERK kinase module as a prime target for the molecular therapy of AML and suggest that both “vertical” and “lateral” combination strategies based on MEK inhibition may produce highly synergistic anti-leukemic effects.Disclosures: No relevant conflicts of interest to declare.
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2008, The American Society of Hematology
2008
