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Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

We investigated the constitutive activation of the MEK/ERK pathway in acute myelogenous leukemia (AML) via a flow cytometric technique to quantitate expression of phosphorylated ERK (p-ERK). A total of 42 AML samples (16 newly diagnosed, 26 relapsed/refractory) were analyzed. Normal bone marrow CD34+ cells (n = 10) had little or no expression of p-ERK, while G-CSF-mobilized CD34+ cells exhibited enhanced p-ERK levels. Markedly elevated p-ERK levels were found in 83.3% of the AML samples, with no differences observed between the newly diagnosed and relapsed/refractory samples. Treatment with a MEK inhibitor resulted in significantly decreased p-ERK levels in both the newly diagnosed and relapsed/refractory samples, which was associated with growth arrest, but not apoptosis induction. In summary, we defined conditions for the analysis of MAPK signaling in primary AML samples. Normal CD34+ cells expressed very low levels of p-ERK, and increased p-ERK levels were found in normal G-CSF-stimulated circulating CD34+ cells. Constitutively high p-ERK levels observed in the majority of AML samples suggest deregulation of this pathway that appears to be independent of disease status. The ability of ERK inhibition to promote growth arrest rather than apoptosis suggests that clinical trials of MEK/ERK inhibitors may be more effective when combined with chemotherapy.

Overexpression of protein kinase C alpha (PKC alpha) promotes Bcl2 phosphorylation and chemoresistance in human acute leukemia cells. The contribution of non-Bcl2 mechanisms in this process is currently unknown. In this report, overexpression of PKC alpha was found not to affect cell proliferation, cell cycle, or activation of mitogen-activated protein kinases. The failure of PKC alpha overexpression to activate non-Bcl2 survival pathways suggested that PKC alpha-mediated chemoresistance requires Bcl2. Supporting this notion, REH/PKC alpha transfectants were found to be as sensitive to HA14-1 (a drug that targets Bcl2 function) as parental cells. In addition, HA14-1 abrogated PKC alpha's ability to protect REH cells from etoposide. These findings suggested that Bcl2 is necessary for the protective function of PKC alpha in REH cells. Since Bcl2 phosphorylation status is negatively regulated by protein phosphatase 2A (PP2A) and PP2A regulates PKC alpha, we investigated whether PKC alpha can conversely regulate PP2A. Overexpression of PKC alpha was found to suppress mitochondrial PP2A activity by a mechanism that, at least in part, involves suppressed expression of the regulatory subunit comprising the Bcl2 phosphatase (ie the PP2A/B56 alpha subunit). The ability of PKC alpha to target both Bcl2 and the Bcl2 phosphatase represents a novel mechanism for chemoresistance.