Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells.

Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment.

MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation.

MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011).

This study was funded by the NCI-K23, NCCTG and CLL Global Foundation.

Shanafelt:Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.