Combination of the AKT Inhibitor Perifosine with the HSP90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin (17-DMAG) Has Synergistic Activity in Multiple Myeloma (MM).

Abstract The serine-threonine kinase AKT is a mediator of tumor proliferation, and its inhibition leads to induction of apoptosis in MM. Heat shock protein-90 (HSP90) is a chaperone protein involved in the refolding of proteins destabilized by stress, including AKT. HSP90 inhibitors have demonstrated in vitro and in vivo activity in MM, and preliminary activity in a phase I clinical trial in MM. We hypothesized that the combination of agents that target two dysregulated pathways in MM, and that interact at the level of AKT will lead to a synergistic cytotoxic activity in MM. MM cell lines with high level of AKT activity (OPM2) and lower AKT activity (multiple dexamethasone-sensitive MM.1S, dexamethasone-resistant MM.1R, and plasma cell leukemia cell line OPM1) were exposed to serial dilutions of perifosine 2-50uM (KRX-0401, Keryx, NY, NY, provided by the NCI) and 17-DMAG 10-200nM (supplied by NCI) alone and in combination for 48 hrs. Inhibition of proliferation was measured using the MTT proliferation assay. Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, CA). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method. A two-sided t-test was used to determine differences in response. Perifosine induced a dose dependent inhibition of proliferation in all cell lines tested with 30uM inducing 49% inhibition as compared to control and 50uM inducing 60% inhibition in MM.1S cells. Perifosine 30uM induced more significant apoptosis in cell lines with high AKT activity (OPM2) with 51% apoptosis as compared to 14.7% in MM.1S cells with lower AKT activity (p=0.001). 17-DMAG demonstrated a dose dependent inhibition of proliferation and induction of apoptosis in all cell lines tested with 17-DMAG 100nM inducing 40% inhibition as compared to control and 200nM inducing 56% inhibition in MM.1S. There was no differential response to 17-DMAG in cell lines tested. The combination of 30uM perifosine and 100nM 17-DMAG resulted in a significant inhibition of proliferation with 76% inhibition as compared to each agent alone (p=0.0001, perifosine alone vs. combination). The combination was synergistic with a combination index of 0.1 according to the Chou-Talalay method. Apoptosis analysis at 48 hrs demonstrated 13.9% apoptosis with perifosine 30uM, 3.1% with 17-DMAG 100nM alone, and 47.9% with the combination of the two agents (p=0.004 combination vs. perifosine). The combination of the AKT inhibitor, perifosine and HSP90 inhibitor, 17-DMAG demonstrated a synergistic anti-proliferative and pro-apoptotic effect on MM cell lines as compared to each agent alone. Cell lines with higher AKT activity were more sensitive to the AKT inhibitor, perifosine. Targeting both the PI3kinase pathway and the heat shock protein response represents an attractive approach to future therapeutic options in relapsed/refractory MM where drug resistance is often a major problem. Furthermore, the differential activity noted among higher AKT activity and lower AKT activity cell lines raises the possibility of tailoring therapy based on AKT expression levels in the future.