AKT INHIBITORS AS CHEMOSENSITIZERS Deborah Defeo-Jones, Raymond Jones, Stan Barnett, Paula Hancock, Kathleen Haskell, Karen Leander, Ron Robinson, Craig W. Lindsley, Zhijian Zhao and Hans E. Huber * Departments of Cancer Research and Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 *
[email protected] INTRODUCTION. The PI3K pathway is upregulated in most human cancers, primarily via activation of growth factor receptors, overexpression of their ligands or inactivation of tumor suppressor PTEN (1). Activation of this pathway provides growth and survival signals and has been postulated to contribute to tumor progression and multi-drug resistance (MDR) in the clinic. The Akt/PKB family of serine/threonine kinases sits a critical juncture in the PI3K pathway, downstream of PTEN but upstream of multiple divergent effector cascades (2). The Akt family consists of three members – Akt1, Akt2 and Akt3 – whose relative contributions to tumorigenesis and MDR are unknown. We identified isozyme-specific inhibitors of Akt1 and Akt2 that, due to an allosteric mechanism of action, are highly specific for Akt over closely related kinases (3). These inhibitors allowed us to probe the role of Akt isozymes in survival signaling and protection from apoptotic stimuli. RESULTS. We find that inhibition of the PI3K/Akt pathway by small molecule Akt inhibitors results in sensitization of many different human tumor cell lines to a variety of apoptotic stimuli, including DNA damaging agents, microtubulebinding agents, targeted therapeutics and radiation. We used Akt1 and Akt2 specific inhibitors (Figure 1) and overexpression of Akt3 to monitor the role individual Akt isozymes play in the cyto-protective effect of this pathway. Under multiple experimental conditions Akt1/2 dual inhibitors or combinations of Akt1 plus Akt2 inhibitors proved to be more effective than inhibitors of either isozyme alone. As an example, the synergistic induction of caspase 3 by combination
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treatment with topoisomerase antagonist, camptothecin, and Akt inhibitors is shown in Figure 2. Fig. 1: Structures of Akt isozyme-specific inhibitors. (A) Akt1 specific compound; Akt1/Akt2 IC50 ratio of 0.03; (B) Akt2 specific; Akt1/Akt2 IC50 ratio of 60.
Fig. 2. Induction of caspase 3 activity in LnCaP cells treated with vehicle, camptothecin, and Akt1 and Akt2 inhibitors, alone or in combination as indicated.
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Dual inhibitors of Akt1 and Akt2 proved to be as effective as PI3K inhibitors, such as LY294002. Akt3, whether endogenous or overexpressed exogenously, was not able to protect cells from apoptotic stimuli, at least in cell culture. mTOR inhibitors have been reported to sensitize cells to apoptotic stimuli and to be particularly effective in PTEN mutant cell lines. With the majority of tested cell lines (LnCaP, HT29, MDA-MB468, MCF7) Akt1/2 dual inhibitors were much more effective at inducing caspase than was rapamycin. The PTEN status did not correlate with the sensitivity of these cell lines to Akt inhibitors. The PI3K/Akt pathway mediates signaling by many different growth factors and cytokines, including insulin. Akt1/2 double knock-out mice are embryonic lethal and Akt2 knock-out mice have a diabetic phenotype. However, we find that intermittent inhibition of Akt1 and Akt2 in rodents is well tolerated. Our data indicate that combination treatments with Akt inhibitors to sensitize tumors to standard chemotherapeutics and targeted agents should be feasible. REFERENCES. 1. Vivanco, I. and Sawyers, C.L. (2002) Nature Reviews Cancer 2, 489-501.
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2. Luo, J., Manning, B.D. and Cantley, L.C. (2003) Cancer Cell 4, 257-262. 3. Barnett, S.F., Defeo-Jones D., Fu, S., Hancock P.J., Haskell K.M., Jones, R.E., Kahana J.A., Kral, A.M., Leander, K., Lee, LL., Malinowski, J., McAvoy, E.M., Nahas, D.D., Robinson, R., Huber, H.E. (2004) Biochemical J.; in press.
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