Abstract 2057: In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL.
Authors: Gomez Fabiola , Chen Lisa , Wierda William , Gandhi Varsha 
Published: August 2014

Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the functional stability of client oncoproteins, which is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins such as Akt, p53, STAT3, Bcr-Abl, and Pim-1. Pim (Pim-1, -2, -3) kinases are constitutively active serine/threonine kinases overexpressed in hematological malignancies such as chronic lymphocytic leukemia (CLL). Pim kinases have several substrates which are involved in gene transcription, protein translation, cell proliferation, and apoptosis. AZD1208 is a small molecule Pim kinase inhibitor currently in clinical trials. The PI3K-dependent activation of AKT (a client protein of HSP90) and the JAK/STAT-dependent induction of PIM are partially overlapping kinase pathways. Therefore, we hypothesized that the mechanism-based combination of 17-AAG (to decrease AKT levels) with AZD1208 (to inhibit Pim kinases) will result in an increase in cytotoxicity in CLL cells. An AZD1208 dose-response experiment was performed in 4 CLL patient samples to determine the optimal cytotoxic concentrations. AZD1208 concentrations of 3 μM and 10 μM were selected to treat 22 additional CLL patient samples for 24 hours. Cell death ranged from 0-30% and 2-45%, respectively, as determined by Annexin-V/PI staining after subtraction of endogenous cell death. All CLL patient samples were sensitive to AZD1208 regardless of their prognostic markers such as IgVH mutation and ZAP70 positivity or cytogenetics such as 17p del (p53 locus) or 11q del (ATM locus). Similarly, prior treatment history or Rai stage did not influence the biological response of AZD1208 in CLL cells. Based on published data, 0.5 μM and 2 μM 17-AAG were selected for the combination treatments with AZD1208 for 24 and 48 hours. Cytotoxicity for these combinations was assessed in 13 CLL blood samples, resulting in additive (n=8) or more than additive (n=3) cell death when using the fractional two-drug combination analysis. Immunoblot analysis was performed in 3 CLL samples probing for HSP90, HSP70, Akt, Pim-1, Mcl-1, Bcl-2, Phospho-4E-BP1 (Thr37/46), 4E-BP1, and β-actin. In agreement with previous work, 17-AAG treatment resulted in an increase of HSP90 and HSP70 protein levels while Akt levels were markedly decreased. In these preliminary immunoblots, Pim-1 and Bcl-2 levels remained unchanged; however, Mcl-1 protein levels decreased in the combination treatments at 24 and 48 hours. Levels for total and phospho-4E-BP1 also declined. Taken together, these preliminary data indicate that AZD1208 (at 3 and 10 μM) has a modest cytotoxic effect in CLL cells. However, when combined with 17-AAG for 24 and 48 hours there was an additive or more than additive effect in majority of CLL samples, with the possible mechanisms of action being through the Akt and 4E-BP1 pathways.

Citation Format: Fabiola Gomez, Lisa Chen, William Wierda, Varsha Gandhi. In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2057. doi:10.1158/1538-7445.AM2013-2057