Abstract C64: Treatment with the Pim protein kinase inhibitor SMI-4a enhances AMPK phosphorylation, decreases Raptor levels, and blocks mTORC1 activity

Abstract

Abstract We have identified a class of small molecule inhibitors of the Pim protein kinases, benzylidene thiazolidine-2-4 diones (J. Med. Chem. (2009) 52:74) with the most potent members having IC50s of 13 nM for Pim-1 and 2.3 µM for Pim-2. Compounds in this chemotype demonstrated selectivity of more than 2500-fold and 400-fold for Pim-1 or Pim-2 respectively while other congeners had equivalent potency towards both isozymes. In vivo, these molecules inhibited Pim kinase autophosphorylation and in a murine model inhibited the growth of subcutaneously implanted murine adenocarcinoma JC cells. One of the members of this chemotype, SMI-4a, has been shown to block the phosphorylation of the mTOR regulatory protein PRAS40 and subsequently the activity of the mTOR pathway (Mol. Cancer. Ther. (2009) 8: 1473; Cancer Biol. Ther. (2009) 8: 846). Now we show that the addition of SMI-4a to malignant cells increases the phosphorylation of AMPKα on Thr 172 in a LKB1-dependent manner, induces the phosphorylation of Raptor on Ser792, decreases the levels of Raptor protein, and inhibits mTORC1 activity. Immunoprecipitation of mTOR from SMI-4a treated cells consistently showed lower levels of bound Raptor and in vitro mTOR kinase assays from treated cells demonstrated a decreased ability to phosphorylate 4E-BP1. Knockdown of PIM-1 via siRNA in K562 leukemic cells showed increased AMPK phosphorylation and decreased Raptor protein levels, further demonstrating an important role for Pim kinase in regulating AMPK phosphorylation and Raptor levels. Additionally, mouse embryo fibroblasts (MEFs) deficient for Pim-1, Pim-2 and Pim-3 kinase (TKO MEFs) showed a significantly increased level of AMPK phosphorylation compared to wild type MEFs, which correlated with decreased mTORC1 activity and increased binding of 4E-BP1 with eIF-4E. The TKO MEFs grew significantly more slowly than wild type. The decreased mTORC1 activity correlated with an increase in the cellular level of AMP in TKO MEFs. Furthermore, the correlation between increased AMPK phosphorylation and a lower level of Raptor protein observed with SMI-4a treatment was also observed in TKO MEFs. Infection of TKO MEFs with lentiviruses expressing Pim 1 or Pim2 was able to reverse these effects, decreasing AMPK phosphorylation, and increasing Raptor protein levels. The cellular activity of mTORC1 was difficult to assess in TKO MEFs as we found substantially lower protein levels of the mTORC1 substrates 4E-BP1 and p70S6K. Akt was readily phosphorylated upon serum stimulation of TKO MEFs and mTORC2 activity was unchanged. Given the role of Pim kinase in regulating mTORC1 activity, we have combined SMI-4a and the mTOR inhibitor rapamycin inducing synergistic blockade of this pathway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C64.