mk-2206 and Cholangiocarcinoma

Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma.. AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.

Topics: Antineoplastic Agents; Benzimidazoles; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Proliferation; Cholangiocarcinoma; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds, 3-Ring; Humans; Mitogen-Activated Protein Kinase Kinases; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; raf Kinases; Signal Transduction; TOR Serine-Threonine Kinases

Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration-dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001-mediated anti-tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK-2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan-AKT inhibitor MK2206 in the CCA cell line TFK-1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK-2206 might be a new, effective strategy for the treatment of CCA.

Topics: Animals; Apoptosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Blotting, Western; Cell Cycle; Cell Proliferation; Cholangiocarcinoma; Drug Synergism; Everolimus; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Immunoprecipitation; Mice; Phosphatidylinositol 3-Kinase; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Stem Cell Assay