Combined Targeting of AKT and mTOR Synergistically Inhibits Formation of Primary Colorectal Carcinoma Tumouroids In Vitro: A 3D Tumour Model for Pre-therapeutic Drug Screening.

Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.

Nörz D ，Mullins CS ，Smit DJ ，Linnebacher M ，Hagel G ，Mirdogan A ，Siekiera J ，Ehm P ，Izbicki JR ，Block A ，Thastrup O ，Jücker M ... -  《-》

Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells.

Grabinski N ，Ewald F ，Hofmann BT ，Staufer K ，Schumacher U ，Nashan B ，Jücker M ... -  《Molecular Cancer》

Combined Targeting of AKT and mTOR Inhibits Proliferation of Human NF1-Associated Malignant Peripheral Nerve Sheath Tumour Cells In Vitro but not in a Xenograft Mouse Model In Vivo.

Schulte A ，Ewald F ，Spyra M ，Smit DJ ，Jiang W ，Salamon J ，Jücker M ，Mautner VF ... -  《-》

Combined targeting of AKT and mTOR using MK-2206 and RAD001 is synergistic in the treatment of cholangiocarcinoma.

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.

Ewald F ，Grabinski N ，Grottke A ，Windhorst S ，Nörz D ，Carstensen L ，Staufer K ，Hofmann BT ，Diehl F ，David K ，Schumacher U ，Nashan B ，Jücker M ... -  《-》

High Sensitivity of Circulating Tumor Cells Derived from a Colorectal Cancer Patient for Dual Inhibition with AKT and mTOR Inhibitors.

Smit DJ ，Cayrefourcq L ，Haider MT ，Hinz N ，Pantel K ，Alix-Panabières C ，Jücker M ... -  《Cells》