Leiomyosarcoma (LMS) are an uncommon group of malignant tumors composed of cells showing smooth muscle differentiation that represent 10-15% of all soft-tissue sarcomas and is therefore one of the most frequent sarcoma subtype. From a genetic point of view, LMS are characterized by numerous genomic alterations whose a large region of deletion on chromosome 10 including the tumor suppressor gene PTEN. It is one of the most frequent aberration (40-50% of cases) and is associated with increased activation of the mTOR pathway. Moreover, in vitro and vivo studies have demonstrated that the mTOR pathway plays a crucial role in LMS tumorigenesis. There are no data about the role of PI3K/mTOR targeting in LMS.
The LMS cell lines have been derived from human surgical STS specimens in our laboratory. Cells were treated for 72hrs with a range of increasing concentrations of Everolimus (mTORC1 inhibitor), BKM120 (PI3KCA inhibitor) and BEZ235 (dual PI3K-mTOR inhibitor) to assess anti-proliferative activity by MTT-assay and apoptosis by AnnexinV/PI staining. Effects on mTOR and MAPK pathway signaling were studied by western blot analysis. The LMS cell lines were exposed to increasing doses of BEZ235 in combination with GDC-0994 (ERK inhibitor) and synergy was assessed by Chou-Talalay combination index (CI). Effects of all inhibitors were observed in mice with subcutaneous LMS cell line-derived xenograft tumors for 3 weeks.
Dose-dependent growth suppression was more clearly induced by BEZ235 (range, 0.001 to 0.1 μM) than by Everolimus and BKM120 (range 0.01 to 1.6 μM). Apoptosis was also much more effectively induced by BEZ235. We examined in vivo antitumor activity of the three drugs in mice inoculated with LMS cells. In vivo, the effects of BEZ235 (70% tumor volume [TV] inhibition versus untreated tumors) was also higher than that of BKM120 and Everolimus (30% and 55% TV inhibition). We assessed the effects of BEZ235 on signaling pathways in all cell lines. Strikingly, mTOR pathway was consistently repressed and BEZ235 markedly induced ERK overactivation. This effect was not observed with BKM120 or Everolimus suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA significantly reduced the enhancing effect of BEZ235 on ERK phosphorylation. Combined treatment with BEZ235 and GDC-0994, a potent ERK inhibitor, resulted in synergistic growth inhibition, apoptosis induction and decrease levels of both phosphorylated proteins of mTOR and ERK pathways in vitro and in vivo.
Targeting PI3K and mTOR simultaneously using BEZ235 effectively inhibits LMS cell growth and prolongs survival of mice in comparison of those treated with BKM120 or Everolimus. However, BEZ235 suppress a negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in LMSs cells. Combining BEZ235 with MEK/ERK inhibitors may be a relevant approach to increase anti-tumor activity and avoid drug resistance.
Citation Format: Benjamin Fourneaux, Vanessa Chaire, Marie Karanian, Audrey Laroche, Antoine Italiano. Dual targeting of PI3K/mTOR leads to MEK/ERK over-activation in leiomyosarcoma through suppression of mTORC2 inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4636.