Abstract #2749: In vitro and in vivo efficacy of the dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in renal cell carcinoma (RCC) (2009)

Introduction: Inhibitors of mTORC1 have shown activity in patients with metastatic RCC. As the PI3-K pathway activates numerous kinases, transcription factors and other proteins associated with cell growth and survival in addition to mTOR, disruption of this pathway upstream of mTOR may be more effective than allosteric inhibition of mTOR alone. To investigate this possibility, the in vitro and in vivo efficacy of the dual PI3-K/mTOR inhibitor NVP-BEZ235 was compared with rapamycin in RCC cell lines. Experimental Design: Five RCC cell lines (786-0, 769-P, A498, Caki-1, Caki-2) were studied. NVP-BEZ235 was used at varying concentrations (25nM-500nM) and 250nM chosen for further in vitro studies based on the complete inhibition of Akt (Ser473) phosphorylation. RCC cells were exposed to DMSO or treated with rapamycin (100nM) or NVP-BEZ235 and either lysed after 24 hours for western blot analysis for phospho-Akt, -S6, -eIF4E, -Mnk1, Cyclin D1, and HIF2\#945; or assayed for proliferation by MTS assay after 48 hours. Growth media was isolated and analyzed for VEGF levels by ELISA. HIF activity in treated cells was determined by transient transfection of a HIF responsive luciferase construct. Xenografts were generated from 786-O cells in nude beige mice and grown to 10mm in size. Mice were then treated with either saline, rapamycin (2.5mg/kg), or NVP-BEZ235 (40mg/kg) once daily by gavage for 21 days. Tumor measurements were taken every other day. Results: Treatment of RCC cell lines with NVP-BEZ235 in vitro resulted in greater reduction in tumor cell proliferation and more complete suppression of Cyclin D1 and HIF2\#945; levels than that achieved with either rapamycin or DMSO. These effects correlated with reduced phosphorylation of eIF4E and Mnk1, neither of which were effected by rapamycin. Despite apparent suppression of cap-dependent translation suggested by the decrease in p-eIF4E, Cyclin D1, and HIF-2\#945; levels, NVP-BEZ235 did not consistently suppress VEGF expression at either the mRNA or protein level more effectively than rapamycin. The reduction of HIF2\#945; levels correlated with reduced HIF activity as determined by luciferase assay. NVP-BEZ235 induced tumor regression in 786-O xenografts that was associated with inhibition of Akt and S6 phosphorylation. In contrast, rapamycin induced only minimal growth arrest. Conclusion: Dual inhibition of PI3-K/mTOR with NVP-BEZ235 induced growth arrest in RCC cell lines both in vitro and in vivo more effectively than inhibition of mTORC1 alone. This growth inhibition was associated with reduction of markers of cap-dependent translation and HIF activity in vitro. Variable effects on circulating VEGF and intracellular VEGF mRNA levels in the setting of diminished HIF activity suggest that treatment with NVP-BEZ235 may result in stabilization of VEGF mRNA. Dual inhibition of PI3-K and mTOR with agents such as NVP-BEZ235 may be a promising strategy for the treatment of RCC. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2749.