Combined mTOR and AKT inhibition in renal cell carcinoma (RCC).

Abstract

e15041 Background: RCC is characterized by VHL abnormalities and upregulation of hypoxia inducible factor (HIF). Inhibition of mTOR, a regulator of HIF, is an established therapy for advanced RCC. mTOR inhibition results in compensatory activation of AKT, a promoter of cell survival and proliferation. Perifosine is an orally bioavailable alkylphospholipid with AKT inhibitory properties. We sought to determine whether the addition of perifosine to an mTOR inhibitor (rapamycin) would synergistically inhibit RCC in preclinical models. Methods: Four clear cell RCC cell lines were studied: 786-O, A498 (VHL mutant), CAKI-1 (VHL wt), and 769-P (VHL methylated). Dose curves of single-agent and combination perifosine + rapamycin were by MTT assay, evaluated by median-effect analysis. Status of phospho-AKT (S473) and HIF-2alpha was by Western blot. Total RNA was isolated from 786-O cells subjected to single-agent and combination treatments. Quantitative real-time PCR (qRT-PCR) was used to examine VEGF mRNA levels and genome-wide expression profiling was performed with Affymetrix HG-U133 Plus 2.0 arrays. Results: CAKI-1, 769-P, and 786-O were sensitive to single-agent perifosine with 50% inhibitory concentrations ranging from 5-10 uM, while A498 was largely insensitive. Perifosine blocked phosphorylation of AKT induced by rapamycin and inhibited HIF-2alpha expression at 72 hrs following treatment in the 786-O and CAKI-1 cell lines. However, combined treatment resulted in subadditive growth inhibition. Rapamycin reduced VEGF expression in all cell lines; however, perifosine markedly induced VEGF expression at 24 hrs in 786-O and CAKI-1, overriding this effect from rapamycin. GeneChip analysis and subsequent pathway modeling revealed an inhibition of the IL-8 pathway by these two agents, concomitant with a 5-fold upregulation of the KLF2 (Kruppel-like factor 2) gene, recently shown to be a suppressor of HIF1alpha. Conclusions: Perifosine is active in select RCC lines and can abrogate the induction of AKT phosphorylation mediated by mTOR inhibition. Combined mTOR and AKT inhibition resulted in the modulation of relevant angiogenesis-related pathways, providing a basis for future clinical investigations. (Egan Family Fund and Landgraf Foundation) No significant financial relationships to disclose.