Preclinical rationale for combination targeted therapy in advanced clear cell renal cell carcinoma (RCC): Abrogation of rapamycin-mediated induction of AKT phosphorylation by perifosine

Abstract

16083 Background: RCCs are highly vascularized tumors characterized by VHL abnormalities resulting in the upregulation of hypoxia inducible factor (HIF). Inhibition of mTOR, an upstream modulator of HIF, is an established therapeutic strategy in advanced RCC. One proposed resistance mechanism for mTOR inhibition is the compensatory upregulation of AKT, a promoter of cell growth and proliferation. Perifosine, an orally bioavailable AKT inhibitor, has clinical activity against RCC. We sought to determine whether treatment with perifosine and an mTOR-inhibitor (rapamycin) would synergistically inhibit RCC in preclinical models. Methods: Four clear cell RCC cell lines were studied: A498, CAKI-1, 769-P, and 786-O. Dose curves of single-agent and combination perifosine + rapamycin were performed by MTT. Combination studies were designed for median-effect. Status of pAKT (S473), pmTOR (S2481) and HIF-2alpha was assessed by Western blot. Cell lines had low basal levels of pAKT and VHL, moderate levels of pmTOR and HIF-2alpha, and no detectable HIF-1alpha. Results: CAKI-1, 769-P, and 786-O were sensitive to perifosine with an IC50 between 5–10 uM, while the IC50 of A498 was > 20 uM. Growth inhibition by rapamycin plateaued by 0.5–1 nM with none of the lines achieving an IC50. Combination treatments were essentially additive at lower doses of perifosine, becoming sub-additive or antagonistic at higher doses. Perifosine blocked phosphorylation of AKT induced by rapamycin. As a single agent and in combination, perifosine inhibited HIF-2alpha expression in the 769-P and CAKI-1 cell lines. Conclusions: Perifosine has high single-agent activity in select RCC lines and can abrogate the induction of AKT phosphorylation mediated by mTOR inhibition. Perifosine + rapamycin was at best additive, suggesting that pAKT is not the sole mechanism of resistance. Studies to explore the roles of VHL and HIF in response to this combination are ongoing. A Southwest Oncology Group trial in RCC patients testing the rational combination of perifosine and the mTOR inhibitor temsirolimus is now under development. No significant financial relationships to disclose.