Abstract A54: Potential mechanisms of resistance to targeted agents in human clear cell renal cell carcinoma

Abstract

Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased >2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a >3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (>80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (<30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to <40% growth inhibition, the combination significantly enhanced anti-proliferative effects (>85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.