Abstract 2852: Lysosomal sequestration of sunitinib may play a role in its resistance

Abstract

Abstract Resistance to tyrosine kinase inhibitors (TKIs) is a major clinical problem. Mechanisms that mediate drug resistance include gene mutations, multidrug efflux and activation of alternative growth factor pathways. Sunitinib, a multi-targeted antiangiogenic TKI, has demonstrated clinical efficacy in advanced renal cell cancer (RCC) and gastrointestinal stromal tumors, but is hampered by resistance. In this study we aimed to unravel mechanisms of sunitinib resistance. Based on its large volume of distribution, we hypothesized that sunitinib concentrations intratumoral would be higher compared to plasma. Indeed we found that in patients harboring different tumors, intratumoral sunitinib concentrations were >30-fold higher than parallel plasma concentrations: 9.5±2.4 µM versus 0.28±0.06 µM (n=3, mean±SEM), respectively, as measured by LC-MS/MS. Subsequently, we studied the in vitro sensitivity to sunitinib (provided by Pfizer Inc.) of 1 RCC and 8 colorectal cancer cell lines and found clinically relevant inhibitory concentrations (IC) on proliferation of IC50 = 1.3±0.1 µM and IC90 = 4.6±0.6 µM sunitinib. In parallel experiments, we induced resistance in vitro in 786-O and HT-29 cells by continuous exposure to increasing concentrations of sunitinib for > 1 year. These resistant tumor cells continued to stably grow on exposures to clinically achieved intratumoral concentrations of 6 µM (786-O) and 12 µM (HT-29) sunitinib, whereas their parental cells died at these concentrations. Microscopy of sensitive and resistant tumor cells revealed sequestration of sunitinib in specific subcellular compartments. Because sunitinib is a hydrophobic (logP=5.2) weak base (pKa = 8.95), we reasoned that it might preferentially accumulate in acidic lysosomes. Indeed, staining with a lysosome-specific fluorescent dye revealed predominant co-localization of sunitinib in lysosomes. In addition, compared to sensitive cells, lysosomal accumulation capacity was increased in resistant cells, which was confirmed by flow cytometry and Western blot analysis (LAMP-1 and LAMP-2 expression). Intracellular concentrations were increased in the resistant tumor cells compared to their parental cells, 4.6±1.1 mM and 2.3±0.4 mM, respectively, and were up to 1000-fold higher than the micromolar concentrations used for in vitro exposure. Growth of resistant cells in drug-free medium resulted in restoration of drug sensitivity and normalization of lysosomal drug accumulation capacity within 12 weeks. In conclusion, our data show that: 1) sunitinib inhibits proliferation of tumor cells in vitro at clinically relevant intratumoral concentrations, 2) tumor cells acquire a transient drug-resistant phenotype under continuous exposure to sunitinib, 3) sunitinib is sequestered in acidic lysosomes and 4) increased lysosomal sequestration may contribute to sunitinib resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2011-2852