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

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

Subcellular localization of several structurally different tyrosine kinase inhibitors

“…The nature of the accumulation of these compounds is highly dependent on the degree to which they are lysosomally accumulated, our data demonstrates that sunitinib and crizotinib are in agreement with the physical properties of lysosomal accumulation. For sunitinib, the lysosomal accumulation has also been demonstrated by using its fluorescent properties which co-localized with the Lysotracker, a marker for lysosomes [7]. Moreover, pre-incubation with Bafilomycin-A prevented trapping of sunitinib in the lysosomes [7,16].…”

“…For sunitinib, the lysosomal accumulation has also been demonstrated by using its fluorescent properties which co-localized with the Lysotracker, a marker for lysosomes [7]. Moreover, pre-incubation with Bafilomycin-A prevented trapping of sunitinib in the lysosomes [7,16]. Sunitinib achieves very high cellular concentrations since it has been shown that 90% of accumulated drug is held within lysosomes within the cell structure.…”

“…Moreover, inhibition may be reversible or irreversible; especially novel third or fourth generation TKIs display irreversible enzyme inhibition [6]. Recently we demonstrated that sunitinib, an inhibitor of the Vascular endothelial growth factor receptor (VEGFR) accumulates in lysosomes [7], while preliminary experiments demonstrated a similar distribution for crizotinib, an ALK-EML4 and cMET inhibitor [5]. In contrast, erlotinib an inhibitor of the epidermal growth factor receptor (EGFR) showed a very low cellular uptake limited to the membrane [8].…”

Mapping of drug distribution in the rabbit liver tumor model by complementary fluorescence and mass spectrometry imaging

“…Fluorescence Imaging Technique Tissue sections were imaged by an AxioZ1 Imager (Zeiss, Feldbach, Switzerland) with a Fluar 5x/0.25 (magnification/numerical aperture) objective equipped with a cooled, sensitive monochrome camera (Zeiss Axiocam MRm). Fluorescence of sunitinib [12] was acquired using a fluorescent filter (FITC, 50 ms exposure, 16-bit image resolution), together with bright field images. To visualize the amount of sunitinib around beads in the tissue, fluorescent image heatmaps were generated using ImageJ (v1.50a, NIH, Bethesda, MD, USA) Lookup Table "Thermal" by adjusting images to 650-2000 pixel brightness (650 pixel and lower: violet, 2000 pixel and higher: red).…”

Blocking stanniocalcin 2 reduces sunitinib resistance in clear cell renal cell carcinoma

“…However, its application has been overshadowed by the emergence of drug resistance. Previous studies have revealed that sunitinib is sequestered in lysosomes as a novel mechanism of drug resistance of ccRCC [26,27]. Therefore, in this study, the relationship between STC2 expression and sunitinib resistance in ccRCC cells was investigated.…”