Abstract 1788: Mechanism of resistance to c-Met tyrosine kinase inhibitors in human melanoma

Abstract

Abstract The over-expression of c-Met and activation by its ligand, hepatocyte growth factor (HGF), are known to cause significant tumor development and metastasis in human melanoma. Hence, c-Met is an attractive target for molecular therapeutic inhibition. To assess c-Met inhibition, we studied the effects of c-Met tyrosine kinase inhibitors (TKIs) in RU melanoma cells and found that JNJ38877605 and SU11274 (c-Met TKIs) inhibited cell growth in RU cells with an IC50 of 0.5 and 1µM, respectively. We then tested the therapeutic effects of JNJ38877605 in vivo. Five million RU melanoma cells were injected subcutaneously into the hind flanks of nude mice. Tumors were allowed to develop for a week after which daily oral treatments of 20 mg/kg JNJ38877605 or vehicle were given. We found that JNJ38877605 decreased the size of RU tumor xenografts in nude mice by 6 fold compared to mice treated with a vehicle control. Unfortunately, while c-Met inhibitors have been successful in clinical trials, acquired resistance to c-Met TKIs (ARQ197) and antibodies (MetMab) has been observed in cancer patients, resulting in tumor recurrence or lack of tumor CR. To determine the mechanism of c-Met TKI resistance, two melanoma cell line models, MU and RU, were exposed to increasing concentrations of SU11274. They proliferated in 7-10 fold higher concentrations of SU11274, and were cross resistant to 12 fold higher concentrations of JNJ38877605. After generating resistant cells, we measured HGF production and the expression levels of p-c-Met (Y1003) and p-c-Met (Y1234/1235) by immunoblotting to compare parental and resistant cells. We found that MU resistant cells exhibited upregulation of p-c-Met (Y1003) (6 fold) and p-c-met (Y1234/1235) (4 fold) compared to parental cells after treatment with HGF. Additionally, p-c-Met (Y1234/1235) was stable for only 30 min in parental cells compared to 60 min in resistant cells, where it was upregulated by 3-fold. This might be due to defects in c-Met ubiquitination resulting in its being recycled back or stabilized onto the membrane. Interestingly, we found that RU parental cells secreted 2 fold more HGF than the resistant cells. This indicates that resistant RU cells may not be as dependent on HGF induced activation in comparison to parental cells. In MU and RU resistant cells, we also found upregulation of p-mTOR (2 and 8 fold, respectively). Upregulation of phospho-pS6kinase (2 fold) and p-4E-BP1 (4 fold), downstream targets of p-mTOR, were also seen in MU cells. MU resistant cells also expressed increases in active β-catenin (protein downstream of Wnt signaling pathway) by 5 fold under no HGF stimulation and 2 fold after 60 min of HGF treatment. Our data indicates that the oral c-Met TKI, JNJ38877605, could be a promising therapeutic option for treating HGF producing melanoma tumors, but its effects could be further enhanced by the addition of mTOR and/or Wnt inhibitors to counteract acquired c-Met TKI resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1788. doi:1538-7445.AM2012-1788