shERBB2 reduced proliferation of BGJ398 RS and the addition of AZD8931 increased this effect, consistent with a requirement for multiple ERBB family members in conferring resistance to BGJ398 (Fig

shERBB2 reduced proliferation of BGJ398 RS and the addition of AZD8931 increased this effect, consistent with a requirement for multiple ERBB family members in conferring resistance to BGJ398 (Fig.3D, center panel). users. These data support the concept that activation of family members is sufficient to bypass dependency on and suggest that concurrent inhibition of these two pathways may be desired when targeting dependent cancers. is observed in squamous cell lung malignancy4, 5, breast cancer6, and amplification of is found in gastric7 and breast cancers8. Activating point mutations of are observed in bladder cancers9, endometrial cancers10 and lung squamous cell carcinoma11. Translocations coupled with amplifications and mutations of have been observed in multiple myeloma12, 13. More recently, high-throughput sequencing systems have identified a variety of gene fusions. and fusions have been recognized in glioblastoma14 and fusions were found in bladder carcinomas and in lung and head and neck squamous cell carcinomas15, 16, 17. Pre-clinical studies have shown that cells harboring FGFR fusions demonstrate dependency on FGFR-mediated signaling, suggesting that malignancy individuals Itgb2 with FGFR fusions may benefit from targeted FGFR kinase inhibition14, 18. Medical trials to test this hypothesis are underway ( As preclinical studies have suggested that triggered FGFRs are potential LTX-315 focuses on for malignancy therapy19, and several selective FGFR inhibitors are under investigation in clinical tests1, 2 with early reports demonstrating medical effectiveness in amplified breast tumor20 and lung malignancy21. NVP-BGJ398 (BGJ398) is an example of a selective, potent and orally bioavailable inhibitor of FGFR1/2/3 (ref. 22). BGJ398 inhibits the proliferation of various FGFR-dependent cell lines at nanomolar concentrations including lung and breast cancers LTX-315 harboring amplification, gastric cancers harboring amplification and bladder cancers with mutations and/or amplifications23. While FGFR inhibition shows considerable clinical promise it is expected that patients who initially respond to FGFR inhibitors will become refractory due to the development of acquired resistance24. Previous studies have shown that activation of some (ref. 27). Despite these initial observations, the mechanisms governing the acquisition of resistance to FGFR inhibitors remain poorly understood. Therefore, an improved understanding of the molecular mechanisms of acquired resistance to FGFR inhibitors will likely provide valuable insight into how best to use this class of agents. To study potential mechanisms of acquired resistance to selective FGFR inhibition, we established resistant cells following long-term exposure to BGJ398. We selected the RT112 bladder malignancy cell collection which harbors both amplification and a fusion as our initial model. Through screening of the activity of 42 membrane receptor tyrosine kinases (RTKs) and mRNA sequencing, we recognized that ERBB2 and ERBB3 are activated in the resistant cells in a ligand dependent fashion. LTX-315 Acquired resistance to FGFR inhibition developed rapidly and was characterized by an Epithelial to Mesenchymal Transition (EMT) along with a switch in dependency from FGFR to ERBB receptor signaling. These results were specific to cell lines with dependency on and were recapitulated using a second FGFR kinase inhibitor, ponatinib. Results Phenotypic changes associated with LTX-315 the acquisition of resistance to the pan-FGFR inhibitor BGJ398 in the RT112 cell collection RT112 cells, which harbor both amplification and the fusion, were rendered resistant to BGJ398 by a series of step-wise increases in drug concentration starting at 4nM (the approximate IC50) until the cells were able to proliferate in 1M BGJ398. We selected this cell collection for our studies given its dependence on the fusion and anecdotal reports of clinical efficacy of FGFR kinase inhibitors in patients with fusions. These cells were termed BGJ398 RS (BGJ398 Resistant Stepwise). 1M was selected as the target final concentration as it is the approximate maximal serum concentration observed in animal and Phase I studies of BGJ398. The cell lines were both insensitive to BGJ398 (Fig.1A) and a second, less specific, FGFR kinase inhibitor ponatinib (Fig.S1). Open in a separate windows Fig. 1 RT112 RS cells are resistant to BGJ398 in vitro and demonstrate EMT-like properties(A) The dependent RT112 human bladder cell collection was made resistant to BGJ398 by growth in increasing concentrations of BGJ398. The resistance was confirmed by dose response curve. Cells were treated with the indicated concentration of BGJ398 for 4 days and growth was measured using the growth proliferation assay and plotted as a percentage of growth relative to untreated control cells. Data points are represented as Mean SD (n=3). (B) Parental and BGJ398 resistant cells display different morphology under light microscope. (C) q-PCR analysis shows.