Abstract Introduction The Fibroblast Growth Factor Receptor (FGFR) has become a key target in urothelial cancer. The FGFR inhibitor (FGFRi) erdafitinib has been approved for clinical use and many others are in development. Unfortunately, responses have shown to last short. Thus, it is urgently required to identify the underlying mechanisms of resistance and establish strategies to overcome it. We designed a comprehensive in vitro study in FGFR3-altered urothelial cancer cell lines after acquiring resistance to FGFRis. In parallel, we monitored the clinical and molecular evolution (through tumor biopsies and ctDNA) of three patients treated with FGFRi at our institution. Experimental Procedures Drug sensitivity to FGFRi (Erdafitinib and AZD4547) was evaluated in bladder cancer cell lines harboring FGFR3 point mutations (pS249C) or rearrangements (FGFR3/BAIAP2L1 or TACC3). After performing 7-days proliferation assays, cell lines showing nM-range sensitivity were long-term treated with high concentrations of both compounds to induce therapeutic resistance. Cell lysates were collected and protein arrays (Human Phospho-RTK and Kinases Array, R&D Systems) were used for the identification of proteins involved in the desensitization to FGFRi. Later, cell lines were treated with selective inhibitors of such kinases. Regarding patients, after providing written consent, tumor tissue was collected retrospectively from the initial diagnosis and prospectively in any new tumor resection performed in daily practice. ctDNA from peripheral blood was also periodically extracted. Results & Conclusions Protein arrays showed an overactivation of phosphorylated forms of proteins involved in PI3K (EGFR and AKT, 4 and 9-fold increase) and MAPK pathways or proteins related to signal transduction (PLCγ, 80-fold increase, among others). When treated with trametinib, IGF1Ri, ipatasertib, everolimus or erlotinib, only trametinib and IGF1Ri demonstrated significant activity in cell cultures. Three patients were included in the clinical cohort of our study (2 males, 1 female). All harbored the mutation p.S249C and initially achieved a partial response. Multiple biopsies (baseline and at tumor progression) were analyzed in case 1, showing a secondary mutation in FGFR3 and molecular alterations in EGFR and IGFR pathways as potential mechanisms of resistance, matching the results previously observed in in vitro models. Studies of the other 2 cases are ongoing and will be presented at the meeting. Combination of FGFRi with additional TKIs could improve the efficacy of these drugs. Further studies and validation in clinical trials are required. Citation Format: Sergio Ruiz-Llorente, Elena Fernández-Sevillano, Paloma Navarro, Juan Francisco Rodríguez-Moreno, Arantzazu Barquín-García, Sandra Amarilla-Quintana, Mónica Yagüe-Fernández, Juan María Roldan-Romero, Raquel Martín, Cristina Rodriguez-Antona, Jesús García-Donas. Mechanisms of resistance to FGFR inhibitors in urothelial cancer cell lines and patients harboring FGFR3 alterations and strategies to overcome it abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5288.