FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma

Background and aims: About 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 are fused to sequences encoded by a diverse array of partner genes (>60), a configuration that causes oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, including FF tyrosine kinase domain mutations, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical and unmet need. Herein, we present the generation of FF-driven murine iCCA models and their exploitation for discovering actionable FF-associated dependencies. Methods: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. Results: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. Conclusions: FF-driven iCCA pathogenesis was successfully modeled in murine Tp53-/-background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for precision-based approaches against human FF+ iCCA.