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

BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing 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, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover 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 into 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 the 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 on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.

Mechanical Stretch of High Magnitude Provokes Axonal Injury, Elongation of Paranodal Junctions, and Signaling Alterations in Oligodendrocytes.

Increasing findings suggest that demyelination may play an important role in the pathophysiology of brain injury, but the exact mechanisms underlying such damage are not well known. Mechanical tensile strain of brain tissue occurs during traumatic brain injury. Several studies have investigated the cellular and molecular events following a static tensile strain of physiological magnitude on individual cells such as oligodendrocytes. However, the pathobiological impact of high-magnitude mechanical strain on oligodendrocytes and myelinated fibers remains under investigated. In this study, we reported that an applied mechanical tensile strain of 30% on mouse organotypic culture of cerebellar slices induced axonal injury and elongation of paranodal junctions, two hallmarks of brain trauma. It was also able to activate MAPK-ERK1/2 signaling, a stretch-induced responsive pathway. The same tensile strain applied to mouse oligodendrocytes in primary culture induced a profound damage to cell morphology, partial cell loss, and a decrease of myelin protein expression. The lower tensile strain of 20% also caused cell loss and the remaining oligodendrocytes appeared retracted with decreased myelin protein expression. Finally, high-magnitude tensile strain applied to 158N oligodendroglial cells altered myelin protein expression, dampened MAPK-ERK1/2 and MAPK-p38 signaling, and enhanced the production of reactive oxygen species. The latter was accompanied by increased protein oxidation and an alteration of anti-oxidant defense that was strain magnitude-dependent. In conclusion, mechanical stretch of high magnitude provokes axonal injury with significant alterations in oligodendrocyte biology that could initiate demyelination.