Abstract B120: Development of PROTACs for targeted degradation of FGFR3-TACC3 oncofusions

Abstract In frame fusions between Fibroblast Growth Factor Receptor 3 (FGFR3) and Transforming Acidic Coiled-Coil containing protein 3 (TACC3) genes on chromosome 4p16.3 have been reported across diverse cancers, including bladder cancer (∼5%) and IDH-wildtype glioblastoma (4.1%), as well as in small subsets of non-small cell lung (∼0.5%), esophageal squamous cell (1.4–2.1%), cervical (1.9%), head and neck (3.7%), and rare nasopharyngeal (2.5%) carcinomas, and other cancer types. These chromosomal translocations generate FGFR3-TACC3 (F3T3) driver oncofusion proteins which promote tumor cell growth and survival. The F3T3-driven cancer cells are critically dependent on the constitutively active oncofusion kinase activity for survival and proliferation, a phenomenon known as "oncogene addiction". While inhibitors of FGFR kinases, such as erdafitinib and fexagratinib, are the treatment of choice for F3T3-driven cancers, most treated patients do not respond to the drug, suggesting insufficient inhibition of the oncofusion activity. Additionally, most tumor responses to FGFR kinase inhibitors are partial and short-lived, followed by development of resistance and disease progression within few months. Based on the oncogene addiction of cancer cells on the F3T3 activity, we hypothesized that rapid ablation of this oncofusion protein can induce robust apoptotic response, leading to significant tumor regression and sustained remissions. To this end, we explored the use of Proteolysis Targeting Chimeric (PROTAC) bifunctional small molecules which can rapidly degrade F3T3 oncofusion protein by harnessing the cell's own proteasome machinery. We synthesized more than 100 novel PROTAC small molecules targeting F3T3 fusion through combination of previously reported TACC3 ligands with various linkers and ligands of E3 ubiquitin ligases CRBN, VHL or XIAP. We evaluated our new anti-F3T3 PROTAC molecules using F3T3-driven bladder (RT4 and RT112) and breast (SUM185PE) cancer cell lines, which express two of the most common oncofusion variants. Several PROTACs were able to degrade the native F3T3 oncofusion protein and suppressed the growth of F3T3-driven cells. One PROTAC (KHS-C4) degraded F3T3 at low nanomolar concentrations, strongly suppressed the oncogenic pathway downstream F3T3 fusion, had excellent predicted pharmacological properties, acceptable in vitro liver microsome stability (Clint = 59 mL/min/kg for human and 318 mL/min/kg for mouse microsomes) and no in vivo toxicity. Importantly, low concentrations of KHS101-C4 (but not of FGFR3 kinase inhibitors) eradicated F3T3-driven cancer cells (but not cancer cells that do not express the F3T3 oncofusion) in vitro. These data suggest that chemically-induced degradation of the F3T3 fusion protein can lead to oncogenic signal ablation sufficiently potent to induce a lethal oncogenic shock and can thus yield more durable responses than FGFR3 kinase inhibitors. KHS-C4 was selected for further evaluation to establish in vivo proof of concept for the PROTAC-based therapeutic strategy against F3T3-driven cancers. Citation Format: Slawomir Andrzejewski, Keita Nakane, Seiya Kitamura, Eugen Dhimolea. Development of PROTACs for targeted degradation of FGFR3-TACC3 oncofusions [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B120.