Abstract 3785: CFON-026 is a macrocycle that disrupts the BTK protein scaffold thereby inhibiting all clinically relevant BTK resistance mutations
Authors: Jos de Man, Joost C.M. Uitdehaag, Freek van Cauter, Sander P.W. van Gemert, Yvonne G.T.H. van Mil, Milan J. Hoffmann, Winfried R. Mulder, Michelle Muller, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri J.P. de Wit, Erik Ensing, Rogier C. Buijsman
Journal: Cancer Research (2025)

Abstract Bruton’s Tyrosine Kinase (BTK) inhibitors are the main treatment in CLL, WM and MCL. The first generation BTK inhibitors bind covalently to Cys481 and include ibrutinib, acalabrutinib and zanubrutinib. However, in CLL, 67% of the patients relapsing after ibrutinib treatment developed a BTKC481S mutation, which causes resistance to covalent BTK inhibitors. Pirtobrutinib was developed as a second generation non-covalent BTK inhibitor, though resistance has emerged via the acquisition of BTK mutant variants BTKT474I and BTKL528W. Clinical trials with other BTK-targeting modalities such as heterobifunctional degraders have identified additional problematic resistance mutations, such as BTKV416L, BTKA428D and BTKM437R. A novel BTK-targeting modality that can combat all clinically relevant BTK mutant variants is therefore needed. CFON-026 is a best-in-class non-covalent BTK inhibitor based on a macrocyclic scaffold. CFON-026 has a unique binding mode in the ATP pocket of BTK, connecting the N-terminal and C-terminal lobe of the kinase fold in a continuous beta-sheet, conferring potent binding and a long target residence time that matches that of covalent inhibitors. The binding mode of CFON-026, as shown by X-ray crystallography, consists of a large number of interactions with the amino acid backbone of BTK, reducing the risk of acquiring resistance via mutations of amino acid side chains. CFON-026 potently inhibits growth of the BTK dependent cell lines TMD8 (IC50 0.45 nM) and REC-1 (IC50 1.7 nM). CFON-026 is also a very potent inhibitor biochemically and cellularly of BTK variants with clinically relevant resistance mutations. CFON-026 is highly cytotoxic to TMD8 mutant cell lines containing endogenous BTK variants (BTKC481S, BTKV416L, BTKT474I and BTKL528W) and inhibits BTKM437R in a HEK293 expression model. So far, we have not identified a BTK mutation that abrogates activity of CFON-026. Here we studied why CFON-026 is effective on ‘kinase dead’ BTK resistance variants such as BTKV416L and BTKL528W. These variants have no enzymatic activity but still propagate an oncogenic signal via recruitment of other kinases on the proximal side of the protein and are generally considered only to be targetable by BTK degraders. X-ray structures show that CFON-026 binding induces conformational rearrangements that disrupt the BTK scaffolding functionality, thereby inhibiting the recruitment and activation of downstream kinases. CFON-026 has good pharmacokinetic properties, metabolic stability and favourable safety characteristics. CFON-026 induces complete tumor regression in a TMD8 xenograft model and is currently progressing towards IND. CFON-026 disrupts the BTK kinase structure, abrogating both kinase activity and scaffold functions. CFON-026 can overcome resistance mutations emerging from all existing covalent and non-covalent BTK inhibitor therapies. Citation Format: Jos de Man, Joost C.M. Uitdehaag, Freek van Cauter, Sander P.W. van Gemert, Yvonne G.T.H. van Mil, Milan J. Hoffmann, Winfried R. Mulder, Michelle Muller, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri J.P. de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a macrocycle that disrupts the BTK protein scaffold thereby inhibiting all clinically relevant BTK resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3785.