Abstract A025: CFON-026, a novel macrocyclic BTK inhibitor disrupts scaffold-dependent signaling and overcomes resistance to BTK-directed therapies

Abstract Bruton’s tyrosine kinase (BTK) inhibitors have transformed the treatment landscape of chronic lymphocytic leukemia (CLL) and other malignant lymphomas, yet resistance to this class of drugs has emerged through various on-target mutations in BTK. We recently identified kinase-impaired mutations that preserve scaffold-dependent B-cell receptor (BCR) signaling despite loss of catalytic activity (Montoya et al. Science 2024). Notably, BTK L528W confers resistance to all FDA-approved BTK inhibitors, and additional mutations may resist degraders in clinical evaluation (Sievers et al. ASH Annual Meeting 2025). These findings highlight the urgent need for therapies capable of binding altered BTK while inhibiting both catalytic and scaffold functions. To overcome BTK inhibitor resistance, we evaluated CFON-026, a novel macrocyclic non-covalent BTK inhibitor developed by Crossfire Oncology. CFON-026 induces conformational rearrangements in the activation loop that disrupt the scaffold function. By targeting both catalytic and scaffold functions, we hypothesized that CFON-026 could inhibit wildtype BTK (WT BTK) while overcoming kinase-impaired resistance mutations, providing an effective stand-alone BTK-targeted therapy for CLL and other lymphomas sensitive to BTK inhibition. Cellular IC50 profiling in TMD8 BTK CRISPR knock-in cells demonstrated sub-10 nM potency across WT, C481S, T474I, and V416L, with retained activity against L528W (60 nM). For context, L528W is highly resistant to pirtobrutinib, with reported IC50 values >5000 nM, corresponding to an approximate ∼80-fold improvement with CFON-026. Intracellular target engagement was confirmed by NanoBRET across BTK mutants. Additionally, binding was validated by SPR, confirming direct target engagement across all forms of BTK. Functionally, CFON-026 suppressed calcium flux across all BTK mutants. Despite stronger NanoBRET binding to WT BTK, CFON-026 more potently suppressed calcium flux in L528W, consistent with scaffold-mediated inhibition independent of catalytic activity. In vivo testing is ongoing in a physiologically relevant BTK L528W transgenic mouse model crossed with Eµ-TCL1 CLL mouse model as well as xenograft models of TMD8 bearing knock-in BTK point mutations. Preliminary in vivo studies show that CFON-026 has favorable stability, pharmacokinetics, and safety, and induces complete tumor regression in a WT BTK TMD8 xenograft model. Collectively, these findings demonstrate that CFON-026 effectively targets both the catalytic and scaffold functions of BTK, enabling potent activity across a spectrum of resistance-associated mutations, including kinase-impaired L528W, and support the continued clinical development of CFON-026 as a next-generation strategy to overcome resistance in CLL and other malignant lymphomas. Citation Format: Erica N. Lamkin, Tim Kong, Eduardo Bravo Jr., Allison Cool, Samantha Sekeres, Joost Uitdehaag, Jos de Man, Freek van Cauter, Sander P.W. van Gemert, Yvonne G.T.H. van Mil, Winfried R. Mulder, Michelle Muller, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri J.P. de Wit, Erik Ensing, Rogier C. Buijsman, Omar Abdel-Wahab, Justin Taylor. CFON-026, a novel macrocyclic BTK inhibitor disrupts scaffold-dependent signaling and overcomes resistance to BTK-directed therapies [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A025.