Paper Details

ABSTRACT

Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet acquired resistance remains an inevitable clinical challenge. While resistance mutations to covalent BTK inhibitors typically arise through catalytic-site mutations such as C481S, recent studies of non-covalent BTK inhibitor resistance have identified kinase-impaired BTK mutants. Studies of these BTK mutations, including L528W and A428D, have revealed a scaffold-dependent B-cell receptor (BCR) signaling despite markedly reduced kinase activity. These mutants evade inhibition by currently approved BTK inhibitors, and BTK A428D additionally confers resistance to BTK degraders in clinical development (zelebrudomide, bexobrutideg, tacabrutideg) because its altered conformation impairs degrader binding. Therapeutic strategies capable of targeting both catalytic and non-catalytic scaffold functions of BTK are therefore urgently needed.

Macrocyclic small molecules possess structural and conformational properties distinct from conventional ATP-competitive kinase inhibitors, enabling stabilization of alternative protein conformations and modulation of protein function beyond catalytic inhibition. We hypothesized that these unique pharmacologic properties could be leveraged to overcome resistance mediated by kinase-impaired BTK mutants, which remain dependent on scaffold-mediated B-cell receptor (BCR) signaling despite loss of catalytic activity. We therefore evaluated CFON-026, a novel macrocyclic non-covalent BTK inhibitor designed to exploit conformational plasticity within the BTK kinase domain. Structural, biophysical, and cellular studies demonstrated that CFON-026 induces remodeling of the BTK activation loop, promoting a conformational state that disrupts scaffold-dependent signaling while preserving high-affinity target engagement and potent activity across both kinase-proficient and kinase-impaired BTK resistance mutations.

Cellular IC₅₀ profiling in BTK-dependent TMD8 CRISPR knock-in models demonstrated sub-10 nM potency against WT BTK and the recurrent resistance mutants C481S, T474I, and V416L while retaining activity against the kinase-impaired L528W (60.2 nM) and A428D (86.7 nM) mutants. BTK L528W exhibits profound resistance to pirtobrutinib (IC₅₀ >5 μM), representing an approximately 80-fold improvement in potency with CFON-026. These findings were confirmed in an additional cell line, HBL1. NanoBRET assays confirmed intracellular target engagement across evaluable BTK variants, while surface plasmon resonance (SPR) demonstrated direct binding to A428D and other conformationally altered mutants that could not be assessed by NanoBRET because of tracer limitations. A high-resolution co-crystal structure of CFON-026 with WT BTK demonstrated backbone-mediated binding and replacement of a conserved water network, providing a structural rationale for its potency against diverse BTK resistance mutants.

Importantly, biochemical target engagement translated into functional suppression of BCR signaling across every resistance genotype tested. CFON-026 completely suppressed BCR-induced calcium flux in WT, C481S, T474I, V416L, L528W, and A428D cells. Despite stronger NanoBRET engagement of WT BTK, inhibition of calcium signaling was even more pronounced in L528W cells, supporting a mechanism extending beyond catalytic inhibition to disruption of scaffold-dependent signaling. Ongoing in vivo studies in physiologically relevant BTK L528W knock-in/Eμ-TCL1 CLL models and TMD8 xenografts demonstrate favorable pharmacokinetic properties, tolerability, and preliminary antitumor activity, including complete tumor regression in WT BTK xenografts.

Collectively, these studies demonstrate that macrocyclic BTK inhibition provides a mechanistically distinct therapeutic strategy capable of overcoming both catalytically active and kinase-impaired resistance mutations. By combining potent catalytic inhibition with disruption of scaffold-dependent BTK signaling, CFON-026 remains active against resistance mechanisms that evade both ATP-competitive inhibitors and targeted protein degraders. These findings support the continued clinical development of CFON-026 as a first-in-class macrocyclic BTK inhibitor for CLL and other BTK-dependent lymphoid malignancies.