Kinase inhibitors are among the most developed targeted therapies for cancer and serve as a frontline treatment replacing chemotherapy for several B-cell malignancies, including chronic lymphocytic leukemia (CLL). Due to its critical role in the proliferation and survival of B-cells, Bruton's Tyrosine Kinase (BTK) is a target for multiple generations of covalent (irreversible) and non-covalent (reversible) small molecule inhibitors. Despite numerous advancements made in the development of targeted therapies, many patients still face relapse due to acquired resistance.
We performed a genomic analysis of CLL patients that relapsed during the phase I/II clinical trial of a novel non-covalent BTKi, pirtobrutinib, and discovered acquired mutations (BTK V416L, A428D, M437R, T474I, L528W) that occur at critical residues within the catalytic kinase domain of BTK and conferred resistance to pirtobrutinib. Using cell-based and molecular assays, we observed that these mutations physically impede drug binding and disrupt the normal kinase activity of BTK but can, upon B-cell receptor stimulation, sustain AKT, ERK, and NFkB signaling and intracellular Ca2+ release. This leads us to believe that BTK is being used as a scaffold for other signaling molecules to phosphorylate PLCG2, the direct downstream target of BTK.
Our data has shown that on-target BTK mutations allow escape from BTK inhibition resulting in BTKi resistance. Even in the absence of BTK catalytic activity, downstream targets are still activated, suggesting an unidentified mechanism of genomic escape. Pinpointing and confirming these unique signaling mechanisms will be paramount in influencing patient treatment options.
