Högskolan i Skövde

Bruton’s tyrosine kinase (BTK) is essential for B-cell receptor signaling and is a major therapeutic target in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Covalent BTK inhibitors such as Ibrutinib and Zanubrutinib have improved patient outcomes, but resistance, mainly due to mutations at the cysteine 481 residue, limits long-term efficacy. The non-covalent BTK inhibitor Pirtobrutinib was developed to overcome this, yet emerging non-cysteine 481 BTK mutations reduce its effectiveness. This study investigated 15 novel non-cysteine 481 BTK mutations for their ability to confer resistance to both covalent and non-covalent inhibitors. BTK-deficient K562 cells were transfected with plasmids encoding each mutant and treated with Ibrutinib, Zanubrutinib, or Pirtobrutinib. Western blotting assessed BTK activation via phosphorylation at Y223 and Y551 residues. Nine variants (V416A, V416E, V416M, A428D, A428V, M437K, M437T, M477K, M477T, M477V, and E513A) remained sensitive to covalent inhibitors but showed resistance to Pirtobrutinib. Resistance was indicated by sustained phosphorylation at Y551 and/or Y223 despite drug exposure, suggesting impaired drug binding or altered BTK conformation. These findings expand the spectrum of resistance-associated BTK mutations and underscore the need for mutation-specific strategies. Future development of next-generation inhibitors or combination therapies will be critical to overcome resistance in CLL and other B-cell malignancies.