Combination of Gatekeeper Mutations and Cysteine 481 Replacement Causes Super Resistance to the Irreversible BTK Inhibitors Ibrutinib, Acalabrutinib and Zanubrutinib

Resistance to the irreversible Bruton´s tyrosine kinase (BTK) inhibitors is the main cause of disease progression in patients with Chronic Lymphocytic Leukemia (Quinquenel et. al. Blood 2019). Cysteine to serine substitution at the position 481 in BTK, is the most common resistance mutation. Other less frequent mutations like the constitutively active phospholipase C-gamma 2 (PLCG2) variant also occur (Woyach et. al. J. Clin. Oncology 2017). Different from many other tyrosine kinase inhibitors, BTK mutations less frequently affect the gatekeeper residue in the kinase domain (Maddocks et. al. JAMA Oncology 2015).

In this study, we have performed mutation scanning with substitutions replacing the gatekeeper residue. We have generated all the possible amino acid substitutions requiring a single nucleotide change in the gatekeeper and several variants requiring 2 or 3 nucleotide substitutions. Selected variants were also combined with substitutions at the C481, which is the binding site of irreversible BTK inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib.

Our results show unexpected, super-resistant variants and demonstrate that concomitant mutations, such as cysteine 481 to serine combined with threonine 474 to isoleucine or methionine, enhanced the resistance to irreversible BTK inhibitors. On the other hand, reversible BTK inhibitors displayed different inhibitory responses against the super-resistant mutants. Binding of the BTK inhibitors was subjected to molecular dynamics predictions, which correlated with the experimental binding data. Based on the available clinical and experimental results, the mechanisms underlying the spectrum of resistance mutations in BTK are presented.