Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant

Abstract

Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.

Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling

“…The regulation of Btk by upstream kinases appears to be complex, and there are certainly differences as to how Btk phosphorylation is regulated in different cell types and signaling pathways. Furthermore, the two sites may be subjected to differences in how tightly they are regulated by phosphatase activity, adding another mechanism for differential regulation (Hamasy et al, 2016).…”

PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors

“…An additional and unexpected observation in this BTK inhibitor resistant line was a decrease in p-BTK at Y223. A recent paper has highlighted that a C481F substitution in BTK leads to catalytic inactivation and subsequent decreased autophosphorylation of BTK [ 48 ].…”

AreBTKandPLCG2mutations necessary and sufficient for ibrutinib resistance in chronic lymphocytic leukemia?

“…A second sample taken when the same patient had developed a BTK C481S mutation, however, showed loss of BTK auto-phosphorylation only upon continuous exposure to ibrutinib, but not with washout of the drug, consistent with conversion to a reversible inhibitor [7], Not all reported BTK mutations have similar functional consequences. When phenylalanine, arginine or tyrosine are substituted for C481, the enzyme has greatly reduced (and in some cases undetectable) autophosphorylation and activation of downstream PLCγ2 in cellular assays [36], raising questions as to how such mutations could drive clinical resistance. Additionally, the reported BTK T316A mutation seen in two ibrutinib resistant patients [11, 34] has also been found in the germline of a patient with a clinical phenotype consistent with a mild form of X linked agammaglobulinemia [37], suggesting that the mutation is a hypomorph.…”