Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.
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.
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Hamasy
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Wang
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Blomberg
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Mohammad
Department of Hematology, Huai'an First People's Hospital, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.
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Yu
Department of Experimental Medical Science, Lund University, Lund, Sweden.
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Vihinen
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Berglöf
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Smith
D000225:Adenine / Q000031:analogs & derivatives; D000077329:Agammaglobulinaemia Tyrosine Kinase; D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D003545:Cysteine; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D010880:Piperidines; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013912:Threonine; D014162:Transfection
D010880:Piperidines; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; C551803:ibrutinib; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D000225:Adenine; D003545:Cysteine