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Cited by 80Chronic Lymphocytic Leukemia ResearchLung Cancer Treatments and MutationsLymphoma Diagnosis and Treatment

BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib

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Estupiñán, H. Yesid, Wang, Qing, Berglöf, Anna, Schaafsma, Gerard C. P., Shi, Yuye, Zhou, Litao, Mohammad, Dara K., Yu, Liang +3 more·2021·Leukemia
DOI 10.1038/s41375-021-01123-6PMC PMC8102192
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Abstract

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

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  • BTK T474I/S have been termed "gatekeeper mutations" as they cause steric interference with binding for ibrutinib or acalabrutinib.

    “BTKT474I/S have been termed “gatekeeper mutations” as they cause steric interference with binding for ibrutinib or acalabrutinib (39, 40).”

    From Resistance Mechanisms and Approach to Chronic Lymphocytic Leukemia after BTK Inhibitor Therapy (2025) · cites this paper as [40]

    The Discussion (page not available in our copy) identifies T474I and T474S as gatekeeper variants and says T474I could sterically interfere with binding of covalent inhibitors. It does not extend that mechanism to T474S: the Discussion says its smaller side chain is less likely to clash, and the Results report that T474S remained sensitive to ibrutinib. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • In vitro studies have shown that cell lines transfected with both C481 and T474 mutations are insensitive to exponentially higher pharmacological serum concentrations of ibrutinib, acalabrutinib, and zanubrutinib.

    “Invitro studies have shown thatcell lines transfected with both C481 and T474mutations are insensitive to exponentially higher pharmacological serum concentrations of ibrutinib, acalabrutinib, and zanubrutinib(40).”

    From Resistance Mechanisms and Approach to Chronic Lymphocytic Leukemia after BTK Inhibitor Therapy (2025) · cites this paper as [40]

    The Results report that transfected cells carrying T474I/C481S or T474M/C481S were highly resistant to ibrutinib, acalabrutinib, and zanubrutinib at elevated concentrations. But the claim does not specify which substitutions: the Results also report that T474A/C481S was sensitive and T474S/C481S was partially sensitive to ibrutinib at a pharmacological concentration. Zanubrutinib also overcame the tested double variants at 64 μM, rather than leaving them wholly insensitive. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

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Authors
Estupiñán, H. Y., Wang, Q., Berglöf, A., Schaafsma, G. C. P., Shi, Y., Zhou, L., Mohammad, D. K., Yu, L., Vihinen, M., Zain, R., & Smith, C. I. E.
Affiliations
  • Karolinska University Hospital (SE)
  • Lund University (SE)
  • Huaian First People’s Hospital (CN)
  • Karolinska Institutet (SE)
ORCID
Venue
Leukemia
Publisher
Springer Science and Business Media LLC
Published
2021-02-01
Volume
35
Issue
5
Pages
1317-1329
ISSN
  • 0887-6924 (ISSN-L)
  • 1476-5551
DOI
10.1038/s41375-021-01123-6
PMC
PMC8102192
OpenAlex
W3127067046
Cited by
80
References
7
Citations by year
2026: 12 · 2025: 15 · 2024: 20 · 2023: 8 · 2022: 17 · 2021: 8
FWCI
8.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Leukemia (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Lund University Publications (Lund University) (submittedVersion)
  • Greater South Information System
Funders
  • Ministerio de Ciencia, Tecnología e Innovación
  • Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS)
  • Jiangsu Commission of Health (H2018085)
  • Cancerfonden
  • Government of Jiangsu Province
  • Medical Research Council
Topics
  • Health Sciences › Medicine › Genetics › Chronic Lymphocytic Leukemia Research
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Lung Cancer Treatments and Mutations
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Lymphoma Diagnosis and Treatment
Keywords
Bruton's tyrosine kinase, BTK inhibitors, ibrutinib, acalabrutinib, zanubrutinib, gatekeeper residue, drug resistance, double mutants, irreversible inhibitors, reversible inhibitors, tyrosine kinase inhibitors, lymphoma
MeSH terms
Agammaglobulinaemia Tyrosine Kinase, Adenine, Animals, Benzamides, Cell Line, Chlorocebus aethiops, Chickens, Cysteine, Humans, Mutation, Piperidines, Pyrazines, Pyrazoles, Pyrimidines, Threonine, Drug Resistance, Neoplasm, COS Cells, Cell Line, Tumor, Protein Kinase Inhibitors, HEK293 Cells

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