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Cited by 1,276Chronic Lymphocytic Leukemia ResearchLymphoma Diagnosis and TreatmentLung Cancer Treatments and Mutations

Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib

Open access
Woyach, Jennifer A., Furman, Richard R., Liu, Ta-Ming, Ozer, Hatice Gulcin, Zapatka, Marc, Ruppert, Amy S., Xue, Ling, Li, Daniel Hsieh-Hsin +17 more·2014·New England Journal of Medicine
DOI 10.1056/nejmoa1400029PMC PMC4144824
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Abstract

BACKGROUND: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS: We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS: We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS: Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

AI snapshot

Written from the paper’s full text.

TL;DR

Acquired resistance to ibrutinib in CLL often involves BTK C481S or PLCγ2 mutations that restore B-cell–receptor signaling. Functional studies show C481S prevents irreversible BTK binding, while PLCγ2 R665W/L845F are gain-of-function and BTK-independent.

Key findings

  • Whole-exome sequencing of six patients with acquired ibrutinib resistance identified BTK C481S in five patients and PLCγ2 mutations in two patients: R665W in Patient 6, and R665W, L845F, plus S707Y in one patient who also had low-frequency BTK C481S.
  • C481S BTK had lower ibrutinib affinity than nonmutant BTK (dissociation constant, 0.2 nM vs. 10.4 nM) and was only reversibly inhibited by ibrutinib; washout experiments in patient cells showed inhibition required continuous drug exposure.
  • PLCγ2 R665W and L845F showed enhanced anti-IgM–mediated calcium flux that was not inhibited by ibrutinib, indicating potential gain-of-function mutations allowing BTK-independent B-cell–receptor signaling.
  • No BTK or PLCγ2 mutations were found in nine patients with prolonged lymphocytosis (≥12 months) on ibrutinib; sequencing depth was >700 reads for BTK C481 and PLCγ2 R665/S707 and >100 reads for PLCγ2 L845.

Methods

  • Whole-exome sequencing was performed on baseline and relapse samples from six patients with acquired resistance to ibrutinib; relapse-specific nonsynonymous mutations were verified by Sanger or Ion Torrent sequencing.
  • Ion Torrent sequencing was used to test for BTK and PLCγ2 mutations in nine patients with prolonged lymphocytosis during ibrutinib therapy.
  • Functional analyses included in vitro kinase assays, flow cytometry, immunoblot assays, and stable transfection into DT40 or HEK 293T cells; statistical methods were detailed in the Supplementary Appendix.

Limitations

  • The paper states that inability to identify small numbers of mutant clones in the presence of large numbers of nonmutant clones may mean such variants were present but not identified; the clinical significance of very low baseline R665W variant reads is unknown and more patients need evaluation.
  • The paper states that other resistance mechanisms are likely present in a subgroup of patients without B-cell–receptor pathway mutations, and work is ongoing to identify alternative mechanisms.
  • Evident from the text: the study is based on a small number of patients (six with acquired resistance and nine with prolonged lymphocytosis), limiting generalizability and statistical power.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1056/nejmoa1400029

Cites

34 of 34 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

17 of 19 cited sources checked · 4 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In patients treated with ibrutinib, a characteristic lymphocytosis develops as CLL cells are mobilized from lymph nodes and the spleen.

    “In patients treated with ibrutinib, a characteristic lymphocytosis develops as CLL cells are mobilized from lymph nodes and the spleen.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] source

      The Results report that lymphocytosis occurred in 77% of patients treated with ibrutinib, and the Introduction describes cell mobilization from nodal and marrow sites as a possible mechanism. Our copy does not identify the spleen as a source of the mobilized CLL cells, so that part of the claim is unsupported. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    Although lymphocytosis typically resolves within 8 months, a subgroup of patients have lymphocytosis that lasts longer than 12 months in the presence of a continued response to ibrutinib.

    “Although lymphocytosis typically resolves within 8 months, a subgroup of patients have lymphocytosis that lasts longer than 12 months in the presence of a continued response to ibrutinib.24”
    Quote in the paperSubstantial claim1 of 1 sources checked
  • Findingcitation

    Dasatinib inhibits nonmutant and C481S mutant BTK with similar efficacy.

    “whereas dasatinib, a reversible inhibitor of BTK,23 inhibited nonmutant and mutant BTK with similar efficacy.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [23] source

      The citation is attached to the description of dasatinib as a reversible BTK inhibitor. The Results establish that dasatinib inhibits BTK, but our copy does not establish that its inhibition is reversible. It also does not test C481S or support the sentence’s comparison of C481S with nonmutant BTK; the mutant it tests is T474I, which was resistant. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    Ibrutinib is an irreversible inhibitor of BTK.

    “Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [6] source

      The section on PCI-32765-induced cytotoxicity states that PCI-32765 is an irreversible BTK inhibitor. Our copy does not name ibrutinib or establish that it is the same compound, so it does not fully support the claim as worded. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [8] source

      The Introduction states that PCI-32765 binds irreversibly to BTK, supporting the inhibitor property. Our copy does not identify PCI-32765 as ibrutinib, so it does not establish that the named drug has that property. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Premisecitation

    At 26 months, the estimated progression-free survival rate among patients treated with ibrutinib is 75%.

    “At 26 months, the estimated progression-free survival rate among patients treated with ibrutinib is 75%.9”
    Quote in the paperNot a substantial claim1 of 1 sources checked
  • May combine two claims. 11
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 1

  • May combine two claims.

    Premisecitation

    Ibrutinib has clinically significant activity in patients with relapsed CLL, with 71% objective complete or partial response and an additional 15 to 20% partial response with persistent lymphocytosis.

    “Ibrutinib has been shown to have clinically significant activity in patients with relapsed CLL, with 71% of patients having an objective complete or partial response and an additional 15 to 20% of patients having a partial response with persistent lymphocytosis.”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    The C481S mutation is not known to cause the X-linked agammaglobulinemia phenotype but instead results in retained catalytic activity.

    “This mutation is not known to cause the X-linked agammaglobulinemia phenotype31 but instead results in retained catalytic activity.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [31] source

      The citation is attached to the claim that C481S is not known to cause the X-linked agammaglobulinemia phenotype. The Mutation Statistics section discusses tolerated substitutions generally, but our copy does not address C481S specifically (searched: C481S, 481, and the phenotype claim). The retained-catalytic-activity clause falls after the citation and is not assessed as attributed to this source. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • The quote only partly states this claim.

    Findingown data

    In Patients 3, 5, and 6, Ion Torrent sequencing identified no mutation in BTK or PLCγ2 in more than 0.2% of reads.

    “In Patients 3, 5, and 6, Ion Torrent sequencing was performed, and no mutation was identified in more than 0.2% of reads”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Patient 6, the decrease in BTK phosphorylation at the time of relapse was similar to the decrease at 11 months of therapy, whereas PLCγ2 showed enhanced phosphorylation at the time of relapse.

    “In Patient 6, the decrease in BTK phosphorylation at the time of the relapse was similar to the decrease at 11 months of therapy, when the patient was having a response to the drug (Fig. 3A), whereas PLCγ2 showed enhanced phosphorylation at the time of relapse”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The C481S mutation allows reversible, but not irreversible, inhibition of BTK by ibrutinib.

    “Ibrutinib washout experiments showed that the C481S mutation allows reversible, but not irreversible, inhibition of BTK by ibrutinib”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In one patient, at baseline BTK phosphorylation was completely abrogated by ibrutinib with either washout or continuous exposure, whereas at relapse ibrutinib inhibited BTK phosphorylation only with continuous exposure.

    “At baseline, BTK phosphorylation was completely abrogated by ibrutinib with either washout or continuous exposure to ibrutinib. At the time of relapse, however, ibrutinib inhibited BTK phosphorylation only with continuous exposure”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After ibrutinib was discontinued in Patient 6, ibrutinib still inhibited BTK phosphorylation but not PLCγ2 phosphorylation.

    “After ibrutinib was discontinued, ibrutinib still inhibited BTK phosphorylation (Fig. 3C) but not PLCγ2 phosphorylation (Fig. 3D).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    The intrinsic affinity of ibrutinib for nonmutant BTK is significantly higher than for C481S mutant BTK (dissociation constant, 0.2 nM vs. 10.4 nM).

    “The intrinsic affinity of ibrutinib for nonmutant BTK was significantly higher than for mutant BTK (dissociation constant, 0.2 nM vs. 10.4 nM)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The R665W and L845F mutations in PLCγ2 are potentially gain-of-function mutations in the presence of B-cell-receptor stimulation and could be relevant as mutations conferring resistance to ibrutinib in patients.

    “These data show that the R665W and L845F mutations in PLCγ2 are potentially gain-of-function mutations in the presence of B-cell–receptor stimulation and could be relevant as mutations conferring resistance to ibrutinib in patients.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Sequencing depth for BTK at C481 and for PLCγ2 at R665 and S707 was more than 700 reads, and the sequencing depth for PLCγ2 at L845 was more than 100 reads.

    “The sequencing depth for BTK at C481 and for PLCγ2 at R665 and S707 was more than 700 reads, and the sequencing depth for PLCγ2 at L845 was more than 100 reads.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    The C481S mutation in BTK is not a second mutation in a previously mutated gene, but rather a primary mutation in a gene that is not recurrently mutated in CLL, and it is mutated at the amino acid required for drug binding.

    “Unlike the mutations in this group, however, the C481S mutation in BTK is not a second mutation in a previously mutated gene, but rather a primary mutation in a gene that is not recurrently mutated in CLL, and it is mutated at the amino acid required for drug binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In a recent preliminary study involving three patients with ibrutinib resistance, sequencing revealed the S707Y mutation in PLCγ2 that we identified in Patient 5 but did not reveal BTK mutations.

    “In a recent preliminary study involving three patients with ibrutinib resistance, sequencing revealed the S707Y mutation in PLCγ2 that we identified in Patient 5 in our study but did not reveal BTK mutations.32”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Burger JA, 2013, Blood source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The inability to identify small numbers of mutant clones in the presence of large numbers of nonmutant clones may mean that such variants were present but were not identified.

    “However, we recognize that our inability to identify small numbers of mutant clones in the presence of large numbers of nonmutant clones may mean that such variants were present but were not identified.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The pattern of a mutation localized to the irreversible binding site of ibrutinib and the immediate downstream kinase is not commonly seen with reversible kinase inhibitors and may be a result of continuous pressure on the drug target.

    “This pattern of a mutation localized to the irreversible binding site of ibrutinib and the immediate downstream kinase is not commonly seen with reversible kinase inhibitors and may be a result of continuous (rather than intermittent) pressure on the drug target.”
    Quote in the paperSubstantial claim
14 of 14

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia.

    “Furthermore, many mutations discovered in PLCγ2 have been linked to the development of complex immune disorders as well as resistance to ibrutinib treatment in chronic lymphocytic leukaemia.”

    From Characterisation of molecular mechanisms for PLCγ2 disease-linked variants (2024) · cites this paper as Woyach et al., 2014

    The Results and Discussion link three PLCγ2 mutations to acquired ibrutinib resistance in patients with chronic lymphocytic leukaemia. The Results also describe one of them, S707Y, as previously reported in an inherited inflammatory disease, but the paper does not link many PLCγ2 mutations to complex immune disorders. Page numbers are unavailable in our copy. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Whole-exome sequencing of six CLL patients with late relapses revealed C481S mutations in BTK of five patients and three distinct mutations in PLCG2 of two patients: L845F, R665W, and S707Y in one patient with tumor cells also harboring a BTK C481S mutation, and PLCG2 R665W as the sole mutation in the other patient.

    “whole-exome sequencing of six CLL patients with late relapses revealed C481S mutations in BTK of five patients and three distinct mutations in PLCG2 of two patients as follows: L845F, R665W, and S707Y in one patient with tumor cells also harboring a BTK C481S mutation and PLCG2 R665W representing the sole mutation in the other patient (29).”

    From The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein (2016) · cites this paper as [29]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • The R665W and L845F mutants of PLCγ2 appeared functionally normal in reconstituted DT40 chicken B cells in the absence of BCR stimulation, but mediated moderately enhanced and markedly prolonged ibrutinib-resistant increases in [Ca2+]i following BCR ligation with anti-IgM.

    “the R665W and L845F mutants of PLC␥ 2 appeared to be functionally normal in reconstituted DT40 chicken B cells in the absence of BCR stimulation, but to mediate moderately enhanced and markedly prolonged ibrutinib-resistant increases in [Ca 2ϩ ] i following BCR ligation with anti-IgM (29).”

    From The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein (2016) · cites this paper as [29]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • Ibrutinib, acalabrutinib, and zanubrutinib bind covalently to residue C481 of BTK, and thus are susceptible to mutations changing C481 to another amino acid, most commonly C481S.

    “Ibrutinib, acalabrutinib, and zanubrutinib bind covalently to residue C481 of BTK, and thus are susceptible to mutations changing C481 to another amino acid, most commonly C481S(19–21).”

    From Resistance Mechanisms and Approach to Chronic Lymphocytic Leukemia after BTK Inhibitor Therapy (2025) · cites this paper as [19]

    The Results report C481S in five patients at relapse and show that it prevents irreversible binding of ibrutinib, conferring relative resistance. Our copy does not address acalabrutinib or zanubrutinib, so it does not establish that those drugs bind C481 or are susceptible to this mutation. The five-patient finding also does not establish that C481S is the most common C481 substitution across all three drugs. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

  • In published studies of disease progression during BTKi therapy, approximately 35% of patients were found to have a BTK mutation alone, an additional 10% had coexisting mutations in PLCG2, 5% had a PLCG2 mutation alone, and the remaining 50% were wild-type for BTK and PLCG2.

    “In published studies of disease progression during BTKi therapy,approximately 35% of patients were found to have aBTK mutation alone, an additional 10% had a coexisting mutations in PLCG2, 5% of cases had a PLCG2mutation alone, and the remaining 50% were wild-type (WT) for BTK and PLCG2 (Figure 2)(12, 19, 21–31).”

    From Resistance Mechanisms and Approach to Chronic Lymphocytic Leukemia after BTK Inhibitor Therapy (2025) · cites this paper as [19]

    The Results section (page unavailable) reports BTK-only, PLCγ2-only, and coexisting mutations among six patients with progressive CLL during ibrutinib therapy. It does not report the claimed 35%/10%/5%/50% distribution across published studies; all six patients in this study had at least one of the two mutations at relapse. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • ALK resistance claim cites a ROS1 mutation study

    Minor

    The text cites reference 27 as an example of resistance through an ALK mutation, but reference 27 is titled as a report of resistance from a CD74-ROS1 mutation.

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Authors
Woyach, J. A., Furman, R. R., Liu, T.-M., Ozer, H. G., Zapatka, M., Ruppert, A. S., Xue, L., Li, D. H.-H., Steggerda, S. M., Versele, M., Dave, S. S., Zhang, J., Yilmaz, A. S., Jaglowski, S. M., Blum, K. A., Lozanski, A., Lozanski, G., James, D. F., Barrientos, J. C., Lichter, P., Stilgenbauer, S., Buggy, J. J., Chang, B. Y., Johnson, A. J., & Byrd, J. C.
Affiliations
  • German Cancer Research Center (DE)
  • Weill Cornell Medicine (US)
  • Pharmacyclics (United States) (US)
  • Janssen (Belgium) (BE)
  • Duke University (US)
  • The Ohio State University (US)
  • Northwell Health (US)
  • Universität Ulm (DE)
ORCID
Venue
New England Journal of Medicine
Publisher
Massachusetts Medical Society
Published
2014-06-12
Volume
370
Issue
24
Pages
2286-2294
ISSN
  • 0028-4793 (ISSN-L)
  • 1533-4406
DOI
10.1056/nejmoa1400029
PMC
PMC4144824
OpenAlex
W2067064694
Cited by
1,275
References
34
Citations by year
2026: 50 · 2025: 77 · 2024: 91 · 2023: 99 · 2022: 102 · 2021: 143 · 2020: 122 · 2019: 99 · 2018: 98 · 2017: 126 · 2016: 118 · 2015: 108 · 2014: 39 · 2012: 1
FWCI
58.67
Type
journal article
Language
en
Open access
Locations
  • New England Journal of Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Institutes of Health (4p01ca081534-16, 5k12ca133250-17, 2p30ca014236-51)
  • National Cancer Institute (P30‐CA014236, R01CA183444, P01CA095426, K12‐CA133250, P50CA140158, K23 CA178183, R01CA177292, P30CA016058, P01-CA-081534)
Topics
  • Health Sciences › Medicine › Genetics › Chronic Lymphocytic Leukemia Research
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Lymphoma Diagnosis and Treatment
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Lung Cancer Treatments and Mutations
Keywords
ibrutinib, Bruton's tyrosine kinase, chronic lymphocytic leukemia, drug resistance, B cell receptor signaling, irreversible kinase inhibitors, relapsed disease
MeSH terms
Agammaglobulinaemia Tyrosine Kinase, Adenine, Aged, Binding Sites, Humans, Middle Aged, Piperidines, Protein-Tyrosine Kinases, Pyrazoles, Pyrimidines, Receptors, Antigen, B-Cell, Recurrence, Leukemia, Lymphocytic, Chronic, B-Cell, Point Mutation, Sequence Analysis, DNA, Drug Resistance, Neoplasm, Phospholipase C gamma, Exome

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