# BTK resistance mutations: C481S, and L528W and T474I after the non-covalent inhibitors

Source: https://onco.cc/biomarkers/btk-c481s/  
OnCo record `btk-c481s` (Biomarker). Data CC BY-NC 4.0, attribute "Data from OnCo (onco.cc)"; commercial use needs a licence.

## TL;DR

A change in the enzyme BTK at the exact point where the drug grips it. The enzyme keeps working and the drug no longer holds, which is the usual reason a BTK inhibitor stops working after it has been working well.

## Summary

Ibrutinib and its covalent successors bind cysteine 481 of BTK irreversibly. Whole-exome sequencing of paired baseline and relapse samples from six patients with acquired resistance found a cysteine-to-serine substitution at that residue in five of them, and three distinct PLCG2 mutations in two; functional work showed that C481S leaves the protein only reversibly inhibited, and that the PLCG2 changes R665W and L845F are gain-of-function lesions producing autonomous B-cell receptor activity. Neither was found in nine patients with prolonged lymphocytosis who were still responding (Woyach 2014).

The answer to C481S is a non-covalent inhibitor that does not need the cysteine. Pirtobrutinib produced an overall response of 73.3% in 247 patients who had already received a covalent BTK inhibitor, with median progression-free survival of 19.6 months (Mato 2023). Resistance to that in turn runs through different residues: the kinase-dead L528W substitution was found in 7 of 13 patients progressing on zanubrutinib against 1 of 24 on ibrutinib, and in two patients it was enriched further under pirtobrutinib, which is cross-resistance rather than a new event; both of those patients responded to venetoclax afterwards (Blombery 2022).

## Fields

- Kind: Biomarker
- Last checked: 2026-09-30
- Also known as: BTK C481S; BTK Cys481Ser; BTK L528W; BTK T474I; BTK resistance mutation; ibrutinib resistance mutation
- Tags: biomarker; resistance; lymphoma

## Sources

- Woyach et al., N Engl J Med 2014: BTK C481S and PLCG2 resistance mutations under ibrutinib: https://doi.org/10.1056/NEJMoa1400029
- Blombery et al., Blood Adv 2022: enrichment of BTK Leu528Trp on zanubrutinib and cross-resistance to pirtobrutinib: https://doi.org/10.1182/bloodadvances.2022008325
- Mato et al., N Engl J Med 2023: pirtobrutinib after a covalent BTK inhibitor (BRUIN, 317 patients): https://doi.org/10.1056/NEJMoa2300696

## Connected records

- biomarkers: [BCL2 G101V and the other venetoclax binding-site mutations](https://onco.cc/biomarkers/bcl2-g101v/)
- cancers: [Mantle cell lymphoma](https://onco.cc/cancers/mantle-cell-lymphoma/), [Marginal zone lymphoma](https://onco.cc/cancers/marginal-zone-lymphoma/), [Non-Hodgkin lymphoma (all types)](https://onco.cc/cancers/non-hodgkin-lymphoma/), [Waldenström macroglobulinaemia](https://onco.cc/cancers/waldenstrom/)
- technologies: [Comprehensive genomic profiling](https://onco.cc/technologies/cgp/), [Liquid biopsy (ctDNA)](https://onco.cc/technologies/liquid-biopsy/)
- targets: [BTK (Bruton tyrosine kinase)](https://onco.cc/targets/btk/), [PLCG2](https://onco.cc/targets/plcg2/)
- drugs: [Acalabrutinib](https://onco.cc/drugs/acalabrutinib/), [Ibrutinib](https://onco.cc/drugs/ibrutinib/), [Pirtobrutinib](https://onco.cc/drugs/pirtobrutinib/), [Venetoclax](https://onco.cc/drugs/venetoclax/), [Zanubrutinib](https://onco.cc/drugs/zanubrutinib/)
- pathways: [B-cell receptor / BTK signalling (to NF-κB)](https://onco.cc/pathways/bcr-signalling/), [Resistance routes: how a blocked pathway comes back](https://onco.cc/pathways/resistance-routes-map/)
- terms: [Cross-resistance](https://onco.cc/terms/cross-resistance/), [Drug resistance (primary and acquired)](https://onco.cc/terms/resistance/), [Next-generation sequencing (NGS)](https://onco.cc/terms/ngs/)

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