Evaluating ibrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, and zanubrutinib in 34 BTK-variants at 7 inhibitor resistance sites

Evaluating ibrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, and zanubrutinib in 34 BTK-variants at 7 inhibitor resistance sites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evaluating ibrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, and zanubrutinib in 34 BTK-variants at 7 inhibitor resistance sites Abdulrahman Hamasy, Qing Wang, Brisejda Rustemi, Eleni Afentaki, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9390771/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Bruton tyrosine kinase (BTK) plays a central role in B-cell receptor signaling, and its inhibition has transformed the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL). Despite the success of BTK inhibitors (BTKi), resistance from acquired BTK mutations poses a significant clinical challenge. Here, we systematically characterized 34 BTK variants associated with 7 sites of resistance, assessing kinase activity and responsiveness to both the covalent (cBTKi), ibrutinib, remibrutinib, rilzabrutinib (reversible cBTKi) and zanubrutinib and the non-covalent (ncBTKi) inhibitor, pirtobrutinib. In contrast to cysteine-481, where all 6 replacements caused by single-nucleotide changes have been reported, this is not the case for substitutions in other locations. Among them, threonine-316-to-alanine (T316A) conferred broad resistance to both cBTKi and ncBTKi; in contrast, substitutions at leucine-528 disrupted catalytic activity while preserving protein expression. Variants at valine-416, alanine-428, methionine-437 and methionine-477, largely maintained sensitivity to cBTKi, whereas glutamate-513 substitutions showed mutation-specific patterns of inhibitor responsiveness. The T316A substitution induced constitutive catalytic activity and, when inherited, was previously reported to cause atypical X-linked agammaglobulinemia suggesting an intricate resistance mode. Notably, the newly approved BTKi, remibrutinib and rilzabrutinib, were ineffective against several resistance-associated variants. These findings highlight the complex, residue-specific nature of BTKi-resistant BTK variants. Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance Biological sciences/Cell biology/Cell signalling Biological sciences/Cancer/Oncogenes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bruton tyrosine kinase (BTK) plays a fundamental role in B-cell biology as a key component of the B-cell receptor (BCR) signaling pathway, which governs B-cell development, maturation, differentiation, and survival 1 . Upon antigen engagement, activation of the BCR triggers a cascade of downstream signaling events mediated by BTK, leading to calcium mobilization, transcriptional activation, and regulation of cellular proliferation and apoptosis 2 . Beyond its role related to B cell receptor, BTK is involved in several additional signaling pathways, including those mediated by chemokine receptors and Toll-like receptors, thereby influencing cell migration, adhesion, and immune regulation 3 , 4 . Dysregulation of BTK-dependent signaling contributes to the pathogenesis of a spectrum of B-cell malignancies, most notably CLL and Waldenström’s macroglobulinemia, where aberrant and constitutive BCR activation sustains the growth, survival, and clonal expansion of malignant B cells 5 . Following the successful experimental development of ibrutinib, the recognition of BTK’s pivotal role in these oncogenic processes provided a strong rationale for the continued development of targeted therapies designed to modulate its activity 6 , 7 . This expanded the number of BTKi, a class of small-molecule agents that bind to and block the catalytic activity of BTK, thereby interrupting pathological BCR signaling 8 . The introduction of BTKi has revolutionized the management of CLL and other B-cell malignancies, representing a major transition from traditional chemoimmunotherapy toward precision-targeted treatment strategies 9 , 10 . By effectively inhibiting BTK-mediated signaling, these agents suppress malignant B-cell proliferation, induce apoptosis, and disrupt interactions between leukemic cells and their supportive microenvironment within lymphoid tissues 11 – 14 . Despite the transformative efficacy of BTKi in B-cell malignancies, the emergence of acquired resistance has become a major clinical challenge, particularly in patients receiving long-term therapy over several years. Resistance mechanisms to BTKi are broadly categorized into those affecting BTK itself and those involving alternative or downstream signaling pathways that restore BCR activity or promote cell survival through BTK-independent mechanisms 15 , 16 . The point mutations affecting the BTK cysteine (C) 481 codon, causing BTK C481-to-serine (S), the most frequent and well-characterized resistance mechanism, are observed in a significant proportion of patients progressing on cBTKi, ibrutinib acalabrutinib or zanubrutinib therapy 17 – 19 . To overcome resistance conferred by acquired BTK C481 variants, next-generation ncBTKi, such as pirtobrutinib, have been developed. These agents bind non-covalently to BTK at sites distinct from the C481 residue, inhibiting both wild-type and C481-mutant BTK 20 . Nevertheless, secondary resistance to ncBTKi has also been documented in variations at other sites. Recently identified mutations such as V416L, A428D, M437R, T474I, T474M and L528W, alter the kinase domain’s structural configuration and reduce affinity for reversible inhibitors 21 – 23 . In vitro characterization of potential resistance-associated BTK variations remains crucial, to further strengthen our understanding of the underlying mechanisms. In this study, we evaluated 34 BTK variants caused by single nucleotide substitutions at 7 residues implicated in therapeutic resistance, to assess their functional activity and sensitivity to both cBTKi and ncBTKi. Materials and methods Plasmids Plasmids encoding BTK substitutions were generated by site-directed mutagenesis and verified by sequencing (Mutagenex, Inc. Suwanee, GA, USA) (Supplementary tabl e1 ). Cell culture and transfections COS-7 and HEK-293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). B7.10 cells, lacking BTK expression, were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 5% chicken serum, 5% glutamine, 50 µM 2-mercaptoethanol, and penicillin-streptomycin as previously described 18 , 19 , 21 . BTK-deficient K562 cells were a kind gift of Dr. Donald B. Kohn, University of California, Los Angeles, CA, and were generated by CRISPR-Cas9–mediated gene editing, as previously described by David H. Gray et al 24 . The cells were grown in RPMI-1640 supplemented with 10% heat-inactivated FBS. All cultures were incubated at 37°C in under 5% CO₂. Plasmids were transiently transfected into COS-7 and HEK-293T cells by using polyethylenimine (PEI) (Polyscience, Inc.). Electroporation of K562 and B7.10 cells was performed by the neon transfection system (Life Technologies, Carlsbad, CA, USA). Western blotting Western blotting Cells were subjected to serum starvation and inhibition, followed by activation with serum and pervanadate for 5 minutes at room temperature. Prior to activation, washout steps were performed three times in serum-free medium to remove residual inhibitors, thereby reducing the risk of off-target effects. Whole-cell lysate was obtained by using modified RIPA buffer supplemented with protease and phosphatase inhibitors. Protein extracts were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with specific primary and secondary antibodies. Signal detection and quantification were performed using the Odyssey imaging system (LI-COR Biosciences GmbH). BTK inhibitors All BTK inhibitors were stored at − 20°C and initially dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, with fresh dilutions prepared in phosphate-buffered saline (PBS) for each experiment. 36 to 48 hours post-transfection, cells were serum-starved for 4 hours, and inhibitors were added during the last 2 hours of starvation. The BTK inhibitors used in this study included ibrutinib (Selleckchem, Houston, TX, USA), zanubrutinib (Chemgood, Glen Allen, VA, USA), and pirtobrutinib, remibrutinib and rilzabrutinib (MedChemExpress, Stockholm, Sweden). Results Expression and catalytic activity of BTK variants We initially studied some of the mutants in COS-7 and HEK293 cells and in the chicken BTK- defective cell line B7.10, as previously described 18 , 19 , 21 . Because human cells would be more relevant, we subsequently generated results using BTK-deficient K562 cells, which lack endogenous BTK expression through gene editing. Cells were transfected with constructs encoding all the BTK variants to allow assessment of kinase activity in an isolated system. A total of 34 amino acid substitutions at key BTK residues contributing to therapeutic resistance were analyzed (Fig. 1 A). Autophosphorylation of tyrosine 223 within the SH3 domain was used as a surrogate marker of BTK catalytic activity 25 , 26 . While the variants were expressed at comparable levels, distinct differences in phosphorylation intensity indicated variable enzymatic activity among the acquired BTK variants (Fig. 1 B). Based on these criteria, we identified 25 out of 34 BTK variants (73%) in our dataset as functionally active. BTK T316A substitution confers resistance to both cBTKi and ncBTKi The SH2 domain of BTK binds to phosphotyrosine-containing motifs on adaptor proteins such as BLNK, an interaction essential for proper B-cell development and signaling 27 . The BTK T316A substitution causes a structurally distinct alteration within the SH2 domain that does not directly affect the ibrutinib-binding site in the kinase domain. This variant has been identified in patients with CLL experiencing disease progression during ibrutinib therapy 28 , 29 . Our results highlight the contribution of non-catalytic domain alterations to therapeutic failure. With the exception of T316A, all other substitutions at this residue retained sensitivity to cBTKi, although their responses to reversible inhibitors varied. The T316A mutation conversely conferred resistance to both cBTKi and ncBTKi, indicating its unique influence on the inhibitor interaction (Fig. 2 ). Substitutions of L528 disrupt the enzyme activity The enzymatically inactive BTK leucine (L) 528-to-tryptophane (W) substitution sustains malignant B-cell survival through non-catalytic functions and activation of compensatory pathways 30 , 31 . These findings, similar to the effect of most of the C481 replacements 18 , underscore the complexity of BTK signaling and reveal that resistance is not solely dependent on catalytic function but also involves kinase-independent mechanisms that sustain oncogenic signaling networks. Substitutions at this residue substantially altered BTK catalytic activity. Among the five variants tested, L528F, L528M, and L528V retained measurable kinase activity. While these active variants demonstrated sensitivity to cBTKi, they displayed reduced responsiveness to the non-covalent inhibitor pirtobrutinib (Fig. 3 ). The BTK variants at residues valine (V) 416, alanine (A) 428, methionine (M) 437 and methionine (M) 477 are sensitive to cBTKi To overcome resistance associated with cBTKi, particularly those by C481 replacements, next-generation ncBTKi, including fenebrutinib, nemtabrutinib, and pirtobrutinib, were developed to engage BTK through hydrogen bonding, ionic, and hydrophobic interactions independent of covalent attachment 32 . Although these agents initially demonstrated substantial efficacy against C481-mutated BTK 32 , 33 , clinical relapses have subsequently been reported. Comprehensive genomic analyses of relapsed or refractory CLL patients revealed the emergence of additional on-target BTK mutations (V416L, A428D, M437R, M477I, T474I, and L528W) alongside downstream PLCγ2 alterations 29 . Importantly, the A428D variant has recently been associated with resistance to BTK degrader therapy in CLL patients 34 . These substitutions are thought to induce conformational changes within the kinase domain that perturb inhibitor binding and regulatory signaling, thereby conferring resistance even to non-covalent BTK inhibitors 21 , 23 . Comparative analysis of phosphorylation profiles across BTK variants at residues V416, A428, M437, and M477, revealed distinct patterns of inhibitor sensitivity. BTK with substitutions at A428, M437, and M477 largely preserved responsiveness to cBTKi, indicating that covalent binding at C481 remains functionally intact, while showing variable sensitivity to reversible agents. In contrast, the BTK variants with alterations at V416 retained sensitivity to cBTKi but exhibited reduced responsiveness to ncBTKi, suggesting that residue-specific conformational alterations in the kinase domain may influence inhibitor binding and catalytic regulation (Fig. 4 and Supplementary Fig. S1 ). BTK variants at E513 cause constitutive activation Evaluation of BTK variants at residue E513 showed that both E513A and E513G substitutions caused constitutive activation and displayed partial sensitivity to ibrutinib. Conversely, they showed resistance to both the cBTKi, zanubrutinib, and the ncBTKi, pirtobrutinib, indicating mutation-specific effects on inhibitor binding (Fig. 4 and Supplementary Fig. S1 ). Limited efficacy of newly approved BTKi remibrutinib and rilzabrutinib against resistance-associated BTK variants Recently, two novel BTK inhibitors have received FDA approval: remibrutinib (Rhapsody), a potent cBTKi approved for chronic spontaneous urticaria 35 , and rilzabrutinib (Wayrilz), a reversible cBTKi approved for adults with persistent or chronic immune thrombocytopenia 36 . To assess their potential efficacy against resistant BTK variants, we evaluated the sensitivities of several clinically relevant mutants in CLL (T316A, M437K, M437V, E513A, and L528V), alongside the common C481S variant. Among these, the M437V mutant remained sensitive to both inhibitors, and the L528V variant retained partial responsiveness to remibrutinib, whereas the other missense variants exhibited resistance to both agents, suggesting limited activity of these newly approved inhibitors against clinically identified resistance-associated BTK mutations (Fig. 5 ). BTKi resistance mutations causing atypical X-linked agammaglobulinemia (XLA) Loss-of-function mutations in the BTK gene cause the disease XLA 37 , 38 , resulting in a differentiation block at the pro-B to pre-B cell transition. Established in 1995, the BTKbase collects all known mutations causing XLA, with the most recent update published in 2023 39 . When analyzing the different acquired mutations, and the single-nucleotide substitution variants tested here, we observed two variants that are known to cause XLA. These are the reported BTKi resistance mutation T316A affecting the SH2 domain 40 , 41 , and the new variant M477R, tested by us in this report, being located in the catalytic SH1 domain. Discussion Comprehensive in vitro characterization of potential resistance-associated BTK variants is essential for understanding their functional and therapeutic implications. Although a substantial number of BTK mutations has been identified in patients receiving BTK inhibitor therapy, many of these variants occur at low frequencies, making it challenging to determine their true clinical relevance. Experimental in vitro validation is one way to critically assess how specific amino acid substitutions affect BTK kinase activity, signaling function, and inhibitor binding. Such investigations not only enhance our understanding of resistance mechanisms but may also guide the rational design of next-generation inhibitors capable of overcoming both common and rare resistance mutations. The number of different replacements causing acquired BTKi resistance, varies considerably. One position is currently unique, the codon for cysteine 481, since all possible seven single-nucleotide changes, including two for serine, have been reported to cause resistance to cBTKi (Fig. 1 A) 42 – 44 . Cysteine 481 carries a nucleophilic thiol side chain forming a covalent bond with cBTKi, and none of the substitutions permits covalent bonding 18 . In contrast, for the frequently mutated L528 position, out of the 5 possible substitutions, only tryptophan replacement has been identified in patients (Fig. 1 A). Of note, of these five substitutions, tryptophan is the largest residue. The other substitutions are either rare or do not cause resistance 30 . Mutation frequencies in genomes are known to be enhanced at CpG sites owing to deamination of 5-methylcytosine to thymine 45 , and in nucleotide repeats causing DNA polymerase slippage during replication 46 . However, such elements are not found in any of the genomic regions affected by BTKi resistance. For the most common cBTKi-acquired resistance variant, C481S, there are two reasons explaining its frequent occurrence. Hence, two different codon changes yield the same cysteine substitution 47 , and secondly C481S is catalytically active 48 . The C481T mutant is also catalytically active 18 , but likely because there is a need for a rare dinucleotide replacement 49 , this variant was so far not reported in any patient. For the 7 sites studied in this report, merely a single replacement for each site (n = 7) has been identified out of 34 possible substitutions (Fig. 1 A). It is possible that only certain replacements yield what could be referred to as “a functional BTK molecule”. Thus, in XLA the majority of amino acid replacements induces instability. However, most of these substitutions affect hydrophobic residues buried in the core of BTK 39 , whereas the BTKi resistance mutations afflict amino acids located on the surface of the catalytic cleft 50 , where replacements more frequently are tolerated in terms of protein stability. Moreover, when a certain missense mutation causing XLA results in the expression of the enzyme, it is common that also other substitutions affecting the same residue are stable 39 . Therefore, we do not favor the idea that differential protein stability is the mechanism underlying the observation that just a single resistance substitution was so far reported for most of the 7 sites. To this end, we have previously studied the gatekeeper residue threonine 474 replacements, both the single-nucleotide substitutions A/I/N/P/S and also more complex alterations 19 . At this site, 2 single-nucleotide substitutions were previously found in patients (Fig. 1 A) 43 . Recently, however, Brown et al. reported the additional T474F/L/M/Y replacements in patients treated with the ncBTKi, pirtobrutinib 23 . Interestingly, these 4 variants could only appear after dinucleotide changes, which, similar to C481T, are expected to be extremely rare. Regarding “functional resistance”, it is well known that mutants do not need to be catalytically active, a case in point being the 5/6 kinase-dead replacements affecting C48 18 . The exact mechanism underlying this form of resistance is not known, but kinase-dead BTK could retain the capacity to assemble a signaling complex. Furthermore, expression of the SRC family kinase HCK (hematopoietic cell kinase) has been suggested as a key component compensating for the lack of BTK’s catalytic activity 51 , 52 . A very interesting resistance variant is T316A located in the SH2 domain. This replacement is unique because it is the only known resistance variant located outside of the kinase domain. T316A was also identified in an Italian patient with dysgammaglobulinemia, differing from classical XLA 40 , as well as in a Japanese patient 41 . In both of these patients the B-cell numbers decreased over time. Also, for BTKi resistance only two patients were so far reported to carry the T316A variant 28 , 29 . Both developed Richter transformation, but in one of the patients only the CLL cells carried the mutation 28 . It was previously reported that T316A drives BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from internal repression 53 . This structure-function analysis is in agreement with our finding of a pronounced constitutive activation induced by the T316A protein in transfected K562 cells. However, also a proline-substitution caused substantial constitutive activity, while lower than T316A, and this variant has not been reported in any patient to date. It could be hypothesized that such an intrinsic activation might be toxic to cells and that this could explain the rarity of patients developing such a resistance to BTKi. The potential noxiousness of T316A, with B-cell numbers decreasing over time in the inherited form 40 , 41 , may be related to the “Goner” concept. This concept was initially developed to explain the fact that BTKi resistance mutations in the PLCG2 gene in CLL patients result in lower variant allele frequencies as compared to BTK gene alterations causing resistance variants 54 , 55 . Because such acquired variants are activated, the toxic constitutive signaling likely causes loss of such B cells. Thus, even if resistance develops, the malignant cells pay a considerable price for carrying such a PLCG2 variant. A special form of resistance is the one previously identified to affect glutamate-513 56 . The authors mutagenized the BTK coding sequence using repair-defective E. coli , reaching an overall nucleotide variant frequency of 90%. They subsequently screened the resulting library for ibrutinib escape mutants in murine BCL1 lymphoma cells. In their report, two of the escape variants, T474M and of E513G, increased BTK autophosphorylation. The T474M substitution together with numerous other replacements of this residue were already investigated by us 19 , so we decided to further study E513 substitutions. Both the novel single-nucleotide replacement to alanine (A) and the previously reported glycine (G) 56 replacement caused very significant constitutive activation (Fig. 1 B). A similar argument, as used for the T316A substitution, may therefore also explain the effect of the E513 replacements. Hence, both of the substitutions E513A/G might be toxic to B cells, and this could be the reason for them not yet being observed as acquired resistance mutations to BTKi in patients. Moreover, if they are noxious, it is also likely that they could cause XLA, possibly in the form of an atypical disease, similar to patients with the T316A variant 40 . While E513 replacements were not reported in BTKi treated patients, nor in XLA, in spite of more than 1000 different mutations having been identified 39 , it could still simply be by chance that they did not yet appear. Besides the structural effect of the replacement with largest aromatic amino acid causing the frequently observed kinase-dead L528W resistance variant, could there be additional mechanisms? Two of the replacements cause significant constitutive activation, namely L528M/V. Thus, a similar reasoning as for T316A could also be made here, namely that this activity could be toxic to cells. Akin to T316A and E513A/G, there was no indication that L528M/V caused toxicity in the short-term transfection experiments using K562. However, as compared to transformed cell lines, primary cells are often more sensitive to mutations and therefore this hypothesis may still hold true. Our assessment of newly FDA-approved BTK inhibitors remibrutinib and rilzabrutinib revealed limited activity against clinically relevant resistance-associated variants. Among tested mutants, only M437V retained sensitivity to both agents, whereas L528V showed partial responsiveness to remibrutinib. These findings underscore the persistent challenge of mutation-caused resistance and suggest that novel next-generation inhibitors may have restricted utility in this context. Collectively, these results emphasize that BTK inhibitor resistance is multifactorial, involving both catalytic and non-catalytic domain mutations, and highlight the use of systematic preclinical assessment of clinically observed BTK variants to predict potential resistance and guide personalized therapeutic strategies in CLL and other B-cell malignancies. Declarations Conflict of interest The authors declare no conflict of interest. Author contributions CIES perceived and conceptualized the project, obtained funding, interpreted data, supervised throughout, wrote part of the discussion and edited the manuscript. RZ interpreted the data, supervised, edited the manuscript and obtained funding. AH, performed most of the experiments with major help from QW. BR, EA, AV and EH performed selected experiments. AB was involved in data interpretation and manuscript editing. MV carried out protein structural analysis. AH wrote the manuscript with edits performed by CIES, RZ and QW. Acknowledgments This work was supported by the Swedish Cancer Society, the Swedish Research Council and the Swedish County Council and Center for Innovative Medicine (CIMED). Data availability statement The dataset generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Smith CIE. From identification of the BTK kinase to effective management of leukemia. Oncogene. 2017; 36: 2045–2053. McDonald C, Xanthopoulos C, Kostareli E. The role of Bruton’s tyrosine kinase in the immune system and disease. Immunology. 2021; 164: 722–736. Ringheim GE, Wampole M, Oberoi K. Bruton’s Tyrosine Kinase (BTK) inhibitors and autoimmune diseases: making sense of BTK inhibitor specificity profiles and recent clinical trial successes and failures. Front Immunol. 2021; 12: 662223. Rip J, de Bruijn MJW, Appelman MK, Singh SP, Hendriks RW, Corneth OBJ. Toll-like receptor signaling drives Btk-mediated autoimmune disease. Front Immunol. 2019;10: 95. Pal Singh S, Dammeijer F, Hendriks RW. Role of Bruton’s tyrosine kinase in B cells and malignancies. Mol Cancer. 2018; 17: 57. Pan Z, Scheerens H, Li SJ, Schultz BE, Sprengeler PA, Burrill LC et al. Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase. ChemMedChem. 2007; 2: 58–61. Ahn IE, Brown JR. Targeting Bruton’s tyrosine kinase in CLL. Front Immunol. 2021; 12: 687458. Mouhssine S, Maher N, Matti BF, Alwan AF, Gaidano G. Targeting BTK in B cell malignancies: from mode of action to resistance mechanisms. Int J Mol Sci. 2024; 25: 3234. Shirley M. Bruton tyrosine kinase inhibitors in B-cell malignancies: their use and differential features. Target Oncol. 2022; 17: 69–84. Kim HO. Development of BTK inhibitors for the treatment of B-cell malignancies. Arch Pharm Res. 2019; 42: 171–181. Nore BF, Vargas L, Mohamed AJ, Brandén LJ, Bäckesjö CM, Islam TC et al. Redistribution of Bruton’s tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases. Eur J Immunol. 2000; 30: 145–154. Burger JA. Targeting the microenvironment in chronic lymphocytic leukemia is changing the therapeutic landscape. Curr Opin Oncol. 2012; 24: 643–649. de Rooij MFM, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood. 2012; 119: 2590–2594. Wiestner A. BCR pathway inhibition as therapy for chronic lymphocytic leukemia and lymphoplasmacytic lymphoma. Hematology Am Soc Hematol Educ Program. 2014; 2014: 125–134. Smith CIE, Burger JA. Resistance mutations to BTK inhibitors originate from the NF-κB but not from the PI3K-RAS-MAPK arm of the B cell receptor signaling pathway. Front Immunol. 2021; 12: 689472. Wiśniewski K, Puła B. A review of resistance mechanisms to Bruton’s kinase inhibitors in chronic lymphocytic leukemia. Int J Mol Sci. 2024; 25: 5246. Brown JR. Resistance to Bruton tyrosine kinase inhibitors. Hematol Oncol Clin North Am. 2025; 39: 951–963. Hamasy A, Wang Q, Blomberg KEM, Mohammad DK, Yu L, Vihinen M et al. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant. Leukemia. 2017; 31: 177–185. Estupiñán HY, Wang Q, Berglöf A, Schaafsma GCP, Shi Y, Zhou L et al. BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib. Leukemia. 2021; 35: 1317–1329. Gomez EB, Ebata K, Randeria HS, Rosendahl MS, Cedervall EP, Morales TH et al. Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor. Blood. 2023; 142: 62–72. Naeem AS, Utro F, Wang Q, Cha J, Vihinen M, Martindale SP et al. Resistance to the non-covalent BTK inhibitor pirtobrutinib. Blood. 2022; 140: 6981–6982. Wang E, Mi X, Thompson MC, Montoya S, Notti RQ, Afaghani J et al. Mechanisms of resistance to noncovalent Bruton’s tyrosine kinase inhibitors. N Engl J Med. 2022; 386: 735–743. Brown JR, Nguyen B, Desikan SP, Won H, Tantawy SI, McNeely SC et al. Genomic determinants of response and resistance to pirtobrutinib in relapsed/refractory chronic lymphocytic leukemia. Blood. 2026; 147: 24–34. Gray DH, Villegas I, Long J, Santos J, Keir A, Abele A et al. Optimizing integration and expression of transgenic Bruton’s tyrosine kinase for CRISPR-Cas9-mediated gene editing of X-linked agammaglobulinemia. CRISPR J. 2021; 4: 191–206. Estupiñán HY, Bouderlique T, He C, Berglöf A, Cappelleri A, Frengen N et al. In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function. Blood Adv. 2024; 8: 1981–1990. Middendorp S, Dingjan GM, Maas A, Dahlenborg K, Hendriks RW. Function of Bruton’s tyrosine kinase during B cell development is partially independent of its catalytic activity. J Immunol. 2003; 171: 5988–5996. Tzeng S, Pai M, Lung F, Wu C, Roller P, Lei B et al. Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia. Protein Sci. 2000; 9: 2377–2385. Sharma S, Galanina N, Guo A, Lee J, Kadri S, Slambrouck C Van et al. Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL. Oncotarget. 2016; 7: 68833–68841. Likold E, Biderman B, Dmitrieva E, Smirnova S, Kislova M, Nikitin E et al. PB1912 BTK and PLCG2 genemutations in chronic lymphocytic leukemia patients with resistance to covalent BTK inhibitor. Hema Sphere. 2023; 7: 3683–3684. Xu B, Liang L, Jiang Y, Zhao Z. Investigating the ibrutinib resistance mechanism of L528W mutation on Brutonʼs tyrosine kinase via molecular dynamics simulations. J Mol Graph Model. 2024; 126: 108623. Nawaratne V, Sondhi AK, Abdel-Wahab O, Taylor J. New means and challenges in the targeting of BTK. Clin Cancer Res. 2024; 30: 2333–2341. Wang Y, Zhang Y, Liu J, Jiang Y, Li J, Shi W. Next-generation Bruton tyrosine kinase inhibitors and degraders in the treatment of B-cell malignancies: advances and challenges. Ann Hematol. 2025; 104: 3929–3941. Molica S, Allsup D. Pirtobrutinib in chronic lymphocytic leukemia: navigating resistance and the personalisation of BTK-targeted therapy. Cancers (Basel). 2025; 17: 2974. Wong RL, Choi MY, Wang H-Y, Kipps TJ. Mutation in Bruton Tyrosine Kinase (BTK) A428D confers resistance to BTK-degrader therapy in chronic lymphocytic leukemia. Leukemia. 2024; 38: 1818–1821. Mullard A. FDA approves BTK inhibitor for chronic hives. Nat Rev Drug Discov. 2025; 24: 815. Press Release: Sanofi's Wayrilz approved in US as first BTK inhibitor for immune thrombocytopenia. Sanofi. https://www.sanofi.com/.( 2025). Accessed 5 Sept 2025. Vetrie D, Vorechovskyt I, Sideras P, Holland J, Davies A, Flinter F et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature. 1993; 316: 226–233. Smith CE, Berglöf A. X-Linked Agammaglobulinemia . In: Adam MP, Bick S, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A (eds). Gene Reviews. Seattle (WA): University of Washington, Seattle, 2001. pp1993–2026. Schaafsma GCP, Väliaho J, Wang Q, Berglöf A, Zain R, Smith CIE et al. BTKbase, Bruton tyrosine kinase variant database in X-linked agammaglobulinemia: looking back and ahead. Hum Mutat. 2023; 2023: 5797541. Graziani S, Di Matteo G, Benini L, Di Cesare S, Chiriaco M, Chini L et al. Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells: XLA diagnosis or not? Clin Immunol. 2008; 128: 322–328. Mitsuiki N, Yang X, Bartol SJW, Grosserichter-Wagener C, Kosaka Y, Takada H et al. Mutations in Bruton’s tyrosine kinase impair IgA responses. Int J Hematol. 2015; 101: 305–313. Woyach JA, Furman RR, Liu T-M, Ozer HG, Zapatka M, Ruppert AS et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014; 370: 2286–2294. Maddocks KJ, Ruppert AS, Lozanski G, Heerema NA, Zhao W, Abruzzo L et al. Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia. JAMA Oncol. 2015; 1: 80–87. Quinquenel A, Fornecker L, Letestu E, Ysebaert I, Fleury C, Lazarian G et al. Brief report lymphoid neoplasia prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study. Blood. 2019; 134: 641–644. Coulondre C, Miller JH, Farabaugh PJ, Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature. 1978; 274: 775–80. Strand M, Prolla TA, Liskay RM, Petes TD. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993; 365: 274–276. Schwartz GW, Shauli T, Linial M, Hershberg U. Serine substitutions are linked to codon usage and differ for variable and conserved protein regions. Sci Rep. 2019; 9: 17238. Ahn IE, Underbayev C, Albitar A, Herman SEM, Tian X, Maric I et al. Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia Key Points. Blood. 2017; 129: 1469–1479. Bazykin GA, Kondrashov FA, Ogurtsov AY, Sunyaev S, Kondrashov AS. Positive selection at sites of multiple amino acid replacements since rat-mouse divergence. Nature. 2004; 429: 558–62. Zain R, Vihinen M. Structure-Function Relationships of Covalent and Non-Covalent BTK Inhibitors. Front Immunol. 2021; 12: 694853. Yang G, Buhrlage SJ, Tan L, Liu X, Chen J, Xu L et al. HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib. Blood. 2016; 127: 3237–3252. Dhami K, Chakraborty A, Gururaja TL, W-K Cheung L, Sun C, DeAnda F et al. Kinase-deficient BTK mutants confer ibrutinib resistance through activation of the kinase HCK. Sci Signal. 2022; 15: eabg5216. Joseph RE, Amatya N, Fulton DB, Engen JR, Wales TE, Andreotti AH. Differential impact of BTK active site inhibitors on the conformational state of full-length BTK. Elife. 2020; 9: e60470. Smith CIE, Zain R. Reduced clone size upon BTK inhibitor resistance mutations relates to toxicity caused by inherited PLCG2 gain-of-function variations. Eur J Haematol. 2024; 113: 130–131. Bunz F. Passengers, drivers, and “goners”. Int J Cancer. 2024; 155: 1696–1698. Wang S, Mondal S, Zhao C, Berishaj M, Ghanakota P, Batlevi CL et al. Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity. JCI Insight. 2019; 4: 127566. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files supplementary.pdf supplementary tabl 1, supplementary figure S1 Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 28 Apr, 2026 Reviewer # 2 agreed at journal 27 Apr, 2026 Reviewer # 1 agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 11 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9390771","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":622398740,"identity":"c83f746e-82dc-44a4-afbf-9a2d2a66e19c","order_by":0,"name":"Abdulrahman Hamasy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYDACCQYDhgcMDAkgNjNEiPkwhItPSwKqFrZkkrXwGON1F//s5m0SiXvs8nTbzx5gLqjZJi8/I+ezwQOGOjmcltw5ViaR8Cy52OxMXgLzjGO3DTfcyN2ckMBwGLdVN3LMJBIOMCduO5BjwMzDdptxg0Tu5gMJDAcSG3DokIdoqU/cdv4NUMu/2/bzZ+Q8Bmqpq8elxQCi5XDithtAW3jbbic23MhhBjqMOQGXuwzvHCu2SDhwHKjljcFh3r7byRvOPDM2SDA4bIjLFrnbzRtvfDhQDXRYjuFjnm+3bee3Jz+W/FFRJ4/T+8jgAJgUADnJgCgNMMB/gCTlo2AUjIJRMPwBAE4kYerzbT9oAAAAAElFTkSuQmCC","orcid":"","institution":"Karolinska institutet","correspondingAuthor":true,"prefix":"","firstName":"Abdulrahman","middleName":"","lastName":"Hamasy","suffix":""},{"id":622398741,"identity":"cbe85c65-3d91-4dda-ad44-1b8932f73c9b","order_by":1,"name":"Qing Wang","email":"","orcid":"","institution":"Karolinska institutet","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Wang","suffix":""},{"id":622398742,"identity":"4134f88a-3512-4586-a28f-68d3a7b09320","order_by":2,"name":"Brisejda Rustemi","email":"","orcid":"","institution":"karolinska institutet","correspondingAuthor":false,"prefix":"","firstName":"Brisejda","middleName":"","lastName":"Rustemi","suffix":""},{"id":622398743,"identity":"869d34f3-8940-4dc2-8645-c8fb60ab87f0","order_by":3,"name":"Eleni Afentaki","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Eleni","middleName":"","lastName":"Afentaki","suffix":""},{"id":622398744,"identity":"aa7cbc17-a77e-42b6-95cf-0208c795df86","order_by":4,"name":"Alex Villa","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Villa","suffix":""},{"id":622398745,"identity":"9af6e1b4-dde3-4292-b97a-6f9b32adccc1","order_by":5,"name":"Md Enamul Haque","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Enamul","lastName":"Haque","suffix":""},{"id":622398746,"identity":"33cac410-c36d-4c4d-a6b9-99587602cd65","order_by":6,"name":"Anna Berglöf","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Berglöf","suffix":""},{"id":622398747,"identity":"ddebc826-6e01-42eb-aeaa-845ec1853fbd","order_by":7,"name":"Mauno Vihinen","email":"","orcid":"","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Mauno","middleName":"","lastName":"Vihinen","suffix":""},{"id":622398748,"identity":"01bf6048-f6a0-43a1-b028-9cac3974b51c","order_by":8,"name":"C.I. Edvard Smith","email":"","orcid":"https://orcid.org/0000-0003-1907-3392","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"C.I.","middleName":"Edvard","lastName":"Smith","suffix":""},{"id":622398749,"identity":"cdf0d04c-4037-41ec-a7df-3912cbfd0a07","order_by":9,"name":"Rula Zain","email":"","orcid":"https://orcid.org/0000-0001-8327-846X","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Rula","middleName":"","lastName":"Zain","suffix":""}],"badges":[],"createdAt":"2026-04-11 23:00:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9390771/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9390771/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107614167,"identity":"527048ff-e7d9-4fe2-b89c-e6166842ea3f","added_by":"auto","created_at":"2026-04-23 09:07:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1850262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional analysis of BTK variant expression and activation. (A) \u003c/strong\u003eSchematic representation of BTK showing the analyzed variants within the SH2 and kinase domains, including the previously characterized C481 and T474 variants. The relative positions of substitutions are indicated along the BTK domain structure. Variants found in patients (red), in cell lines (blue) or synthetic (black). Including the published C481 and T474\u003csup\u003e18,19\u003c/sup\u003e(green).\u003cstrong\u003e (B) \u003c/strong\u003eBTK-deficient K562 cells expressing BTK variants were analyzed 48 hours post-transfection. Following 4 hours of serum starvation, cells were stimulated with serum and pervanadate for 5 minutes at room temperature. BTK activation was assessed by immunoblot analysis of phosphorylation at Y223, quantified as the ratio of phosphorylated Y223 to total BTK protein. The constitutively active variants are in grey and the inactive variants are in red.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/e4813f48c84b55032232e386.jpg"},{"id":107707029,"identity":"913af103-60f1-46e8-9ccb-46b8200b4a7d","added_by":"auto","created_at":"2026-04-24 09:19:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":420785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT316A acquired variant uniquely confers broad BTK inhibitor resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBTK-deficient K562 cells were transfected with catalytically active BTK T316 variants. Forty-eight hours after transfection, cells were serum-starved for 4 hours, treated with BTK inhibitors for 2 hours, and then stimulated for 5 minutes at room temperature. Variants were inhibited at pharmacologically relevant concentrations of ibrutinib (0.5 μM), pirtobrutinib (0.1 μM), and zanubrutinib (0.5 μM). BTK expression and phosphorylation at Y223 and Y551 were assessed by immunoblotting. Unlike other substitutions which remain sensitive to irreversible inhibitors, the T316A variant uniquely confers resistance to both covalent and non-covalent BTK inhibitors, indicating a unique mechanism of therapeutic resistance.\u003c/p\u003e","description":"","filename":"figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/0a0ff023d3a5a8e1f9cfd02a.jpg"},{"id":107614168,"identity":"a460467e-a10a-4e1b-9a42-3fd51ca0a4ac","added_by":"auto","created_at":"2026-04-23 09:07:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL528F, L528M and L528V variants are active but show selective inhibitor resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBTK-deficient K562 cells expressing enzymatically active L528 variants were serum-starved for 4 hours, treated with BTK inhibitors (0.5 μM of ibrutinib, 0.1 μM pirtobrutinib or 0.5 μM zanubrutinib) for 2 hours, and subsequently stimulated for 5 minutes. Immunoblot analysis of BTK expression and phosphorylation at Y223 and Y551 revealed that L528F, L528M and L528V retained measurable kinase activity. The active variants remained sensitive to covalent inhibitors ibrutinib and zanubrutinib, but showed reduced responsiveness to the non-covalent inhibitor pirtobrutinib.\u003c/p\u003e","description":"","filename":"figures5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/cbfaaefb6d0fbbc3cd72f02e.jpg"},{"id":107614171,"identity":"e2be0a50-dade-41a2-ba92-619f5a8f8ba7","added_by":"auto","created_at":"2026-04-23 09:07:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":517009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of the inhibitors to BTK\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVisualisation of BTK inhibitor binding to BTK in the partial kinase domain cocrystal structures for ibrutinib, zanubrutinib and pirtobrutinib complexes (complete kinase domain in Supplementary Fig. S1). The residues investigated with variations are in red (V416, A428, M437, M477, E513 and L528). Previously studied T474\u003csup\u003e19\u003c/sup\u003e and C481\u003csup\u003e18 \u003c/sup\u003eare in blue. The structures were superimposed and visualized with UCSF ChimeraX (UCSF ChimeraX: Tools for structure building and analysis). Resistance annotations indicate resistance (R) or partial resistance (PR) to covalent and non-covalent BTK inhibitors.\u003c/p\u003e","description":"","filename":"figures6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/a7dee7e048e6832da6667285.jpg"},{"id":107614170,"identity":"1d2500fe-1e21-4742-901b-4a8de391ba30","added_by":"auto","created_at":"2026-04-23 09:07:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":337539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistance-associated BTK variants show differential sensitivity to remibrutinib and rilzabrutinib\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sensitivities of BTK variants T316A, M437K, M437V, E513A, L528V, and C481S to the recently FDA-approved inhibitors remibrutinib (irreversible) and rilzabrutinib (reversible) were evaluated. BTK-deficient K562 cells expressing these variants were serum-starved for 4 hours, treated with 0.5 μM of inhibitors for 2 hours, and subsequently stimulated for 5 minutes. M437V was fully sensitive to both inhibitors, L528V was partially responsive to remibrutinib, and all other variants were resistant.\u003c/p\u003e","description":"","filename":"figures7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/b5643acf71029eb0999334b7.jpg"},{"id":107709076,"identity":"e0c6c6bb-da89-45dc-afd8-26e1304ad2a0","added_by":"auto","created_at":"2026-04-24 09:34:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3688174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/66a8f05d-397e-4bd7-84bd-c286e9436710.pdf"},{"id":107614172,"identity":"9c3e493e-c16c-4231-894b-3bd04558d5c9","added_by":"auto","created_at":"2026-04-23 09:07:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":239028,"visible":true,"origin":"","legend":"supplementary tabl 1, supplementary figure S1","description":"","filename":"supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9390771/v1/e970fb5eed485c455d515ed1.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Evaluating ibrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, and zanubrutinib in 34 BTK-variants at 7 inhibitor resistance sites","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBruton tyrosine kinase (BTK) plays a fundamental role in B-cell biology as a key component of the B-cell receptor (BCR) signaling pathway, which governs B-cell development, maturation, differentiation, and survival\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Upon antigen engagement, activation of the BCR triggers a cascade of downstream signaling events mediated by BTK, leading to calcium mobilization, transcriptional activation, and regulation of cellular proliferation and apoptosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Beyond its role related to B cell receptor, BTK is involved in several additional signaling pathways, including those mediated by chemokine receptors and Toll-like receptors, thereby influencing cell migration, adhesion, and immune regulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Dysregulation of BTK-dependent signaling contributes to the pathogenesis of a spectrum of B-cell malignancies, most notably CLL and Waldenstr\u0026ouml;m\u0026rsquo;s macroglobulinemia, where aberrant and constitutive BCR activation sustains the growth, survival, and clonal expansion of malignant B cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFollowing the successful experimental development of ibrutinib, the recognition of BTK\u0026rsquo;s pivotal role in these oncogenic processes provided a strong rationale for the continued development of targeted therapies designed to modulate its activity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This expanded the number of BTKi, a class of small-molecule agents that bind to and block the catalytic activity of BTK, thereby interrupting pathological BCR signaling\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The introduction of BTKi has revolutionized the management of CLL and other B-cell malignancies, representing a major transition from traditional chemoimmunotherapy toward precision-targeted treatment strategies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. By effectively inhibiting BTK-mediated signaling, these agents suppress malignant B-cell proliferation, induce apoptosis, and disrupt interactions between leukemic cells and their supportive microenvironment within lymphoid tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the transformative efficacy of BTKi in B-cell malignancies, the emergence of acquired resistance has become a major clinical challenge, particularly in patients receiving long-term therapy over several years. Resistance mechanisms to BTKi are broadly categorized into those affecting BTK itself and those involving alternative or downstream signaling pathways that restore BCR activity or promote cell survival through BTK-independent mechanisms\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The point mutations affecting the BTK cysteine (C) 481 codon, causing BTK C481-to-serine (S), the most frequent and well-characterized resistance mechanism, are observed in a significant proportion of patients progressing on cBTKi, ibrutinib acalabrutinib or zanubrutinib therapy\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo overcome resistance conferred by acquired BTK C481 variants, next-generation ncBTKi, such as pirtobrutinib, have been developed. These agents bind non-covalently to BTK at sites distinct from the C481 residue, inhibiting both wild-type and C481-mutant BTK\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Nevertheless, secondary resistance to ncBTKi has also been documented in variations at other sites. Recently identified mutations such as V416L, A428D, M437R, T474I, T474M and L528W, alter the kinase domain\u0026rsquo;s structural configuration and reduce affinity for reversible inhibitors\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vitro characterization of potential resistance-associated BTK variations remains crucial, to further strengthen our understanding of the underlying mechanisms. In this study, we evaluated 34 BTK variants caused by single nucleotide substitutions at 7 residues implicated in therapeutic resistance, to assess their functional activity and sensitivity to both cBTKi and ncBTKi.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003ePlasmids encoding BTK substitutions were generated by site-directed mutagenesis and verified by sequencing (Mutagenex, Inc. Suwanee, GA, USA) (Supplementary tabl\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003ee1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and transfections\u003c/h3\u003e\n\u003cp\u003eCOS-7 and HEK-293T cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). B7.10 cells, lacking BTK expression, were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 5% chicken serum, 5% glutamine, 50 \u0026micro;M 2-mercaptoethanol, and penicillin-streptomycin as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. BTK-deficient K562 cells were a kind gift of Dr. Donald B. Kohn, University of California, Los Angeles, CA, and were generated by CRISPR-Cas9\u0026ndash;mediated gene editing, as previously described by David H. Gray \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e. The cells were grown in RPMI-1640 supplemented with 10% heat-inactivated FBS. All cultures were incubated at 37\u0026deg;C in under 5% CO₂. Plasmids were transiently transfected into COS-7 and HEK-293T cells by using polyethylenimine (PEI) (Polyscience, Inc.). Electroporation of K562 and B7.10 cells was performed by the neon transfection system (Life Technologies, Carlsbad, CA, USA).\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eCells were subjected to serum starvation and inhibition, followed by activation with serum and pervanadate for 5 minutes at room temperature. Prior to activation, washout steps were performed three times in serum-free medium to remove residual inhibitors, thereby reducing the risk of off-target effects. Whole-cell lysate was obtained by using modified RIPA buffer supplemented with protease and phosphatase inhibitors. Protein extracts were separated by SDS\u0026ndash;polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with specific primary and secondary antibodies. Signal detection and quantification were performed using the Odyssey imaging system (LI-COR Biosciences GmbH).\u003c/p\u003e\n\u003ch3\u003eBTK inhibitors\u003c/h3\u003e\n\u003cp\u003eAll BTK inhibitors were stored at \u0026minus;\u0026thinsp;20\u0026deg;C and initially dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, with fresh dilutions prepared in phosphate-buffered saline (PBS) for each experiment. 36 to 48 hours post-transfection, cells were serum-starved for 4 hours, and inhibitors were added during the last 2 hours of starvation. The BTK inhibitors used in this study included ibrutinib (Selleckchem, Houston, TX, USA), zanubrutinib (Chemgood, Glen Allen, VA, USA), and pirtobrutinib, remibrutinib and rilzabrutinib (MedChemExpress, Stockholm, Sweden).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExpression and catalytic activity of BTK variants\u003c/h2\u003e \u003cp\u003eWe initially studied some of the mutants in COS-7 and HEK293 cells and in the chicken BTK- defective cell line B7.10, as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Because human cells would be more relevant, we subsequently generated results using BTK-deficient K562 cells, which lack endogenous BTK expression through gene editing. Cells were transfected with constructs encoding all the BTK variants to allow assessment of kinase activity in an isolated system. A total of 34 amino acid substitutions at key BTK residues contributing to therapeutic resistance were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Autophosphorylation of tyrosine 223 within the SH3 domain was used as a surrogate marker of BTK catalytic activity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. While the variants were expressed at comparable levels, distinct differences in phosphorylation intensity indicated variable enzymatic activity among the acquired BTK variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on these criteria, we identified 25 out of 34 BTK variants (73%) in our dataset as functionally active.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBTK T316A substitution confers resistance to both cBTKi and ncBTKi\u003c/h3\u003e\n\u003cp\u003eThe SH2 domain of BTK binds to phosphotyrosine-containing motifs on adaptor proteins such as BLNK, an interaction essential for proper B-cell development and signaling\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The BTK T316A substitution causes a structurally distinct alteration within the SH2 domain that does not directly affect the ibrutinib-binding site in the kinase domain. This variant has been identified in patients with CLL experiencing disease progression during ibrutinib therapy\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our results highlight the contribution of non-catalytic domain alterations to therapeutic failure. With the exception of T316A, all other substitutions at this residue retained sensitivity to cBTKi, although their responses to reversible inhibitors varied. The T316A mutation conversely conferred resistance to both cBTKi and ncBTKi, indicating its unique influence on the inhibitor interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSubstitutions of L528 disrupt the enzyme activity\u003c/h3\u003e\n\u003cp\u003eThe enzymatically inactive BTK leucine (L) 528-to-tryptophane (W) substitution sustains malignant B-cell survival through non-catalytic functions and activation of compensatory pathways\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These findings, similar to the effect of most of the C481 replacements\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, underscore the complexity of BTK signaling and reveal that resistance is not solely dependent on catalytic function but also involves kinase-independent mechanisms that sustain oncogenic signaling networks.\u003c/p\u003e \u003cp\u003eSubstitutions at this residue substantially altered BTK catalytic activity. Among the five variants tested, L528F, L528M, and L528V retained measurable kinase activity. While these active variants demonstrated sensitivity to cBTKi, they displayed reduced responsiveness to the non-covalent inhibitor pirtobrutinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe BTK variants at residues valine (V) 416, alanine (A) 428, methionine (M) 437 and methionine (M) 477 are sensitive to cBTKi\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo overcome resistance associated with cBTKi, particularly those by C481 replacements, next-generation ncBTKi, including fenebrutinib, nemtabrutinib, and pirtobrutinib, were developed to engage BTK through hydrogen bonding, ionic, and hydrophobic interactions independent of covalent attachment\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Although these agents initially demonstrated substantial efficacy against C481-mutated BTK\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, clinical relapses have subsequently been reported. Comprehensive genomic analyses of relapsed or refractory CLL patients revealed the emergence of additional on-target BTK mutations (V416L, A428D, M437R, M477I, T474I, and L528W) alongside downstream PLCγ2 alterations\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Importantly, the A428D variant has recently been associated with resistance to BTK degrader therapy in CLL patients\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These substitutions are thought to induce conformational changes within the kinase domain that perturb inhibitor binding and regulatory signaling, thereby conferring resistance even to non-covalent BTK inhibitors\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eComparative analysis of phosphorylation profiles across BTK variants at residues V416, A428, M437, and M477, revealed distinct patterns of inhibitor sensitivity. BTK with substitutions at A428, M437, and M477 largely preserved responsiveness to cBTKi, indicating that covalent binding at C481 remains functionally intact, while showing variable sensitivity to reversible agents. In contrast, the BTK variants with alterations at V416 retained sensitivity to cBTKi but exhibited reduced responsiveness to ncBTKi, suggesting that residue-specific conformational alterations in the kinase domain may influence inhibitor binding and catalytic regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBTK variants at E513 cause constitutive activation\u003c/h2\u003e \u003cp\u003eEvaluation of BTK variants at residue E513 showed that both E513A and E513G substitutions caused constitutive activation and displayed partial sensitivity to ibrutinib. Conversely, they showed resistance to both the cBTKi, zanubrutinib, and the ncBTKi, pirtobrutinib, indicating mutation-specific effects on inhibitor binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLimited efficacy of newly approved BTKi remibrutinib and rilzabrutinib against resistance-associated BTK variants\u003c/h2\u003e \u003cp\u003eRecently, two novel BTK inhibitors have received FDA approval: remibrutinib (Rhapsody), a potent cBTKi approved for chronic spontaneous urticaria\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and rilzabrutinib (Wayrilz), a reversible cBTKi approved for adults with persistent or chronic immune thrombocytopenia\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To assess their potential efficacy against resistant BTK variants, we evaluated the sensitivities of several clinically relevant mutants in CLL (T316A, M437K, M437V, E513A, and L528V), alongside the common C481S variant. Among these, the M437V mutant remained sensitive to both inhibitors, and the L528V variant retained partial responsiveness to remibrutinib, whereas the other missense variants exhibited resistance to both agents, suggesting limited activity of these newly approved inhibitors against clinically identified resistance-associated BTK mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBTKi resistance mutations causing atypical X-linked agammaglobulinemia (XLA)\u003c/h2\u003e \u003cp\u003eLoss-of-function mutations in the \u003cem\u003eBTK\u003c/em\u003e gene cause the disease XLA\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, resulting in a differentiation block at the pro-B to pre-B cell transition. Established in 1995, the BTKbase collects all known mutations causing XLA, with the most recent update published in 2023\u003csup\u003e39\u003c/sup\u003e. When analyzing the different acquired mutations, and the single-nucleotide substitution variants tested here, we observed two variants that are known to cause XLA. These are the reported BTKi resistance mutation T316A affecting the SH2 domain\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and the new variant M477R, tested by us in this report, being located in the catalytic SH1 domain.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eComprehensive in vitro characterization of potential resistance-associated BTK variants is essential for understanding their functional and therapeutic implications. Although a substantial number of BTK mutations has been identified in patients receiving BTK inhibitor therapy, many of these variants occur at low frequencies, making it challenging to determine their true clinical relevance. Experimental in vitro validation is one way to critically assess how specific amino acid substitutions affect BTK kinase activity, signaling function, and inhibitor binding. Such investigations not only enhance our understanding of resistance mechanisms but may also guide the rational design of next-generation inhibitors capable of overcoming both common and rare resistance mutations.\u003c/p\u003e \u003cp\u003eThe number of different replacements causing acquired BTKi resistance, varies considerably. One position is currently unique, the codon for cysteine 481, since all possible seven single-nucleotide changes, including two for serine, have been reported to cause resistance to cBTKi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Cysteine 481 carries a nucleophilic thiol side chain forming a covalent bond with cBTKi, and none of the substitutions permits covalent bonding\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, for the frequently mutated L528 position, out of the 5 possible substitutions, only tryptophan replacement has been identified in patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Of note, of these five substitutions, tryptophan is the largest residue. The other substitutions are either rare or do not cause resistance\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMutation frequencies in genomes are known to be enhanced at CpG sites owing to deamination of 5-methylcytosine to thymine\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and in nucleotide repeats causing DNA polymerase slippage during replication\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, such elements are not found in any of the genomic regions affected by BTKi resistance. For the most common cBTKi-acquired resistance variant, C481S, there are two reasons explaining its frequent occurrence. Hence, two different codon changes yield the same cysteine substitution\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, and secondly C481S is catalytically active\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The C481T mutant is also catalytically active\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, but likely because there is a need for a rare dinucleotide replacement\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, this variant was so far not reported in any patient.\u003c/p\u003e \u003cp\u003eFor the 7 sites studied in this report, merely a single replacement for each site (n\u0026thinsp;=\u0026thinsp;7) has been identified out of 34 possible substitutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It is possible that only certain replacements yield what could be referred to as \u0026ldquo;a functional BTK molecule\u0026rdquo;. Thus, in XLA the majority of amino acid replacements induces instability. However, most of these substitutions affect hydrophobic residues buried in the core of BTK\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, whereas the BTKi resistance mutations afflict amino acids located on the surface of the catalytic cleft\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, where replacements more frequently are tolerated in terms of protein stability. Moreover, when a certain missense mutation causing XLA results in the expression of the enzyme, it is common that also other substitutions affecting the same residue are stable\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Therefore, we do not favor the idea that differential protein stability is the mechanism underlying the observation that just a single resistance substitution was so far reported for most of the 7 sites. To this end, we have previously studied the gatekeeper residue threonine 474 replacements, both the single-nucleotide substitutions A/I/N/P/S and also more complex alterations\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. At this site, 2 single-nucleotide substitutions were previously found in patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Recently, however, Brown et al. reported the additional T474F/L/M/Y replacements in patients treated with the ncBTKi, pirtobrutinib\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Interestingly, these 4 variants could only appear after dinucleotide changes, which, similar to C481T, are expected to be extremely rare.\u003c/p\u003e \u003cp\u003eRegarding \u0026ldquo;functional resistance\u0026rdquo;, it is well known that mutants do not need to be catalytically active, a case in point being the 5/6 kinase-dead replacements affecting C48\u003csup\u003e18\u003c/sup\u003e. The exact mechanism underlying this form of resistance is not known, but kinase-dead BTK could retain the capacity to assemble a signaling complex. Furthermore, expression of the SRC family kinase HCK (hematopoietic cell kinase) has been suggested as a key component compensating for the lack of BTK\u0026rsquo;s catalytic activity\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA very interesting resistance variant is T316A located in the SH2 domain. This replacement is unique because it is the only known resistance variant located outside of the kinase domain. T316A was also identified in an Italian patient with dysgammaglobulinemia, differing from classical XLA\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, as well as in a Japanese patient\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In both of these patients the B-cell numbers decreased over time. Also, for BTKi resistance only two patients were so far reported to carry the T316A variant\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Both developed Richter transformation, but in one of the patients only the CLL cells carried the mutation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It was previously reported that T316A drives BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from internal repression\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This structure-function analysis is in agreement with our finding of a pronounced constitutive activation induced by the T316A protein in transfected K562 cells. However, also a proline-substitution caused substantial constitutive activity, while lower than T316A, and this variant has not been reported in any patient to date. It could be hypothesized that such an intrinsic activation might be toxic to cells and that this could explain the rarity of patients developing such a resistance to BTKi.\u003c/p\u003e \u003cp\u003eThe potential noxiousness of T316A, with B-cell numbers decreasing over time in the inherited form\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, may be related to the \u0026ldquo;Goner\u0026rdquo; concept. This concept was initially developed to explain the fact that BTKi resistance mutations in the \u003cem\u003ePLCG2\u003c/em\u003e gene in CLL patients result in lower variant allele frequencies as compared to \u003cem\u003eBTK\u003c/em\u003e gene alterations causing resistance variants\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Because such acquired variants are activated, the toxic constitutive signaling likely causes loss of such B cells. Thus, even if resistance develops, the malignant cells pay a considerable price for carrying such a PLCG2 variant.\u003c/p\u003e \u003cp\u003eA special form of resistance is the one previously identified to affect glutamate-513\u003csup\u003e56\u003c/sup\u003e. The authors mutagenized the \u003cem\u003eBTK\u003c/em\u003e coding sequence using repair-defective \u003cem\u003eE. coli\u003c/em\u003e, reaching an overall nucleotide variant frequency of 90%. They subsequently screened the resulting library for ibrutinib escape mutants in murine BCL1 lymphoma cells. In their report, two of the escape variants, T474M and of E513G, increased BTK autophosphorylation. The T474M substitution together with numerous other replacements of this residue were already investigated by us\u003csup\u003e19\u003c/sup\u003e, so we decided to further study E513 substitutions. Both the novel single-nucleotide replacement to alanine (A) and the previously reported glycine (G)\u003csup\u003e56\u003c/sup\u003e replacement caused very significant constitutive activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A similar argument, as used for the T316A substitution, may therefore also explain the effect of the E513 replacements. Hence, both of the substitutions E513A/G might be toxic to B cells, and this could be the reason for them not yet being observed as acquired resistance mutations to BTKi in patients. Moreover, if they are noxious, it is also likely that they could cause XLA, possibly in the form of an atypical disease, similar to patients with the T316A variant\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. While E513 replacements were not reported in BTKi treated patients, nor in XLA, in spite of more than 1000 different mutations having been identified\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, it could still simply be by chance that they did not yet appear.\u003c/p\u003e \u003cp\u003eBesides the structural effect of the replacement with largest aromatic amino acid causing the frequently observed kinase-dead L528W resistance variant, could there be additional mechanisms? Two of the replacements cause significant constitutive activation, namely L528M/V. Thus, a similar reasoning as for T316A could also be made here, namely that this activity could be toxic to cells. Akin to T316A and E513A/G, there was no indication that L528M/V caused toxicity in the short-term transfection experiments using K562. However, as compared to transformed cell lines, primary cells are often more sensitive to mutations and therefore this hypothesis may still hold true.\u003c/p\u003e \u003cp\u003eOur assessment of newly FDA-approved BTK inhibitors remibrutinib and rilzabrutinib revealed limited activity against clinically relevant resistance-associated variants. Among tested mutants, only M437V retained sensitivity to both agents, whereas L528V showed partial responsiveness to remibrutinib. These findings underscore the persistent challenge of mutation-caused resistance and suggest that novel next-generation inhibitors may have restricted utility in this context. Collectively, these results emphasize that BTK inhibitor resistance is multifactorial, involving both catalytic and non-catalytic domain mutations, and highlight the use of systematic preclinical assessment of clinically observed BTK variants to predict potential resistance and guide personalized therapeutic strategies in CLL and other B-cell malignancies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eCIES perceived and conceptualized the project, obtained funding, interpreted data, supervised throughout, wrote part of the discussion and edited the manuscript. RZ interpreted the data, supervised, edited the manuscript and obtained funding. AH, performed most of the experiments with major help from QW. BR, EA, AV and EH performed selected experiments. AB was involved in data interpretation and manuscript editing. MV carried out protein structural analysis. AH wrote the manuscript with edits performed by CIES, RZ and QW.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the Swedish Cancer Society, the Swedish Research Council and the Swedish County Council and Center for Innovative Medicine (CIMED).\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe dataset generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmith CIE. From identification of the BTK kinase to effective management of leukemia. Oncogene. 2017; 36: 2045\u0026ndash;2053.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDonald C, Xanthopoulos C, Kostareli E. The role of Bruton\u0026rsquo;s tyrosine kinase in the immune system and disease. Immunology. 2021; 164: 722\u0026ndash;736.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRingheim GE, Wampole M, Oberoi K. Bruton\u0026rsquo;s Tyrosine Kinase (BTK) inhibitors and autoimmune diseases: making sense of BTK inhibitor specificity profiles and recent clinical trial successes and failures. Front Immunol. 2021; 12: 662223.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRip J, de Bruijn MJW, Appelman MK, Singh SP, Hendriks RW, Corneth OBJ. Toll-like receptor signaling drives Btk-mediated autoimmune disease. Front Immunol. 2019;10: 95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePal Singh S, Dammeijer F, Hendriks RW. Role of Bruton\u0026rsquo;s tyrosine kinase in B cells and malignancies. Mol Cancer. 2018; 17: 57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan Z, Scheerens H, Li SJ, Schultz BE, Sprengeler PA, Burrill LC \u003cem\u003eet al.\u003c/em\u003e Discovery of selective irreversible inhibitors for Bruton\u0026rsquo;s tyrosine kinase. ChemMedChem. 2007; 2: 58\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn IE, Brown JR. Targeting Bruton\u0026rsquo;s tyrosine kinase in CLL. Front Immunol. 2021; 12: 687458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMouhssine S, Maher N, Matti BF, Alwan AF, Gaidano G. Targeting BTK in B cell malignancies: from mode of action to resistance mechanisms. Int J Mol Sci. 2024; 25: 3234.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShirley M. Bruton tyrosine kinase inhibitors in B-cell malignancies: their use and differential features. Target Oncol. 2022; 17: 69\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HO. Development of BTK inhibitors for the treatment of B-cell malignancies. Arch Pharm Res. 2019; 42: 171\u0026ndash;181.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNore BF, Vargas L, Mohamed AJ, Brand\u0026eacute;n LJ, B\u0026auml;ckesj\u0026ouml; CM, Islam TC \u003cem\u003eet al.\u003c/em\u003e Redistribution of Bruton\u0026rsquo;s tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases. Eur J Immunol. 2000; 30: 145\u0026ndash;154.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurger JA. Targeting the microenvironment in chronic lymphocytic leukemia is changing the therapeutic landscape. Curr Opin Oncol. 2012; 24: 643\u0026ndash;649.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Rooij MFM, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ \u003cem\u003eet al.\u003c/em\u003e The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood. 2012; 119: 2590\u0026ndash;2594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiestner A. BCR pathway inhibition as therapy for chronic lymphocytic leukemia and lymphoplasmacytic lymphoma. Hematology Am Soc Hematol Educ Program. 2014; 2014: 125\u0026ndash;134.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith CIE, Burger JA. Resistance mutations to BTK inhibitors originate from the NF-κB but not from the PI3K-RAS-MAPK arm of the B cell receptor signaling pathway. Front Immunol. 2021; 12: 689472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiśniewski K, Puła B. A review of resistance mechanisms to Bruton\u0026rsquo;s kinase inhibitors in chronic lymphocytic leukemia. Int J Mol Sci. 2024; 25: 5246.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown JR. Resistance to Bruton tyrosine kinase inhibitors. Hematol Oncol Clin North Am. 2025; 39: 951\u0026ndash;963.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamasy A, Wang Q, Blomberg KEM, Mohammad DK, Yu L, Vihinen M \u003cem\u003eet al.\u003c/em\u003e Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant. Leukemia. 2017; 31: 177\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstupi\u0026ntilde;\u0026aacute;n HY, Wang Q, Bergl\u0026ouml;f A, Schaafsma GCP, Shi Y, Zhou L \u003cem\u003eet al.\u003c/em\u003e BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib. Leukemia. 2021; 35: 1317\u0026ndash;1329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomez EB, Ebata K, Randeria HS, Rosendahl MS, Cedervall EP, Morales TH \u003cem\u003eet al.\u003c/em\u003e Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor. Blood. 2023; 142: 62\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaeem AS, Utro F, Wang Q, Cha J, Vihinen M, Martindale SP \u003cem\u003eet al.\u003c/em\u003e Resistance to the non-covalent BTK inhibitor pirtobrutinib. Blood. 2022; 140: 6981\u0026ndash;6982.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang E, Mi X, Thompson MC, Montoya S, Notti RQ, Afaghani J \u003cem\u003eet al.\u003c/em\u003e Mechanisms of resistance to noncovalent Bruton\u0026rsquo;s tyrosine kinase inhibitors. N Engl J Med. 2022; 386: 735\u0026ndash;743.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown JR, Nguyen B, Desikan SP, Won H, Tantawy SI, McNeely SC \u003cem\u003eet al.\u003c/em\u003e Genomic determinants of response and resistance to pirtobrutinib in relapsed/refractory chronic lymphocytic leukemia. Blood. 2026; 147: 24\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGray DH, Villegas I, Long J, Santos J, Keir A, Abele A \u003cem\u003eet al.\u003c/em\u003e Optimizing integration and expression of transgenic Bruton\u0026rsquo;s tyrosine kinase for CRISPR-Cas9-mediated gene editing of X-linked agammaglobulinemia. CRISPR J. 2021; 4: 191\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstupi\u0026ntilde;\u0026aacute;n HY, Bouderlique T, He C, Bergl\u0026ouml;f A, Cappelleri A, Frengen N \u003cem\u003eet al.\u003c/em\u003e In BTK, phosphorylated Y223 in the SH3 domain mirrors catalytic activity, but does not influence biological function. Blood Adv. 2024; 8: 1981\u0026ndash;1990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiddendorp S, Dingjan GM, Maas A, Dahlenborg K, Hendriks RW. Function of Bruton\u0026rsquo;s tyrosine kinase during B cell development is partially independent of its catalytic activity. J Immunol. 2003; 171: 5988\u0026ndash;5996.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTzeng S, Pai M, Lung F, Wu C, Roller P, Lei B \u003cem\u003eet al.\u003c/em\u003e Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia. Protein Sci. 2000; 9: 2377\u0026ndash;2385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma S, Galanina N, Guo A, Lee J, Kadri S, Slambrouck C Van \u003cem\u003eet al.\u003c/em\u003e Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL. Oncotarget. 2016; 7: 68833\u0026ndash;68841.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLikold E, Biderman B, Dmitrieva E, Smirnova S, Kislova M, Nikitin E \u003cem\u003eet al.\u003c/em\u003e PB1912 BTK and PLCG2 genemutations in chronic lymphocytic leukemia patients with resistance to covalent BTK inhibitor. Hema Sphere. 2023; 7: 3683\u0026ndash;3684.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, Liang L, Jiang Y, Zhao Z. Investigating the ibrutinib resistance mechanism of L528W mutation on Brutonʼs tyrosine kinase via molecular dynamics simulations. J Mol Graph Model. 2024; 126: 108623.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNawaratne V, Sondhi AK, Abdel-Wahab O, Taylor J. New means and challenges in the targeting of BTK. Clin Cancer Res. 2024; 30: 2333\u0026ndash;2341.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhang Y, Liu J, Jiang Y, Li J, Shi W. Next-generation Bruton tyrosine kinase inhibitors and degraders in the treatment of B-cell malignancies: advances and challenges. Ann Hematol. 2025; 104: 3929\u0026ndash;3941.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolica S, Allsup D. Pirtobrutinib in chronic lymphocytic leukemia: navigating resistance and the personalisation of BTK-targeted therapy. Cancers (Basel). 2025; 17: 2974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong RL, Choi MY, Wang H-Y, Kipps TJ. Mutation in Bruton Tyrosine Kinase (BTK) A428D confers resistance to BTK-degrader therapy in chronic lymphocytic leukemia. Leukemia. 2024; 38: 1818\u0026ndash;1821.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMullard A. FDA approves BTK inhibitor for chronic hives. Nat Rev Drug Discov. 2025; 24: 815.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePress Release: Sanofi's Wayrilz approved in US as first BTK inhibitor for immune thrombocytopenia. Sanofi. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sanofi.com/.(\u003c/span\u003e\u003cspan address=\"https://www.sanofi.com/.(\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e2025). Accessed 5 Sept 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVetrie D, Vorechovskyt I, Sideras P, Holland J, Davies A, Flinter F \u003cem\u003eet al.\u003c/em\u003e The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature. 1993; 316: 226\u0026ndash;233.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith CE, Bergl\u0026ouml;f A. \u003cem\u003eX-Linked Agammaglobulinemia\u003c/em\u003e. In: Adam MP, Bick S, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A (eds). Gene Reviews. Seattle (WA): University of Washington, Seattle, 2001. pp1993\u0026ndash;2026.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaafsma GCP, V\u0026auml;liaho J, Wang Q, Bergl\u0026ouml;f A, Zain R, Smith CIE \u003cem\u003eet al.\u003c/em\u003e BTKbase, Bruton tyrosine kinase variant database in X-linked agammaglobulinemia: looking back and ahead. Hum Mutat. 2023; 2023: 5797541.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraziani S, Di Matteo G, Benini L, Di Cesare S, Chiriaco M, Chini L \u003cem\u003eet al.\u003c/em\u003e Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells: XLA diagnosis or not? Clin Immunol. 2008; 128: 322\u0026ndash;328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitsuiki N, Yang X, Bartol SJW, Grosserichter-Wagener C, Kosaka Y, Takada H \u003cem\u003eet al.\u003c/em\u003e Mutations in Bruton\u0026rsquo;s tyrosine kinase impair IgA responses. Int J Hematol. 2015; 101: 305\u0026ndash;313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoyach JA, Furman RR, Liu T-M, Ozer HG, Zapatka M, Ruppert AS \u003cem\u003eet al.\u003c/em\u003e Resistance mechanisms for the Bruton\u0026rsquo;s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014; 370: 2286\u0026ndash;2294.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaddocks KJ, Ruppert AS, Lozanski G, Heerema NA, Zhao W, Abruzzo L \u003cem\u003eet al.\u003c/em\u003e Etiology of ibrutinib therapy discontinuation and outcomes in patients with chronic lymphocytic leukemia. JAMA Oncol. 2015; 1: 80\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuinquenel A, Fornecker L, Letestu E, Ysebaert I, Fleury C, Lazarian G \u003cem\u003eet al.\u003c/em\u003e Brief report lymphoid neoplasia prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study. Blood. 2019; 134: 641\u0026ndash;644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoulondre C, Miller JH, Farabaugh PJ, Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature. 1978; 274: 775\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrand M, Prolla TA, Liskay RM, Petes TD. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993; 365: 274\u0026ndash;276.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwartz GW, Shauli T, Linial M, Hershberg U. Serine substitutions are linked to codon usage and differ for variable and conserved protein regions. Sci Rep. 2019; 9: 17238.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn IE, Underbayev C, Albitar A, Herman SEM, Tian X, Maric I \u003cem\u003eet al.\u003c/em\u003e Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia Key Points. Blood. 2017; 129: 1469\u0026ndash;1479.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBazykin GA, Kondrashov FA, Ogurtsov AY, Sunyaev S, Kondrashov AS. Positive selection at sites of multiple amino acid replacements since rat-mouse divergence. Nature. 2004; 429: 558\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZain R, Vihinen M. Structure-Function Relationships of Covalent and Non-Covalent BTK Inhibitors. Front Immunol. 2021; 12: 694853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang G, Buhrlage SJ, Tan L, Liu X, Chen J, Xu L \u003cem\u003eet al.\u003c/em\u003e HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib. Blood. 2016; 127: 3237\u0026ndash;3252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhami K, Chakraborty A, Gururaja TL, W-K Cheung L, Sun C, DeAnda F \u003cem\u003eet al.\u003c/em\u003e Kinase-deficient BTK mutants confer ibrutinib resistance through activation of the kinase HCK. Sci Signal. 2022; 15: eabg5216.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoseph RE, Amatya N, Fulton DB, Engen JR, Wales TE, Andreotti AH. Differential impact of BTK active site inhibitors on the conformational state of full-length BTK. Elife. 2020; 9: e60470.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith CIE, Zain R. Reduced clone size upon BTK inhibitor resistance mutations relates to toxicity caused by inherited PLCG2 gain-of-function variations. Eur J Haematol. 2024; 113: 130\u0026ndash;131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBunz F. Passengers, drivers, and \u0026ldquo;goners\u0026rdquo;. Int J Cancer. 2024; 155: 1696\u0026ndash;1698.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Mondal S, Zhao C, Berishaj M, Ghanakota P, Batlevi CL \u003cem\u003eet al.\u003c/em\u003e Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity. JCI Insight. 2019; 4: 127566.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9390771/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9390771/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBruton tyrosine kinase (BTK) plays a central role in B-cell receptor signaling, and its inhibition has transformed the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL). Despite the success of BTK inhibitors (BTKi), resistance from acquired BTK mutations poses a significant clinical challenge. Here, we systematically characterized 34 BTK variants associated with 7 sites of resistance, assessing kinase activity and responsiveness to both the covalent (cBTKi), ibrutinib, remibrutinib, rilzabrutinib (reversible cBTKi) and zanubrutinib and the non-covalent (ncBTKi) inhibitor, pirtobrutinib. In contrast to cysteine-481, where all 6 replacements caused by single-nucleotide changes have been reported, this is not the case for substitutions in other locations. Among them, threonine-316-to-alanine (T316A) conferred broad resistance to both cBTKi and ncBTKi; in contrast, substitutions at leucine-528 disrupted catalytic activity while preserving protein expression. Variants at valine-416, alanine-428, methionine-437 and methionine-477, largely maintained sensitivity to cBTKi, whereas glutamate-513 substitutions showed mutation-specific patterns of inhibitor responsiveness. The T316A substitution induced constitutive catalytic activity and, when inherited, was previously reported to cause atypical X-linked agammaglobulinemia suggesting an intricate resistance mode. Notably, the newly approved BTKi, remibrutinib and rilzabrutinib, were ineffective against several resistance-associated variants. These findings highlight the complex, residue-specific nature of BTKi-resistant BTK variants.\u003c/p\u003e","manuscriptTitle":"Evaluating ibrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, and zanubrutinib in 34 BTK-variants at 7 inhibitor resistance sites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 09:07:08","doi":"10.21203/rs.3.rs-9390771/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-28T23:25:48+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-27T09:36:49+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-13T21:06:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-13T16:21:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T15:05:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-13T14:59:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2026-04-11T22:59:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"03678f4f-bf6e-4459-9f7b-e975dd8decb7","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66232950,"name":"Biological sciences/Cancer/Cancer therapy/Cancer therapeutic resistance"},{"id":66232951,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":66232952,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2026-04-23T09:07:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 09:07:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9390771","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9390771","identity":"rs-9390771","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}