{"doi":"10.1002/hon.3164_403","title":"Drug‐resistance mutations in BTK occur in distinct enzymatic classes and are overcome by BTK degradation","abstract":"Introduction: Increasing use of covalent Bruton tyrosine kinase (BTK) inhibitors ibrutinib, acalabrutinib, and zanubrutinib as well as noncovalent inhibitors nemtabrutinib and pirtobrutinib, have elucidated a series of acquired BTK mutations, some of which can confer cross-resistance to other BTK inhibitors in patients with B-cell malignancies. Our biochemical and biological assays revealed that in addition to impeding drug binding, some BTK drug resistance mutations have diminished or absent enzymatic function and reduced autophosphorylation of tyrosine 223 (Y223), a marker of BTK activation. Our data suggest that recurrent kinase domain BTK mutations occur in distinct enzymatic groups: certain BTK mutations are kinase proficient (T474I/F and C481S mutations), while others reduce enzymatic activity (M437R mutation), and some mutations render BTK kinase dead (V416L, C481Y/R/F and L528W, see Figure). Despite their lack of BTK activation, upon B-cell receptor (BCR) stimulation kinase dead BTK mutants showed enhanced activation of downstream BCR signals and hyperactivated calcium release. Methods: To identify the signaling mechanisms of kinase dead BTK mutants, we generated CRISPR-CAS9 knockin mutant cells and utilize several orthogonal proteomic approaches in BTK-dependent human lymphoma B cells expressing WT or mutant (C481S, V416L, T474I and L528W) BTK. We performed global phosphoproteomics, kinobead assays, BTK immunoprecipitation mass spectrometry studies, and 2D differential gel electrophoresis to unbiasedly elucidate a novel scaffolding function of BTK. Results: Collectively our data revealed enhanced physical interactions of kinase dead BTK with protein kinases HCK and ILK as well as activation of multiple signaling moieties in malignant B cells. Given the newly discovered scaffolding function of BTK, we decided to test strategies to eliminate, rather than inhibit mutant BTK proteins. While other PROTAC degraders of BTK have been demonstrated to target wild-type or C481S-mutant forms of BTK in preclinical studies, we now demonstrate the first clinical-stage (clinicaltrials.gov #NCT04830137) degrader of BTK, NX-2127, which binds to each drug resistant BTK mutant proteoform (as demonstrated by biophysical and structural data) and induces their proteasomal degradation causing inhibition of BCR signaling. Treatment of chronic lymphocytic leukemia (CLL) patients with NX-2127 achieves >80% degradation of BTK and we demonstrate proof-of-concept therapeutic benefit in patients with CLL (Figure). The research was funded by: S.M. is supported by the Ruth L. Kirschstein National Research Service Award for Individual Predoctoral Fellows (1F31CA275378). J.T. is supported by the NCI/NIH (K08CA230319), the Doris Duke Charitable Foundation and the Edward P. Evans Foundation. Keyword: Chronic Lymphocytic Leukemia (CLL) Conflicts of interests pertinent to the abstract. A. Alencar Consultant or advisory role: Kite, SeaGen, Epizyme, Janseen, BeiGene, Incyte, TG Therapeutics, Lilly, and Genenetch Research funding: LOXO, Beigene, and Incyte O. Abdel-Wahab Consultant or advisory role: H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics, Janssen, Envisagenics Inc., AIChemy, Harmonic Discovery Inc., and Pfizer Boulder Research funding: H3B Biomedicine, Nurix Therapeutics, Minovia Therapeutics, and LOXO Oncology","journal":"Hematological Oncology","year":2023,"id":408403,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.959,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1188082,"name":"J. Bourcie","orcid":null,"position":1,"is_corresponding":false},{"id":1187658,"name":"Arun Sondhi","orcid":"0000-0002-4748-5522","position":2,"is_corresponding":false},{"id":908949,"name":"Jacob Jahn","orcid":"0000-0003-0745-7049","position":3,"is_corresponding":false},{"id":799040,"name":"Alvaro J. Alencar","orcid":"0000-0002-1720-9295","position":4,"is_corresponding":false},{"id":75043,"name":"Omar Abdel-Wahab","orcid":"0000-0002-3907-6171","position":5,"is_corresponding":false},{"id":1187659,"name":"Janet Taylor","orcid":"0000-0001-7148-5437","position":6,"is_corresponding":false},{"id":1188081,"name":"Simone Montoya","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T01:21:18.414003Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}