Diverse signaling and vulnerabilities accompany distinct BTK mutations associated with clinical resistance to COVALENT, non-COVALENT and protacs targeting BTK in MYD88 mutated lymphomas.

Abstract Background: BTK is a key mediator of BCR signaling that promotes survival in various B-cell malignancies. Mutations in MYD88, frequently observed in Waldenström's Macroglobulinemia (WM; 95–97%), primary CNS lymphoma (70–80%), ABC subtype of DLBCL (40%), marginal zone lymphoma (5–10%), and chronic lymphocytic leukemia (5–15%) trigger BTK mediated pro-survival signaling. Covalent (cBTK-i) and non-covalent (ncBTK-i) BTK kinase inhibitors have transformed treatment paradigms in many of these diseases. However, resistance often emerges via BTK mutations: C481S confers resistance to cBTK-i, while ncBTK-i resistance arises from mutations that include V416L, A428D, M437R, T474I, and L528W. Proteolysis targeting chimeras (PROTACs), which degrade BTK offer a promising salvage strategy. Several BTK PROTACs (BGB-16673, NX-2127, NX-5948) are active in patients with acquired BTK mutations associated with resistance to cBTK-i and ncBTK-i. Yet, recent studies (Wong et al, Leuk. 2024; 38:1818-21, Wang et al, EHA 2025; PF558) report that the BTK A428D mutation confers resistance even to these investigational PROTACs, representing a major therapeutic gap. Methods: We developed an expansive array of engineered cell model systems using ABC DLBCL (TMD8) and WM (BCWM.1) lymphoma cells to express wild-type or mutated BTK (C481S, V416L, A428D, M437R, T474I, L528W). Transcriptomic alterations were analyzed by RNA-seq. Protein expression and activation of key signaling molecules (BTK, HCK, LYN, PLCγ2, ERK, STAT3, NF-κB) were assessed by immunoblotting. Sensitivity to cBTK-i (zanubrutinib); ncBTK-i (pirtobrutinib); BTK PROTACs (BGB-16673, NX-2127, NX-5948); and a novel oral compound (DFCI-002-06) developed and characterized by us (Liu et al, Blood 2024; 144 Supp1: 834) that degrades HCK, LYN, and BTK were evaluated using CellTiter-Glo and apoptosis assays. Results: Engineered TMD8 and BCWM.1 lines expressing various BTK mutations showed expected resistance profiles: C481S conferred resistance to covalent BTKi, while all other mutations conferred resistance to pirtobrutinib. RNA-seq and signaling analyses revealed that each BTK mutation induced a distinct pro-survival signaling signature. Notably, despite reduced BTK Y223 phosphorylation in V416L, A428D, and L528W mutants—suggestive of reduced kinase activity—downstream pathways remained active, implicating compensatory mechanisms. HCK expression and activation were markedly elevated in BCWM.1 cells expressing A428D, M437R, and L528W. In TMD8 cells, HCK activation was increased with M437R and T474I but decreased with C481S, A428D, and L528W. PLCγ2 and STAT3 activation was increased in M437R, T474I, and L528W, but decreased in A428D. LYN and p-LYN levels were elevated in BCWM.1 cells with A428D, M437R, and L528W, but reduced in TMD8 A428D and BCWM.1 V416L cells. NF-κB (p65) and IκBα activation varied by mutation and cell line, i.e. both were decreased in M437R expressing cells; increased in BCWM.1 V416L; and elevated in A428D expressing TMD8 but suppressed in BCWM.1 cells. Upon treatment, all PROTACs efficiently degraded BTK in wild-type and C481S-, V416L-, M437R-, and T474I-expressing cells. In L528W-mutant cells, BTK degradation was achieved by BGB-16673, NX-2127, and NX-5948, but not by DFCI-002-06. In A428D expressing TMD8 and BCWM.1, DFCI-002-06 robustly degraded HCK, LYN and BTK and led to suppression of downstream survival signaling, and induced greater cytotoxicity and apoptosis versus BGB-16673, NX-2127, and NX-5948 which showed resistance. Conclusions: Our findings showed diverse signaling and vulnerabilities that accompany distinct BTK mutations associated with clinical resistance to covalent, non-covalent and PROTACs targeting BTK. We identified HCK and LYN dependent alternative pro-survival signaling in V416L, A428D, and L528W BTK mutant expressing cells which were deficient for BTK Y223 kinase signaling. Importantly, we observed that the DFCI-002-06 which degrades HCK, LYN and BTK broadly overcame resistance—including in BTK A428D expressing lymphoma cells that show resistance to BGB-16673, NX-2127, and NX-5948. Our findings highlight the necessity to consider individual BTK mutations in treatment planning and support development of DFCI-002-06 for MYD88 mutated B-cell lymphomas with acquired resistance to BTK-inhibitors.