Mechanisms of acquired resistance to BTK and BCL2 inhibitors reveal clinically actionable vulnerabilities in B-cell lymphoma

Background: BCL2 inhibitors (i) and BTK-directed agents, including BTK degraders (d), are essential components of modern therapy for B-cell lymphomas. However, acquired resistance often limits long-term benefits. Identifying the molecular mechanisms that underlie resistance and understanding reciprocal drug sensitivities may help guide the rational sequencing of treatments. Here, we investigated the mechanisms of secondary resistance to the BCL2-i venetoclax and the BTK-d bexobrutideg (NX-5948) in models of marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). Methods: Derivatives of Karpas1718 (MZL) and REC1 (MCL) with acquired resistance were developed through chronic exposure to increasing doses of venetoclax or bexobrutideg. Parental and resistant lines were characterized using drug sensitivity assays, apoptosis and cell cycle analyses, molecular profiling, and immunoblotting of apoptotic regulators and BCR signaling components. Results: Venetoclax-resistant models from both lymphoma subtypes exhibited broad cross-resistance to other BCL2-i and upregulation of BCL-xL, with MZL cells also resistant to MCL1-i. Regulation of anti-apoptotic proteins varied among models: BCL-xL increased in both venetoclax-resistant models, whereas MCL1 increased only in Karpas1718. All venetoclax-resistant cell lines retained sensitivity to BTK-d, revealing a potential therapeutic vulnerability. Resistance to BTK-d was highly model-specific. In Karpas1718-resistant cells, no BTK degradation occurred after bexobrutideg exposure, and these cells were also resistant to BGB-16673, another BTK-d. Nonetheless, they remained sensitive to BTK-i and all BCL2-i, suggesting that switching to a different drug class could restore their response. Conversely, REC1 derivatives became resistant to both BTK-d and BTK-i. Since resistance was linked to continued drug-induced BTK degradation, these findings indicate a BTK-independent resistance mechanism. Interestingly, the bexobrutideg-resistant REC1 cells were also resistant to BCL2-i. No mutations were observed in BTK, PLCG2, or BCL2 . Conclusions: In two distinct B-cell lymphoma models, resistance to venetoclax and BTK-d developed through target-specific and model-specific adaptations involving anti-apoptotic reprogramming, altered BCR signaling dependence, and incomplete BTK degradation. Notably, cross-drug sensitivities persisted in several contexts: venetoclax-resistant MZL and MCL cells remained susceptible to BTK degradation, while BTK-d-resistant MZL cells continued to respond to BCL2 inhibition. These insights emphasize clinically actionable strategies for treatment sequencing and support precision approaches to overcome therapeutic resistance in B-cell lymphoma.