Combination strategies overcome treatment resistance driven by gain‐of‐function bcl10 mutations in diffuse large b‐cell lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL), the most common lymphoma diagnosis, has heterogeneous biology and prognosis. Frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), used regardless of subtype, cures ∼60%, but relapsed/refractory patients have persistently poor prognosis. Efforts to improve frontline and establish subtype-targeted therapy have largely failed. Post-hoc analysis of the negative PHOENIX trial suggested younger patients with specific subtypes benefited from adding a Bruton’s tyrosine kinase inhibitor (BTKi) to R-CHOP, but BN2 (LymphGen)/Cluster 1 (Chapuy clusters) cases did not. BCL10, encoding a key oncoprotein promoting NF-kB activation through formation of the CBM complex with CARD11 and MALT1, is mutated in DLBCL (∼5%), clustering strongly in BN2 (∼40%). While these gain-of-function mutations alter CBM complex dynamics and promote BTKi resistance, therapeutic strategies to overcome them are minimally defined. We find resistance to multiple drug classes is driven by BCL10 mutants, but rational combinations can restore treatment sensitivity, informing biomarker-driven strategies for clinical translation. Methods: We established gain-of-function models for two classes of BCL10 mutants: a recurrent R58Q CARD-domain alteration and truncations of the ST-rich domain. We probed impact through RNA-seq and transcription-factor (TF) analyses. We assessed deregulation of cytokines and downstream signaling, performed drug screens and validations and assessed drug combinations. Results: Both mutation classes promote constitutive CBM activation (NF-kB: p65/IKBα; AP-1: JNK/cJUN). Consistent with CBM biology, RNA-seq showed activation of IL6-JAK-STAT3 and TNFα signaling via NF-kB but implicated novel cytokines including CXCL10 and IL7, confirmed by ELISA assays. BCL10 mutant biology converged on TFs downstream of CBM (RELA, JUN) and cytokine signaling (STAT1, STAT2). As previously reported, BCL10 mutants drove BTKi resistance, including the non-covalent inhibitor pirtobrutinib. We sought drug vulnerabilities using the TargetMol Epigenetic library, revealing additional BCL10-mutant resistances. Confirmed across multiple lines in dose-response viabilities, these targets included PI3K, BCL2, PIM, and AKT. Given lack of single drug activities in DLBCL, we sought rational combinations. MALT1 activity is critical in BCL10-mutant cells and the combination of MALT1i with BTKi was additive. MALT1i-independent combinations showed striking synergy between BTKi and BCL2i (pirtobrutinib+venetoclax), even though both independently are resistant, informing a rational combination with non-covalent BTKis. Conclusions: Gain-of-function BCL10 mutants drive oncogenesis and drug resistance through persistent signaling and cytokine activation which is overcome by novel drug combinations. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.