Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and the activated B cell-like subtype (ABC-DLBCL) is the most aggressive form and harbors frequent mutations of immune signaling pathways that culminate in constitutive activation of CARD11-MALT1-BCL10 (CBM) complex and downstream NF-κB pathway. CBM complexes form large macromolecular structures due to signal-induced polymerization of the BCL10 subunit, which is affected by recurrent somatic mutations in ABC-DLBCLs. Through biochemical, structural and functional dissection of these mutations, we find that BCL10 mutations fall into two functionally distinct classes: missense mutations of the BCL10 CARD domain (hotspot R58Q) and truncation of its C-terminal tail (hotspot E140X). To explore the functional consequences of BCL10 mutations, we established reporter systems to evaluate their impact on MALT1 and NF-𝜅B activities which are BCL10 downstream signaling cascades. We found that almost all mutants induced aberrantly strong NF-𝜅B and MALT1 activities in lymphoma cells as compared to WT BCL10, indicating the gain-of-function effect of BCL10 mutations. By performing immunohistochemistry staining of p65 in a set of tumor tissue microarray from DLBCL patients (n=298), we revealed that BCL10 mutant tumors have significantly (Mann-Whitney p<0.0001) increased p65 nuclear staining score compared to BCL10 WT tumors, suggesting enhanced NF-𝜅B activity. To investigate the biochemical impact of BCL10 mutants on CBM complex formation, we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 proteins and found that both BCL10R58Q and BCL10E140X manifested faster and more spontaneous polarization compared to BCL10WT. Surprisingly, through mapping the BCL10-MALT1 interaction, we found that truncating mutation (E140X) abrogated a novel protein interaction motif through which MALT1 inhibits BCL10 polymerization, thus unleashing spontaneous CBM filament formation and inducing addiction to MALT1 activity. In marked contrast, the CARD missense mutation (R58Q) on BCL10 filament interface not only does not disrupt but enhances filament formation and it also alters CBM complex kinetics forming glutamine network structures that stabilize BCL10 filaments, but this still may require the upstream signal to activate MALT1. Importantly, we found that BCL10 mutant cells were less dependent on upstream CARD11 activation in MALT1 activation, NF-𝜅B signaling and cell growth assays performed in ABC-DLBCL lines. Furthermore, in vitro and in vivo xenograft studies revealed that BCL10 mutant lymphomas are resistant to BTK inhibitors, whereas BCL10 truncating (E140X) but not missense CARD (R58Q) mutants were hypersensitive to MALT1 protease inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL and further precision can be achieved by tailoring therapy (e.g. MALT1 inhibitors that are currently being tested in clinical trials) according to specific biochemical effects of distinct mutation classes. Citation Format: Min Xia, Liron David, Matthew R Teater, Johana Gutierrez, Xiang Wang, Cem Meydan, Andrew Lytle, Graham Slack, David Scott, Ozlem Onder, Kojo Elenitoba-Johnson, Nahuel Zamponi, Leandro Cerchietti, Tianbao Lu, Ulrike Philippar, Lorena Fontan, Hao Wu, Ari Melnick. BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A28.