Oncogenic CARD11 Mutations and Autonomous BCR Signaling Act As Functionally Equivalent Alternative Drivers in ABC-DLBCL

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.1619-1619
• Main Authors: Eken, Janneke, Havenaar, Fleur R.M., Quinten, Edwin, De Groen, Ruben A.L., Sepulveda-Yanez, Julieta, Vermaat, Joost S.P., van Bergen, Cornelis A.M., Veelken, Hendrik
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-204704

Diffuse large B-cell lymphoma of activated B-cell type (ABC-DLBCL) is characterized by chronic signaling of the B-cell receptor (BCR) pathway with constitutive NF-kB activation. We have recently identified autonomous, i.e. antigen-independent, signaling originating from individual BCR complexes as the cause of BCR pathway activation in the majority of ABC-DLBCL cases (Eken et al., J Exp Med 2024). As a pure immunological driver, autonomous BCR signaling cannot be recognized by sequence analysis nor be readily added to proposed genetic DLBCL subclassifications. Besides autonomous BCR signaling, recurrent activating mutations of various members of the BCR signaling cascade, in particular CD79 and CARD11, and in the TLR pathway member MYD88 contribute to the ABC phenotype. Intriguingly, lack of autonomous BCR signaling is associated with a homozygous CARD11 L251P mutation in the OCI-Ly3 ABC-DLBCL cell line. We therefore investigated the function of recurrent CARD11 mutations in the CARD11 wild-type ABC-DLBCL cell line TMD8. TMD8 cells express a BCR with intermediate-high autonomous signaling and are highly sensitive to the BTK inhibitor acalabrutinib (IC50 = 0.005 nM) by the MTS viability assay. TMD8 cells were gene edited by CRISPR/cas9 homology-directed repair to express the presumed oncogenic CARD11 coiled-coil region mutations L251P, D230N, K215N, and R337Q, cloned, and analyzed by sanger sequencing. Introduction of a hemizygous CARD11L251P/- allele converted TMD8 cells to full acalabrutinib resistance (IC50 >62 μM). Hemizygous and homozygous R337Q mutations had negligible effects (CARD11R337Q/-: IC50 = 0.003 nM; CARD11R337Q/R337Q: 0.007 nM). Homozygous K215N and D230N mutations induced partial resistance to acalabrutinib (CARD11K215N/K251N clones: IC50 = 0.25-0.42 nM; CARD11D230N/D230N: IC50 = 0.127 nM). Monoallelic mutations had variable but less pronounced effects (CARD11K215N/WT clones: IC50 = 0.007-0.184 nM; CARD11D230N/WT: IC50 = 0.009 nM). In accordance with the observed sensitivities of CARD11-edited TMD8 clones to BTK inhibition, only CARD11L251P/- clones were able to survive knock-out of their autonomously signaling BCR by CRISPR/cas9-mediated non-homologous end-joining. After BCR knock-out, an IgM-negative population of app. 20% remained detectable in CARD11L251P/- TMD8 cells by flow cytometry for at least 4 weeks of cell culture. For all other CARD11-mutant clones, IgM-negative cells consistently amounted to