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

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 CARD11^L251P/- allele converted TMD8 cells to full acalabrutinib resistance (IC₅₀ >62 μM). Hemizygous and homozygous R337Q mutations had negligible effects (CARD11^R337Q/-: IC₅₀ = 0.003 nM; CARD11^R337Q/R337Q: 0.007 nM). Homozygous K215N and D230N mutations induced partial resistance to acalabrutinib (CARD11^K215N/K251N clones: IC₅₀ = 0.25-0.42 nM; CARD11^D230N/D230N: IC₅₀ = 0.127 nM). Monoallelic mutations had variable but less pronounced effects (CARD11^K215N/WT clones: IC₅₀ = 0.007-0.184 nM; CARD11^D230N/WT: IC₅₀ = 0.009 nM).

In accordance with the observed sensitivities of CARD11-edited TMD8 clones to BTK inhibition, only CARD11^L251P/- 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 CARD11^L251P/- 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 <1%, even after successful retroviral transduction with the non-signaling OCI-Ly3 IgG BCR. Furthermore, competitive co-culture of three independent TMD8 CARD11^L251P/- clones with mock gene-edited control clones failed to showed any growth advantages.

In a reciprocal driver-targeting approach, OCI-Ly3 were transduced with the TMD8 BCR and a CRISPR/cas9-resistant but otherwise wild-type CARD11 allele. CRISPR/cas9-mediated knock-out of the CARD11^L251P alleles increased acalabrutinib sensitivity from an IC₅₀ >96 μM in CARD11^L251P/L251P/tgCARD11^WT OCI-Ly3 cells to 0.002-7.2 μM in CARD11^ko/ko/tgCARD11^WT cells. All tested gene-modified TMD8 and OCI-Ly3 clones remained equally sensitive to downstream BCR signaling inhibition with the MALT1 inhibitor MI2.

In conclusion, we have developed an in vitro system to quantify the effects of defined mechanisms of BCR pathway activation in ABC-DLBCL. Our reciprocally controlled results demonstrate that the strongly activating CARD11 L251P mutation and a BCR with sufficiently strong autonomous signalling provide functionally equivalent but alternative oncogenic growth and survival signals in experimental systems of ABC-DLBCL. Other recurrent mutations of the coiled-coil CARD11 region, even with a CADD-PHRED score of up to 5, have weaker or virtually undetectable effects. In the presence of these mutations, a BCR without autonomous signaling activity fails to rescue knock-out of the autonomous BCR signaling activity, indicating insufficiency of tonic BCR signaling. The low prevalence of the CARD11 L251P mutation emphasizes the importance of antigen-independent autonomous BCR signaling as a frequent non-genetic immunological driver mechanism and the need for detailed dissection of the relative functional contribution of genetic and immunological drivers, including the zygosity of driver mutations, in order to develop optimal individualized targeted therapy for DLBCL patients.