Alternative oncogenic drivers and sensitivity to BTK inhibition in CRISPR/Cas gene-edited human DLBCL cell models.

Diffuse large B-cell lymphoma (DLBCL) can be subclassified by phenotype into germinal center B-cell-like and activated B-cell-like (ABC) subtypes and by recurrent potentially oncogenic mutations into 5 to 7 genetic clusters. In ABC-DLBCL, potentially oncogenic mutations frequently occur in genes involved in B-cell receptor (BCR) signaling and NF-κB activation. Autonomous BCR signaling acts as an alternative immunologic driver predominantly in ABC-type DLBCL that cannot be captured by either subclassification system. The relative functional contribution and interdependence of these mechanistically diverse oncogenic drivers have not been completely defined. To directly compare the effects of autonomously signaling BCR and signalosome-activating CARD11 mutations on NF-κB activation and survival of ABC-DLBCL, we reciprocally exchanged these driver mechanisms in the MYD88L265P-mutated ABC-DLBCL cell lines TMD8 and OCI-Ly3. Only CARD11L251P (not CARD11K215N, CARD11D230N, and CARD11R337Q) compensated TMD8 cells for the loss of autonomous BCR signaling, as indicated by survival of BCR knockout and conversion to complete resistance to acalabrutinib. Transduction of the TMD8 BCR rescued OCI-Ly3 cells from replacing the CARD11L215P variant with CARD11WT. The autonomous TMD8 BCR signal provided a slight growth advantage over CARD11L251P-driven cells in both reciprocal systems. Unsupervised clustering of genetically engineered TMD8 and OCI-Ly3 clones demonstrated tight clustering with their parental cells and only minor alterations of cellular pathways. Only the strongest signalosome-activating mutation has functional near-equivalency to an autonomously signaling BCR for NF-κB activation and growth and survival in ABC-DLBCL. Quantifying the effects of co-occurring potential NF-κB-activating mechanisms is essential to predict Bruton tyrosine kinase (BTK) inhibition sensitivity in individual ABC-DLBCL cases.