BTK kinase activity is dispensable for the survival of diffuse large B-cell lymphoma

Inhibitors targeting Bruton's tyrosine kinase (BTK) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treat-ment as a result of mutations in BTK. Here, by a combination of structural modeling, in vitro assays, and deep phospho-tyrosine proteomics, we demonstrated that four clinically observed BTK mutations-C481F, C481Y, C481R, and L528W -inactivated BTK kinase activity both in vitro and in diffused large B-cell lymphoma (DLBCL) cells. Paradoxically, we found that DLBCL cells harboring kinase-inactive BTK exhibited intact B cell receptor (BCR) signaling, unperturbed transcrip-tion, and optimal cellular growth. Moreover, we determined that DLBCL cells with kinase-inactive BTK remained addicted to BCR signaling and were thus sensitive to targeted BTK degradation by the proteolysis-targeting chimera. By per-forming parallel genome-wide CRISPR-Cas9 screening in DLBCL cells with WT or kinase-inactive BTK, we discovered that DLBCL cells with kinase-inactive BTK displayed increased dependence on Toll-like receptor 9 (TLR9) for their growth and/or survival. Our study demonstrates that the kinase ac-tivity of BTK is not essential for oncogenic BCR signaling and suggests that BTK's noncatalytic function is sufficient to sus-tain the survival of DLBCL.