Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling

• Published in: Molecular cancer therapeutics 2024-01, Vol.23 (1), p.35-46
• Main Authors: Li, Wei, Sano, Renata, Apatira, Mutiah, DeAnda, Felix, Gururaja, Tarikere, Yang, Muhua, Lundgaard, Greta, Pan, Chin, Liu, Jing, Zhai, Yongjiao, Yoon, Woo Hyun, Wang, Longcheng, Tse, Chris, Souers, Andrew J, Lee, Chih-Hung
• Format: Article
• Language: English
• Subjects: Agammaglobulinaemia Tyrosine Kinase; Drug Mechanisms; Hematological Cancers; Humans; Lymphoma, B-Cell - drug therapy; Phospholipase C gamma - metabolism; Preclinical Models; Protein Kinase Inhibitors - pharmacology; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell - metabolism; Signal Transduction; Small Molecule Agents; Small Molecule Therapeutics; Tyrosine Kinase Inhibitors
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-22-0642

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only. Hypothesizing that differing binding modes may have differential impact on the B-cell receptor (BCR) signaling pathway, we evaluated the activities of multiple BTKis in B-cell lymphoma models in vitro and in vivo. We demonstrated that, although all three types of BTKis potently inhibited BTK-Y223 autophosphorylation and phospholipase C gamma 2 (PLCγ2)-Y1217 transphosphorylation, hinge-only binders were defective in inhibiting BTK-mediated calcium mobilization upon BCR activation. In addition, PLCγ2 activation was effectively blocked by back-pocket and H3 pocket binders but not by hinge-only binders. Further investigation using TMD8 cells deficient in Rac family small GTPase 2 (RAC2) revealed that RAC2 functioned as a bypass mechanism, allowing for residual BCR signaling and PLCγ2 activation when BTK kinase activity was fully inhibited by the hinge-only binders. These data reveal a kinase activity-independent function of BTK, involving RAC2 in transducing BCR signaling events, and provide mechanistic rationale for the selection of clinical candidates for B-cell lymphoma indications.