Abstract 3232: Development of novel Btk inhibitor, CB988 targering ibrutinib-resistant Btk C481S mutant

Introduction: Bruton's tyrosine kinase (Btk) is a member of the Tec family of cytoplasmic tyrosine kinase. Btk plays a crucial role in the BCR signaling, essential for B-cell development, and Btk has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved Btk inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of Btk to inhibit Btk enzymatic activity, but recent studies suggested that the C481S mutation in Btk disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent Btk inhibitor is highly demanded to overcome ibrutinib resistance. Material and methods: We produced two conformationally different Btks, an activated form of Btk (Btk[A]) and an unactivated form of Btk (Btk[U]) by treating Btk protein with ATP (autophosphorylation) or with Lambda Protein Phosphatase (dephosphorylation), respectively. To evaluate cellular potency of Btk inhibitors, phosphorylations of Btk and PLC- γ were analyzed by Western blotting in Ramos cells, a human Burkitt’s lymphoma cell line. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate anti-tumor efficacy of Btk inhibitors. The ibrutinib-resistant Btk[C481S] mutant was produced to assess efficacy of compounds against the drug resistant enzyme. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: Based on structure activity relationship studies, we identified a novel Btk inhibitor, CB988 as a highly selective non-covalent Btk inhibitor. CB988 exhibited remarkable activity for the both conformations of Btks with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.78 and 0.09 nM for Btk[A] and Btk[U], respectively). These results suggested that CB988 preferentially binds to an inactive conformation of Btk. In cellular assays, CB988 strongly reduced Tyr223 phosphorylation of Btk at nano-molar concentration, and decreased phosphorylation of PLC-γ in Ramos cells. Furthermore, CB988 significantly reduced the proliferation of OCI-Ly10 cells. More importantly, CB988 potently inhibited the ibrutinib-resistant Btk[C481S] mutant in vitro (IC50 = 0.6 nM), which differs from other covalent Btk inhibitors. Conclusions: Btk[C481S] mutation have been reported in ibrutinib relapsed CLL and MCL patients. Novel non-covalent Btk inhibitor, CB988 strongly inhibited Btk[C481S]