Docirbrutinib (AS-1763), a novel non-COVALENT BTK inhibitor, demonstrates efficacy in BTK inhibitor-resistant mutant cells Free

Background: Bruton's tyrosine kinase inhibitors are a standard therapy for chronic lymphocytic leukemia (CLL). Despite their efficacy, covalent BTK inhibitors suffer from drug resistance driven by mutation at C481 in BTK. To address this, noncovalent BTK inhibitors (ncBTKi) have been developed, however, resistance can still emerge through mutations such as T474I and L528W. These resistant mutations in BTK emphasize the importance of developing novel inhibitors capable of targeting multiple resistant variants. Previously, we demonstrated that docirbrutinib (AS-1763) is a potent and highly selective ncBTKi, orally available and effective against both wild-type (WT) and multiple resistant BTK mutants, including those resistances to covalent BTKi and ncBTKi. This work extends our previous findings by examining the efficacy of docirbrutinib in human diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK mutations, both alone and in combination with a BCL2 inhibitor.

Study Design and Methods: We investigated the effects of docirbrutinib on BCR signaling using HEK293 or OCI-Ly10 cells expressing various BTK mutants. For L528W kinase dead mutant, we generated OCI-Ly10 cells overexpressing L528W mutant to hijack the BCR signaling. The off-rate of docirbrutinib was measured using the rapid dilution assay. Apoptosis was assessed in OCI-Ly10 cells harboring mutant BTK using a Annexin V Apoptosis Detection Kit, and data were acquired by flow cytometry. Synergistic effects between docirbrutinib and venetoclax in OCI-Ly10 cells were evaluated using isobologram analysis.

Results: The enzymatic activity of BTK is regulated by upstream kinases, which phosphorylate Tyr551 and induce a conformational change of BTK. Compounds that bind to a Src-like inactive conformation of BTK can sequester Tyr551 from these kinases, thereby allowing Tyr551 to remain unphosphorylated. Docirbrutinib potently inhibited phosphorylation at Tyr551 as well as Tyr223, an autophosphorylation site, suggesting that docirbrutinib binds to a Src-like inactive conformation of BTK, as predicted by our docking model. Inhibitors that bind to the inactive conformation of kinases have previously been reported to exhibit slow off-rates. To investigate this property for docirbrutinib, we performed a rapid dilution assay. As anticipated, the rate of product formation following docirbrutinib treatment was markedly reduced, demonstrating that docirbrutinib acts as a slow off-rate inhibitor. The inhibitory potency of docirbrutinib against BTK mutants was evaluated in OCI-Ly10 cells harboring resistance mutations. Docirbrutinib demonstrated strong antiproliferative activity against BTK WT, C481S, and T474I mutants. Overexpression of the L528W mutant in OCI-Ly10 cells conferred resistance to both ibrutinib and pirtobrutinib; however, docirbrutinib retained its antiproliferative activity in these cells. This effect is likely attributable to the tight binding of docirbrutinib to the Src-like inactive conformation of BTK L528W, which may disrupt its scaffold function and thereby inhibit cell survival signaling.

Given the known clinical synergy between BTK inhibitors and venetoclax, we investigated the synergistic antiproliferative effects of docirbrutinib and venetoclax in OCI-Ly10 cells using isobologram analysis. The results demonstrated a synergistic effect between docirbrutinib and venetoclax. We also assessed apoptosis induced by the combination of docirbrutinib and venetoclax in BTK-mutant OCI-Ly10 cells. The combination treatment resulted in a significant increase in apoptosis compared to either agent alone. In ex vivo treatment of three L528W-mutated CLL samples, the combination of docirbrutinib and venetoclax reduced cell mean viability more effectively than venetoclax alone at 72 hours (35.5% vs 50.3%).

Conclusions: Docirbrutinib is a novel ncBTKi characterized by a slow off-rate that enables sustained BTK inhibition, potentially leading to prolonged therapeutic effects similar to those of cBTKi. Profiling across a panel of BTK mutants demonstrated that docirbrutinib is effective against both wild-type and multiple resistant BTK variants, including the kinase-dead L528W mutant. In combination with venetoclax, docirbrutinib induced significant cell death in BTK-mutant OCI-Ly10 cells and primary CLL. The detailed mechanism underlying these effects will be presented. Ongoing clinical trial is testing efficacy of docirbrutinib in CLL patients with WT or mutant BTK.