Abstract 2021: ONO-WG-307, a novel, potent and selective inhibitor of Bruton's tyrosine kinase (Btk), results in sustained inhibition of the ERK, AKT and PKD signaling pathways

Abstract

Purpose: Btk is a key regulator of B cell receptors (BCR) which play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk may be effective in the treatment of B-cell lymphoma. Response between cell survival and BCR signaling is implicated in different types of B-cell lymphoma. Therefore, we evaluated the inhibitory effect of ONO-WG-307 on Btk-dependent signal transduction by large-scale and quantitative phosphoproteome analysis to a depth of more than 10,000 phosphorylation sites. Methods: Two tumor cell lines (sensitive and non-sensitive) were treated with ONO-WG-307. After 72h, cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay. P-Btk was used as a marker for Btk activity determined by Western blot and Flow Cytometry. Quantitative phosphoproteomics were enabled by differential SILAC labeling of lymphoma cells. After 1 hr incubation with ONO-WG-307, the total cellular proteins were digested and phosphopeptides were enriched by a combination of strong cation exchange and immobilized metal affinity chromatography. Site-specific phosphorylations were identified and by LC-MS analysis on a LTQ-Orbitrap-Velos mass spectrometer followed by bioinformatic processing. Results: Btk is potently and selectively inhibited by ONO-WG-307 in an in vitro Btk kinase assay, with an IC50 in the subnanomolar range whereas IC50 values for other tyrosine kinases (Lck, Lyn and Fyn) were above 1 µM. The concentration required for growth inhibition for the sensitive cells was 3.59 nmol/L (IC50), whereas considerably higher concentrations were required to suppress growth of non-sensitive cells. Surprisingly, inhibition of cellular Btk and ERK phosphorylation by ONO-WG-307 levels of P-Btk and P-ERK were similar in both sensitive and non-sensitive cells. However, our quantitative phosphoproteome studies revealed selective suppression of Akt-mediated signaling and cellular protein kinase D activity in sensitive compared to non-sensitive cells.Conclusion: These results suggest that the selective ONO-WG-307 inhibition of “responder” cell growth might be due to a critical role of Btk-mediated signaling through Akt and protein kinase D. These data shed new light on the cellular mode-of-action of Btk inhibition and support the potential clinical utility of ONO-WG-307 in B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2021. doi:1538-7445.AM2012-2021

Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement

“…Tirabrutinib (GS-4059/ONO-4059) binds irreversibly to C481 within the ATP binding pocket of BTK. 20 , 21 The chemical structure of tirabrutinib is shown in Figure 1A . A biotinylated probe was designed that binds in the same BTK pocket as the parent tirabrutinib molecule.…”

Novel Bruton's tyrosine kinase inhibitors currently in development

“…In vitro, ONO-WG-307 exhibited differential antiproliferative effects in the nanomicromolar range against multiple NHL/CLL cell lines 158. Quantitative phosphoproteome studies with both sensitive and nonsensitive cells revealed selective ONO-WG-307 suppression of Akt-mediated signaling and cellular protein kinase D activity in sensitive compared to nonsensitive cells 159. These findings suggest a critical role of Btk-mediated signaling through Akt and protein kinase D. Orally administered ONO-WG-307 (50 mg/kg) induced a dose-dependent inhibition of tumor growth in an ABC-DLBCL xenograft (TMD-8) model and moderate antitumor activity in an FL xenograft model 158.…”

Bruton’s tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib

“…ONO-4059 (formerly known as ONO-WG-307) is a potent (IC 50 = 2 nM) reversible BTK inhibitor that blocks autophosphorylation of Tyr223 [Yasuhiro et al 2012]. In vitro, ONO-4059 diminishes proliferation or FL, MCL, and CLL cell lines and shows therapeutic promise in combination with rituximab [Kozaki et al 2011[Kozaki et al , 2012.…”