ONO-4059, A Novel Bruton’s Tyrosine Kinase (Btk) Inhibitor: Synergistic Activity In Combination With Chemotherapy In a ABC-DLBCL Cell Line

Bruton’s tyrosine kinase (Btk) is a key regulator of the B-cell receptor (BCR) signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. Recent studies indicate that targeting Btk is effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC₅₀ in the sub-nmol/L range. The activated B-cell-like (ABC) sub-type of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and a substantial unmet need still exists. The standard treatment with Rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) for ABC-DLBCL in particular being associated with a poorer outcome. The combination of ONO-4059 with the Bcl-2 inhibitor, ABT-263 has demonstrated greater apoptosis in ABC-DLBCL cell lines than that seen with single agent treatment (Ryohei K. et al., EHA 2013 meeting). To explore rational combinations, ONO-4059 combined with chemotherapy drugs was evaluated in a ABC-DLBCL cell line.

ABC-DLBCL cell line (TMD-8) was incubated with ONO-4059 monotherapy (1-1000 nmol/L) and in combination with various agents that target various pathways thought to be involved in DLBCL. Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay. Apoptosis was determined by AnnexinV-FITC/7AAD (AnnV+/7AAD-) staining.

ONO-4059 inhibited the TMD-8 cell growth and Btk phosphorylation in a concentration-dependent manner, with an IC₅₀ of 3.59 for 72 hr and 23.9 nmol/L for 4 hr respectively. Furthermore, the decrease in Btk phosphorylation subsequently down-regulated Erk phosphorylation. At the 48 hr time-point, 7% of apoptosis occurred in TMD-8 cells at 10 nmol/L, an earlier time-point than that observed for dead cell. After the combination of 10 nmol/L of ONO-4059 with 25 nmol/L of doxorubicin, 500 nmol/L of etoposide, 1.5 nmol/L of vincristine and 1000 nmol/L of dexamethasone, the increased apoptosis ratio was observed, 25, 20, 17 and 29%, respectively. Synergy was observed when ONO-4059 was combined with the CHOP regimen.

Recent studies indicate that targeting Btk induces BIM expression, the pro-apoptotic protein, following inhibition of Erk phoshphorylation. Our results demonstrate that treatment with ONO-4059 in combination with chemotherapy results in a synergistic effect, inducing apoptosis and is more effective compared with respective monotherapies. ONO-4059 is currently being developed in a Phase I clinical trial for the treatment of B-cell malignancies. Additional combination studies are underway using the TMD-8 xenograft model, testing ONO-4059 in combination with various agents.

Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Yoshizawa:Ono Pharmaceutical CO., Ltd: Employment. Birkett:Ono Pharma UK: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.