The B Cell Receptor Antagonist R406 Alters The Balance Between Anti-Apoptotic and Pro-Apoptotic Signals To Strongly Synergize With ABT-199 In Diffuse Large B Cell Lymphoma Cells

SYK plays an essential role in B cell receptor (BCR) mediated survival in Diffuse Large B Cell Lymphoma (DLBCL). In a previous clinical investigation, fostamatinib, a potent Syk inhibitor, showed evidence of anti-tumour activity with an overall response rate of ∼20% in a cohort of DLBCL patients (JW Friedburg et al Blood. 2010, 115:2578-85). This finding prompted us to investigate the mechanism of action of fostamatinib in DLBCL. Our previous work demonstrated R406, the metabolic active form of fostamatinib, functions as a BCR antagonist in DLBCL cells. Treatment with R406 reduced MCL1 and down-regulated Bfl-1 expression, leading to apoptosis induction in sensitive cells. Due to R406’s impact on apoptosis machinery, here we explored the ability of R406 to combine with a selective Bcl2 inhibitor, ABT-199 in DLBCL cells.

Drug combinations were tested in DLBCL cells and synergy scores were determined using a 6x6 dose matrix followed by analysis via Chalice software (Zalicus). ABT-199 strongly synergized with R788, the prodrug of R406, in multiple R406 sensitive, but not resistant cell lines. In contrast, little effect was observed in combinations of R788 with other reagents, including ibrutinib (PCI-32765), bortezomib, CAL-101 and dasatinib. Flow-cytometry apoptosis analysis confirmed that R406 enhanced ABT-199-induced apoptosis in both ABC subtype and GCB subtype DLBCL cells. Further studies demonstrated R406 not only decreased anti-apoptotic proteins, but also activated pro-apoptotic proteins. Specifically, R406 suppressed ERK-dependent phosphorylation of BIM on S69, resulted in stabilization of BIM. In addition, R406 induced dephosphorylation of BAD at S136 in sensitive but not resistant cells, likely through inhibition of AKT kinase activity. Furthermore, treatment of R406 also upregulated other pro-apoptotic BH3-only proteins such as HRK1 and BMF1, which may partially contribute to the observed synergy.

In conclusion, we have demonstrated R406 strongly synergizes with ABT-199 in vitro, likely by altering the balance between anti-apoptotic and pro-apoptotic signals in DLBCL cells. This may provide a potential combination therapy to be tested in DLBCL.