Abstract 2598: Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma
Authors: Kuo Hsu-Ping , Hsieh Sidney , Chang Betty 
Published: August 2015

Abstract
Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent B-lymphocyte neoplasm (Morton, Blood 2006). The activated B-cell (ABC) subtype is associated with poorer prognosis (Lenz, NEJM 2008). Over 20% of ABC-DLBCL cases have mutations in the CD79B or CD79A subunits of the B-cell receptor (BCR), generating chronically active BCR signaling (Staudt, ASH 2012). Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton's tyrosine kinase, an essential BCR pathway enzyme. Phase 2 trial results of single-agent ibrutinib in DLBCL showed efficacy in the ABC subtype, with an overall response rate of 40% and 60% in CD79B-mutant ABC-DLBCL (Wilson, ASH 2012). Identification of MYD88 L265P mutations in ABC-DLBCL revealed a link between Toll-like receptor (TLR) signaling and ABC-DLBCL (Ngo, Nature 2011). The findings that a TLR 7/8/9 antagonist selectively inhibits cell survival of MYD88 L265P-mutated ABC-DLBCL cells (Bhagat, AACR 2014) emphasize the importance of TLR signaling in ABC-DLBCL. We hypothesized that TLR-related kinases/pathways important to ABC-DLBCL may be unknown and tested for synergy between ibrutinib and inhibitors targeting these pathways in ABC-DLBCL cells.
Methods: Combination effects of ibrutinib and TLR9 antagonists, a TAK1 inhibitor, or an endosomal TLR inhibitor were tested in ABC-DLBCL cell lines with MYD88 mutations. TMD-8, HBL-1, and OCI-LY10 cell lines were treated with inhibitors or antagonists alone or in combination with ibrutinib for 3 days; cell growth effects were determined by the CellTiter-Glo luminescent cell viability assay (Promega). The combination index (C.I.), a drug interactivity measurement, was calculated with CalcuSyn. Synergy scores were calculated by the Chalice Analyzer (Horizon CombinatoRx).
Results: We identified synergistic inhibitory effects on ABC-DLBCL cell growth between ibrutinib and TLR9 antagonists as well as the TAK1 inhibitor. Different TLR9 antagonist combinations significantly enhanced ibrutinib killing effects in ABC-DLBCL cells in the presence or absence of class A, B, and C TLR9 agonists. Stimulation with TLR9 agonists increased resistance of ABC-DLBCL cells. Strong synergy was also identified between ibrutinib and an inhibitor of endocytic TLRs (TLR3, 7, 8, 9) with an average C.I. of 0.11 and synergy score of 4.22. Our assays demonstrated that an endosomal TLR inhibitor possesses similar ibrutinib synergistic effects as other TLR9 antagonists. Similar results were observed for inhibition of the downstream TLR signaling molecule TAK1.
Conclusions: We provide evidence of strong synergistic effects between ibrutinib and inhibitors targeting TLR signaling. Survival of ABC-DLBCL cell lines with MYD88 mutations requires signals through both TLR and BCR. Combined, these results provide rationale for targeting both pathways simultaneously in MYD88-mutated ABC-DLBCL.
Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Betty Chang. Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2598. doi:10.1158/1538-7445.AM2015-2598