Abstract The aberrant activation of B cells receptor (BCR) signaling plays an essential role in the pathogenesis of B-cell lymphomas. Targeting BCR signaling by inhibiting Bruton's tyrosine kinase (BTK) has been shown to be a successful strategy in treating B-cell lymphomas including mantle-cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Ibrutinib, an oral, covalent BTK inhibitor achieved high overall response rate (ORR) and prolonged duration of responses in CLL and MCL patients. In contrast, ibrutinib was reported to have much lower response rate in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) and the responses in DLBCL was restricted to the activated B-cell (ABC) subtype. These differences highlight the need to understand the bypass signaling/pathway to BTK inhibition. In this study, we identified CD40L-CD40 pathway as one of the potential bypass pathways to BTK inhibitory therapy. We showed that CD40 was consistently expressed in mature B cells, whilst CD40L expression levels were heterogenic in tissue microarray (TMA) with 150 lymphoma samples. In these samples, circa 57% of lymphoma tissues have medium or strong level of CD40L expression in TMA, indicating the existence of CD40L-CD40 signaling in human lymphoma tissues. Consistent with the response rate of ibrutinib in patients, in 68 non-GCB type of DLBCL, in which aberrant activation of NF-kB pathway is important for the pathogenesis, about 60% samples showed CD40L expression. Co-culture of Rec-1 or TMD8 cells with HEK293/CD40L attenuated the anti-proliferation activity of ibrutinib in vitro. This observation was also confirmed with recombinant soluble CD40L protein. Detailed mechanistic analysis revealed that CD40L-CD40 signaling activated the NF-κB and MAPK pathway and offset the inhibition of ibrutinib. Additionally, activating NF-κB pathway with PKC agonist abolished the cell killing effects of ibrutinib. These data suggest CD40L may be used as a potential biomarker for studying clinical responses to BTK inhibitors. Additionally, our results support combination strategies of BTK inhibitors with agents blocking CD40 or downstream NF-κB and MAPK pathway in patient population where CD40L-CD40 pathway is activated. Citation Format: Zhijian Sun, Lusong Luo. CD40L-CD40 signaling on B-cell lymphoma response to BTK inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1298.