Abstract 2686: Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia
Authors: Dadashian Eman L. , Herman Sarah , Wiestner Adrian 
Published: July 2017

Abstract
Introduction: The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is clinically active in lymphoproliferative diseases driven by B-cell receptor (BCR) and Toll-like receptor (TLR) signaling, including chronic lymphocytic leukemia (CLL) and Waldenstrom macroglobulinemia (WM), respectively. However, deep remissions are uncommon and resistance to single agent has been described. The hallmark of WM is an activating mutation in MYD88 in the TLR signaling pathway. While mutations in MYD88 are uncommon in CLL, our previous study identified gene signatures indicative of active BCR and TLR signaling in CLL cells residing in lymphoid tissues (Herishanu, Blood 2011). Further, TLR9 activating CpG oligonucleotides induce proliferation and extend CLL cell survival in vitro. These observations suggest that BCR and TLR signaling may cooperate to activate CLL cells in the tissue microenvironment. Here, we tested the hypothesis that targeting both BCR and TLR signaling could improve therapy for CLL.
Methods: CLL PBMCs were treated with ibrutinib and/or an IRAK1/4 inhibitor (Calbiochem) for 1h and then stimulated with soluble αIgM, CpG, or both. We quantified changes in phosphorylation of BTK, PLCγ2 and ERK (BCR pathway) and STAT3 and STAT1, as well as total IRAK1 (TLR pathway).
Results: As expected, ibrutinib inhibited phosphorylation of BTK, PLCγ2 and ERK (P&lt;.05) and decreased the survival of CLL cells stimulated with αIgM (P=.001). CpG stimulated TLR signaling degrades IRAK1 and the stimulates cytokine secretion that can, in autocrine fashion, activate STAT phopsphorylation. The IRAK1/4 inhibitor effectively inhibited TLR signaling resulting in stabilization of IRAK1 (P=.002), decreased phosphorylation of STAT1/3 (P=.04) and decreased viability compared to CpG stimulated but not IRAK inhibitor treated cells. Ibrutinib had no effect on CpG-induced IRAK1 degradation, but was comparable to IRAK1/4 inhibition in reducing STAT phosphorylation, suggesting that inhibition of BTK can antagonize downstream effects of TLR activation but not upstream IRAK dependent steps. In contrast, IRAK1/4 inhibition had no effect on αIgM-induced BCR activation. Next we evaluated the effect of dual BCR and TLR activation, modelling co-operative activation of both pathways in the tumor microenvironment. Under these in vitro conditions, ibrutinib prevented BCR activation and partial TLR activation, while IRAK1/4 affected only the TLR pathway. When ibrutinib and the IRAK1/4 inhibitor were combined, activation of both BCR and TLR signaling was prevented resulting in a significant reduction in CLL cell viability (P=.01) compared to co-activation of both pathways.
Conclusion: While ibrutinib partially inhibited TLR signaling, an IRAK1/4 inhibitor was required for full inhibition of the pathway. The combination of BTK and IRAK1/4 inhibition for the treatment of lymphoproliferative diseases warrants further investigation.
Citation Format: Eman L. Dadashian, Sarah Herman, Adrian Wiestner. Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2686. doi:10.1158/1538-7445.AM2017-2686