Ibrutinib Inhibits Both B-Cell Receptor and Toll-like Receptor Signaling in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) cells rely on signals from the microenvironment to for cell activation, proliferation and survival. Microarray analysis of CLL cells harvested from patient-matched lymph node and peripheral blood demonstrated an up-regulation of B-cell receptor (BCR) signaling in the tissue microenvironment (Herishanu, Blood 2011). Concurrent with increased BCR signaling a dynamic increase in NF-κB signaling was also noted in the activated CLL cells in the tissue microenvironment (Herishanu, Blood 2011).  As NF-κB can be activated downstream of multiple signaling pathways, in addition to the BCR, we sought to determine the role of alternative pathways on NF-κB activation. In gene expression analysis of CD19+ selected CLL cells, we found that in addition to an increase in BCR signaling the Toll-like receptor (TLR) signaling pathway was also significantly up-regulated in CLL cells in the lymph node microenvironment compared to circulating CLL cells.

Activation of TLR signaling can cooperate with BCR signaling to overcome anergy and promote the expansion of auto-reactive B-cells (Leadbetter, Nature 2002). In addition, TLR signaling can induce proliferation of CLL cells and upregulates co-stimulatory molecules that may make CLL cells more effective antigen presenting cells. The latter effect has been explored as a therapeutic strategy to enhance the efficacy of immunotherapy of CLL by rendering CLL cells more susceptible to attack by cytotoxic T-cells (Spaner and Masellis, Leukemia 2007).  We hypothesize that inhibition of TLR signaling could contribute directly to anti-leukemic effects by inhibiting survival and proliferation pathways.

To test the effect of TLR signaling in CLL, CLL PBMCs were stimulated in vitro with a CpG oligonucleotide, which activates TLR9, for 6 hours. Increased pIκBα expression along with significant increases in pSTAT3 (P=0.001), IL-10 production (P=0.008) and up-regulation of the cell surface activation markers CD54, CD69 and CD86 (P<0.001) were observed, consistent with activation of TLR signaling. In order to evaluate the effect of inhibiting TLR signaling on CLL cells, we utilized an inhibitor of IRAK1/4 (EMD Chemicals), which acts directly downstream of MyD88.  As expected, inhibition of IRAK1/4 significantly reduced TLR signaling in CpG stimulated CLL cells, resulting in decreased NF-κB signaling, IL-10 secretion (P=0.03) and phosphorylation of STAT3 (P<0.001) in drug treated compared to vehicle treated cells.  Concurrently, inhibition of proliferation (as measured by Ki67) and tumor cell activation (CD69 and CD86 expression) was observed (P<0.05), demonstrating a direct anti-leukemic effect.

Recently, the BTK inhibitor ibrutinib was shown to be clinically active in Waldenstrom’s macrogloublinemia (WM), a chronic B-cell malignancy not dissimilar to CLL that is characterized by mutations in the TLR adaptor MyD88 (Treon, NEJM 2012; Treon, NEJM 2015). We therefore investigated whether ibrutinib could inhibit TLR signaling in CLL cells.  Indeed, even at doses as low as 100nM, ibrutinib significantly inhibited CpG dependent activation of NF-κB signaling (P<0.05), downstream secretion of IL-10 (P<0.05) and phosphorylation of STAT3 (P<0.01). Additionally, proliferation (Ki67) and cellular activation induced by CpG were inhibited by ibrutinib treatment (P<0.05). Notably, both the inhibition of TLR signaling and the anti-tumor effect of ibrutinib at 100nM was comparable to inhibition achieved with 10µM of the IRAK1/4 inhibitor.

In summary, TLR signaling is engaged in CLL cells in the lymph node microenvironment and upregulates proliferation and survival pathways. Ibrutinib appears to be as effective as a direct IRAK inhibitor in blocking TLR signaling in CLL cells, suggesting that treatment with ibrutinib is sufficient to inhibit both BCR and TLR signaling.

This work was supported by the Intramural Research Program of National Heart Lung and Blood Institute of the National Institutes of Health.