TLR Signaling Is Activated in Lymph Node–Resident CLL Cells and Is Only Partially Inhibited by Ibrutinib

Abstract

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells driven by B-cell receptor (BCR) signaling and activated primarily in the lymph node. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib effectively inhibits BCR-dependent proliferation and survival signals and has emerged as a breakthrough therapy for CLL. However, complete remissions are uncommon and are achieved only after years of continuous therapy. We hypothesized that other signaling pathways that sustain CLL cell survival are only partially inhibited by ibrutinib. In normal B cells, Toll-like receptor (TLR) signaling cooperates with BCR signaling to activate prosurvival NF-kB. Here, we show that an experimentally validated gene signature of TLR activation is overexpressed in lymph node-resident CLL cells compared with cells in the blood. Consistent with TLR activation, we detected phosphorylation of NF-kB, STAT1, and STAT3 in lymph node-resident CLL cells and in cells stimulated with CpG oligonucleotides in vitro. CpG promoted IRAK1 degradation, secretion of IL10, and extended survival of CLL cells in culture. CpG-induced TLR signaling was significantly inhibited by both an IRAK1/4 inhibitor and ibrutinib. Although inhibition of TLR signaling was incomplete with either drug, the combination achieved superior results, including more effective inhibition of TLRmediated survival signaling. Our data suggest an important role for TLR signaling in CLL pathogenesis and in sustaining the viability of CLL cells during ibrutinib therapy. The combination of ibrutinib with a TLR pathway inhibitor could provide superior antitumor activity and should be investigated in clinical studies. Significance: CLL relies on the concomitant cooperation of B-cell receptor and Toll-like receptor signaling; inhibition of both pathways is superior to inhibition of either pathway alone.

Interleukin‐1 receptor‐associated kinase 4 inhibitor interrupts toll‐like receptor signalling and sensitizes chronic lymphocytic leukaemia cells to apoptosis

“…At a concentration of 10 μmol/l, IRAK4i reduced cell viability in 41 out of 43 samples analysed while it had no or a protective effect in two samples (the percentage of cell viability for each CLL sample is reported in Table SI). These data suggested that CLL cells incubated in vitro might still partially depend on TLR signalling for their survival, likely reminiscent of previous microenvironmental interactions occurring in lymph nodes (Dadashian et al , ) strongly indicating the TLR signalling pathway as a novel druggable target in CLL. However, among the 41 ‘responsive’ cases (i.e.…”

“…As constitutive TLR signalling has been shown to be crucial for the survival of CLL cells even in the absence of this genetic drive, which is rarely observed in CLL, we reasoned that interfering with the TLR pathway may induce a potential therapeutic effect. Indeed, a recent paper showed that a dual IRAK1/4 inhibitor was able to block TLR stimulation in CLL cells (Dadashian et al , ). In addition, Gimenez et al () very recently reported that the IRAK4 inhibitor ND2158 disrupted inflammatory pathways and delayed tumour development in CLL.…”

“…Our data fully support a role for IRAK4i in disrupting the protective effect of previous or concomitant TLR stimulation of CLL cells in vitro . Interestingly, IRAK4i induces apoptosis directly in peripheral blood‐derived CLL cells likely as a consequence of minimal constitutive signalling activation of circulating CLL cells reminiscent of tissue microenvironment interactions which may include TLR as well as IL‐1 receptor family activation (Dadashian et al , ). In detail, IRAK4i was able to upregulate P53 and to activate caspase‐3 and annexin V thus inducing apoptosis.…”

“…Toll‐like receptors (TLR) are prototypic receptors of the innate immune system and act as co‐stimulatory molecules in the context of BCR stimulation (Kremlitzka et al , ). Notably, TLR are expressed by clonal cells in several B‐cell malignancies including CLL where TLR signalling is activated in lymph node‐resident leukaemic cells (Dadashian et al , ). In vitro stimulation of TLR can modulate leukaemic cell viability and proliferation (Muzio et al , ; Ntoufa et al , ).…”

“…Two recent papers demonstrated that targetting IRAK1 and/or IRAK4 with different kinase inhibitors disrupted TLR signalling in CLL cells leading to cell death (Dadashian et al , ; Gimenez et al , ). Along this line, here we tested the activity of a different IRAK4‐specific cell‐permeable indolo[2,3‐c]quinoline‐derivative compound (Tumey et al , ) in primary CLL cells as well as cell lines, alone or in combination with two different drugs, the BTK inhibitor ibrutinib and the purine analogue fludarabine.…”