TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target

Abstract

CLL remains incurable despite BCR-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate, which is activated by unmethylated CpG-DNA. Here, we show that plasma from CLL patients contains significantly more unmethylated DNA than plasma from healthy controls (p<0.0001) and that cell-free DNA levels correlate with the prognostic markers CD38, b2-microglobulin and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (TTFT: p=0.003, HR=4.0). We went on to show that TLR9 expression was associated with in-vitro CLL cell migration (p<0.001) and intracellular endosomal TLR9 strongly correlated with aberrant surface expression ((sTLR9); r=0.9). In addition, lymph node-derived CLL cells showed increased sTLR9 (p=0.016) and RNA sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility and inflammation in sTLR9hi cells. Mechanistically, the TLR9 agonist, ODN2006, promoted CLL cell migration (p<0.001) that was mediated, by p65 NF-kB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NSG mouse xenograft model. Finally, we showed that dual targeting of TLR9 and BTK was strongly synergistic (median CI=0.2 at ED50), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

“…Previously our group had reported that stimulating CLL cells with a TLR9 agonist, ODN2006, significantly increased CLL cell migration in 2D transwell systems [ 38 ]. We investigated if CLL migration was a FAK-dependent process by establishing whether TLR9 agonism induced p-FAK and by pharmacological inhibition of FAK using defactinib.…”

Homotypic targeting of immunomodulatory nanoparticles for enhanced peripheral and central immunity

“…The TLR9 receptors are localized on the membrane of endosomal compartments, where they interact with internalized CpG ODNs for signaling activation. 46 , 47 , 48 , 49 We therefore examined the subcellular localization of CpG FAM NP and CpG FAM NP@M R . LysoTracker is a fluorescent dye that labels acidic organelles such as the endolysosomes.…”

The Immunomodulatory Functions of BTK Inhibition in the Central Nervous System

“… 42 The pathogenetic activation of BCR signaling and TLR signaling synergistically promote the proliferation and survival of CLL cells. 50 Targeting BTK, a critical component of BCR signaling, has emerged as a promising therapeutic approach against CLL. Although BTK is also involved in TLR pathway, the single use of BTK inhibitors only partly dampened the activation of TLR pathway in CLL cells, which accounted for the low complete remission rate for CLL patients who received BTK inhibition treatment.…”