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
