Tlr9 Expression In Chronic Lymphocytic Leukemia Identifies A Promigratory Subpopulation And Novel Therapeutic Target

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-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 that is activated by unmethylated cytosine guanine dinucleotideDNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, beta(2)-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P5.003). Wealso show that TLR9 expression was associated with in vitro CLL cellmigration (P <.001), and intracellular endosomal TLR9 strongly correlatedwith aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9(hi) and sTLR9(lo) CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9(hi) cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cellmigration (P <.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 aNOD/Shi-scid/IL-2R gamma(null) mouse xenograft model. Finally, weshowed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), 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.