TY - JOUR
T1 - TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target
AU - Kennedy, Emma
AU - Coulter, Eve
AU - Halliwell, Emma
AU - Profitos-Peleja, Nuria
AU - Walsby, Elisabeth
AU - Clark, Barnaby
AU - Phillips, Elizabeth H
AU - Burley, Thomas A
AU - Mitchell, Simon
AU - Devereux, Stephen
AU - Fegan, Christopher D
AU - Jones, Christopher I
AU - Johnston, Rosalynd
AU - Chevassut, Tim
AU - Schulz, Ralph
AU - Seiffert, Martina
AU - Agathanggelou, Angelo
AU - Oldreive, Ceri
AU - Davies, Nicholas
AU - Stankovic, Tatjana
AU - Liloglou, Triantafillos
AU - Pepper, Chris
AU - Pepper, Andrea G S
N1 - © 2021 by The American Society of Hematology.
PY - 2021/6/3
Y1 - 2021/6/3
N2 - 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 dinucleotide-DNA. 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, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .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, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB 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 NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed 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.
AB - 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 dinucleotide-DNA. 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, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .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, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB 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 NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed 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.
KW - Animals
KW - Cell Movement/drug effects
KW - Female
KW - Gene Expression Regulation, Leukemic/drug effects
KW - Humans
KW - Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
KW - Male
KW - Mice
KW - Mice, Inbred NOD
KW - Mice, SCID
KW - Neoplasm Proteins/agonists
KW - Oligodeoxyribonucleotides/pharmacology
KW - Signal Transduction/drug effects
KW - Toll-Like Receptor 9/agonists
KW - Xenograft Model Antitumor Assays
U2 - 10.1182/blood.2020005964
DO - 10.1182/blood.2020005964
M3 - Article (journal)
C2 - 33512408
SN - 0006-4971
VL - 137
SP - 3064
EP - 3078
JO - Blood
JF - Blood
IS - 22
ER -