Differential Effects of TLR1/2 and TLR2/6 Signaling on Normal and Premalignant Hematopoietic Stem and Progenitor Cells
Authors: Monlish Darlene A. , Greenberg Zev J. , Bhatt Sima T. , Ralphs Dagmar , Keller John L. , Bendesky Lauren , Schuettpelz Laura G. 
Published: October 2019

Abstract
Prior studies from our lab and others have demonstrated a role for Toll-like receptor 2 (TLR2) in regulating both normal and premalignant hematopoietic stem and progenitor cells (HSPCs), however the contributions of its binding partners, TLR1 and TLR6, remain unknown. In CD34+ bone marrow cells of patients with myelodysplastic syndrome (MDS), increased TLR2 was associated with lower-risk disease, elevated rates of apoptosis associated with improved prognosis, and enhanced survival. Conversely, increased levels of TLR6, but not TLR1, was associated with higher-risk disease and an increased percentage of bone marrow blasts (Zeng et al., Exp Cell Res 2016 and Wei et al., Leukemia 2013). These data suggest that there may be heterodimer-specific effects of TLR2 signaling on HSPCs influencing disease progression.
To elucidate the unique contributions of the heterodimer pairs in MDS pathogenesis and leukemogenesis, we utilized a well-established mouse model of MDS that expresses the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter. The "NHD13" mice recapitulate many of the salient features of human MDS and succumb to cytopenias or leukemia by 14 months of age (Lin et al., Blood 2005). Importantly, we observed significantly increased expression of TLRs 1, 2, and 6 on the c-Kit+, Sca-1+, Lineage- ("KSL") HSPCs of the NHD13 mice, similar to the increased expression of these TLRs on CD34+ cells of MDS patients. To begin to delineate the heterodimeric differences, NHD13 mice were treated chronically with either PAM2CSK4 (PAM2), a TLR2/6-specific agonist, or PAM3CSK4 (PAM3), a TLR1/2-specific agonist, to assess the effects on cytopenias and survival. After five months of treatment, a significant increase was observed in the total number of white blood cells in NHD13 mice treated with PAM2 (p=0.007), but not PAM3 (vs. vehicle (water)-treated controls), a finding that was not recapitulated in wild-type (WT) controls. On the contrary, a significant decrease in the total number of platelets in both NHD13 and WT mice treated with PAM3 was observed as compared to vehicle-treated controls (p=0.024 and p=0.011, respectively). Further supporting the existence of heterodimer-specific differences, death was expedited in NHD13 mice treated with PAM2 as compared to those treated with PAM3 (p=0.019), with a median survival of 243 days vs. 338 for the PAM3-treated cohort. The cause of death, as determined by a hematopathologist based on cytology and blast percentage, was most often due to leukemia.
To investigate the potential mechanism through which enhanced TLR2/6 signaling accelerates leukemogenesis and death in NHD13 mice, the HSPCs of premalignant NHD13 mice treated with PAM2 or PAM3 were characterized by flow cytometry and evaluated for cell cycling and cell death. Both the total number and frequency of KSL cells were significantly increased in NHD13 mice treated with PAM2 (p=0.007 and p<0.0001, respectively), but not PAM3, vs. water-treated controls. No significant changes were noted in either cell cycling or apoptosis following agonist treatment. A microarray of bone marrow KSL cells revealed that stimulation of the TLR2/6 pathway is associated with an activated c-Myc signature, suggesting that enhanced signaling through this pathway, but not TLR1/2, may enhance leukemogenesis via Myc activation. Further, the expression levels of six downstream targets of c-Myc, including BAX, APEX1, ODC1, FKBP4, NCL, and HSPD1, were significantly increased in both WT and NHD13 mice following PAM2 treatment. Evaluation of serum cytokines also revealed heterodimer-specific alterations, including increased IL-6 levels in NHD13 mice treated with PAM2, but not PAM3. These data corroborate numerous previous reports linking IL-6 to MDS pathogenesis and transformation to acute myeloid leukemia. Ongoing studies involving mass cytometry, IL-6knockout mice, and pharmacological inhibitors of both IL-6 and c-Myc aim to further elucidate the mechanism through which TLR2/6-specific activation accelerates leukemogenesis and death in the NHD13 mouse model of MDS. These studies hope to inform more targeted therapeutics that could potentially delay MDS progression and reduce off-target effects.

Disclosures
No relevant conflicts of interest to declare.