Toll like Receptor 2/6 but Not 1/2 Signaling Promotes Leukemogenesis in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and cellular dysplasia with high rates of transformation to leukemia. The only cure is hematopoietic stem cell transplant, which is associated with significant morbidity and mortality, thus there is a need to better delineate the pathogenesis of MDS and identify novel targets for treatment. Recent studies suggest that deregulated innate immune signaling, and, in particular, enhanced toll like receptor (TLR) signaling, may contribute to the pathogenesis of MDS. The TLRs are a family of pattern recognition receptors that play a central role in innate immunity. Specifically, TLR2 and its binding partners, TLR1 and TLR6, are markedly elevated in the CD34+ cells of patients with MDS compared to healthy controls (Wei et al, Leukemia 2013). Thus, we hypothesize that enhanced TLR2 signaling may contribute to the pathogenesis of MDS.

To elucidate the contribution of TLR2 signaling to MDS, we used a well-characterized mouse model of MDS (mice expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter) to determine the effects of loss or gain of TLR2 signaling on disease progression. These "NHD13" mice have cytopenias, dysplasia, marrow hypercellularity, and increased apoptosis by 4-7 months of age; transgenic mice die by 14 months of age of leukemia or cytopenias (Lin et al, Blood 2005).Of note, similar to human HSCs, we find increased expression of TLR2 by flow cytometry on the HSCs of NHD13 mice compared to controls.

Given the association of enhanced TLR2 signaling with MDS, we predicted that loss of TLR2 would improve outcomes in NHD13 mice. To this end, these mice were crossed to Tlr2^-/- mice to generate 4 groups: NHD13⁺;Tlr2^-/-; NHD13⁺;Tlr2^+/+, NHD13^-;Tlr2^-/- and NHD13^-;Tlr2^+/+. Surprisingly, loss of TLR2 is associated with significantly worse survival with a median survival of 313 days for NHD13⁺;Tlr2^-/-, and 367 days for NHD13⁺;Tlr2^+/+(p=0.01; with the majority of mice dying from leukemia); and loss of TLR2 did not improve cytopenias. Ongoing experiments are aimed at determining the effects of TLR2 loss on HSC cycling, apoptosis, and function in the NHD13 mice and their wild-type (WT) sibling controls.

Conversely, we are asking how stimulation of TLR2 affects disease by treating NHD13 mice chronically with a TLR1/2 agonist (PAM₃CSK₄) or a TLR2/6 agonist (PAM₂CSK₄) or water control. NHD13 mice receiving PAM₂CSK₄ had elevated white blood cell and hemoglobin counts after five months as compared to mice receiving PAM₃CSK₄ and control mice, while mice receiving PAM₃CSK₄ demonstrated a higher incidence of thrombocytopenia, suggesting that TLR2 signaling may contribute to cytopenias. Strikingly, however, we found that treatment with the TLR2/6 agonist (PAM₂CSK₄) significantly (p < 0.0001) accelerates the time to leukemia and death (but not the TLR1/2 agonist, PAM₃CSK₄), with deaths occurring as early as 70 days of life, mostly from leukemia. After four months of treatment, 100% of control and 93% of PAM₃CSK₄ treated NHD13 mice were alive, while only 37.5% of PAM₂CSK₄ treated NHD13 mice were alive (p= 0.0002).

This raises the intriguing possibility that there are heterodimer-specific effects of TLR2 signaling on premalignant HSCs. Historically, the association of TLR2 with TLR1 versus TLR6 was thought to expand the ligand spectrum without altering downstream signaling (Farhat et al, J Leuk Biol 2008), however recent data (and our own findings) suggest that there may be heterodimer-specific differences (Rolf et al, Euro J of Immunology 2015). Furthermore, while expression of TLR2 itself correlates with low-risk disease and longer survival, high expression of TLR6 is associated with higher-risk disease and increased marrow blasts (Wei et al, Leukemia 2013). We are currently exploring the mechanisms behind the heterodimer-specific effects of TLR2 signaling on leukemogenesis and survival in the NHD13 mice by elucidating the effects of PAM₃CSK₄ versus PAM₂CSK₄ on HSC cycling, apoptosis, and function, as well as determining the downstream signaling pathways (utilizing mass cytometry) and target genes that are uniquely activated by the different agonists. Ultimately, an understanding of TLR1/2 versus TLR2/6 stimulation effects on WT and premalignant HSCs is critical to the development of targeted therapies toward this pathway.