Loss of Toll-like Receptor 2 Results in Accelerated Leukemogenesis in the NUP98-HOXD13 Mouse Model of MDS

The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. Stem cell transplantation is the only curative treatment available to a subset of patients and is associated with significant morbidity and mortality; thus, new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll-like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Increased TLR2 signaling has been associated with low-risk disease, higher rates of apoptosis associated with improved prognosis, and longer survival (Zeng et al ., Exp Cell Res 2016; Wei et al., Leukemia 2013), suggesting TLR2 may, in fact, have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS to shape efforts to target this pathway in patients.

To elucidate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter). These " NHD13" mice recapitulate many features of human MDS and die of leukemia or cytopenias (Lin et al ., Blood 2005). Similar to CD34+ cells in humans, we found a nearly two-fold increase of TLR2 on NHD13 HSCs as compared to controls. NHD13 mice were crossed to Tlr2^-/- mice to generate NHD13;Tlr2^-/-, NHD13;Tlr2^+/+, Tlr2^-/-, and wild-type (WT) groups. Interestingly, loss of TLR2 in NHD13 mice did not confer an improvement in peripheral cytopenias, but was associated with accelerated leukemogenesis and significantly worse survival (p=0.016, with a median survival of 335 days in NHD13;Tlr2^-/- mice vs. 370 days for NHD13;Tlr2^+/+ mice; in both cohorts, leukemia was the primary cause of death- 12/20 for NHD13;Tlr2^-/- and 10/13 for NHD13;Tlr2^+/+). An even more significant acceleration of leukemogenesis and death was observed in NHD13 mice crossed to mice lacking myeloid differentiation primary response gene 88 (MyD88), an adaptor required for most TLR signaling, including TLR2 (p<0.0001, with a median survival of 218 days in NHD13;MyD88^-/- mice vs. 370 days in NHD13 mice).

To investigate the potential mechanism by which TLR2 signaling loss accelerates leukemogenesis in NHD13 mice, hematopoietic stem and progenitor cells (c-Kit+, Sca-1+, Lin-; HSPCs) of premalignant NHD13;Tlr2^+/+ and NHD13;Tlr2^-/- mice were evaluated for cell cycling, cell death, and DNA damage. While loss of TLR2 in the NHD13 mice did not confer a significant difference in HSPC cycling, it did reduce cell death (HSPCs of NHD13⁺;Tlr2^+/+ mice display 18.4% Annexin V+ cells, while those of NHD13⁺;Tlr2^-/- mice display only 6.2%, p=0.0008). An assessment of caspase-1 and caspase-3/7 activities showed a significant reduction in activated caspase-1 in the HSPCs of NHD13;Tlr2^-/- mice compared to NHD13; Tlr2^+/+ mice (average luminescence units: 7310 vs. 10164), suggesting that TLR2 specifically promotes inflammasome-dependent cell death in the NHD13 mice. RNA sequencing of sorted HSPCs also demonstrated an enrichment of apoptosis-related gene pathways in the NHD13 compared to the NHD13;Tlr2^-/- cells. Moreover, the accumulated HSPCs in the NHD13;Tlr2^-/- mice showed elevated levels of γH2AX staining as compared to NHD13; Tlr2^+/+ mice and WT controls, indicating TLR2 loss promotes the survival of damaged HSPCs.

As apoptosis of HSPCs is characteristic of low-risk MDS and is lost as the disease progresses to leukemia, these data suggest that some TLR2 signaling may be protective against leukemic transformation via promotion of apoptosis of damaged premalignant HSCs. Further supporting this idea, we found that in 38% of cases evaluated, TLR2 surface expression was lost from the malignant cells upon transformation to acute leukemia in the NHD13 mice. Thus, down-regulation of TLR2 may be a later event in the progression of MDS to acute leukemia, and complete inhibition of this pathway may, in fact, facilitate the survival of pre-leukemic HSPCs. Ongoing experiments are aimed at elucidating the mechanism by which TLR2 signaling promotes premalignant cell death in MDS and understanding how TLR2 is down-regulated upon transformation to leukemia.