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The use of immunotherapy to treat patients with myelodysplastic syndromes (MDS) shows promise but is limited by our incomplete understanding of the immunologic milieu. In solid tumors, CD141Hiconventional dendritic cells (CD141HicDCs) are necessary for antitumor immunosurveillance and the response to immunotherapy. Here, we found that CD141HicDCs are reduced in MDS bone marrow and based on the premise established in solid tumors, we hypothesized that reduced numbers of CD141HicDCs are associated with inferior overall survival in MDS patients. We found that MDS patients with reduced numbers of CD141HicDCs, but not other DC populations, showed reduced overall survival. To examine the basis for reduction in CD141HicDCs, we found fewer numbers of progenitors committed to DC differentiation in the MDS bone marrow and these progenitors expressed lower levels of interferon regulatory factor-8 (IRF8), a master regulator of CD141HicDC differentiation. To rescue impaired CD141HicDC differentiation, we used pharmacologic inhibition of lysine-specific demethylase 1A (LSD1) to promote CD141HicDC differentiation by MDS progenitors. These data reveal a previously unrecognized element of the MDS immunologic milieu. Epigenetic regulation of CD141HicDC differentiation offers an intriguing opportunity for intervention and a potential adjunct to immunotherapy for patients with MDS.

Background:Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hiconventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141HicDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141HicDCs due to impaired myeloid differentiation. Methods:Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results:We found fewer CD141HicDCs (p<0.0001), CD1c+cDCs (p<0.005) and plasmacytoid DCs (CD123+pDCs; p<0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141HicDCs had superior survival (p < 0.05). No differences in survival were seen in patients stratified by the CD1c+cDCs (p = 0.96) and CD123+pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141HicDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p<0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141HicDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p<0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141HicDCs compared to those with higher levels of IRF8 (n=10; p<0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141HicDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8expression in myeloid leukemia cells, would induce CD141HicDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+cells, and in HD and MDS CD34+progenitors (p<0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p<0.05) at a region -70 kb to the IRF8transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+cells increased the number of mature CD141HicDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+cells increased the number of CD141HicDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+cells from Irf8knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+cells resulted in increased numbers of CD141HicDCs in vitro(p<0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+cells did not result in differentiation of CD141HicDCs. These data suggest that LSD1 inhibition drives CD141HicDCs differentiation through IRF8. Conclusion:These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141HicDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients.