Overexpression of Histone Demethylase KDM6B in the Hematopoietic Compartment Promotes the Effect of Innate Immune Activation and Results in Myelodysplastic Syndrome (MDS)-like Phenotype in Mice

Abstract

The histone demethylase KDM6B regulates transcriptional activity of genes involved in innate immune signals and controls differentiation of hematopoietic cells. Our laboratory previously reported that KDM6B is overexpressed in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) of over 50% MDS pts (Leukemia 2013). Pharmacologic targeting of KDM6B has shown pre-clinical activity in several neoplasms such as glioma and ALL. It still remains to be elucidated whether KDM6B overexpression directly alters hematopoiesis, in order to test the hypothesis that overexpression of KDM6B contributes to aberrant epigenetic and innate immune signal in MDS and thus forms an important pathological mechanism and potential therapeutic target.Here we report an animal model of KDM6B overexpression in mouse hematopoietic compartment (Vav-KDM6B). In 20-wk old mice, KDM6B overexpression led to mild phenotype including reduced white cell count (WBC) in peripheral blood (PB) (15% p<0.05 N=47) and decreased LSK in BM (20% p<0.05 N=12). When exposed to chronic low dose Toll-like receptor ligand lipopolysaccharide (LPS) (6mg every other day, i.p. 6 wks), phenotype severity was more profound in Vav-KDM6B mice than in wildtype (WT) mice. First, hematopoietic cell numbers in both PB and BM were significantly reduced: LPS led to a 40% decrease of PB WBC (p<0.01 N=17) and a 38% decrease of BM cell (p<0.01 N=13 Fig 1A) in KDM6B mice, compared to 20% and 28% reduction respectively in WT mice treated by LPS. Second, both number and repopulating function of BM long term (LT) HSC were severely compromised: LPS led to decrease of BM LT-HSC by 52% (p<0.01 N=13) in KDM6B mice compared to a 35% decrease in WT mice. Functionally, colonies formed in methylcellulose by LSKs of LPS treated KDM6B mice decreased by 75% (p<0.01 N=13) at 2nd plating, whereas LPS led to a reduction of 60% for WT-LSK colony formation at 2nd plating. Similar effect was also observed when BM repopulating capacity was evaluated by chimerism (CD45.2:CD45.1) in competitive transplantation assay (p<0.05 N=11). Third, increase of megakaryocytic dysplasia was documented in BM of LPS treated KDM6B mice compared to WT counterparts (Fig 1B).To identify KDM6B overexpression mediated molecular signals under innate immune stimuli, we performed genome-wide transcriptome study using BM LSK of treated mice. Results reveal that over 130 genes were more profoundly activated by LPS in LSK of Vav-KDM6B than in WT LSK. Gene set enrichment assays (GSEA) indicated that major molecular pathways related to these genes include MDS-relevant signals, such as inflammatory and complement innate immune signals, MAPK pathway, as well as activated gene signature of RPS14 knockout in human BM CD34+ cell. We also performed RNA-Seq analysis in BM CD34+ HSPCs of MDS (N=26) and healthy controls (N=10). Results confirmed that a significant proportion of these genes (N>20) are overexpressed in MDS patients. Furthermore, we demonstrated in mouse BM cells that KDM6B overexpression mediates histone H3K4 and H3K27 methylation at promoters of several key innate immune genes towards transcription activation.We next evaluated effect of pharmacological KDM6B targeting using a highly specific KDM6B inhibitor (GSK-J4). At relatively low-dose (0.5mM), GSK-J4 could increase in vitro colony formation of LSKs of LPS treated KDM6B mice by 80% (p<0.05) at 2nd plating. Injection of GSK-J4 (160mg once a week, 5 weeks) resulted in 50% increase of WBM cells in LPS treated Vav-KDM6B mice (p<0.05). These results suggest that GSK-J4 can improve compromised hematopoiesis of Vav-KDM6B mice. We next treated primary BM HSPCs isolated from patients with low risk MDS (N=11) with GSK-J4. Results indicated that at 0.5mM, GSK-J4 could improve colony formation of MDS CD34+ cells (p=0.05). Flow cytometry analysis using cells from colonies indicated that GSK-J4 led to increased myeloid mark CD11b (p=0.08) and reduced apoptotic mark annexin-V (p<0.05). The same concentration of GSK-J4 did not affect colony formation of normal BM CD34+ HSPCs isolated from healthy donors (N=4).Taken together, our results indicate that overexpression of KDM6B mediates the hyperactivation of innate immune signals and has a functional impact in BM HSPCs, supporting role for KDM6B overexpression in MDS pathogenesis. Furthermore, our study also indicates that pharmacological inhibition of KDM6B may have therapeutic potential in MDS. [Display omitted] DisclosuresKhoury:Kiromics: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Pfizer: Research Funding.