Hematopoietic Stem Cell Signatures Research Articles

Transcriptomic Profile of Lin−Sca1+c-kit (LSK) cells in db/db mice with long-standing diabetes

BackgroundThe Lin−Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored.MethodsIn this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA.ResultsWe uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype.ConclusionOur findings highlight that chronic diabetes adversely alters HSCs’ homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.

Single-Cell Transcriptomics Points to a Role of Stemness and Bone Marrow Niche Interactions in the Poor Response to Induction Therapy in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis with a 15-20% relapse rate. The induction therapy response is the strongest predictor of relapse and shows great variation between patients. However, whether intra-patient leukemic cell heterogeneity plays a role for the induction therapy response has not yet been investigated. We studied 13 patients with T-ALL (11 pediatric and 2 young adult, median follow-up 37 months). Of these, three were good responders (end of induction (EOI) minimal residual disease (MRD) undetectable/non-quantifiable by flowcytometry and PCR), while ten had a poor response (EOI MRD>0.01%, (range 0.05-97%). Of the ten poor responders, three experienced relapse and one died from resistant disease. Bone marrow samples from diagnosis (n=13) and paired samples from relapse (n=3) or MRD (n=4, MRD >5%) were subjected to combined single-cell RNA sequencing, single-cell surface marker profiling by antibody-derived tag (ADT)-sequencing, and single-cell TCR sequencing, using the 10X Genomics platform. A published healthy bone marrow dataset was used for cell annotation and clear distinction was observed between healthy cell subsets and leukemic blasts. TCR sequencing data revealed a hyperexpanded clone in six patients, with identical clonal sequences at diagnosis and relapse(n=2)/MRD(n=1). Surface marker profiling using a panel of 134 surface antigens showed good correspondence between leukemia-associated immunophenotypes found by ADT-sequencing and by flow cytometry for 16 common clinically used markers. To examine factors associated with a poor therapy response, we compared average gene expression of leukemic cells in poor and good responders at diagnosis using differential gene expression (DGE) and gene set enrichment analysis (GSEA). At diagnosis, poor responders showed upregulation of genes involved in extracellular matrix (ECM) degradation/organization, including the adhesion molecules ITGA5 and PECAM1, and MMP11, while showing downregulation of cell cycle genes. Accordingly, the fraction of cells predicted to be in G0/G1 phase at diagnosis of poor responders positively correlated with EOI MRD (r=0.52, p=0.07, Spearman's rank correlation). To investigate if this high fraction of quiescent cells in poor responders was associated with a more stem-like cell state, we examined the expression of a published hematopoietic stem cell (HSC) signature (Forsberg et al, 2010) that we previously selected for investigation in BCP-ALL (Modvig et al, Molecular Oncology, 2022). Indeed, patients with a poor response had a higher percentage of leukemic blasts with an HSC signature module score above zero (p=0.028, Kolmogorov-Smirnov, distribution in Figure 1), suggestive of stemness as a contributing factor to therapy resistance. To define molecular changes in leukemic blasts during therapy, we compared the leukemic blast gene expression profiles in matched diagnostic and MRD samples using DGE and GSEA. This revealed that the leukemic blasts within the first month of therapy downregulated cell cycle genes, while upregulating chemokine receptors, such as CXCR4. To identify specific genes associated with high MRD, we combined genes upregulated in poor responders (n=314, p<0.01, DGE) with genes associated with EOI MRD levels (n=98, r>0.8 and p<0.001, rank regression). This resulted in a 30-gene signature, comprising several genes previously associated with cancer stem cells, such as MICAL3, ELF4, and NLRP3, and genes encoding proteins involved in cancer drug resistance. These included sialyl transferases, acid ceramidase ( ASAH1) and zyxin ( ZYX). ZYX silencing was previously shown to downregulate the anti-apoptotic protein BCL-2 in leukemia. Accordingly, the percentage of cells with detectable BCL2 expression at diagnosis correlated with EOI MRD (r=0.58, p=0.04), supporting a role of BCL-2 inhibitor therapy in T-ALL. In conclusion, our data suggest that therapy-resistant cells are more stem-like and quiescent at diagnosis, display upregulation of genes involved in bone marrow niche ECM organization and adhesion, and genes related to drug resistance. Likewise, residual leukemic blast following therapy show less proliferation and CXCR4 upregulation. Thus, single-cell transcriptomics provides important molecular insights into therapy-resistant leukemic cells and could comprise a valuable prognostic tool in T-ALL.

Epigenetic Classification of Acute Myeloid Leukemia Revealed By Genome-Wide Chromatin Profiling

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease and is predominantly defined by genetic abnormalities and morphology. However, many lines of evidence suggest the role of epigenetic alterations in the pathogenesis and the classification of AML, whereas no large-scale comprehensive analyses of both genetic and epigenetic alterations in AML have been reported. Methods We enrolled a total of 1,564 primary AML cases (524 from Japanese and 1,040 from Swedish cohorts), whose diagnostic samples were subjected to ATAC sequencing together with targeted capture sequencing using an in-house gene panel including 331 known AML driver genes and an additional 1,158-1,317 probes for copy number detection. We also performed RNA (N=1,398) and H3K27ac-targeted ChIP sequencing (N=80). Normal bone marrow samples from 25 donors as well as 15 remission samples were analyzed by ATAC-seq as a control. Results ATAC-seq revealed approximately 300,000 reproducible peaks in primary AML. Most peaks were found in gene-distal regions, and explained a large variance across samples. Among all peaks, 72% were not detected in control samples and thus, specific to AML. Analysis of the correlations between ATAC-seq accessibility and gene expression revealed a subset of regulatory elements that regulate nearby gene expression; each peak was linked to a median of 1 gene (0-31), while each gene was associated with a median of 11 peaks (0-110). We next estimated the cellular composition of the bulk AML samples by deconvolution analysis using ATAC-seq data. The predominance of hematopoietic stem cell (HSC), monocyte, and erythroid signatures was associated with French-American-British (FAB) subtypes of minimal differentiation/without maturation subtypes (M0/M1), myelomonocytic/monoblastic/monocytic subtypes (M4/M5), and erythroid subtype (M6), respectively, suggesting bulk ATAC-seq of AML samples reflected the differentiation potential of leukemic cells. The ATAC-based clustering identified 11 distinct epigenetic subgroups, including three well-known genetic classes defined by gene fusions, such as PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11. By contrast, some genetic classes defined by a single mutation, such as NPM1 or CEBPA mutation, were further classified into multiple distinct epigenetic subgroups. For example, the NPM1-mutated subtype was separated into four subgroups based on the varying degrees of ATAC signatures associated with HSC and monocytes. Each subgroup of NPM1-mutated AML showed different clinical characteristics such as white blood cell counts, age, and prognosis, and co-mutation pattern in FLT3, DNMT3A, and IDH1/ IDH2 mutations. Gene ontology analysis revealed that the subgroup of NPM1-mutated AML with high HSC signature showed upregulated TGF-β signaling, while one with monocytic signature showed upregulated TNF-α signaling and interferon-γ response. Next, we evaluated transcription factor (TF) binding sites in a genome-wide manner, using TF motifs and foot printing in upregulated ATAC-peaks in each subgroup and revealed distinct profiles of activated TFs in each epigenetic subgroup of NPM1-mutated AML. In the subgroup characterized by high HSC signature, HSC-related TFs that belong to MECOM, HOX, and RUNX families, were more active. By contrast, other TFs, such as IRF, CEBP, and JUN/FOS families, were more enriched in the subgroup showing high monocyte signature. By developing a prediction model for these subgroups of NPM1-mutated AML based on the gene expression profile, we reproduced these subgroups and validated clinical and molecular features in an external Beat AML cohort. Utilizing ex vivo drug sensitivity dataset in the Beat AML, we identified candidate drugs showing subgroup-specific effectiveness such as panobinostat for NPM1-mutated AML with monocytic differentiation. Finally, ChIP-seq analysis identified a number of super-enhancers in the vicinity of leukemia-related TFs and oncogenes. Conclusion Through comprehensive profiling of chromatin accessibility and gene mutations/expression in a large cohort of AML patients, we demonstrate the epigenetic heterogeneity and identify novel AML subgroups that had unique epigenetic and clinical features. Our findings highlight the role of epigenetic alterations in AML pathogenesis and impact on molecular classification of AML.

Identifying cancer subtypes based on embryonic and hematopoietic stem cell signatures in pan-cancer.

Cancer cells with stem cell-like properties may contribute to cancer development and therapy resistance. The advancement of multi-omics technology has sparked interest in exploring cancer stemness from a multi-omics perspective. However, there is a limited number of studies that have attempted to subtype cancer by combining different types of stem cell signatures. In this study, 10,323 cancer specimens from 33 TCGA cancer types were clustered based on the enrichment scores of six stemness gene sets, representing two types of stem cell backgrounds: embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs). We identified four subtypes of pan-cancer, termed StC1, StC2, StC3 and StC4, which displayed distinct molecular and clinical features, including stemness, genome integrity, intratumor heterogeneity, methylation levels, tumor microenvironment, tumor progression, responses to chemotherapy and immunotherapy, and survival prognosis. Importantly, this subtyping method for pan-cancer is reproducible at the protein level. Our findings indicate that the ESC signature is an adverse prognostic factor in cancer, while the HSC signature and ratio of HSC/ESC signatures are positive prognostic factors. The subtyping of cancer based on ESC and HSC signatures may provide insights into cancer biology and clinical implications of cancer.

Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

New-targeted therapy for leukemia based on Endoplasmic Reticulum Stress

The unfolded protein response (UPR) is an evolutionarily conserved adaptive pathway, which is activated by the stress of the endoplasmic reticulum (ER). ER stress often occurs due to the high protein demand in cells and protein folding errors in several diseases, such as different cancers and autoimmune diseases. UPR is mediated by three primary arms called inositol-requiring enzyme-1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6α (ATF6α). Given that homeostasis in protein synthesis is frequently deregulated in cancers, UPR plays a critical role in controlling survival and cell death. In addition,, resistance to apoptosis is mediated by the pro-survival mechanism of ER stress in cancer cells. Recent evidence highlighted the deregulation of UPR signaling in hematopoietic stem cells (HSCs) and leukemic cells, so that targeting UPR-driven pro-survival pathways may present new therapeutic benefits in leukemia. In this review article, we aim to provide an updated knowledge on the role of UPR as a novel therapeutic target in leukemia. We first define the different types of leukemia and their challenges with current treatments, and then explore the contribution of UPR to leukemia pathogenesis and treatment. Finally, UPR targeting strategies in pre-clinical and clinical trials of patients with leukemia will be presented.

CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs.During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker.Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive.In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature.Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. [Display omitted] DisclosuresFang: EdiGene, Inc.: Current Employment.

Unraveling Heterogeneity of Aged Hematopoietic Stem Cells By Single-Cell RNA Sequence Analysis

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging.scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age.Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene.Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Intrapatient Complex Transcriptional Responses to Conventional Chemotherapy in Relapsed Acute Myeloid Leukemia Revealed By Single Cell RNA-Seq Analysis

Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. DisclosuresHonda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

Gene expression profiling of CD34(+) cells from patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (JAK2), Calreticulin (CALR) and MPL (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase TET2, polycomb group protein ASXL1 and histone-lysine N-methyltransferase EZH2 genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in JAK2V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of NANOG and E2F4 transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase SUV39H1, SWI/SNF complex subunit SMARCC2, SMARCE2, chromatin remodeling complex subunit SS18, tubulin β (TUBB) and cyclin dependent kinase CDK1. Among the upregulated epigenetic markers, there was a ~10-fold increase in MYB expression in JAK2V617F negative samples. A significant increase in total CD34 counts in JAK2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in JAK2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver (FL)-Derived Hematopoietic Stem Cells (HSCs) at Single Cell Resolution

Intro: The complex and tightly regulated process of human hematopoietic development culminates in the production of hematopoietic stem cells (HSCs), which subsequently acquire functional competence and undergo expansion in the fetal liver (FL). The establishment of a high-resolution molecular signature of FL HSCs provides insights into HSC biology with potential utility in the purification and expansion of engraftable HSCs ex vivo and the generation of HSCs from pluripotent cell sources. To profile HSCs at this developmental stage, we performed CITE-Seq, a technique that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies to simultaneously map transcriptional and protein-level expression in individual cells. To connect expression profiles with functional engraftment, we have coupled this with transplantation assays in immunocompromised mice. Methods: In these studies, three populations of human FL cells were used: CD34- cells, CD34+ cells and CD34+ cells furtherenrichedby expression of GPI-80, a marker tightly linked to engraftment potential, to explicitly identify HSCs capable of long-term engraftment. These populations were stained with a panel of oligo-tagged antibodies, processed via the 10X Genomics platform, and sequenced (26,407 total cells). Results: Transplantation experiments using the same sorted fractions that were assayed by CITE-seq revealed superior engraftment potential of the GPI-80+ fraction, and thus enrichment for bona fide HSCs at the functional level. This functional signature coincided with enrichment for known HSC markers such as ITGA6 (CD49f), PROCR (EPCR), CD164, MLLT3, HLF, CLEC9A and HMGA2 at the transcriptional level. As such, by profiling &amp;gt;7000 GPI-80+ cells, we have achieved unprecedented resolution of the engraftable HSC compartment within the FL. Combined analysis of all captured FL fractions accurately recapitulated the hematopoietic landscape of the FL at this developmental stage, representing the expected hematopoietic lineages and cell types. To gain further insight into FL HSCs, we next focused on the CD34+ HSC/progenitor compartment where we tracked cluster dynamics upon functional HSC enrichment between the CD34+ bulk and GPI-80+ sample. We noted a prominent (4-fold) increase in a cluster marked by enrichment for genes including RGCC, LMNA, VIM, ID1 and ID3 as well as components of the AHR pathway, suggesting that this expression profile strongly correlates with engraftment potential. Notably, LMNA is expressed in postnatal HSCs and its expression has been shown to decrease upon aging (Grigoryan et al., 2018). In line with this, we also found that LMNA is more highly expressed in our prenatal FL HSPCs compared to postnatal HSPCs. This data suggests a potential role for LMNA in endowing FL HSCs with their superior engraftment potential compared to postnatal HSCs. To complement our transcriptomic characterization of engraftment potential, we also collected cell surface marker expression data based on sequencing of a series of antibody-derived tags (ADTs). This additional layer of information uncovered expression patterns that weren't readily apparent based on mRNA expression data and inspired us to use this ADT information to gate out populations of interest via in silico sorting. This enabled us to compare the transcriptional profiles associated with well-described HSC enrichment signatures to assess whether they represent equivalent cell populations. We compared CD34+CD90+CD49f+ (~Notta et al., 2011) vs CD34+CD133+GPI-80+ (~Sumide et al., 2018) vs CD34+EPCR+ (~Subramaniam et al., 2019) in silico sorted cells and found a strong overlap in enriched genes, suggesting that the transcriptomic signature corresponding to engraftment potential identified in this work is not exclusive to GPI-80+ sorted cells but represents engraftable HSCs beyond this enrichment strategy. Conclusion: We have achieved unprecedented resolution of the engraftable HSC compartment in the human FL. Combining transcriptional profiling of the engraftment potential of FL HSCs with cell surface level expression data provides an in-depth characterization of this unique and developmentally relevant HSC source. This data will be valuable in optimizing the purification and expansion of engraftable HSCs ex vivo as well as in guiding the in vitro generation of HSCs from pluripotent cell sources. Disclosures No relevant conflicts of interest to declare.

AML-079: The PNT Domain of ERG Regulates Hematopoietic Stemness and is Required for the Maintenance of ERG-Induced Leukemia

The ETS transcription factor ERG has a crucial role in hematopoiesis. It is essential for hematopoietic stem cell maintenance and megakaryopoiesis. Increased expression of ERG is associated with poor prognosis in acute myeloid leukemia. Previous studies by us and others established ERG as an oncogene in human leukemia. The PNT domain of ERG is a conserved domain that, in some other ETS factors, is required for protein-protein interactions, but its function for ERG is unknown. To examine whether the PNT domain of ERG is essential for hematopoietic stem and progenitor cells (HSPC) transformation, murine fetal liver-derived-HSPC were transduced with human WT and deleted PNT (DelPNT) domain ERG and cultured in methylcellulose. In re-plating assays, the PNT domain was not essential for HSPC self-renewal yet, DelPNT-ERG-transduced cells demonstrated increased proliferation capacity. Flow cytometry analysis showed decreased expression of hematopoietic stem markers for DelPNT-ERG-transduced cells. Consistent with these findings, RNA sequencing revealed enrichment of genes associated with myeloid differentiation and loss of hematopoietic stem cell signature in HSPC transduced with DelPNT-ERG. Transduction-transplantation assays in C57B6 mice suggested that the PNT domain is not essential for leukemia development. Nevertheless, time to leukemia development was significantly delayed for delPNT-ERG-transduced mice. Gene expression analysis showed enrichment for cell cycle and Myc target genes in leukemia induced by WT-ERG as compared to DelPNT-ERG. The proximity map of ERG-interacting protein suggested that the PNT domain is essential for ERG association with the SWI-SNF complex, which was previously shown to mediate the viability and proliferation of leukemia cells, at least in part by the maintenance of Myc expression. To further validate this finding, we conducted ChIP-sequencing on myeloid progenitor cell line and revealed decreased intensities of histone modifications associated with an active enhancer at the Myc locus in cells transduced with DelPNT-ERG as compared to WT-ERG. These results suggest that in pre-leukemic conditions, the PNT domain has a role in preserving the stem cell phenotype associated with ERG, thereby preventing premature differentiation. Furthermore, ERG interaction with the SWI-SNF complex was significantly dependent on the PNT domain and may be essential for leukemia maintenance.

3142 – GLUCOCORTICOID-INDUCED LEUCINE ZIPPER (GILZ) INTRINSICALLY REGULATES HEMATOPOIETIC STEM CELL FUNCTION

The maintenance of hematopoietic stem cells (HSC) is linked to their quiescent state, while HSC proliferation is associated with differentiation and a loss of long-term stem cell potential. The balance between HSC quiescence and proliferation is tightly regulated by intrinsic and extrinsic cues in the bone marrow. Endogenous glucocorticoid hormones (GC) regulate HSC homing via control of CXCR4 expression. Glucocorticoid-Induced Leucine Zipper (GILZ) is a gene rapidly induced by GC. It mediates many of GC' anti-proliferative and anti-inflammatory effects in several cell types. We found that GILZ is expressed in HSC and addressed its role in hematopoiesis using GILZ knock-out (KO) mice. At steady state, young GILZ-KO mice did not show alteration in HSC number and lineage commitment compared to WT mice. However, competitive transplantation studies revealed a significant decrease in the frequency and number of GILZ-KO compared to WT HSC, suggesting that GILZ-deficient HSC have competitive disadvantage compared to WT cells. RNAseq analysis of gene expression revealed several deregulated cellular pathways implicated in HSC function in GILZ KO compared to WT HSC. Importantly, Gene Set Enrichment Analysis showed a significant depletion of the HSC signature and an enrichment in the mTOR signalling signature in GILZ deficient HSC. Consistently, GILZ deficient HSC showed enhanced proliferation as revealed by flow cytomery analysis of Ki67 expression and DNA content. Mice transplanted with GILZ deficient cells showed significantly earlier mortality compared to WT cells-transplanted mice in 5-FU treatment experiments, demonstrating a cell-intrinsic role of GILZ in HSC function. Overall, these data identify GILZ is a novel regulator of HSCs function.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p &lt; 2.2e-16; HSC-CLP: R= 0.53; p &lt; 2.2e-16; HSC-CMP: R = 0.39, p &lt;2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p &lt; 2.2e-16; HSC-CLP: R= -0.38; p &lt; 2.2e-16; HSC-CMP: R = -0.67, p &lt;2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p &lt; 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

Transcriptional, Protein-Level and Functional Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells at Single Cell Resolution

Intro: In the mouse, hematopoietic stem cells (HSCs) can be isolated and characterized at single cell resolution using a well-defined panel of markers. While it is possible to enrich for human HSCs using a panel of associated markers, similar resolution has not been attained. By profiling HSCs residing in the human fetal liver (FL) using a novel technique called CITE-Seq that combines single cell RNA sequencing (scRNAseq) and cell surface marker interrogation using oligo-tagged antibodies, we aimed to establish an accurate molecular signature of engraftable human HSCs shortly after they arise in development. As HSCs are defined functionally, we have coupled this transcriptomic and protein-level characterization with transplantation assays in immunocompromised NOD scid gamma (NSG) mice to connect expression profiles of cell subsets with functional engraftment. Methods: CITE-Seq was performed on human FL cells (week 19) that showed robust engraftment capability in NSG mice. CD34+ and CD34- cells were magnetically separated and stained with a panel of 19 oligo-tagged antibodies that were deemed relevant to characterize HSCs, including classical HSC markers but also novel targets that were identified in a previous pilot scRNAseq experiment conducted on CD34+ FL cells. From the CD34+ fraction, we sorted live-gated cells (CD34+bulk) as well as a population of cells that was further enriched based on the expression of GPI-80, a marker tightly linked to engraftment potential (CD34+GPI-80+, ~3%). CD34-GlycophorinA(GYPA)- cells were also sorted to assay for the presence of CD34- HSCs. These fractions were then loaded onto the 10x Genomics platform for capture of single cells and subsequent reverse transcription and amplification of both mRNAs and antibody-derived tags (ADTs). Results: Both mRNA and ADT libraries were successfully sequenced, yielding 29-43,000 reads/cell for the mRNA portion and &gt;1,500 reads/cell for the ADT fraction. After quality control and filtering, this effort resulted in 8,775 CD34+bulk cells, 7,279 CD34+GPI-80+ cells, and 6,937 CD34-GYPA- cells available for further analysis. Simultaneous transplantation experiments of the fractions assayed by CITE-seq revealed superior engraftment potential of the CD34+GPI-80+ fraction, confirming enrichment for bona fide HSCs at the functional level. This was also reflected in the scRNAseq data where we found enrichment for known HSC markers such as VNN2 (GPI-80), PROM1 (CD133), PROCR (EPCR), THY1 (CD90), ITGA6 (CD49f), HMGA2, CLEC9A and HLF in the CD34+GPI-80+ fraction compared to CD34+bulk cells. As our pilot studies revealed considerable differences in transcriptional expression (via scRNAseq) as compared to protein-level expression (via cell surface marker expression), integration of the transcriptomic and cell surface marker expression data will further refine the signature of engraftable HSCs. Both layers of information at single cell resolution will allow for the identification of novel markers or unique combinations of markers that are directly correlated with engraftment potential. Conclusion: By isolating the GPI-80+ population within the CD34+ fraction in human FL, we have achieved unprecedented resolution of the signature of engraftable HSCs as confirmed by transplantation experiments. The in-depth characterization of this compartment as well as the surrounding CD34+ and CD34- cells within the FL is expected to yield valuable insights with respect to several biological questions. This data can be directly harnessed in improving the purification and expansion of engraftable HSCs as well as in guiding the in vitro generation of HSCs from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

Innate Immune Signal-Regulated Hematopoiesis

Adult bone marrow (BM) had been long thought to be an immune-privileged organ where little immune reactions occur upon immunological challenges, and thus to form an advantageous environment to preserve long-lived hematopoietic and immune cells, e.g., hematopoietic stem cells (HSCs) that maintain lifelong hematopoiesis. They are mostly kept in quiescence, i.e., very slowly dividing within the steady state BM microenvironment, often referred to as niche, which consists of various type of non-hematopoietic cells such as endothelial cells, mesenchymal stromal cells1. In contrast, recent studies have suggested that a broad range of immunological and inflammatory responses occur in BM and largely influence HSC function2. Upon hematopoietic challenges, e.g., infection, inflammation, cancer, both HSCs and the surrounding niche cells can sense hematopoietic demand signals and integrate it to hematopoiesis via direct (HSC-mediated) and indirect (niche-mediated) sensing mechanisms. As a consequence, primitive HSC and their differentiated progenitors (HSPCs) migrate to inflamed organs, proliferate and differentiate into specific cell lineages that are locally consumed and to be replenished. Infection is one of hemato-immunological challenges that are highly conserved in evolution and relevant to pathogenesis of many diseases, e.g., cancer. Host defense against infection is initiated by rapid but relatively non-specific responses that involve innate immune effector cells, e.g., macrophages, granulocytes, and then is followed by slower but specific responses that involve acquired immunity. Recent studies have shown that not only immune cells but also HSPCs express innate immune sensors, such as Toll-like receptors (TLRs), and the ligation of receptors results in secretion of pro-inflammatory cytokines, cell migration, proliferation and differentiation into myeloid lineage cells (King, Nat Rev Immunol 2016). We have also shown that systemic infection of gram negative bacterial activates quiescent HSCs to proliferation through its cognate receptor, TLR4, and eventually impairs their hematopoietic repopulating ability3. More recently, we have found that intestinal tissue damage activates early hematopoiesis in BM via microbial signals and direct early HSPCs to inflamed lymph node to produce myeloid cells and promote tissue repair. Given the fact that innate immune cells are epigenetically programmed with innate immune memory upon sensitization ("training") infection to resist future infectious insults4, and that HSPCs are long-lived and immune-responsive, it has been demonstrated that upon exposure to pathogen, HSPCs also are able to memorize infection through metabolic and epigenetic changes, and build hemato-immune system with better protection to subsequent pathogen insults5. Taken together, these findings define the BM not as an immune-privileged reservoir, but rather as an organ of active immune reactions where immature HSPCs are capable of adapting the demand signal to hematopoiesis in response to hemato-immunological challenges, and of being trained by innate immune activation to reconstitute host defense with more resistance against future infection. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature. 2014 Jan 16;505(7483):327-34 Takizawa H, Boettcher S, Manz MG. Demand-adapted regulation of early hematopoiesis in infection and inflammation.Blood. 2012 Mar 29;119(13):2991-3002. Takizawa H, Fritsch K, Kovtonyuk LV, et al. Pathogen-Induced TLR4-TRIF Innate Immune Signaling in Hematopoietic Stem Cells Promotes Proliferation but Reduces Competitive Fitness.Cell Stem Cell. 2017 Aug 3;21(2):225-240.e5. Netea MG, Joosten LA, Latz E, et al. Trained immunity: A program of innate immune memory in health and disease.Science. 2016 Apr 22;352(6284):aaf1098. Kopf M, Nielsen PJ. Training myeloid precursors with fungi, bacteria and chips. Nat Immunol. 2018 Apr;19(4):320-322. Disclosures No relevant conflicts of interest to declare.

Bmi1 counteracts hematopoietic stem cell aging by repressing target genes and enforcing the stem cell gene signature

Polycomb-group proteins are critical regulators of stem cells. We previously demonstrated that Bmi1, a component of polycomb repressive complex 1, defines the regenerative capacity of hematopoietic stem cells (HSCs). Here, we attempted to ameliorate the age-related decline in HSC function by modulating Bmi1 expression. The forced expression of Bmi1 did not attenuate myeloid-biased differentiation of aged HSCs. However, single cell transplantation assays revealed that the sustained expression of Bmi1 augmented the multi-lineage repopulating capacity of aged HSCs. Chromatin immunoprecipitation-sequencing of Bmi1 combined with an RNA sequence analysis showed that the majority of Bmi1 direct target genes are developmental regulator genes marked with a bivalent histone domain. The sustained expression of Bmi1 strictly maintained the transcriptional repression of their target genes and enforced expression of HSC signature genes in aged HSCs. Therefore, the manipulation of Bmi1 expression is a potential approach against impairments in HSC function with aging.