A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment.

Understanding how differentiation programs originate from the gene-expression 'landscape' of hematopoietic stem cells (HSCs) is crucial for the development of new clinical therapies. We mapped the transcriptional dynamics underlying the first steps of commitment by tracking transcriptome changes in human HSCs and eight early progenitor populations. We found that transcriptional programs were extensively shared, extended across lineage-potential boundaries and were not strictly lineage affiliated. Elements of stem, lymphoid and myeloid programs were retained in multilymphoid progenitors (MLPs), which reflected a hybrid transcriptional state. By functional single cell analysis, we found that the transcription factors Bcl-11A, Sox4 and TEAD1 (TEF1) governed transcriptional networks in MLPs, which led to B cell specification. Overall, we found that integrated transcriptome approaches can be used to identify previously unknown regulators of multipotency and show additional complexity in lymphoid commitment.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

The evolution of cellular deficiency in GATA2 mutation.

Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56(bright) NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8(+) memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making.

Single-cell analysis of early B-lymphocyte development suggests independent regulation of lineage specification and commitment in vivo.

To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early B-lineage cells from Pax-5-deficient animals crossed to a B-lineage-restricted reporter mouse, allowing us to identify B-lineage-specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promoter-controlled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified B-lineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5-deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5- and Ebf-1-deficient progenitors, it was possible to identify a set of B-cell-restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.

Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

Load and lock: the molecular mechanisms of B-lymphocyte commitment.

The maturation of B-lymphoid cells from hematopoietic stem cells in the bone marrow is a complex process under control of interplay between extrinsic and intrinsic factors. In addition to serving as a model for normal cell differentiation, disturbances in this process results in immunodeficiencies and malignancies, such as leukemia and lymphoma, making the subject of high relevance for modern medicine. Although the process of B lymphopoiesis has been under intense investigation, recent methodological developments within the area of cell sorting, genome-wide expression, and DNA-binding analysis has allowed for a rapid development of the understanding of B-lymphocyte differentiation. This has suggested that the path to B-lymphoid cell fate may be initiated by lineage priming reflected in the expression of lymphoid associated genes already in multipotent hematopoietic progenitors. Upon differentiation, the gene expression profile is changed to involve an increasing number of B-lineage-restricted genes linked to loss of alternative developmental potentials and B-lineage commitment. This review focuses on the molecular regulation of early B-lymphoid development and aims to provide an up to date summary of the current status of the research area.

IL-7 mediates Ebf-1-dependent lineage restriction in early lymphoid progenitors.

Deficiencies in the IL-7 signaling pathway result in severe disruptions of lymphoid development in adult mice. To understand more about how IL-7 deficiency impacts early lymphoid development, we have investigated lineage restriction events within the common lymphoid progenitor (CLP) compartment in IL-7 knockout mice. This revealed that although IL-7 deficiency had a minor impact on the development of LY6D(-) multipotent CLPs, the formation of the lineage restricted LY6D(+) CLP population was dramatically reduced. This was reflected in a low-level transcription of B-lineage genes as well as in a loss of functional B-cell commitment. The few Ly6D(+) CLPs developed in the absence of IL-7 displayed increased lineage plasticity and low expression of Ebf-1. Absence of Ebf-1 could be linked to increased plasticity because even though Ly6D(+) cells develop in Ebf-1-deficient mice, these cells retain both natural killer and dendritic cell potential. This reveals that IL-7 is essential for normal development of Ly6D(+) CLPs and that Ebf-1 is crucial for lineage restriction in early lymphoid progenitors.

Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.

Aging of the hematopoietic stem cell compartment is believed to contribute to the onset of a variety of age-dependent blood cell pathophysiologies. Mechanistic drivers of hematopoietic stem cell (HSC) aging include DNA damage accumulation and induction of tumor suppressor pathways that combine to reduce the regenerative capacity of aged HSCs. Such mechanisms do not however account for the change in lymphoid and myeloid lineage potential characteristic of HSC aging, which is believed to be central to the decline of immune competence and predisposition to myelogenous diseases in the elderly. Here we have prospectively isolated functionally distinct HSC clonal subtypes, based on cell surface phenotype, bearing intrinsically different capacities to differentiate toward lymphoid and myeloid effector cells mediated by quantitative differences in lineage priming. Finally, we present data supporting a model in which clonal expansion of a class of intrinsically myeloid-biased HSCs with robust self-renewal potential is a central component of hematopoietic aging.

In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.

p38 Mitogen-activated protein kinase/signal transducer and activator of transcription-3 pathway signaling regulates expression of inhibitory molecules in T cells activated by HIV-1-exposed dendritic cells.

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1-primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules.

A dose-dependent role for EBF1 in repressing non-B-cell-specific genes.

In the absence of early B-cell factor 1 (EBF1), B-cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 and its DNA-binding partner Runx1 by evaluating B lymphopoiesis in single (EBF1(het) and Runx1(het)) and compound haploinsufficent (Ebf1(+/-) Runx1(+/-), ER(het)) mice. Here, we demonstrate that decreased Ebf1 gene dosage results in the inappropriate expression of NK-cell lineage-specific genes in B-cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ER(het) mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ER(het) mice and in Ebf1(-/-) Pax5(-/-) fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B-cell identity by directing repression of non-B-cell-specific genes.

Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity.

To investigate molecular events involved in the regulation of lymphoid lineage commitment, we crossed lambda5 reporter transgenic mice to Rag1-GFP knockin mice. This allowed us to subfractionate common lymphoid progenitors and pre-pro-B (fraction A) cells into lambda5(-)Rag1(low), lambda5(-)Rag1(high), and lambda5(+)Rag1(high) cells. Clonal in vitro differentiation analysis demonstrated that Rag1(low) cells gave rise to B/T and NK cells. Rag1(high) cells displayed reduced NK-cell potential with preserved capacity to generate B- and T-lineage cells, whereas the lambda5(+) cells were B-lineage restricted. Ebf1 and Pax5 expression was largely confined to the Rag1(high) populations. These cells also expressed a higher level of the surface protein LY6D, providing an additional tool for the analysis of early lymphoid development. These data suggest that the classic common lymphoid progenitor compartment composes a mixture of cells with relatively restricted lineage potentials, thus opening new possibilities to investigate early hematopoiesis.

Interleukin-7-induced Stat-5 acts in synergy with Flt-3 signaling to stimulate expansion of hematopoietic progenitor cells.

The development of lymphoid cells from bone marrow progenitors is dictated by interplay between internal cues such as transcription factors and external signals like the cytokines Flt-3 ligand and Il-7. These proteins are both of large importance for normal lymphoid development; however, it is unclear if they act in direct synergy to expand a transient Il-7R(+)Flt-3(+) population or if the collaboration is created through sequential activities. We report here that Flt-3L and Il-7 synergistically stimulated the expansion of primary Il-7R(+)Flt-3(+) progenitor cells and a hematopoietic progenitor cell line ectopically expressing the receptors. The stimulation resulted in a reduced expression of pro-apoptotic genes and also mediated survival of primary progenitor cells in vitro. However, functional analysis of single cells suggested that the anti-apoptotic effect was additive indicating that the synergy observed mainly depends on stimulation of proliferation. Analysis of downstream signaling events suggested that although Il-7 induced Stat-5 phosphorylation, Flt-3L caused activation of the ERK and AKT signaling pathways. Flt-3L could also drive proliferation in synergy with ectopically expressed constitutively active Stat-5. This synergy could be inhibited with either receptor tyrosine kinase or MAPK inhibitors suggesting that Flt-3L and Il-7 act in synergy by activation of independent signaling pathways to expand early hematopoietic progenitors.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.

Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication.

BACKGROUND: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow. RESULTS: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development), OP-9 cells (strongly supportive of B cell development), pro-B and pre-B cells as well as mature peripheral B-lineage cells, we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further, the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells. CONCLUSIONS: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways.