New insight into the causes, consequences, and correction of hematopoietic stem cell aging.

Aging of hematopoietic stem cells (HSCs) is characterized by lineage bias, increased clonal expansion, and functional decrease. At the molecular level, aged HSCs typically display metabolic dysregulation, upregulation of inflammatory pathways, and downregulation of DNA repair pathways. Cellular aging of HSCs, driven by cell-intrinsic and cell-extrinsic factors, causes a predisposition to anemia, adaptive immune compromise, myelodys, plasia, and malignancy. Most hematologic diseases are strongly associated with age. But what is the biological foundation for decreased fitness with age? And are there therapeutic windows to resolve age-related hematopoietic decline? These questions were the focus of the International Society for Experimental Hematology (ISEH) New Investigator Committee Fall 2022 Webinar. This review touches on the latest insights from two leading laboratories into inflammatory- and niche-driven stem cell aging and includes speculation on strategies to prevent or correct age-related decline in HSC function.

Iron homeostasis governs erythroid phenotype in polycythemia vera.

Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Translational research for bone marrow failure patients.

Bone marrow failure syndromes encompass a range of inherited and acquired hematological diseases that result in insufficient blood cell production, which leads to severe complications including anemia, weakening of the immune system, impaired coagulation, and increased risk of cancer. Within inherited bone marrow failure syndromes, a number of genetically distinct diseases have been described including Shwachman-Diamond syndrome and Fanconi anemia. Given the genetic complexity and poor prognosis of these inherited bone marrow failure syndromes, there is increasing interest in both characterizing the genetic landscapes of these diseases and developing novel gene therapies to effectively monitor and cure patients. These topics were the focus of the winter 2021 International Society for Experimental Hematology New Investigator Webinar, which featured presentations by Dr. Akiko Shimamura and Dr. Paula Río. Here, we review the topics covered within this webinar.

Hematopoietic Stem Cell Heterogeneity Is Linked to the Initiation and Therapeutic Response of Myeloproliferative Neoplasms.

The implications of stem cell heterogeneity for disease pathogenesis and therapy are poorly defined. JAK2V617F+ myeloproliferative neoplasms (MPNs), harboring the same mutation in hematopoietic stem cells (HSCs), display diverse phenotypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These chronic malignant disorders are ideal models to analyze the pathological consequences of stem cell heterogeneity. Single-cell gene expression profiling with parallel mutation detection demonstrated that the megakaryocyte (Mk)-primed HSC subpopulation expanded significantly with enhanced potential in untreated individuals with JAK2V617F+ ET, driven primarily by the JAK2 mutation and elevated interferon signaling. During treatment, mutant HSCs were targeted preferentially in the Mk-primed HSC subpopulation. Interestingly, homozygous mutant HSCs were forced to re-enter quiescence, whereas their heterozygous counterparts underwent apoptosis. This study provides important evidence for the association of stem cell heterogeneity with the pathogenesis and therapeutic response of a malignant disease.

Rational design and characterisation of a linear cell penetrating peptide for non-viral gene delivery.

The design of a non-viral gene delivery system that can release a functional nucleic acid at the intracellular destination site is an exciting but also challenging proposition. The ideal gene delivery vector must be non-toxic, non-immunogenic, overcome extra- and intra-cellular barriers, protect the nucleic acid cargo from degradation with stability over a range of temperatures. A new 15 amino acid linear peptide termed CHAT was designed in this study with the goal of delivering DNA with high efficiency into cells in vitro and tissues in vivo. Rational design involved incorporation of key amino acids including arginine for nucleic acid complexation and cellular uptake, tryptophan to enhance hydrophobic interaction with cell membranes, histidine to facilitate endosomal escape and cysteine for stability and controlled cargo release. Six linear peptides were synthesised with strategic sequences and amino acid substitutions. Data demonstrated that all six peptides complexed pDNA to produce cationic nanoparticles less than 200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for endosomal escape. Peptide 4, now termed CHAT, was non-cytotoxic, traversed the plasma membrane of breast and prostate cancer cell lines, and elicited reporter-gene expression following intra-tumoural and intravenous delivery in vivo. CHAT presents an exciting new peptide for the delivery of nucleic acid therapeutics.

Lineage commitment of hematopoietic stem cells and progenitors: insights from recent single cell and lineage tracing technologies.

Blood production is essential to maintain human health, and even small perturbations in hematopoiesis can cause disease. Hematopoiesis has therefore been the focus of much research for many years. Experiments determining the lineage potentials of hematopoietic stem and progenitor cells (HSPCs) in vitro and after transplantation revealed a hierarchy of progenitor cell states, where differentiating cells undergo lineage commitment-a series of irreversible changes that progressively restrict their potential. New technologies have recently been developed that allow for a more detailed analysis of the molecular states and fates of differentiating HSPCs. Proteomic and lineage-tracing approaches, alongside single-cell transcriptomic analyses, have recently helped to reveal the biological complexity underlying lineage commitment during hematopoiesis. Recent insights from these new technologies were presented by Dr. Marjorie Brand and Dr. Allon Klein in the Summer 2019 ISEH Webinar, and are discussed in this Perspective.

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program.

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro-B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell-programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation.

Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery.

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were <100nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors.

Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential.

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus.

Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice.

Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg(+/Mld2) mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl(-/-) Erg(+/Mld2) compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.

Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome.

Down syndrome is characterized by multiple phenotypic manifestations associated with trisomy of chromosome 21. The transient myeloproliferative disorder and acute megakaryocytic leukemia associated with Down syndrome are uniquely associated with mutations in the transcription factor GATA1; however, the identity of trisomic genes on chromosome 21 that predispose to these hematologic disorders remains unknown. Using a loss-of-function allele, we show that specific reduction to functional disomy of the Erg gene corrects the pathologic and hematologic features of myeloproliferation in the Ts(17(16))65Dn mouse model of Down syndrome, including megakaryocytosis and progenitor cell expansion. Our data provide genetic evidence establishing the need for Erg trisomy for myeloproliferation in Ts(17(16))65Dn mice and imply that increased ERG gene dosage may be a key consequence of trisomy 21 that can predispose to malignant hematologic disorders in Down syndrome.

Murine hematopoietic blast colony-forming cells and their progeny have distinctive membrane marker profiles.

Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a Kit(+) ScaI(+) phenotype distinguishing them from most lineage-restricted progenitor cells. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of Kit(-) Flt3R(+) cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1(+) and Mac1(+) cells but BL-CFC-F colonies also contained a significant population of B220(+) and IL-7R(+) cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system.

Dual requirement for the ETS transcription factors Fli-1 and Erg in hematopoietic stem cells and the megakaryocyte lineage.

Fli-1 and Erg are closely related members of the Ets family of transcription factors. Both genes are translocated in human cancers, including Ewing's sarcoma, leukemia, and in the case of Erg, more than half of all prostate cancers. Although evidence from mice and humans suggests that Fli-1 is required for megakaryopoiesis, and that Erg is required for normal adult hematopoietic stem cell (HSC) regulation, their precise physiological roles remain to be defined. To elucidate the relationship between Fli-1 and Erg in hematopoiesis, we conducted an analysis of mice carrying mutations in both genes. Our results demonstrate that there is a profound genetic interaction between Fli-1 and Erg. Double heterozygotes displayed phenotypes more dramatic than single heterozygotes: severe thrombocytopenia, with a significant deficit in megakaryocyte numbers and evidence of megakaryocyte dysmorphogenesis, and loss of HSCs accompanied by a reduction in the number of committed hematopoietic progenitor cells. These results illustrate an indispensable requirement for both Fli-1 and Erg in normal HSC and megakaryocyte homeostasis, and suggest these transcription factors may coregulate common target genes.

The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells.

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.

Mechanisms of lymphangiogenesis: targets for blocking the metastatic spread of cancer.

The lymphatic vasculature is an important route of metastatic spread in cancer and recent studies have demonstrated that lymphangiogenesis (the growth of lymphatic vessels) associated with tumors promotes metastasis via the lymphatics. Therefore, the molecular mechanisms that drive lymphangiogenesis are attractive targets for development of novel therapeutics designed to restrict cancer metastasis. Such therapeutics would be of high priority as metastasis is the most lethal aspect of tumor biology. Research over the past seven years has identified protein growth factors and cell surface receptors that signal for lymphangiogenesis during embryonic development, in adult tissues and in cancer. Proteases that process and thereby activate lymphangiogenic growth factors have also been defined. Lymphangiogenic growth factors, the enzymes that activate them and the cell surface receptors signalling for growth of lymphatic vessels are prime targets for anti-lymphangiogenic drugs designed to restrict cancer metastasis. Agents targeting some of these proteins have already shown promise for blocking tumor lymphangiogenesis and lymphatic metastasis in animal models. This article focuses on current and emerging targets for blocking these processes that have been defined in recent studies of the molecular mechanisms controlling lymphangiogenesis. Strategies to block the actions of these proteins in cancer are also explored.