Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response.

Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

The impact of clonal diversity and mosaicism on haematopoietic function in Fanconi anaemia.

Recent advances have facilitated studies of the clonal architecture of the aging haematopoietic system, and provided clues to the mechanisms underlying the origins of hematopoietic malignancy. Much less is known about the clonal composition of haematopoiesis and its impact in bone marrow failure (BMF) disorders, including Fanconi anaemia (FA). Understanding clonality in FA is likely to inform both the marked predisposition to cancer and the rapid erosion of regenerative reserve seen with this disease. This may also hold broader lessons for haematopoietic stem cell biology in other diseases with a clonal restriction. In this review, we focus on the conceptual basis and available tools to study clonality, and highlight insights in somatic mosaicism and malignant evolution in FA in the context of haematopoietic failure and gene therapy.

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

Extracellular vesicles impose quiescence on residual hematopoietic stem cells in the leukemic niche.

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.

Concise Review: Adaptation of the Bone Marrow Stroma in Hematopoietic Malignancies: Current Concepts and Models.

The bone marrow stroma maintains hematopoiesis and coordinately regulates regenerative responses through dynamic interactions with hematopoietic stem and progenitor cells. Recent studies indicate that stromal components in the bone marrow of leukemia patients undergo a process of successive adaptation that in turn exerts dramatic effects on the hematopoietic stem cell compartment and promotes leukemic drug resistance. Therefore, functional changes in discrete marrow stromal populations can be considered an aspect of leukemia biogenesis in that they create an aberrant, self-reinforcing microenvironment. In this review, we will describe the current understanding of the remodeling of the hematopoietic stem cell niche following invasion by leukemia cells. We place emphasis on existing evidence of how mesenchymal stem cells and their progeny facilitate neoplastic growth and describe available models and analytical techniques to understand the conversion of the niche toward disease persistence. Stem Cells 2018;36:304-312.

Immunosuppressive therapy for pediatric aplastic anemia: a North American Pediatric Aplastic Anemia Consortium study.

Quality of response to immunosuppressive therapy and long-term outcomes for pediatric severe aplastic anemia remain incompletely characterized. Contemporary evidence to inform treatment of relapsed or refractory severe aplastic anemia for pediatric patients is also limited. The clinical features and outcomes for 314 children treated from 2002 to 2014 with immunosuppressive therapy for acquired severe aplastic anemia were analyzed retrospectively from 25 institutions in the North American Pediatric Aplastic Anemia Consortium. The majority of subjects (n=264) received horse anti-thymocyte globulin (hATG) plus cyclosporine (CyA) with a median 61 months follow up. Following hATG/CyA, 71.2% (95%CI: 65.3,76.6) achieved an objective response. In contrast to adult studies, the quality of response achieved in pediatric patients was high, with 59.8% (95%CI: 53.7,65.8) complete response and 68.2% (95%CI: 62.2,73.8) achieving at least a very good partial response with a platelet count ≥50×109L. At five years post-hATG/CyA, overall survival was 93% (95%CI: 89,96), but event-free survival without subsequent treatment was only 64% (95%CI: 57,69) without a plateau. Twelve of 171 evaluable patients (7%) acquired clonal abnormalities after diagnosis after a median 25.2 months (range: 4.3-71 months) post treatment. Myelodysplastic syndrome or leukemia developed in 6 of 314 (1.9%). For relapsed/refractory disease, treatment with a hematopoietic stem cell transplant had a superior event-free survival compared to second immunosuppressive therapy treatment in a multivariate analysis (HR=0.19, 95%CI: 0.08,0.47; P=0.0003). This study highlights the need for improved therapies to achieve sustained high-quality remission for children with severe aplastic anemia.

Extracellular vesicles in the hematopoietic microenvironment.

Self-renewal and differentiation are defining characteristics of hematopoietic stem and progenitor cells, and their balanced regulation is central to lifelong function of both blood and immune systems. In addition to cell-intrinsic programs, hematopoietic stem and progenitor cell fate decisions are subject to extrinsic cues from within the bone marrow microenvironment and systemically. Yet, many of the paracrine and endocrine mediators that shape hematopoietic function remain to be discovered. Extracellular vesicles serve as evolutionarily conserved, constitutive regulators of cell and tissue homeostasis, with several recent reports supporting a role for extracellular vesicles in the regulation of hematopoiesis. We review the physiological and pathophysiological effects that extracellular vesicles have on bone marrow compartmental function while highlighting progress in understanding vesicle biogenesis, cargo incorporation, differential uptake, and downstream effects of vesicle internalization. This review also touches on the role of extracellular vesicles in hematopoietic stem and progenitor cell fate regulation and recent advances in therapeutic and diagnostic applications of extracellular vesicles in hematologic disorders.

Mesenchymal Stromal Cell-derived Extracellular Vesicles Promote Myeloid-biased Multipotent Hematopoietic Progenitor Expansion via Toll-Like Receptor Engagement.

Mesenchymal stromal cells (MSCs) present in the bone marrow microenvironment secrete cytokines and angiogenic factors that support the maintenance and regenerative expansion of hematopoietic stem and progenitor cells (HSPCs). Here, we tested the hypothesis that extracellular vesicles (EVs) released by MSCs contribute to the paracrine crosstalk that shapes hematopoietic function. We systematically characterized EV release by murine stromal cells and demonstrate that MSC-derived EVs prompt a loss of HSPC quiescence with concomitant expansion of murine myeloid progenitors. Our studies reveal that HSPC expansion by MSC EVs is mediated via the MyD88 adapter protein and is partially blocked by treatment with a TLR4 inhibitor. Imaging of fluorescence protein-tagged MSC EVs corroborated their cellular co-localization with TLR4 and endosomal Rab5 compartments in HSPCs. The dissection of downstream responses to TLR4 activation reveals that the mechanism by which MSC EVs impact HSPCs involves canonical NF-κB signaling and downstream activation of Hif-1α and CCL2 target genes. Our aggregate data identify a previously unknown role for MSC-derived EVs in the regulation of hematopoiesis through innate immune mechanisms and illustrate the expansive cell-cell crosstalk in the bone marrow microenvironment.

Understanding the bone marrow microenvironment in hematologic malignancies: A focus on chemokine, integrin, and extracellular vesicle signaling.

Signaling between leukemia cells and nonhematopoietic cells in the bone marrow microenvironment contributes to leukemia cell growth and survival. This complicated extrinsic mechanism of chemotherapy resistance relies on a number of pathways and factors, some of which have yet to be determined. Research on cell-cell crosstalk the bone marrow microenvironment in acute leukemia was presented at the 2016 annual Therapeutic Advances in Childhood Leukemia (TACL) investigator meeting. This review summarizes the mini-symposium proceedings and focuses on chemokine signaling via the cell surface receptor CXCR4, adhesion molecule signaling via integrin α4, and crosstalk between leukemia cells and the bone marrow microenvironment that is mediated through extracellular vesicles.

Pearson marrow pancreas syndrome in patients suspected to have Diamond-Blackfan anemia.

Pearson marrow pancreas syndrome (PS) is a multisystem disorder caused by mitochondrial DNA (mtDNA) deletions. Diamond-Blackfan anemia (DBA) is a congenital hypoproliferative anemia in which mutations in ribosomal protein genes and GATA1 have been implicated. Both syndromes share several features including early onset of severe anemia, variable nonhematologic manifestations, sporadic genetic occurrence, and occasional spontaneous hematologic improvement. Because of the overlapping features and relative rarity of PS, we hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. Here, we evaluated patient DNA samples submitted for DBA genetic studies and found that 8 (4.6%) of 173 genetically uncharacterized patients contained large mtDNA deletions. Only 2 (25%) of the patients had been diagnosed with PS on clinical grounds subsequent to sample submission. We conclude that PS can be overlooked, and that mtDNA deletion testing should be performed in the diagnostic evaluation of patients with congenital anemia.

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

Fetal origins of hematopoietic failure in a murine model of Fanconi anemia.

Hematopoietic failure is the predominant clinical manifestation of Fanconi anemia (FA), a rare, recessively inherited disorder. Mutations in 1 of 15 genes that coordinately function in a complex pathway to maintain DNA integrity also predispose patients to constitutional defects in growth and development. The hematologic manifestations have been considered to reflect the progressive loss of stem cells from the postnatal bone marrow microenvironment. Ethical concerns preclude the study of human hematopoiesis in utero. We report significant late gestational lethality and profound quantitative and qualitative deficiencies in the murine Fancc(-/-) fetal liver hematopoietic stem and progenitor cell pool. Fancc(-/-) fetal liver hematopoietic stem and progenitor cells revealed a significant loss of quiescence and decline in serial repopulating capacity, but no substantial difference in apoptosis or levels of reactive oxygen species. Our studies suggest that compromised hematopoiesis in Fancc(-/-) animals is developmentally programmed and does not arise de novo in bone marrow.

Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system.

Cell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Dynamic Tracking of Native Polyclonal Hematopoiesis in Adult Mice.

Hematopoietic dysfunction has been associated with a reduction in the number of active precursors. However, precursor quantification at homeostasis and under diseased conditions is constrained by the scarcity of available methods. To address this issue, we optimized a method for quantifying a wide range of hematopoietic precursors. Assuming the random induction of a stable label in precursors following a binomial distribution, the estimation depends on the inverse correlation between precursor numbers and the variance of precursor labeling among independent samples. Experimentally validated to cover the full dynamic range of hematopoietic precursors in mice (1 to 105), we utilized this approach to demonstrate that thousands of precursors, which emerge after modest expansion during fetal-to-adult transition, contribute to native and perturbed hematopoiesis. We further estimated the number of precursors in a mouse model of Fanconi Anemia, showcasing how repopulation deficits can be segregated into autologous (cell proliferation) and non-autologous causes (lack of precursor). Our results support an accessible and reliable approach for precursor quantification, emphasizing the contemporary perspective that native hematopoiesis is highly polyclonal.

Inflammatory recruitment of healthy hematopoietic stem and progenitor cells in the acute myeloid leukemia niche.

Inflammation in the bone marrow (BM) microenvironment is a constitutive component of leukemogenesis in acute myeloid leukemia (AML). Current evidence suggests that both leukemic blasts and stroma secrete proinflammatory factors that actively suppress the function of healthy hematopoietic stem and progenitor cells (HSPCs). HSPCs are also cellular components of the innate immune system, and we reasoned that they may actively propagate the inflammation in the leukemic niche. In two separate congenic models of AML we confirm by evaluation of the BM plasma secretome and HSPC-selective single-cell RNA sequencing (scRNA-Seq) that multipotent progenitors and long-lived stem cells adopt inflammatory gene expression programs, even at low leukemic infiltration of the BM. In particular, we observe interferon gamma (IFN-γ) pathway activation, along with secretion of its chemokine target, CXCL10. We show that AML-derived nanometer-sized extracellular vesicles (EVAML) are sufficient to trigger this inflammatory HSPC response, both in vitro and in vivo. Altogether, our studies indicate that HSPCs are an unrecognized component of the inflammatory adaptation of the BM by leukemic cells. The pro-inflammatory conversion and long-lived presence of HSPCs in the BM along with their regenerative re-expansion during remission may impact clonal selection and disease evolution.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Uncovering the Genetic Etiology of Inherited Bone Marrow Failure Syndromes Using a Custom-Designed Next-Generation Sequencing Panel.

Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for ∼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.