PLAG1 dampens protein synthesis to promote human hematopoietic stem cell self-renewal.

Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.

Musashi-2 attenuates AHR signalling to expand human haematopoietic stem cells.

Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.

OTUD7A Regulates Neurodevelopmental Phenotypes in the 15q13.3 Microdeletion Syndrome.

Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.

Aberrant Clonal Hematopoiesis following Lentiviral Vector Transduction of HSPCs in a Rhesus Macaque.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control 1.

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

Producing megakaryocytes from a human peripheral blood source.

BACKGROUND: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.

RNAi screen identifies Jarid1b as a major regulator of mouse HSC activity.

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.

RNA binding protein-directed control of leukemic stem cell evolution and function.

Strict control over hematopoietic stem cell decision making is essential for healthy life-long blood production and underpins the origins of hematopoietic diseases. Acute myeloid leukemia (AML) in particular is a devastating hematopoietic malignancy that arises from the clonal evolution of disease-initiating primitive cells which acquire compounding genetic changes over time and culminate in the generation of leukemic stem cells (LSCs). Understanding the molecular underpinnings of these driver cells throughout their development will be instrumental in the interception of leukemia, the enabling of effective treatment of pre-leukemic conditions, as well as the development of strategies to target frank AML disease. To this point, a number of precancerous myeloid disorders and age-related alterations are proving as instructive models to gain insights into the initiation of LSCs. Here, we explore this myeloid dysregulation at the level of post-transcriptional control, where RNA-binding proteins (RBPs) function as core effectors. Through regulating the interplay of a myriad of RNA metabolic processes, RBPs orchestrate transcript fates to govern gene expression in health and disease. We describe the expanding appreciation of the role of RBPs and their post-transcriptional networks in sustaining healthy hematopoiesis and their dysregulation in the pathogenesis of clonal myeloid disorders and AML, with a particular emphasis on findings described in human stem cells. Lastly, we discuss key breakthroughs that highlight RBPs and post-transcriptional control as actionable targets for precision therapy of AML.

Spermidine metabolism regulates leukemia stem and progenitor cell function through KAT7 expression in patient-derived mouse models.

Acute myeloid leukemia (AML) is a devastating disease initiated and maintained by a rare subset of cells called leukemia stem cells (LSCs). LSCs are responsible for driving disease relapse, making the development of new therapeutic strategies to target LSCs urgently needed. The use of mass spectrometry-based metabolomics profiling has enabled the discovery of unique and targetable metabolic properties in LSCs. However, we do not have a comprehensive understanding of metabolite differences between LSCs and their normal counterparts, hematopoietic stem and progenitor cells (HSPCs). In this study, we used an unbiased mass spectrometry-based metabolomics analysis to define differences in metabolites between primary human LSCs and HSPCs, which revealed that LSCs have a distinct metabolome. Spermidine was the most enriched metabolite in LSCs compared with HSPCs. Pharmacological reduction of spermidine concentrations decreased LSC function but spared normal HSPCs. Polyamine depletion also decreased leukemic burden in patient-derived xenografts. Mechanistically, spermidine depletion induced LSC myeloid differentiation by decreasing eIF5A-dependent protein synthesis, resulting in reduced expression of a select subset of proteins. KAT7, a histone acetyltransferase, was one of the top candidates identified to be down-regulated by spermidine depletion. Overexpression of KAT7 partially rescued polyamine depletion-induced decreased colony-forming ability, demonstrating that loss of KAT7 is an essential part of the mechanism by which spermidine depletion targets AML clonogenic potential. Together, we identified and mechanistically dissected a metabolic vulnerability of LSCs that has the potential to be rapidly translated into clinical trials to improve outcomes for patients with AML.