Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in ...early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid–restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid–restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.
•CD10-ve PreProB-progenitors are the earliest fetal B-lymphoid–restricted progenitors and are enriched in fetal BM.•Fetal PreProB-progenitors have a unique ontogeny-related developmental gene expression program distinct from their rare adult counterparts.
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Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: ...an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.
Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 ...of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.
Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of ...enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.
Advances in treatment of childhood leukaemia has led to vastly improved survival rates, however some subtypes such as those characterised by MLL gene rearrangement (MLL-r), especially in infants, ...continue to have high relapse rates and poor survival. Natural history and molecular studies indicate that infant acute lymphoblastic leukaemia (ALL) originates in utero, is distinct from childhood ALL, and most cases are caused by MLL-r resulting in an oncogenic MLL fusion protein. Unlike childhood ALL, only a very small number of additional mutations are present in infant ALL, indicating that MLL-r alone may be sufficient to give rise to this rapid onset, aggressive leukaemia in an appropriate fetal cell context. Despite modifications in treatment approaches, the outcome of MLL-r infant ALL has remained dismal and a clear understanding of the underlying biology of the disease is required in order to develop appropriate disease models and more effective therapeutic strategies.
•MLL rearranged infant ALL arises in utero and has a dismal prognosis.•The developmental stage/site-specific context of leukaemia initiation is important.•Human fetal cell-specific models of infant ALL are needed to study the disease.•This will allow the development of much needed newer targeted therapies.
Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in ...the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.
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•Yolk-sac-derived macrophages are the most abundant hematopoietic cells in the AGM•Cx3cr1 mediates yolk-sac macrophage progenitor recruitment to the AGM niche•AGM macrophages dynamically interact with emerging intra-aortic hematopoietic cells•Pro-inflammatory AGM macrophages are positive regulators of HSC generation
HSC-independent macrophages derive from the early yolk-sac stages of embryonic hematogenesis. Mariani and colleagues demonstrate that specific pro-inflammatory embryonic HSC-independent macrophages recruited to the AGM (AGM-aMs) are crucial components of the AGM microenvironment, dynamically interact with emerging hematopoietic cells, and enhance HSC generation.
Long term survival rates for childhood cancers is steadily increasing, however cancer survivors can experience fertility problems as a consequence of chemotherapy treatment. This is particularly ...problematic for young boys, for whom no fertility preservation treatment is yet established. Here, we have determined the effects on prepubertal mouse testis of three commonly used chemotherapy drugs; cyclophosphamide (using its active metabolite phosphoramide mustard), cisplatin and doxorubicin, exposing testicular fragments to a clinically relevant range of concentrations in vitro. All three drugs induced a specific and highly significant loss of germ cells, including spermatogonial stem cells. In contrast, there was no significant effect on somatic cells, for either Sertoli or interstitial cells. Time course analysis of cleaved Caspase-3 expression showed a significant increase in apoptosis eight hours prior to a detectable decrease in germ cell numbers following exposure to phosphoramide mustard or cisplatin, although this pattern was not seen following doxorubicin-exposure. Moreover, analysis of DNA damage at 16 h showed increased γH2AX expression in response to all three drugs. Overall, results show that cisplatin, doxorubicin and cyclophosphamide all specifically induce loss of germ cells, including of spermatogonial stem cells, in the prepubertal mouse testis at concentrations relevant to human therapeutic exposures.
Although advances in the treatment of childhood acute lymphoblastic leukaemia (ALL) mean that about 90% of children with ALL are now leukaemia-free 5 years on from their diagnosis, the prognosis for ...the subset of ALL that occurs within the first year of life, referred to as infant ALL, remains dismal. The most common genetic driver of infant ALL is a chromosomal translocation that leads to rearrangement of the Mixed Lineage Leukaemia (MLL) gene. In infant ALL, the MLL-rearrangement invariably arises in utero, and this single mutational hit in fetal life is sufficient to give rise to a leukaemia that is uniquely aggressive and treatment-refractory, even when compared to leukaemia in older children with the same MLL-rearrangement. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Moreover, the contribution of the particular human fetal haematopoietic progenitor in which the translocation occurs remains unexplored. In this DPhil project, I aimed to address the hypothesis that fetal-specific gene expression programmes cooperate with the most common MLL-rearrangement in infants, MLL-AF4, to initiate and maintain infant ALL. I further aimed to identify the human fetal haematopoietic progenitor(s) that provide the permissive cellular context for leukaemia initiation. I found that CRISPR/Cas9 editing of primary human fetal liver (FL) cells to produce an MLL-AF4/t(4;11) chromosomal translocation gave rise to a leukaemia that replicated the aggressive phenotypic features of infant ALL, designated CRISPRMLL-AF4+ ALL. Using direct comparison of the transcriptomes of human fetal and adult haematopoietic progenitor cells, I identified a fetal-specific gene signature and further showed that CRISPRMLL-AF4+ ALL and MLL-AF4 infant ALL patients, but not MLL-AF4 childhood ALL patients, maintain the expression of these fetal genes. Finally, I found that, in-keeping with the B-ALL phenotype of MLL-AF4 infant ALL, lymphoid primed progenitors provided the most permissive cellular context to give rise to CRISPRMLL-AF4+ ALL.
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