Blood and immune development in human fetal bone marrow and Down syndrome.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs.

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.

Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation.

Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis.

Lmo2 induces hematopoietic stem cell-like features in T-cell progenitor cells prior to leukemia.

LIM domain only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells (HSCs) and early T-cell precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.

Genome-wide association studies of Down syndrome associated congenital heart defects.

Congenital heart defects (CHDs) are the most common structural birth defect and are present in 40-50% of children born with Down syndrome (DS). To characterize the genetic architecture of DS-associated CHD, we sequenced genomes of a multiethnic group of children with DS and a CHD (n=886: atrioventricular septal defects (AVSD), n=438; atrial septal defects (ASD), n=122; ventricular septal defects (VSD), n=170; other types of CHD, n=156) and DS with a structurally normal heart (DS+NH, n=572). We performed four GWAS for common variants (MAF>0.05) comparing DS with CHD, stratified by CHD-subtype, to DS+NH controls. Although no SNP achieved genome-wide significance, multiple loci in each analysis achieved suggestive significance (p<2×10-6). Of these, the 1p35.1 locus (near RBBP4) was specifically associated with ASD risk and the 5q35.2 locus (near MSX2) was associated with any type of CHD. Each of the suggestive loci contained one or more plausible candidate genes expressed in the developing heart. While no SNP replicated (p<2×10-6) in an independent cohort of DS+CHD (DS+CHD: n=229; DS+NH: n=197), most SNPs that were suggestive in our GWASs remained suggestive when meta-analyzed with the GWASs from the replication cohort. These results build on previous work to identify genetic modifiers of DS-associated CHD.

FERM domain mutations induce gain of function in JAK3 in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an incurable disease where most patients succumb within the first year of diagnosis. Both standard chemotherapy regimens and mAbs directed against ATLL tumor markers do not alter this aggressive clinical course. Therapeutic development would be facilitated by the discovery of genes and pathways that drive or initiate ATLL, but so far amenable drug targets have not been forthcoming. Because the IL-2 signaling pathway plays a prominent role in ATLL pathogenesis, mutational analysis of pathway components should yield interesting results. In this study, we focused on JAK3, the nonreceptor tyrosine kinase that signals from the IL-2R, where activating mutations have been found in diverse neoplasms. We screened 36 ATLL patients and 24 ethnically matched controls and found 4 patients with mutations in JAK3. These somatic, missense mutations occurred in the N-terminal FERM (founding members: band 4.1, ezrin, radixin, and moesin) domain and induced gain of function in JAK3. Importantly, we show that these mutant JAK3s are inhibited with a specific kinase inhibitor already in human clinical testing. Our findings underscore the importance of this pathway in ATLL development and offer a therapeutic handle for this incurable cancer.

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies.

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow-stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFß stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics. See related commentary by Derecka and Crispino, p. 263. This article is highlighted in the In This Issue feature, p. 247.

Accelerated epigenetic aging in newborns with Down syndrome.

Accelerated aging is a hallmark of Down syndrome (DS), with adults experiencing early-onset Alzheimer's disease and premature aging of the skin, hair, and immune and endocrine systems. Accelerated epigenetic aging has been found in the blood and brain tissue of adults with DS but when premature aging in DS begins remains unknown. We investigated whether accelerated aging in DS is already detectable in blood at birth. We assessed the association between age acceleration and DS using five epigenetic clocks in 346 newborns with DS and 567 newborns without DS using Illumina MethylationEPIC DNA methylation array data. We compared two epigenetic aging clocks (DNAmSkinBloodClock and pan-tissue DNAmAge) and three epigenetic gestational age clocks (Haftorn, Knight, and Bohlin) between DS and non-DS newborns using linear regression adjusting for observed age, sex, batch, deconvoluted blood cell proportions, and genetic ancestry. Targeted sequencing of GATA1 was performed in a subset of 184 newborns with DS to identify somatic mutations associated with transient abnormal myelopoiesis. DS was significantly associated with increased DNAmSkinBloodClock (effect estimate = 0.2442, p < 0.0001), with an epigenetic age acceleration of 244 days in newborns with DS after adjusting for potential confounding factors (95% confidence interval: 196-292 days). We also found evidence of epigenetic age acceleration associated with somatic GATA1 mutations among newborns with DS (p = 0.015). DS was not associated with epigenetic gestational age acceleration. We demonstrate that accelerated epigenetic aging in the blood of DS patients begins prenatally, with implications for the pathophysiology of immunosenescence and other aging-related traits in DS.

Transitions in lineage specification and gene regulatory networks in hematopoietic stem/progenitor cells over human development.

Human hematopoiesis is a dynamic process that starts in utero 18-21 days post-conception. Understanding the site- and stage-specific variation in hematopoiesis is important if we are to understand the origin of hematological disorders, many of which occur at specific points in the human lifespan. To unravel how the hematopoietic stem/progenitor cell (HSPC) compartment changes during human ontogeny and the underlying gene regulatory mechanisms, we compare 57,489 HSPCs from 5 different tissues spanning 4 developmental stages through the human lifetime. Single-cell transcriptomic analysis identifies significant site- and developmental stage-specific transitions in cellular architecture and gene regulatory networks. Hematopoietic stem cells show progression from cycling to quiescence and increased inflammatory signaling during ontogeny. We demonstrate the utility of this dataset for understanding aberrant hematopoiesis through comparison to two cancers that present at distinct time points in postnatal life-juvenile myelomonocytic leukemia, a childhood cancer, and myelofibrosis, which classically presents in older adults.

A human fetal liver-derived infant MLL-AF4 acute lymphoblastic leukemia model reveals a distinct fetal gene expression program.

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.

H3K79me2/3 controls enhancer-promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

Enforced expression of E47 has differential effects on Lmo2-induced T-cell leukemias.

LIM domain only-2 (LMO2) overexpression in T cells induces leukemia but the molecular mechanism remains to be elucidated. In hematopoietic stem and progenitor cells, Lmo2 is part of a protein complex comprised of class II basic helix loop helix proteins, Tal1and Lyl1. The latter transcription factors heterodimerize with E2A proteins like E47 and Heb to bind E boxes. LMO2 and TAL1 or LYL1 cooperate to induce T-ALL in mouse models, and are concordantly expressed in human T-ALL. Furthermore, LMO2 cooperates with the loss of E2A suggesting that LMO2 functions by creating a deficiency of E2A. In this study, we tested this hypothesis in Lmo2-induced T-ALL cell lines. We transduced these lines with an E47/estrogen receptor fusion construct that could be forced to homodimerize with 4-hydroxytamoxifen. We discovered that forced homodimerization induced growth arrest in 2 of the 4 lines tested. The lines sensitive to E47 homodimerization accumulated in G1 and had reduced S phase entry. We analyzed the transcriptome of a resistant and a sensitive line to discern the E47 targets responsible for the cellular effects. Our results suggest that E47 has diverse effects in T-ALL but that functional deficiency of E47 is not a universal feature of Lmo2-induced T-ALL.

LMO2 induces T-cell leukemia with epigenetic deregulation of CD4.

In this study, we present a remarkable clonal cell line, 32080, derived from a CD2-Lmo2- transgenic T-cell leukemia with differentiation arrest at the transition from the intermediate single positive to double positive stages of T-cell development. We observed that 32080 cells had a striking variegated pattern in CD4 expression. There was cell-to-cell variability, with some cells expressing no CD4 and others expressing high CD4. The two populations were isogenic and yet differed in their rates of apoptosis and sensitivity to glucocorticoid. We sorted the 32080 line for CD4-positive or CD4-negative cells and observed them in culture. After 1 week, both sorted populations showed variegated CD4 expression, like the parental line, showing that the two populations could interconvert. We determined that cell replication was necessary to transit from CD4(+) to CD4(-) and CD4(-) to CD4(+). Lmo2 knockdown decreased CD4 expression, while inhibition of intracellular NOTCH1 or histone deacetylase activity induced CD4 expression. Enforced expression of RUNX1 repressed CD4 expression. We analyzed the CD4 locus by Histone 3 chromatin immunoprecipitation and found silencing marks in the CD4(-) cells and activating marks in the CD4(+) population. The 32080 cell line is a striking model of intermediate single positive to double positive T-cell plasticity and invokes a novel mechanism for LMO2's oncogenic functions.

LIM domain only-2 (LMO2) induces T-cell leukemia by two distinct pathways.

The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.