Modeling human yolk sac hematopoiesis with pluripotent stem cells

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac–derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

ALKBH5 Modulates Hematopoietic Stem and Progenitor Cell Energy Metabolism through m 6a Modification-Mediated RNA Stability

During hematopoietic differentiation from hematopoietic stem cells (HSCs) to mature blood cells, cells undergo a metabolic shift from glycolysis to mitochondrial respiration. The mechanisms by which hematopoietic cells adjust their energy metabolism are still under investigation. N6-mehyladenosine (m 6A) mRNA modification has been reported to regulate numerous fundamental cellular processes through control of RNA stability or translational efficiency. The fat mass and obesity-associated protein (FTO), an m 6A m and m 6A mRNA demethylase, has been reported to affect cellular metabolism in acute myeloid leukemia (AML). ALKBH5, the specific RNA m 6A demethylase, controls oncogene expression in AML. ALKBH5 becomes highly expressed in hematopoietic progenitors during hematopoietic development but the physiological role of RNA m 6A demethylase during hematopoiesis remains unknown. To investigate the function of the RNA m 6A demethylase ALKBH5 in hematopoiesis, we generated Vav-iCre +; Alkbh5fl/fl (vcAlkbh5-/-) mice, resulting in deletion of Alkbh5 specifically in the hematopoietic system. vcAlkbh5-/-mice showed no hematopoietic defects at steady states up to 12 months of age. We applied TimeLapse-seq on lineage-depleted bone marrow cells of WT and vcAlkbh5-/- mice to determine whether loss of ALKBH5 perturbed mRNA stability and/or RNA turnover. Ogdh mRNA was the most destabilized transcript resulting in significantly reduced OGDH protein levels. OGDH is the rate-limiting enzyme in the tricarboxylic acid (TCA) cycle. Inhibition of OGDH subsequently induces production of L-2-hydroxyglutarate (L-2-HG), whose metabolism is closely coupled to energy metabolism through inhibition of oxygen consumption. L-2-HG, the enantiomer of D-2-HG, inhibits the function of a-ketoglutarate (a-KG)-dependent enzymes, including TET and KDM enzymes. We measured L- and D-2-HG in the plasma of WT and vcAlkbh5-/- mice by chiral derivatization to distinguish the two enantiomers. Although D-2-HG levels were similar in the plasma of WT and vcAlkbh5-/- mice, L-2-HG levels were significantly increased in the plasma of vcAlkbh5-/- mice. We therefore determined the function of Jumonji C-domain lysine demethylases (JmjC-KDMs) by measuring histone methylation: H3K9me3, H3K27me3 and H3K36me3 modifications were all significantly increased in Alkbh5-deficient hematopoietic cells. We next sought to understand whether reduction of OGDH expression and resulting increased L-2-HG levels production could impair energy metabolism via perturbation of the TCA cycle and oxidative phosphorylation (OXPHOS) in the mitochondria. We isolated lineage negative hematopoietic stem and progenitor cells (HSPCs) from WT and vcAlkbh5-/- mice and subjected these to the Seahorse ATP Rate Assay. Comparing oxygen consumption rate (OCR) data and the kinetics of the Extracellular Acidification Rate (ECAR) of both groups, we found that less ATP was produced by mitochondria of the vcAlkbh5-/- cells, while ATP produced by glycolysis showed no difference between the two groups. In the meantime, the ultrastructure of mitochondria in the Alkbh5-deficient cells remains normal. We next determined whether the attenuated energy metabolism of Alkbh5-deficient HSPCs was functionally relevant by testing HSPC function in competitive transplantation assays. Interestingly, vcAlkbh5-/- cells showed a significant competitive defect at all differentiation stages except in phenotypic long-term HSCs (LT-HSCs). This suggests that LT-HSCs, thought to preferentially rely on glycolysis as opposed to OXPHOS for their energy source, are protected from loss of ALKBH5 and OGDH. In conclusion, our study demonstrates that ALKBH5 modulates energy metabolism by regulating mRNA stability of metabolic enzymes through its m 6A demethylation activity during hematopoiesis. This finding links Alkbh5 expression kinetics to the metabolic shift from glycolysis to mitochondrial OXPHOS during hematopoietic development. Disclosures No relevant conflicts of interest to declare.

USB1 Is a miRNA Deadenylase That Regulates Hematopoietic Development

Poikiloderma with neutropenia (PN)is an autosomal-recessive bone marrow failure (BMF) syndrome in which patients harbor homozygous or compound heterozygous mutations in the human gene C16orf57, which encodes the evolutionarily conserved RNA 3' to 5' exonuclease U6 biogenesis 1 (USB1). USB1 is required for the proper maturation of U6 and U6atac snRNAs, core components of the spliceosome, and consequently, splicing defects have been observed in yeast and zebrafish models with USB1 deficiency. However, lymphoblastoid cells from PN patients do not exhibit reduced U6 snRNA levels and have normal pre-mRNA splicing, establishing PN as a singular BMF syndrome, where the underlying genetic cause has been identified but the molecular mechanisms leading to tissue failure remain obscure. To investigate the role of USB1 in a physiological context, we utilized CRISPR/Cas9 to create human embryonic stem cells (hESCs) containing a frequently occurring c.531_del_A loss-of-function mutation in the USB1 gene (USB1_c.531_del_A hESCs). USB1_c.531_del_A hESCs have normal karyotype, normal growth rate, and retain pluripotency, indicating that clinically-relevant mutations in USB1 are not deleterious in undifferentiated hESCs. To elucidate the role of USB1 during hematopoiesis, we performed serum-free hematopoietic differentiations to derive hematopoietic progenitor cells from WT and USB1_c.531_del_A hESCs. The formation of definitive hematopoietic progenitors (CD45+) was decreased in USB1 mutant cells compared to WT cells, and definitive colony potential analysis showed compromised colony formation in USB1 mutants. These observations indicate that loss-of-function mutations in USB1 negatively influence hematopoiesis. Additionally, as PN is associated with severe non-cyclic neutropenia, we analyzed the potential of neutrophil formation in WT and USB1 mutant cells. USB1 mutants have reduced formation of CD15+/CD66b+ lineages, indicating abnormal neutrophil development. Conditional expression of WT USB1 in USB1_c.531_del_A mutant cells rescued these phenotypes, leading to normal hematopoietic development. Interestingly, USB1 mutants showed no reduction in the overall levels of U6 and U6atac snRNAs, similar to what was observed in patient cells. To identify other possible targets of USB1, we sequenced the transcriptome and miRome of WT and USB1 mutant cells in different stages of hematopoietic development. Through these analyses, we demonstrate that hematopoietic failure in USB1 mutants is caused by dysregulated miRNA levels during blood development, due to a failure to remove destabilizing 3' end oligo(A) tails added by PAPD5/7. Moreover, we demonstrate that modulation of oligoadenylation through genetic or chemical inhibition of PAPD5/7 rescues the defective hematopoiesis observed in USB1 mutants. This work indicates USB1 acts as a miRNA deadenylase and suggests PAPD5/7 inhibition as a potential therapy for PN. Disclosures Parker: Faze Therapeutics: Other: Co-founder.

Reference Mapping Pediatric Leukemia Using Single Cell Multiomic Atlas of Pediatric Hematopoiesis

Acute lymphoblastic leukemia is the most common pediatric cancer and leading cause of cancer related mortality in pediatric populations. A key challenge to bridge better therapies to patients that fail conventional therapy are to understand their tumor landscape and aberrations in cell signaling, particularly in relation to normal hematopoietic development. To address this gap, we produced a unified reference map of pediatric T, B, and myeloid cell development from the HSPC using single cell RNA-seq and single-cell ATAC-seq on healthy pediatric bone marrow and thymus. We employed 6 different FACS sorting strategies in order to capture rare, but informative, progenitor cell states, including those of the CCR9+ CD34+ CD1A- CD4- CD8- early-T-cell precursor, CD34+ CD1A- CD4- CD8- pro-T cell, and CD34+ CD1A+ CD4- CD8- pre-T cell and Lin-CD34+CD38- multipotent, lymphoid, and myeloid progenitors from the bone marrow. We mapped leukemic cells from patients from 4 different subtypes of pediatric leukemia (T-ALL, ETP-ALL, MPAL, AML) to our healthy reference and found that our reference map can distinguish between subtle differences in transcriptome and epigenome that were undetectable using surface marker or canonical gene expression. Notably, using trajectories inferred from our healthy reference map, we discovered a large amount of inter-tumoral and intra-tumoral heterogeneity, with leukemic blasts from different patients and different populations within any one patient projecting to different cell states along normal development. Finally, we mapped engrafted leukemic cells from patient derived xenografts (PDX) back to our healthy reference. While we observed patient-specific transcriptomic shifts in engrafted versus primary leukemic blasts, we found that the overall transcriptomic hierarchy is maintained in the most PDX, with engrafted cells projecting to near-identical stages of arrest along our healthy hematopoietic trajectory. Interestingly, for PDX that projected to different areas in development compared to primary sample, we discovered alterations in expression of key transcription factors that regulate hematopoietic development. Our single cell multi-omic reference map of pediatric hematopoiesis serves as a valuable reference for mapping RNA-seq and ATAC-seq data back to nearest healthy precursors in normal hematopoietic development. On-going analysis is utilizing single cell transcriptomic, chromatin accessibility data from additional leukemic patients, including patients with B-ALL, to determined key genes and regulators that are altered in comparison to nearest healthy cell-types. In addition, population level signatures learned from healthy reference are being tested in bulk-transcriptomic ALL datasets. We are eager to present the results of these analyses at ASH. *CC and JX, as well as, DTT and KT contributed equally to this work Figure 1 Figure 1. Disclosures Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Lymphoblasts from Minimal Residual Disease in B-ALL Have a Transcriptional and Chromatin Accessibility Profile Shifted Towards Earlier Hematopoietic Progenitor Biology

Background: Prognosis for children and adults with B-cell acute lymphoblastic leukemia (B-ALL), the most common pediatric malignancy, is heavily dependent on minimal residual disease (MRD) levels at the end of induction. Rates of detectable MRD are dependent on patient age and genomic subgroup. For patients with detectable MRD, the primary therapeutic adjustment is intensification of therapy if possible. Despite the incredible advances in MRD detection and clear associations with prognosis, uncovering the underlying biology of MRD could provide critical insight into mechanisms of therapy resistance and suggest research avenues for precise therapeutic approaches. Methods: We collected samples from paired diagnostic and MRD time points from four patients with B-ALL, with MRD ranging from 0.15-0.84%. Based upon clinical MRD immunophenotype, we performed FACS sorting of lymphoblasts from each sample in order to isolate the rare MRD cells, and performed single-cell RNA sequencing (scRNA-seq) on all four pairs and single-cell ATAC sequencing (scATAC-seq) on two of these pairs. Data were compared across time points and integrated with publicly available datasets studying normal hematopoietic development. Raw scRNA-seq and scATAC-seq data were preprocessed using the Cell Ranger pipeline (10x Genomics, v.3.1.0). Seurat V4 (Hao et al., Cell 2021), Signac (Stuart et al., bioRxiv 2020), and MetaCell (Baran et al., Genome Biology 2019) were used to perform quality control (QC), normalize, identify highly variable features, perform dimension reduction, identify both discretized cell clusters and transient meta cells, and ultimately, visualize using uniform manifold approximation and projection (UMAP). Results: Post scRNA-seq QC, the median number of cells per datasets was 2772, the median number of genes per dataset was 13855 and the median number of UMIs per cell was 6518. Post scATAC-seq QC, the median number of cells per datasets was 1157, the median number of peaks per dataset was 76190 and the median number of peaks per cell was 26642. We compared the diagnosis to MRD time points within each of the four cases. In each case, the transcriptionalprofile of the lymphoblasts changed from diagnosis to MRD timepoints (Fig. 1a), with specific transcriptional clusters enriched in the sorted MRD samples (Fig. 1b). To understand this difference, we compared the time points to publicly available datasets of normal hematopoietic development. First, we compared our data to published bulk RNA-seq data from sorted, well-defined hematopoietic development states (Corces et al., Nature Genetics 2016). We performed principal component analysis (PCA) on this data, and projected our generated scRNA-seq data onto the same principal component (PC) space. While the first PC captures the systematic artifact between the bulk and single-cell data, the second PC captures the developmental pseudo time. In each case, cells from sorted MRD show transcriptional profiles shifterd towards earlier progenitor cells on development pseudo time (Fig. 1c). We performed a similar analysis using scATAC-seq from a paired diagnosis and MRD sample, showing that the chromatin accessibility profile of MRD lymphoblasts reveals the same directional change towards patterns seen in earlier hematopoietic progenitor cells. We repeated a similar data integration on a published scRNA-seq dataset from well annotated normal hematopoietic developmental states (Granja et al. Nature Biotechnology 2019), which again revealed that the transcriptional patterns of MRD lymphoblasts align more closely with common lymphoid progenitors than pre-B cells. (Fig. 1d). Conclusions: Using FACS sorting to isolated rare populations of MRD and subsequent single-cell analyses, we show that therapeutic pressure results in residual disease with an earlier progenitor transcriptional profile, likely determined by altered chromatin accessibility profile. Moving forward, we will explore the drivers of these changing transcriptomic and epigenomic profiles through expanding our number of cases to confirm initial findings, optimizing ATAC sequencing approaches to study the rare cell populations present in low-level MRD, and exploring specific transcription factor motifs driving these differences. Our findings have the potential to inform research into rational therapeutic approaches directed toward the specific biology of low level MRD in B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

ABSTRACTRecent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1 null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafish runx1-/- lines we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-GFP+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1- /- embryos. Interestingly, genetic inactivation of both gata2b and its paralog, gata2a, did not affect hematopoiesis. However, knocking out runx1 and any three of the four alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.

A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development.

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.