Hematopoietic Stem And Progenitor Cells Compartment Research Articles

Branching Trajectories and Diversification of Clonal Escape in Aplastic Anemia Revealed By Single-Cell Genomics

Acquired aplastic anemia (AA) is a bone marrow failure resulting from an autoimmune T-cell attack against hematopoietic stem and progenitor cells (HSPCs). Several escape mechanisms within the HSPC compartment have been reported: i) adaptive alterations causing loss of high-risk HLA allele of AA development [deletion or uniparental disomy (UPD) of chromosome 6p including HLA cluster region, and loss-of-function (LOF) mutations in HLA gene], ii) clonal expansion of paroxysmal nocturnal hemoglobinuria (PNH) clone resulting from PIGA mutation, and iii) presence of clonal hematopoiesis of indeterminate potential (CHIP) mutations (i.e., BCOR/ BCORL1, DNMT3A, ASXL1). Multiple types of alterations often co-occur, however due to the inherent limitations of conventional methods, it is not known if i) these alterations are in the same or independent clones, ii) what is the mutational hierarchy, and iii) what is the cell of origin. We addressed these questions using single-cell and bulk genomic sequencing. We developed custom single-cell panels covering HLA class I and II genes, along with 14 genes commonly mutated in AA and myelodysplastic syndromes. To identify copy number variations and UPD, we included 197 single nucleotide variants (SNVs) frequent across all ethnic backgrounds on chromosomes 6, 7, 8, 20, and 21. The TotalSeq™-D panel (BioLegend), was included to target 42 surface antigens and the samples were prepared on the Tapestri platform (Missionbio). High-resolution HLA genotyping was done using AlloSeq Tx 17 hybrid capture NGS assay (CareDx), and for confirmatory bulk sequencing we applied a custom duplex UMI NGS panel (TWIST Bioscience). We examined ten patients with severe AA (pediatric: n=6, age range 2-15 years; adult: n=4, 26-67), seven patients with refractory cytopenia of childhood (RCC, age range 2-16 years), who presented a hypocellular marrow and absence of a genetic predisposition syndrome, and one healthy control. All 17 patients had at least one of the HLA-A or HLA-B risk alleles linked to AA development. A total of 136,505 cells (median 4,467/run) were successfully genotyped across the samples. Single-cell trajectory analysis revealed that all identified clones arose independent of each other (Figure 1). UPD6p clones were found in five patients with three having multiple UPD6p clones carrying different breakpoints, pointing to clonal diversification. Additionally, three cases had LOF mutations in HLA-A or HLA-B (two having coexisting UPD6p). In a patient with two UPD6p and two HLA-A mutant clones, we verified that HLA-A risk allele (A*02:01) was lost in each instance. Multi-omic analysis revealed that these clones had both granulocytic and non-granulocytic (incl. lymphocytic) surface phenotypes, suggesting their early origin within hematopoietic ontogeny. Seven patients had nine independent clones with PIGA mutations (0.3-11.3% of the cells), consistent with its tendency to generate clonal mosaicism. Clones with mutations in BCOR/BCORL1, ASXL1, DNMT3A, and TET2 were identified in six, seven, four, and two patients, respectively. We next analyzed how many patients carry multiple clones and found that 65% (11/17) have concomitant clones, with the majority (9/11) belonging to different types of clonal escape (Figure 1). Finally, we found that 4/6 patients with adaptive HLA loss (representing true clonal escape from T-cell autoimmunity) also carried CHIP clones, indicating that adaptive hematopoiesis does not protect from the acquisition of CHIP mutations. In conclusion, we show that patients with AA across age spectrum and some patients with RCC share similar clonal backgrounds, with multiple functionally redundant alterations arising independently from coexisting CHIP clones. For the first time at a single-cell level, we were able to resolve the multifaceted clonal mosaic generated by the immunological forces at play in AA, and precisely unravel their immunoediting consequences.

Cooperation between SF3B1 and JAK2V617F Mutations Accelerates Fibrotic Progression in Myeloproliferative Neoplasms By Enhancing STAT1 Signaling

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1 mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1 K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1 mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies. To address this, we applied two complimentary single-cell multiomic methods to study 15 individuals (healthy donor [HD, n=5], JAK2V617F-single mutated [J+, n=5] and JAK2V617F- SF3B1K666double mutated MPN cases [JS+, n=5]). First, we characterised the cellular landscape of JS+ MPN using CITE-seq/10X genomics platform. Then, we analyzed SF3B1-mutant ( SF3B1mut) disease-driving hematopoietic stem and progenitor cells (HSPCs) and their associated aberrant splicing signatures using TARGET-seq, which permitted single-cell genotyping with allelic resolution and intra-patient comparison of mutant versus wild-type (wt) cells. In an analysis of 109,498 cells, we observed abnormally expanded erythroid (ERP), megakaryocyte (MK) and unexpectedly, eosinophil-basophil-mast cell (EBM) progenitors in JS+ as compared with J+ or HD HSPCs (Fig 1A). Two HSC clusters were present, one of which (HSC_mpp2) principally comprised of JS+ cells and had a distinct molecular program (Fig 1A); upregulation in MK (eg. PF4, VWF) and EBM lineage signature genes (eg. TPSB2), interferon signaling genes but downregulation in apoptosis genes and canonical stemness markers (eg. CD133). These signatures suggest the JS+ driven HSC cluster may be MK/EBM-transcriptionally primed and may therefore, promote expansion of these abnormal progenitors which are implicated in driving fibrosis development. Through genotyping of >5000 HSPCs, we resolved the clonal architecture of JS+ MPN, observing that in most cases double mutant cells were present and clonally dominant, with mutant SF3B1 the initiating event. Mutations arose from the HSC level and genotypes were equally distributed in the HSPC compartment. Highlighting the importance of single-cell analysis, and not apparent at the bulk level, JAK2V617F and SF3B1K666mutationsresided in separate clones for one case with a WHO fibrosis score 0 in contrast to the other 4 cases with double mutant cells and fibrosis scores 1-3. This reinforces the cooperative effect of thesemutationsto drive fibrosis. We performed full-length transcriptomics of select HSPCs to study SF3B1mut-specific aberrant splicing events (ASE). In a global splicing analysis, more events were detected in JAK2V617F- SF3B1K666 double mutant than single mutant HSPCs. Retained intron (RI) events were the most prevalent ASE, followed by skipped exon and alternative 3' splice site events. We identified previously described SF3B1mut-specific ASE ( MAP3K7, ERGIC3 and SEPTIN6) confirming the validity of our dataset to study new ASE. Inter- and intra-patient comparison of SF3B1mut versus SF3B1wt cells uncovered a number of novel biologically relevant ASE, including two RI events in STAT1. These RI events were reduced in SF3B1mut HSPCs with the predicted consequence of increased STAT1 expression and activation, of direct relevance to MPN pathobiology since STAT1 is known to play a key role in enhancing megakaryopoiesis in MPN (Fig 1B). We validated these events using long-read Nanopore sequencing of single-cell cDNA libraries and have developed a cell model system with transient expression of SF3B1K666 mutation versus SF3B1wt in HEK293T to corroborate STAT1 ASE occurring as a direct consequence of mutant SF3B1. In further support of upregulated STAT1 activation, we observed upregulation of STAT1 downstream targets in abnormally expanded JS+ HSPCs. In conclusion, this analysis resolved the clonal architecture and hematopoietic cellular composition of JS+ MPN, identifying abnormally expanded and transcriptionally primed HSPC populations with abnormal splicing of STAT1 enhancing JAK-STAT signaling, uncovering the mechanism by which these mutations promote an accelerated fibrotic phenotype.

Single-Cell Whole-Genome Sequencing after Chemotherapeutic Treatment Reveals Cell Type-Specific Mutational Burdens

Introduction The hematopoietic system is especially vulnerable to chemotherapeutic treatment-related long-term consequences, including clonal and malignant hematopoiesis. These diseases are caused by the acquisition of driver mutations in hematopoietic stem and progenitor cells (HSPCs). An increased rate of mutation accumulation, as identified in HSPCs of patients who receive chemotherapeutic treatment, can therefore result in disrupted blood production. Accordingly, childhood cancer survivors have a five-fold increased risk of secondary cancers, amongst which therapy-related myeloid neoplasms (t-MN) are one of the most prevalent. Here, we aim to increase the understanding of the origin of t-MN through the characterization of the short- and long-term mutational consequences of chemotherapy in different hematopoietic stem and progenitor cell types. Methods We studied the genome-wide mutation burden in the HSPC compartment at the time of an initial cancer diagnosis, following chemotherapeutic treatment, and upon a second cancer diagnosis. We performed whole-genome sequencing (WGS) of single HSPCs, including hematopoietic stem cells (HSCs, CD34+CD38-CD45RA-CD90+), multipotent progenitor cells (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitor cells (CMPs, CD34+CD38+CD45RA-), and granulocyte-monocyte progenitor cells (GMPs, CD34+CD38+CD45RA+). In order to obtain enough DNA for WGS, we performed clonal expansion of single cells or primary template-directed amplification of the DNA of one cell. Next, we normalized the mutation burden in each cell to the expected number of mutations (Osorio et al. Cell Reports 2018). In addition, we performed both whole genome and transcriptome sequencing of individual HSCs/MPPs. Results First, we investigated pediatric ten t-MN patients with a latency between the initial and second cancer diagnosis spanning 1-12 years. We applied WGS to clonally expanded HSPCs (CD34+CD38-CD45RA-) and found that the relative increase in mutation burden in HSPCs is restricted to the first five years after initial treatment. To gain more insight into the heterogeneous HSPC compartment, we subsequently investigated cell-type specific mutational loads based on immunophenotypic markers. So far, we have included three pediatric cancer patients with comparable treatment regimens and diagnoses, namely medium risk acute lymphoblastic leukemia. Here, we found that the mutation burden is lower in HSCs when compared to the progenitor cell types (Figure 1A). When comparing the mutation load in the HSCs to the linear mutation accumulation observed in HSCs and MPPs of healthy donors, we identified a fraction of cells overlapping with the expected mutation load based on the age of the patient (Figure 1B). These data suggest that HSCs are more protected against chemotherapy-induced mutagenesis than progenitor cells, although this protective effect varies between individual cells. Therefore, we also performed both whole genome and transcriptome sequencing from a single cell, to characterize the transcriptional state of HSCs/MPPs showing a relatively high or low mutation load within their respective cell type. Conclusions Our results indicate that primitive hematopoietic stem cells are more protected against chemotherapy-induced mutations than the more proliferative progenitor cells. In addition, we find that in t-MN patients with a long latency (>5 years), the mutation load in HSPCs returns to the expected baseline. Based on our data, we hypothesize that the more damaged progenitor cells may be replaced over time, fueled by the undamaged primitive stem cell pool. Thus, our results may explain why the risk of t-MN drops 5-7 years after treatment end.

Modeling Cell Population Dynamics in Early Hematopoiesis Reveals Mechanisms of Myeloid Bias Mediated By NFκB-Dysregulation

Alterations in hematopoiesis are associated with immune dysregulation and perturbations in NFκB signaling. Aging, for example, is correlated with increased myeloid and decreased lymphoid cell output, as well as elevated levels of NFκB activity in hematopoietic stem and progenitor cells (HSPC) and inflammatory cytokines in the bone marrow. Further, patients with myelodysplastic syndrome (MDS) show increased expression of components of the NFκB signaling pathway in HSPC and mesenchymal cells, in addition to increased production of inflammatory cytokines. Despite being implicated in these contexts, it remains unknown how elevated NFκB signaling might affect the production and abundance of certain HSPC subsets. Here we interrogate how increased NFκB activity may alter the developmental dynamics within the HSPC compartment to give rise to a biased progenitor population. To investigate how myeloid cell overproduction arises in the bone marrow, we developed mathematical models to recapitulate HSPC population dynamics. To address the role of NFκB in the process, we leveraged data from the IκB - mouse model ( Nfkbia +/- Nfkbib -/- Nfkbie -/-), which has constitutively elevated NFκB activity. Cell counts of HSPC subpopulations demonstrated a myeloid biased alteration in multipotent progenitor (MPP) abundances in the IκB -mouse, reminiscent of what occurs with aging. Fitting the cell population dynamics model to these data suggested that accelerated differentiation of Short-Term HSC (STHSC) with skewing in their fate commitment is insufficient to account for the increase in MPP2 and MPP3 cells. To explore additional mechanisms contributing to myeloid bias, we utilized single-cell RNA sequencing (scRNAseq) data from IκB - versuscontrol HSPC. We first used pseudotime reconstruction to position cells along a continuous differentiation trajectory from HSCs to erythrocyte-megakaryocyte- (MPP2), myeloid- (MPP3), and lymphoid- (MPP4) primed progenitors and then fit these data to a partial differential equation (PDE) model of population dynamics. This analysis suggested not only a loss of lymphoid fate specification amongst HSC but also an increase in the expansion of early myeloid primed cells to produce more MPP3 cells. Transplantation of wildtype HSPC into a lethally irradiated IκB - mouse additionally showed that an inflamed microenvironment is sufficient to drive myeloid bias. Utilizing the same mathematical models to describe these data revealed similar perturbations in population dynamics of the transplanted HSPC. Finally, the scRNAseq pseudotime analysis identified differentially expressed genes along the differentiating lineages that indicated decreased HSC commitment towards the lymphoid fate and increased proliferation of early myeloid primed cells, supporting the model predictions. We next leveraged scRNAseq datasets from HSPC isolated from MDS patients and healthy aged-matched controls to explore if these features of NFκB-mediated immune dysregulation are clinically relevant. These data demonstrated myeloid bias in progenitors at the transcriptional level and suggested elevated NFκB signaling amongst the HSPC from MDS patients. Finally, applying the same mathematical modeling and gene expression analyses developed for the murine datasets revealed common perturbations in developmental dynamics of MDS HPSC as the IκB - mouse models. These analyses motivate further study of the downstream consequences of immune dysregulation on hematopoiesis and the identification of perturbations in NFκB signaling in pathological contexts.

Dual Targeting of HDM2 and PPM1D As a Novel Therapeutic Approach for Myelofibrosis

Dysregulation of TP53 in myeloproliferative neoplasms (MPNs) has been hypothesized to be a key driver of disease progression (Kubesova et al, Leukemia. 2018;32 (2):450-61). Targeting of double minute protein 2 (HDM2), a negative regulator of TP53, results in activation of the TP53 pathway which induces cell death in hematopoietic stem and progenitor cells (HSPCs). Nutlins, antagonists of HDM2, are currently being evaluated in multiple MPN clinical trials with promising results to date. Protein Phosphatase Mg2+/Mn2+ dependent 1D protein (PPM1D) is another key negative regulator of the TP53 pathway that dephosphorylates TP53 and stabilizes HDM2 (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). We and others have previously reported the presence of PPM1D mutations in MPNs that result in overexpression of the protein (Grinfeld et al, N Engl J Med. 2018 Oct 11;379 (15):1416-30). We have also reported overexpression of PPM1D in the mononuclear cells (MNC) of a subset of MPN patients not harboring PPM1D mutations. We hypothesize that inhibition of PPM1D in myelofibrosis (MF) would target the MF HSPC and sensitize MF HSPCs to HDM2 inhibitors resulting in further depletion of MF HSPCs. To further characterize PPM1D expression in the MF HSPC compartment, we utilized quantitative real time-PCR, to assess PPM1D transcript levels in CD34+ cells from 28 MF patients and compared these transcript levels to levels in CD34+ normal control cells (n=6). Forty-three percent (12/28) of the MF samples had overexpression of PPM1D (>1.5 fold increase) and the fold increase ranged from 1.8-59. To identify whether PPM1D is a viable therapeutic target for MF HSPCs, we utilized hematopoietic colony assays to test the activity of GSK2830371, an allosteric inhibitor of PPM1D (PPM1Di) alone and in combination with a low dose HDM2 inhibitor (HDM2i). Eight samples from distinct MF patients were tested under these conditions. There was not a significant reduction in the number of JAK2V617F+ colonies upon treatment with single agent HDM2i (50 nM) (p>0.05) or PPM1Di (1uM) (p>0.05). Combination of PPM1Di (1 uM) + HDM2i (50 nM) resulted in decreased JAK2V617F+ colony formation in all 8 samples (p<0.005) (Fig. 1). The reduction in JAK2V617F+ colony number was significant compared to either single agent treatment with HDM2i (p<0.001) or PPM1Di (p<0.05) (Fig. 1). Four samples had persistence of or an increase in JAK2 wild-type (WT) colony number with combination treatment. The MF samples utilized were from a heterogenous population of patients that all had WT TP53 and PPM1D but included patients with high risk myeloid mutations (in ASXL1, NRAS), complex cytogenetics and prior exposure to standard MF directed therapies. Additionally, to validate the on-target effect of PPM1Di, we showed that treatment of leukemia cell lines with PPM1Di led to increased protein levels of phosphorylated-p53 and p21 and resulted in decreased viability and increased apoptosis. To determine the effect of PPM1Di +/- HDM2i on distinct subclones we employed a multi-omics approach that allows for single cell mutation and epitope analysis of a clonally complex patient sample. We analyzed MNCs from a JAK2V617F, NRASG12D, TET2T1884A, ASXL1W960* MF patient treated in suspension culture with PPM1Di (1uM) alone, low dose HDM2i (50 nM) alone and PPM1Di (1uM) + low dose HDM2i (50 nM) in combination. Compared to vehicle alone or either drug alone, a marked effect was seen with the combination approach whereby the percent of WT cells increased by nearly 5 fold (4% to 19%) and 4/6 mutated clones were decreased in size, demonstrated by the fishplot (Fig. 2). These results show that the combination of the HDM2i + PPM1Di depletes multiple MF clones, while allowing for the emergence of more WT cells. Furthermore these studies demonstrate the utility of single cell genotyping to evaluate the effects of novel therapeutic strategies on MF clonal architecture. These studies indicate that PPM1Di sensitizes MF HSPCs to an HDM2i and is a promising strategy to treat MF patients. To this purpose, we are currently developing a novel clinical grade PPM1D targeting agent. Dual targeting of PPM1D and HDM2 has the potential to further deplete multiple MF HSPC clones while allowing the persistence of WT cells. Additional combinatorial mutation + epitope studies are underway to determine degree of MF HSPC subpopulation depletion utilizing this treatment strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omics Profiling of JMML HSPCs Reveals Onco-Fetal Reprogramming and Identifies Novel Prognostic Biomarkers and Therapeutic Targets in High-Risk JMML

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of early childhood, which is characterized by highly heterogenous clinical outcomes ranging from spontaneous resolution to early relapse after hematopoietic stem cell transplantation (HSCT). Established clinical risk factors as well as characteristic mutational patterns do not fully explain this heterogeneity. Recently, we and others identified DNA methylation subgroups in JMML, which were shown to be the only significant factor for predicting overall survival (Schönung et al., Clinical Cancer Research 2021). Together, these findings suggest a functional role for DNA methylation in the molecular pathogenesis of JMML. To elucidate the impact of aberrant DNA methylation on pathogenesis and disease progression, we leveraged a multi-modal molecular analysis approach. We integrated flow cytometry data with single-cell RNA-seq and ultra-low input whole-genome bisulfite sequencing (WGBS) data in order to dissect the hematopoietic system across patients from all epigenetic subgroups (n=8). With this approach, we aimed to identify novel candidate prognostic biomarkers and therapeutic targets for high-risk JMML patients. We observed conservation of epigenetic subgroups already in the hematopoietic stem and progenitor cell (HSPC) compartment, suggesting that differential methylation might be key to understand pathogenesis and course of the disease in JMML. In addition, single-cell transcriptomics revealed disease-specific aberrations to be most pronounced in the HSPC compartment relative to all other hematopoietic cell types. Consequently, we focused our work on JMML HSPCs, which revealed a high degree of immunophenotypic, transcriptomic and methylomic heterogeneity across JMML risk groups. This included the expression of proto-oncogenes belonging to the AP-1 family, CTNNB1 and MECOM (EVI1). Furthermore, we observed divergent lineage priming across JMML subgroups which affected lymphoid, myeloid and erythroid differentiation programs. Remarkably, subgroup-specific aberrations comprised upregulation of fetal developmental programs in HSPCs from high-risk JMML patients. This is of particular interest regarding the cell-of-origin in JMML, since mutations activating the RAS signaling pathway have been identified in newborn blood samples from children who later developed JMML (Behnert et al., Leukemia 2022). Comparing the methylome data of HSPCs from JMML patients with HSPCs from healthy individuals at different developmental stages (fetal, perinatal, juvenile and adult) suggested progressive epigenomic reprogramming of leukemic HSPCs towards high-risk JMML. As a consequence, we observed signatures of both accelerated aging and aberrant activation of onco-fetal programs. This included the downregulation of postnatal HSPC markers such as AVP and vice versa the upregulation of prenatal markers such as HMGA2 in high-risk HSPCs, which was associated with differential methylation of these genes. Moreover, binding motifs of central hematopoietic and developmental transcription factors such as RUNX1, FLI1, CDX4, and several HOX genes were not only enriched in differentially methylated regions but were also found to be differentially methylated themselves. Systematic comparison of JMML HSPCs with their healthy nearest normal counterparts revealed disease-specific expression of several cell surface markers. The expression patterns appeared to be regulated in a subgroup-specific manner, which nominates these factors as novel prognostic biomarkers in JMML. Moreover, we identified surface markers that were not only differentially methylated but also known drug targets in other entities, which is why we selected these genes as high confidence candidates for further functional validation. In a preclinical patient-derived xenograft (PDX) mouse model, targeted treatment with a therapeutic antibody against one of these candidates efficiently depleted high-risk JMML HSPCs and improved survival of JMML-PDX mice. In conclusion, the personalized molecular analysis of JMML HSPCs disentangled disease-specific heterogeneity and provided novel insights into tumorigenesis. Subgroup-specific epigenetic dysregulation included onco-fetal reprogramming of JMML HSPCs and revealed novel prognostic biomarkers and promising novel drug targets specifically for high-risk JMML.

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Oral Trypanosoma cruzi Acute Infection in Mice Targets Primary Lymphoid Organs and Triggers Extramedullary Hematopoiesis.

During the acute phase of Chagas disease, Trypanosoma cruzi circulation through the bloodstream leads to high tissue parasitism in the host. In primary lymphoid organs, progenitor cell reduction paralleled transient immunosuppression. Herein we showed that acute oral infection in mice promotes diffuse parasitism in bone marrow cells at 14 and 21 days post-infection (dpi), with perivascular regions, intravascular regions, and regions near the bone being target sites of parasite replication. Phenotypic analysis of hematopoietic differentiation in the bone marrow of infected mice showed that the cell number in the tissue is decreased (lineage-negative and lineage-positive cells). Interestingly, analysis of hematopoietic branching points showed that hematopoietic stem and progenitor cells (HSPCs) were significantly increased at 14 dpi. In addition, the pool of progenitors with stem plasticity (HSC-MPP3), as well as multipotent progenitors (MPPs) such as MPP4, also showed this pattern of increase. In contrast, subsequent progenitors that arise from MPPs, such as common lymphoid progenitors (CLPs), lymphoid-primed MPPs (LMPPs), and myeloid progenitors, were not enhanced; conversely, all presented numeric decline. Annexin V staining revealed that cell death increase in the initial hematopoietic branching point probably is not linked to CLPs and that myeloid progenitors decreased at 14 and 21 dpi. In parallel, our investigation provided clues that myeloid progenitor decrease could be associated with an atypical expression of Sca-1 in this population leading to a remarkable increase on LSK-like cells at 14 dpi within the HSPC compartment. Finally, these results led us to investigate HSPC presence in the spleen as a phenomenon triggered during emergency hematopoiesis due to mobilization or expansion of these cells in extramedullary sites. Splenocyte analysis showed a progressive increase in HSPCs between 14 and 21 dpi. Altogether, our study shows that the bone marrow is a target tissue in T. cruzi orally infected mice, leading to a hematopoietic disturbance with LSK-like cell bias accounting on HSPCs possibly affecting myeloid progenitor numbers. The LMPP and CLP reduction converges with defective thymocyte development. Lastly, it is tempting to speculate that the extramedullary hematopoiesis seen in the spleen is a mechanism involved in the hematological maintenance reported during the acute phase of oral T. cruzi infection.

Single Cell Analysis Elucidates the Maturation of Human Stem and Progenitor Cell Function from Fetal through Adult Hematopoiesis

Hematopoiesis continually replenishes the supply of circulating blood cells from embryonic development through the entirety of human lifespan. Although all hematopoietic lineages are produced throughout life, biases in lineage output occur at various stages, including lymphoid bias in childhood and myeloid bias in adulthood. Furthermore, many blood disorders demonstrate marked biases in age of onset, such as bone marrow failure disorders, clonal hematopoiesis of indeterminate potential, and numerous hematologic malignancies. A lack of insight into the normal physiologic changes occurring in the hematopoietic stem and progenitor cell (HSPC) compartments during development and maturation fundamentally limits our understanding of how age biased blood disorders arise. Two major unresolved questions are: (1) what changes in the molecular regulation of hematopoietic lineage commitment occur over the course of human life? And (2) are certain HSPC states present only during specific ages of life, and if so, do age-specific HSPCs have distinct biology?To address these questions we performed single cell RNA sequencing (scRNAseq) on human HSPCs from first and second trimester fetal liver hematopoiesis, and bone marrow hematopoiesis spanning childhood into mature adulthood. In total, HSPC samples were obtained from 14 distinct human donors. Dimensionality reduction and marker gene analysis identified uncommitted hematopoietic stem cells (HSCs) and the developmental trajectories of each lineage emanating from multipotent HSCs. We then identified the genes activated upon commitment to each hematopoietic lineage during fetal, childhood, and mature adult hematopoiesis using the Population Balance Analysis and Stationary Optimal Transport algorithms, followed by Elastic Net gene regression. For each lineage we determined the putative transcription factor network that is consistently active in driving commitment to that lineage throughout life, but surprisingly also found the existence of adjunctive transcription factor networks that only drove lineage commitment at specific ages.We next used unbiased clustering of scRNAseq data to identify 21 distinct subtypes in the HSPC compartment across human life. Using marker gene analysis and singleCellNet algorithm comparisons to an existing human adult bone marrow scRNAseq data set, we hierarchically ordered and annotated these HSPC subtypes ranging from uncommitted HSCs to lineage committed progenitor cells. We found that cellular distribution within the HSPC compartment amongst these subtypes varied markedly throughout human lifetime, with higher representation of HSCs in fetal life, predominance of lymphoid progenitors in childhood, and higher representation of myeloid progenitors in adulthood. Focusing on the distribution of cells among HSC subtypes over human life, we identified an HSC subtype exclusive to mid-gestation that was not present in early fetal or postnatal timepoints. This HSC subtype had a characteristic immunophenotype and was enriched for expression of early response transcription factors and mRNA decay factors. We functionally validated that this mid-gestation-specific HSC subtype was phenotypically unique using colony formation assays and xenotransplantation. Mid-gestation-specific HSCs were more clonogenic with a greater number of multi-lineage outcomes, and also demonstrated increased multilineage engraftment capacity compared to other HSC subtypesOur findings reveal that the intrinsic biology of hematopoietic lineage commitment fundamentally changes over the course of the human lifetime, and define and validate age-specific HSPC subtypes. In particular, the biology of the mid-gestation-specific HSC we identified has potential applications for improving engraftment and multi-lineage reconstitution in hematopoietic cell transplantation. DisclosuresRegev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

BackgroundThe identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies.ResultsIn healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy.To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes.Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models.ConclusionWe have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. DisclosuresDick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Single Cell Deconvolution of Myeloblasts Reveals Depletion of HSCs and Expansion of Inflammatory Granulocyte-Monocyte Progenitors (GMPs) Associated with Adverse Outcomes in Chronic Myelomonocytic Leukemia (CMML)

Single Cell Deconvolution of Myeloblasts Reveals Depletion of HSCs and Expansion of Inflammatory Granulocyte-Monocyte Progenitors (GMPs) Associated with Adverse Outcomes in Chronic Myelomonocytic Leukemia (CMML)

Single-Cell RNA Sequencing Analysis Reveals Mechanisms of Initiation and Progression in Chronic Myelomonocytic Leukemia

Despite advances in the genetic characterization of chronic myelomonocytic leukemia (CMML), the molecular mechanisms that drive the disease during its distinct phases remain unclear. To uncover vulnerabilities in CMML that could be therapeutically targeted to halt its evolution, we sought to dissect at the single-cell level the cellular and transcriptomic changes that occur in the hematopoietic system at the time of CMML's initiation and its progression after hypomethylating agent (HMA) therapy.To evaluate the molecular mechanisms underlying CMML maintenance, we performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin -)CD34 + hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) mononuclear cells (MNCs) isolated from untreated CMML patients (n=5 and 6, respectively) and age-matched healthy donors (HDs; n=2 and 3, respectively). Our integrated analysis revealed that CMML Lin -CD34 + HSPCs had a predominant granulomonocytic differentiation route with an increased frequency of early and committed myeloid-monocytic progenitors at the expense of HSCs and megakaryocyte/erythroid progenitors (Fig. 1a). Differential expression analysis among the clusters revealed that most transcriptomic differences occurred in CMML HSCs, which were characterized by the upregulation of genes involved in oxidative phosphorylation, type I interferon (IFN) and IFNγ response, myeloid development, and inflammatory signaling and had downregulated expression of genes involved in TNFα-mediated NF-κB activation (Fig. 1b). These data suggest that CMML HSCs undergo metabolic reprogramming and demand a higher level of mitochondrial activity to maintain activated monocytic differentiation in response to inflammatory signaling.Consistent with these results, scRNA-seq analysis of MNCs isolated from the same HD and CMML BM samples showed that monocytes were significantly increased at the expense of erythroid precursors and B cells in CMML (Fig. 1c). CMML monocytes had upregulated genes involved in IFNγ response, oxidative phosphorylation, MYC targets, NF-κB activation, and inflammation (e.g., S100A9, CCL3, IL1B). Interestingly, among the anti-apoptotic BCL2 family members, only the NF-κB transcriptional target BCL2A1 was significantly overexpressed.To investigate the mechanisms of resistance to HMA therapy, we performed integrated scRNA-seq analysis of sequential Lin -CD34 + cells and BM MNCs isolated from CMML patients at the time of disease initiation and progression after HMA therapy failure. CMML progression was driven by a significant expansion of lympho-myeloid progenitors (LMPPs) at the expense of earlier HSCs , which exacerbated myelomonocytic differentiation in the HSPC compartment (Fig. 1d). Expanded LMPPs were characterized by higher levels of IFNγ response, NF-κB survival signaling, and cell cycle regulators. Accordingly, scRNA-seq analysis of MNCs cells from the same patients showed significantly increased frequencies of monocytes and a reduction of naïve CD4 +/CD8 + T cells and effector memory CD8 + T cells. Differential expression analysis of the 2 sample groups in the monocyte population identified five different cellular clusters, one of which emerged only at progression (Fig. 1e). This population was characterized by high expression levels of inflammatory cytokines and the anti-apoptotic modulators MCL1 and BCL2A1. Together, these data suggest that CMML progression arises from immature myeloid progenitors at the stem cell level and that downstream monocytes undergo transcriptomic rewiring and acquire survival mechanisms that induce therapy resistance and further accelerate disease progression.In conclusion, our results elucidate the differentiation hierarchies and transcriptional programs associated with CMML's initiation and its progression after HMA therapy. Our data suggest that therapies targeting downstream effectors of NF-kB-mediated survival signaling could overcome treatment failure. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; KAHR Medical Ltd: Honoraria; Jazz: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astellas Health: Honoraria; NOVA Research: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astra Zeneca: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Hydrogel-Based 3D Culture Model for Down Syndrome Associated Transient Myeloproliferative Disorder Using Customized Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD.iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E).To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P<0.05). In colony forming unit (CFU) assay, the 3D generated HSPCs gave rise to a 2.0-fold higher number of CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colonies compared to 2D, with 2.0-fold decreased number of BFU-E (erythroid) colonies and a similar number of CFU-GM (granulocyte, macrophage) colonies (Fig. 1G). Thus, the low modulus synthetic matrix promoted hematopoietic differentiation producing higher percentage of early HSPCs as compared to the 2D culture system.We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K).In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. [Display omitted] DisclosuresBarwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Abstract 3201: Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen

Abstract Growing evidence suggests that tumor progression can interfere with normal hematopoiesis and skew the host system to undergo myeloid biased changes. Hematopoietic stem and progenitor cells (HSPCs) are a rare population of precursor cells giving rise to progeny that replenish blood cells throughout life. HSPCs are mainly localized in the bone marrow niche in healthy adults, however pathophysiological conditions such as cancer can cause extramedullary hematopoiesis in organs like the spleen. In this study, we investigated tumor-driven phenotypic and molecular alterations in the HSPC compartment at a single-cell level during extramedullary hematopoiesis using single-cell multiomics. We used a B16-F10 melanoma mouse model to uncover the tumor-mediated changes. Mice with tumor-burden showed splenomegaly and alterations in the composition of the HSPC compartment in the spleen consistent with extramedullary hematopoiesis. We then isolated four HSPC populations, including Lin- Sca1+ cKit+ (LSK), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocyte-erythrocyte progenitor (MEP) cells from the spleen and bone marrow of melanoma-burdened mice using the BD FACSMelody™ Cell Sorter. Next, we used an integrated workflow on the BD Rhapsody™ Single-Cell Analysis System for the downstream single-cell multiomics analysis. We utilized oligo-conjugated antibodies for NGS-based sample multiplexing and protein detection (BD® Single-Cell Multiplexing Kit, BD® AbSeq Assay), along with whole transcriptome analysis for a comprehensive analysis of mRNA expression. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization enabled delineation of the developmental trajectories of HSPC populations and exploration of phenotypic and transcriptomic changes across different hematopoietic subpopulations and across distinct anatomic sites. The analysis also identified unique hematopoietic cell clusters present during melanoma-driven extramedullary hematopoiesis in the spleen. Overall, our findings highlight melanoma-driven perturbations in hematopoiesis by utilizing advanced single-cell multiomics technology. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD-23277 (v1.0) 1120 BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved. Citation Format: Nihan Kara, Nikolay Samusik, Xiaoshan Shi, Chip Lomas, Stephanie Widmann, Aaron J. Tyznik. Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3201.

Novel Roles of Separase in Hematopoiesis

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. In recent years, cancer genome sequencing has revealed several loss of function cohesin mutations in early stages of myeloid, hematologic and epithelial malignancies. These mutations affect the self-renewal and differentiation capacity of HSCs indicating cohesin as a tumor suppressor. Our prior studies indicated that Separase haploinsufficiency acts synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas. In contrast to cohesin proteins, the function of the cohesin protease, Separase in hematopoiesis and hematological malignancies has not been well defined.Here we examined the effect of Espl1 heterozygosity using our hypomorphic mouse model (mEspl1 +/hyp ) that has reduced germline Separase activity. The impact of heterozygous loss of mEspl1 allele on total number of HSCs was analyzed by staining with hematopoietic stem and progenitor cells (HSPCs) markers and FACS analysis of cells isolated from bone marrow. Our analysis showed that Separase mutant mice have significantly increased (~2 fold) immunophenotypic HSPCs compared to wild type littermates. To examine whether reduced Separase level elevates self-renewal capacity of HSCs in vivo , we performed competitive transplantation assays to compare differentiation and self-renewal capacity of HSCs in mEspl1+/hyp and WT littermate controls. The outcomes with significantly higher donor derived reconstitution suggested an expansion of HSPCs compartment in mutant mice. In addition, we also observed an altered hematopoietic differentiation with an increase in percentage of donor-derived T- and B-cells in Separase mutant mice. The increased number of HSCs could be a consequence of change in autonomous function of the cells due to reduced level of Separase. To test the cell autonomous function of mEspl1+/hyp , in vitro colony forming unit (CFU) assays were performed in methylocellulose medium. mEspl1+/hyp cells showed significantly higher serial re-plating capacity compared to WT littermate control cells, supporting the notion of cell autonomous function as a probable cause of the observed phenotype.Considering the role of cohesin on gene expression, the effect of heterozygous loss of Separase was analyzed with global transcriptional profiling. Several hematopoietic fingerprint genes like Tle1, Smarca1, Scl2a10, H19, Krt2-8 and Galnt3 were found upregulated. Other genes important in HSC function such as Runx1, Satb1, Hoxa9 and Pbx1 were also upregulated in mEspl1+/hyp mice. The transcriptional profiling identified genes involved in several biological processes such as leukocyte differentiation, cell apoptosis, and chromatin modifications. However, mechanism and direct gene targets of Separase in hematopoiesis or other biological pathways are yet to be identified. To study further on transcriptional regulatory roles and to characterize the direct targets of Separase, ChIP-Seq (cohesin and Separase) and RNA-Seq experiments are being performed in WT and mEspl1+/hyp HSCs.With these studies we hope to better understand the role of cohesin in association with Separase on hematopoiesis particularly the mechanisms that control the fate of HSCs, bone marrow engraftment and function; how cohesin mutations may give rise to malignant transformation, and if this pathway can be targeted for therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Eltrombopag Specifically Expands Hematopoietic Multipotent Progenitors in Human Aplastic Anemia

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been recently added to first-line immunosuppressive therapy (IST) in immune aplastic anemia (AA), significantly improving hematologic recovery and survival (Townsley et al. NEJM 2017), corroborating preclinical data suggesting a direct effect of EPAG on hematopoietic precursors. Here, we analysed the in vivo effects of EPAG on the composition of the hematopoietic stem and progenitor cell (HSPC) compartment after hematologic recovery in human AA patients treated with IST combined with EPAG. Twelve patients with severe immune aplastic anemia who achieved hematologic response six months after initial treatment were invited to participate in the study. Six patients were treated with EPAG associated with cyclosporine and six were treated with rabbit antithymocyte globulin and cyclosporine, but treatment choice was not determined by this study. 25 mL of marrow aspirate was obtained from each patient for multiparametric analysis in an 8-color flow cytometry. Mononuclear cells were separated by gradient density centrifugation and CD34+ cells were purified using magnetic column beads. We used a 9-antibody panel to identify HSPC subpopulations, as follows: hematopoietic stem cells (HSC), CD34+ CD38- CD45RA- CD90+ CD49f+; multipotent progenitor cells (MPP), CD34+ CD38- CD45RA- CD90- CD49f-; multilymphoid progenitor cells (MLP), CD34+ CD38+ CD45RA+ CD10+ CD7-; common myeloid progenitor cells (CMP), CD34+ CD38+ CD45RA- CD135+ CD10- CD7-; granulocyte-monocyte progenitor cells (GMP), CD34+ CD38+ CD45RA+ CD135+ CD10- CD7-; and megakaryocytic-erythroid progenitor cells (MEP), CD34+ CD38+ CD45RA- CD135- CD10- CD7-. Purified marrow CD34+ cells were also assessed in methylcellulose medium and colonies were counted 14 days after triplicate plating. Cell populations and colony formation assay were compared between the two groups using a two-tailed non-parametric Mann-Whitney test. The percentage of CD34+ of total nucleated bone marrow cells was 1.2% (range, 0.28-2.9%) in patients who received IST/EPAG and 0.2% (0.11-0.65%) in those who received IST alone (P&amp;lt;0.005). The percentage of MPPs cells was the only one significantly expanded in patients who received IST/EPAG as compared to IST alone (P&amp;lt;0,05). There was no statistical difference in the number of colony-formation units between groups. Townsley et al. (NEJM 2017) reported on the CD34+ compartment reconstitution three and six months after initial therapy, but did not compare this recovery to patients not receiving EPAG. Additionally, they did not indicate which primitive cells are directly affected by EPAG. It was speculated that EPAG may act on HSPC proliferation via the THPO-MPL signalling pathway, supported by previously evidence that loss-of-function mutations in the THPO gene result in aplastic anemia (Dasouki et al. Blood 2013). Here, we compared for the first time the HSPC compartment reconstitution in AA patients treated with either IST alone or EPAG added to IST. We found that marrow CD34+ cell numbers were higher when EPAG was added to IST, indicating a positive effect on the HSPC compartment. More importantly, we found that MPPs were the only HSPC subset to be significantly increased when patients were treated with EPAG. The role of the THPO-MPL signalling pathway in HSPCs is not entirely clear, but our findings are in agreement with recent observations. In xenotransplant models, Matsuoka et al (Cell Transplantation 2017) found that human CD34+ MPL- cells are able to sustain long-term engraftment, whereas MPL+ cells showed deficient repopulation capacity. Likewise, Cocault et al (Experimental Hematology 2016) found that in human CD34+CD38- cells, MPL is preferentially expressed in CD90-CD49f- cells. Recent observations in refractory AA patients suggest that EPAG may expand quiescent preexisting abnormal clones in the marrow (Winkler et al. Blood 2019). However, in our cohort, we did not observe any aberrant HSPC immunophenotype suggesting the expansion of abnormal clones. In conclusion, we found that in patients with immune aplastic anemia, EPAG significantly expands CD34+ cells and specifically expands hematopoietic multipotent progenitor cells. Our findings support the hypothesis that in vivo the THPO-MPL signalling pathway plays a role in human MPP proliferation, but not in HSCs. Disclosures Scheinberg: Novartis Brasil S.A.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Calado:Novartis Brasil S.A.: Speakers Bureau.

Dnmt3a Mutation Confers a Selective Advantage Specifically to Cells within the Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment

The mechanisms by which specific somatic mutations promote the competitive advantage of hematopoietic stem and progenitor cells (HSPCs), particularly in the context of clonal hematopoiesis (CH), is poorly understood. CH in the healthy aging population has been suggested to originate from hematopoietic stem cells (HSCs), supported by data showing that somatic mutations occur at similar variant allele frequencies (VAF) across mature hematopoietic cells of all hematopoietic lineages, as well as HSPCs. However, this data could also be consistent with CH-associated somatic mutations conferring long-term self-renewal potential onto multipotent progenitor cells. We recently developed an inducible knockin mouse model (Dnmt3aR878H/+Mx1-Cretg/o) to recapitulate the hotspot mutation DNMT3AR882H observed in human CH and AML (Loberg et al., Leukemia 2019) in order to empirically determine the cell type(s) within the HSPC compartment that gain a competitive advantage following acquisition of a CH-associated somatic mutation. In this model, we observed that the Dnmt3aR878H mutation expands multiple populations within the HSPC compartment with distinct kinetics. At 6 weeks post-induction of the mutation, we observed significant expansion of multipotent progenitor populations MPP2 and MPP3, both in frequency and in total cell number, compared to Mx1-Cre controls. At 6 months post-induction of the mutation, we observed significant expansion of long-term (LT)-HSCs, short-term (ST)-HSCs, and MPP3 cells. Furthermore, serial colony-forming unit (CFU) assays revealed that Dnmt3aR878H-mutant LT-HSCs, ST-HSCs, and MPP3 cells, but not MPP4 cells, had increased replating capacity compared to their control counterparts. This data suggested that several distinct populations of HSPCs might be capable of gaining an enhanced competitive advantage with the Dnmt3aR878H mutation. To determine which of the expanded Dnmt3aR878H-mutant HSPC populations gained a selective advantage in vivo, we performed competitive transplantation studies of purified subpopulations of HSPCs. Upon transplant, Dnmt3aR878H-mutant LT-HSCs demonstrated a competitive engraftment advantage compared to control LT-HSCs. In contrast, Dnmt3aR878H-mutant ST-HSCs and MPP3 cells showed significantly impaired engraftment compared to their control counterparts. These data definitively demonstrate that LT-HSCs are the cells driving Dnmt3aR878H-mutant CH. To uncover the molecular mechanisms by which this mutation confers a competitive advantage specifically to LT-HSCs, we performed RNA-seq on Dnmt3aR878H-mutant and Mx1-Cre control LT-HSCs, ST-HSCs, MPP3, and MPP4 cells at 6 weeks post-induction of the mutation. Our analysis revealed that LT-HSCs had the greatest number of transcriptional alterations when comparing Dnmt3aR878H-mutant to control cells (412 genes), while ST-HSCs, MPP3 and MPP4 cells had few significantly differentially expressed genes when comparing Dnmt3aR878H-mutant to control cells (15, 3 and 4 genes, respectively). Of the 412 genes significantly differentially expressed in LT-HSCs, 396 were downregulated and 16 were upregulated in Dnmt3aR878H-mutant versus control cells. Downregulated genes were enriched in signatures of mature hematopoietic cell differentiation and function, including heme metabolism, immune system processes, and inflammatory response, all consistent with a mechanism of impaired differentiation capacity of Dnmt3aR878H-mutant LT-HSCs. Upregulated genes included several associated with lipid biosynthesis, metabolism and glycosylation (Vldlr, Gcnt2, Fut8, Uprt, Scd), genes relevant to HSC function (Angpt1, Kit), and the tumor suppressor Rasa1, indicating that these mechanisms and processes may directly contribute to the selective advantage of Dnmt3aR878H-mutant LT-HSCs. Collectively, our data demonstrate that the LT-HSC compartment specifically and selectively gains a competitive advantage upon acquiring a somatic mutation that commonly occurs in CH and AML. Furthermore, Dnmt3aR878H does not confer long-term in vivo self-renewal potential onto ST-HSCs or multipotent progenitor cells. Given the cell-context specificity of Dnmt3aR878H mutation, mechanistic studies focused on the processes highlighted above should be performed specifically within LT-HSCs to identify therapeutic targets to prevent CH. Disclosures Trowbridge: Fate Therapeutics: Patents &amp; Royalties: patent license.

Single-Cell RNA Sequencing Reveals Distinct Hematopoietic Stem Cell Hierarchies in MDS

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

Long-Term Hydroxyurea Use Is Associated with Lower Levels of Hematopoietic Stem and Progenitor Cells in Patients with Sickle Cell Disease

Background Sickle cell disease (SCD) is curable by transplantation and potentially by gene therapy, and is generally treated by a combination of blood transfusion and Hydroxyurea (HU). Characterizing the hematopoietic system in SCD patients is important because the long-term effect of HU treatment are not known, and because of lower than expected efficacy of transduction and transplantation in Hematopoietic Stem and Progenitor Cells (HSPCs) in recent gene therapy trials. Previous studies have shown that the number of bone marrow (BM) CD34+ cells is elevated in SCD patients and that HU treatment is associated with decreased level of CD34+ cells in the peripheral blood (PB) and BM relative to steady state patients. However, hematopoiesis in SCD patients naive or treated with HU or transfusion remains poorly understood. Here, we report on the characterization of the HSPC compartment in patients with SCD by prospective isolation of 49f+ long-term Hematopoietic Stem Cells (49f+LT-HSCs), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs). Methods After obtaining consent, PB and/or BM were collected from 69 patients with HBSS/SB0, aged 12 to 45years, and 25 healthy adult African American controls. Patients were divided into chronic transfusion therapy (n=19), HU (n=31) and naïve (n=19) groups. Frozen mono-nuclear cells were analyzed by flow cytometry on a BD LSRII using CD 49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. Results FACS analysis revealed that the number PB CD34+ cells was 2.5 CD34+/uL of blood in the HU group as compared to 19 CD34+/uL in the exchange and naive groups, and 7.3 CD34+/uL in the control group (q-value &amp;lt;0.05 in all cases). Analysis with additional markers revealed that the decrease in circulating HSPCs in the HU group affected the entire hematopoietic system since the number of 49f+LT-HSCs, MPPs, CMPs, MEPs were all significantly lower in the HU group. The decrease in cell number in the HU group, however, was not homogeneous. The proportion of LT-HSCs was higher in the HU and transfusion groups when compared to the naive and control groups. The HU group also had the lowest proportion of MPPs and GMPs, as well as the highest proportion of MEPs. We then investigated hematopoiesis as a function of the length of HU treatment to elucidate the long-term treatment effect of this cytotoxic agent. Patients &amp;gt; 18 years of age that had been treated on HU for at least three years exhibited a strong statistically significant negative correlation between years on HU and CD34+/uL (R2 = 0.41), LT-HSC/uL (R2 = 0.35), MPP/uL (R2 =0.43), CMP/uL (R2 = 0.37), MEP/uL (R2 = 0.25) and GMP/uL (R2 0.39, p&amp;lt;0.01 in all cases). Importantly there was no correlation between WBC counts, age, HU dose, or serum erythropoietin level versus the numbers of any HSPC/uL. Lastly, we compared the number of HSPCs in paired PB and BM samples 10 controls and 4 SCD patients. This revealed that the numbers of CD34+, HSCs, MPPs, CMPs, GMPs and MEPs in the PB and BM were well correlated (r2 in 0.6-0.8 range) suggesting that in first approximation, results obtained in the PB reflect changes in the BM rather than changes in egress of HSPCs from the BM. Discussion We have observed lower numbers of circulating CD34+,49f+LT- HSCs, MPPs, CMP, GMP and MEPs in individuals with HBSS on HU therapy when compared to naive, chronic transfusion and, to a lesser extent, controls. Furthermore we observed subtle differences in the proportion of various circulating stem and progenitor cells (HSPCs) suggesting that the various treatments affects hematopoiesis in complex ways. The strong negative correlations between the length of HU treatment and the numbers of HSPCs can be explained either by decreased cell mobilization to the periphery, or by a depletion of the HSPC numbers in the BM overtime. Most patients undergoing gene therapy trials are currently taken off HU and placed on transfusion therapy for several months to increase CD34+ cell collection and LT-HSC transduction efficiency. We observed a greater number of circulating 49f+LT-HSC/uL of blood in the transfusion group than in the HU group, but the proportion of 49f+LT-HSC relative to the number of CD34+ were similar in both groups. Functional studies may help determine whether 49f+LT-HSCs from the transfusion group are qualitatively different from of the HU group and more amenable to gene therapy. Figure Disclosures Manwani: Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Minniti:Doris Duke Foundation: Research Funding.

Acute congenital Toxoplasma gondii infection alters fetal hematopoiesis via interferon-gamma

Abstract In adulthood, infection drives cytokine-mediated inflammation that can directly influence hematopoietic stem and progenitor cell (HSPC) function and differentiation, but the effects of maternal infection on the trajectory of fetal hematopoietic and immune cells have not been elucidated. Toxoplasma gondii is a TORCH pathogen that can be vertically transmitted upon acute infection during pregnancy, with dire consequences for the developing fetus. Maternal IFNγ production during congenital infection is required to prevent vertical transmission and promote parasite clearance, but at the cost of lowered birth weights and premature abortion. Here, we investigated the effects of acute congenital toxoplasmosis on fetal hematopoiesis. Our examination of fetal hematopoiesis in response to maternal T. gondii infection of varying virulence revealed that discrete HSPC populations, including subsets of fetal hematopoietic stem cells, B1 precursors, and myeloid progenitors are fundamentally altered in response to congenital toxoplasmosis. To assess the ability of IFNγ to mediate these observed changes and directly cross the placental barrier, we performed epistatic crosses of mice deficient for IFNγ receptor. Our results indicate that maternal IFNy produced in response to T. gondii infection may drive observed changes to the fetal HSPC compartment and that fetal HSPCs respond directly to maternal sources of IFNγ in a manner distinct from adult HSPCs. Ongoing experiments attempt to resolve the mechanisms by which vertical transmission and maternal inflammation perturb the establishment of the fetal immune system, and the direct role of IFNγ in mediating these phenomena.