HSC Compartment Research Articles

ETV6 Represses TNF during Stress Hematopoiesis and Regulates HSC Self Renewal

INTRO: ETS Variant Transcription Factor 6 (ETV6), formerly known as Translocation ETS Leukemia (TEL), functions primarily as a transcriptional repressor and is highly expressed in mouse and human HSPCs where it is essential for the development and maintenance of adult hematopoiesis. Nevertheless, it has remained poorly understood how ETV6 regulates the HSC compartment. In 2015, our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies (most often B-acute lymphoblastic leukemia), a condition now known as thrombocytopenia 5 (T5). METHODS: To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis and promote leukemogenesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human T5-associated variant (R359X). We used complementary in vitro and in vivo assays to comprehensively characterize the numbers and functions of Etv6R355X/+ and Etv6+/+ hematopoietic progenitors. RESULTS:Etv6R355X/+ mice display several of the cardinal features of T5, including abnormally rounded platelets with increased area compared to platelets from wild-type Etv6+/+ littermates. Further, Etv6R355X/+ platelets exhibit significantly delayed and incomplete clot formation as measured through clot retraction assays. Together, these findings indicate that this novel model will serve as a viable means to study the human disease. We observe that 3-month-old Etv6R355X/+mice maintain normal frequencies and numbers of lineage-sca1+cKit+ (LSK) cells in the bone marrow (BM); however, at 12 months of age, they exhibit a significant decrease in frequency in this compartment. At each of these timepoints, there are variable shifts in the proportions and/or numbers of certain HSC subpopulations. To examine whether the Etv6R355X/+mutation impacts HSC function, we carried out in vitrosEtv6R355X/+BM cells generate more colonies than wild-type cells in the first two rounds of plating, but significantly fewer or no colonies by the fourth plating. Similarly, Etv6R355X/+LSK cells exhibit significantly impaired engraftment when challenged via competitive transplantation, with near complete failure to reconstitute irradiated recipients by the tertiary transplant. Etv6R355X/+LSKs home to the bone marrow comparably to Etv6+/+cells, indicating that homing does not account for the defect in repopulation capacity. To investigate how ETV6 functions in HSCs, we performed RNA-seq of Etv6R355X/+LSKs and flow cytometry-purified HSPC subpopulations from 12-month-old mice, which revealed upregulation of genes associated with an active stem cell phenotype, downregulation of genes supporting stem cell quiescence, and upregulated expression of Tnf with enrichment for the Hallmark TNFa Signaling via NFKB geneset. Subsequent experiments using CUT&RUN of Etv6+/+LSK cells revealed several known and novel ETV6 target genes, including Tnf, a cytokine implicated in regulating stress hematopoiesis. The sites bound by ETV6 at the Tnf locus aligned with ATAC-seq peaks generated from HPC5 cells, a mouse bone marrow progenitor cell line, suggesting that this is a region of open chromatin and active transcriptional regulation. Corroborating these findings, examination of Etv6R355X/+HSPCs at 6 weeks post-transplant revealed increased cell cycling and intracellular TNF production. CONCLUSION: From these studies, we conclude that ETV6 regulates HSC maintenance. Further, it binds to a regulatory sequence within the Tnf locus, whereby it represses TNF expression. The loss or reduction of this repression in Etv6R355X/+HSCs is associated with increased cell cycling under conditions of hematopoietic stress and a loss of self-renewal capacity. Studies are ongoing to determine how these changes promote leukemogenesis and contribute to the other features of T5.

Clonal Trajectories and Therapeutic Targeting of High-Risk SF3B1-Mutant Myelodysplastic Syndromes

Myeloid neoplasms are driven by acquisition of clonal genetic lesions, yet it remains unclear how these lesions cooperate to transform hematopoietic stem and progenitor cells (HSPCs) programs and modify drug responses. The integration of genomic profiling with clinical and cytogenetic data in the recent IPSS-M has significantly improved risk stratification of patients with myelodysplastic syndromes (MDS). Spliceosome mutations in SF3B1 are common initiating events in MDS and confer favorable prognosis, while secondary mutations in RUNX1 and STAG2 transform this low-risk condition into high-risk MDS or secondary acute myeloid leukemia. To understand how these events cooperate with SF3B1 mutations, we modeled these clonal trajectories in primary human HSPCs. SF3B1 K700E mutation was introduced in CD34+ cells isolated from cord blood or peripheral blood of healthy donors, using CRISPR/Cas9 editing and AAV6-mediated homology directed repair. By introducing an intronic fluorescent reporter, this system allows for the tracking and isolation of SF3B1-mutant cells for molecular and functional analysis. Edited cells were heterozygous for the K700E mutation and recapitulated alternative 3' splice site selection of genes previously identified in SF3B1-mutant patients. To model disease progression, we introduced secondary RUNX1 (S-R), STAG2 (S-S), or control AAVS1 (S-A) mutations into SF3B1 K700E knock-in CD34+ HSPCs, leading to a reduction of RUNX1 or STAG2 expression in SF3B1-mutant cells (50% and 90%, respectively). The S-R and S-S trajectories induced divergent alterations in lineage specification of SF3B1-mutant HSPCs. S-R promoted myeloid skewing at the expense of the erythroid lineage, while S-S induced a block in differentiation, impairing both myeloid and erythroid differentiation. Consistently, introduction of secondary RUNX1 or STAG2 mutations in an induced pluripotent stem cell (iPSC-HPCs) model of SF3B1-mutant MDS recapitulated S-R myeloid expansion and S-S maturation arrest identified in primary HSPCs. S-R and S-S genotypes resulted in divergent transcriptional programs including inflammation, immune response, and myeloid differentiation. However, both high-risk clonal trajectories expanded the immature CD34+CD38- HSCs/multipotent progenitors (MPPs). S-R selectively expanded CD34+CD38-CD133+ HSCs, whereas S-S expanded the more mature CD34+CD38-CD133- population. These data suggest that, despite divergent molecular pathways, high-risk mutations converge on expansion of stem cell potential of SF3B1-mutant HSPCs. To understand how genetic (co-mutation patterns) and epigenetic (HSC state) heterogeneity can influence drug responses, we profiled a panel of spliceosome inhibitors and 166 FDA approved and investigational compounds. Both S-R and S-S genotypes conferred decreased response to single and combinatorial agents conventionally used for the treatment of myeloid neoplasms. High-risk genotypes maintained elevated sensitivity to SF3B inhibition, but conferred differential response to novel classes of spliceosome modulators, with STAG2 but not RUNX1 loss selectively promoting response to type I PRMTs inhibitor MS023. By contrast, CHK1 inhibitor Prexasertib was highly selective for SF3B1-mutant cells irrespective of co-mutations, inhibiting growth and cell cycle progression. Taken together these data indicate that the presence of SF3B1 mutation offers therapeutic vulnerabilities that can be leveraged to target high-risk genotypes, and different SF3B1 co-mutated genes can further modulate drug response of SF3B1-mutant cells. In conclusion, progression from low-risk SF3B1-mutant MDS to high-risk disease is mediated by molecularly distinct trajectories driven by RUNX1 and STAG2 mutations that converge on expansion of the HSC compartment. Moreover, clonal progression is associated with genotype-specific drug responses and increased resistance to standard agents, and ongoing studies are elucidating how genetic and epigenetic states affect therapeutic responses.

3211 – MULTIOMIC ANALYSIS OF BONE MARROW HEMATOPOIETIC STEM/PROGENITOR CELLS REVEALS A POTENTIAL EARLY STAGE OF MALIGNANCY OF CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL

Clonal hematopoiesis of indeterminate potential (CHIP) increases the risk to develop hematological cancers such as acute myeloid leukemia (AML) tenfold. Since AML has been shown to originate from a hematopoietic stem/progenitor cell (HSPC) we compared the transcriptional landscape of bone marrow HSPCs from a healthy donor and a CHIP carrier (mutations in DNMT3A, 1.1% VAF and TET2, 5.3% VAF) by performing single cell RNA and ATAC sequencing. Cell frequency analysis of HSCP subpopulations, defined by analysis of conserved genes in clusters after dimensional reduction, showed that the proportion of HSC was twofold higher in the CHIP donor whereas the lymphocyte progenitor and pre-B cells compartment was fourfold lower, indicating an overall myeloid bias in the CHIP donor. Gene Ontology analysis of differentially expressed genes in the HSC compartment showed an upregulation of genes involved in metabolic processes in CHIP HSCs, among which genes that are associated with the onset of AML such as MEIS1 and PBX1. Furthermore, the expression levels of genes that are involved in niche retention, such as CD44 and VLA-4 were significantly higher in CHIP HSCs, suggesting alterations in the architecture of CHIP bone marrow. In addition to differences in gene expression, chromatin accessibility analysis showed unique genomic peaks in the CHIP HSC. Analysis of these sequences for binding motifs of transcription factors showed an overabundance of GATA, CEBP and RUNX1 motifs, all believed to play major roles in the onset of AML. The transcriptional landscapes of HSPC from healthy and CHIP donors show distinct differences, indicating that even the absence of signs of hematological disease, changes associated with a possible onset of malignancy can already be observed and provide possible targets for early intervention. Clonal hematopoiesis of indeterminate potential (CHIP) increases the risk to develop hematological cancers such as acute myeloid leukemia (AML) tenfold. Since AML has been shown to originate from a hematopoietic stem/progenitor cell (HSPC) we compared the transcriptional landscape of bone marrow HSPCs from a healthy donor and a CHIP carrier (mutations in DNMT3A, 1.1% VAF and TET2, 5.3% VAF) by performing single cell RNA and ATAC sequencing. Cell frequency analysis of HSCP subpopulations, defined by analysis of conserved genes in clusters after dimensional reduction, showed that the proportion of HSC was twofold higher in the CHIP donor whereas the lymphocyte progenitor and pre-B cells compartment was fourfold lower, indicating an overall myeloid bias in the CHIP donor. Gene Ontology analysis of differentially expressed genes in the HSC compartment showed an upregulation of genes involved in metabolic processes in CHIP HSCs, among which genes that are associated with the onset of AML such as MEIS1 and PBX1. Furthermore, the expression levels of genes that are involved in niche retention, such as CD44 and VLA-4 were significantly higher in CHIP HSCs, suggesting alterations in the architecture of CHIP bone marrow. In addition to differences in gene expression, chromatin accessibility analysis showed unique genomic peaks in the CHIP HSC. Analysis of these sequences for binding motifs of transcription factors showed an overabundance of GATA, CEBP and RUNX1 motifs, all believed to play major roles in the onset of AML. The transcriptional landscapes of HSPC from healthy and CHIP donors show distinct differences, indicating that even the absence of signs of hematological disease, changes associated with a possible onset of malignancy can already be observed and provide possible targets for early intervention.

Effects of Caloric Restriction on the Epigenetic Landscape of Hematopoietic Stem Cells

During aging, alterations of hematopoietic stem cells are associated with functional decline of the blood system. Caloric restriction (CR) interventions have been reported to improve adult stem cells in other tissue types during aging so we sought to evaluate the effects of CR on the aged HSC compartment. We find significant epigenetic alterations in HSCs isolated from aged mice after life-long CR compared to ad libitum fed aged mice. We further evaluated the epigenetic landscapes and functional potential of aged HSCs shortly after allowing life-long CR mice access to ad libitum food. We uncover epigenetic modification associated with functional alterations of the HSCs, defining potential mechanisms by which restrictions in food consumption affect the aging hematopoietic compartment.

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Specificity and Heterogeneity of Trained Immunity in Hematopoietic Stem and Progenitor Cells

When innate immune cells and hematopoietic stem and progenitor cells (HSPCs) are exposed to pathogenic agents, they develop heightened responses to subsequent infection through a process called trained immunity. After exposure to a pathogen, bone marrow derived macrophages (BMDMs) generated from trained HSPCs are capable of enhanced pathogen clearance and cytokine production, while exhibiting persistent metabolic rewiring. Because some models of HSPC trained immunity are dependent on interferon gamma (IFNγ) signaling, and our lab has described extensive changes in HSC self-renewal and differentiation upon IFNγ exposure, we hypothesized that persistent IFNγ signaling induced by chronic infection results in reprogramming of HSPCs, causing improved non-specific immunity. To test our hypothesis, we generated a chimeric mouse model of trained immunity by transplanting control or M. avium-exposed HSPCs into naïve recipient mice. Mice that received M. avium trained HSPCs had decreased bacterial load, less splenomegaly, and fewer granulomas upon subsequent M. avium infection, indicating improved immunity. Furthermore, BMDMs generated from mice trained with a single dose of recombinant IFNγ (rIFNγ) exhibited increased pathogen clearance and metabolic profiles ex vivo. To test if rIFNγ training was sufficient to induce HSPC trained immunity in vivo, we isolated HSPCs from rIFNγ-exposed mice and challenged the recipients 4 months later. These in vivo experiments demonstrated that rIFNγ training was insufficient to induce substantial protection against subsequent M. avium challenge, but still induced BMDM metabolic rewiring four months post HSPC training. Collectively, our studies indicate that there are degrees of training that occur upon IFNγ exposure, likely related to the concentration and duration of the primary stimulus.To assess the specificity of cross protection of HSPC trained immunity, we utilized our chimeric mouse model and tested two different training and infection pathogens: M. avium and influenza. When we challenged M. avium-trained HSPC recipients with influenza, we found that although there was mildly decreased lung histopathology and increased production of IFNγ and TNFα, mice succumbed to infection like untrained controls. When we swapped the order of pathogens, we observed that mice receiving influenza-trained HSPCs produced BMDMs with increased killing capability and systemically higher IL-6 and RANTES levels, but these features were insufficient to significantly reduce bacterial CFU counts upon M. avium challenge. These transplant experiments indicate that trained immunity encoded in HSPCs is pathogen specific.To dissect the mechanism of M. avium-induced trained immunity in HSPCs, we performed RNAseq analysis on M. avium-trained HSPCs post-transplant and cross referenced it with RNAseq and WGBS data on primary M. avium-exposed HSPCs. These studies showed consistent differences in cellular signaling, metabolism, immunity, and antigen processing and presentation in the trained HSPCs, indicating that epigenetic and transcriptional reprogramming induced by M. avium exposure is durable following transplant and secondary challenge.To ascertain whether transcriptional changes are homogeneous throughout the HSC compartment, we completed scRNA-seq on naïve and M. avium-exposed hematopoietic cells. We found that genes upregulated upon M. avium exposure in HSCs, including Batf2 and Cxcl9, were induced in a subset of HSPCs, indicating that there is a heterogeneous response to training within the HSPC pool. Strikingly, the trained immunity signature was maintained in neutrophils and macrophages but lost in mature B cells, indicating specific propagation of genetic signatures induced by training in certain lineages. Finally, we found an emerging population of HSCs with B cell gene signatures upon M. avium exposure. Emergence of this HSPC subpopulation may suggest the development of a cell that acts as a direct intermediate between HSC and B cells following training.Our work shows that trained immunity induced by M. avium and persistent IFNγ signaling is pathogen-specific and heterogeneous among primitive HSPCs. Emergence of specific responder cell populations within the HSPC pool may be responsible for enhanced protection against specific infection stimuli, whereas the presence of non-responders may insure long term health of the HSPC pool. DisclosuresNo relevant conflicts of interest to declare.

Elevated Central Carbon Metabolism - a Hallmark for Senescent Cells in Aging Human Hematopoietic Stem Cell Compartment

Comprehensive proteomic studies of HSC derived from bone marrow of healthy human subjects (n = 59) in different age groups (range: 20 - 72 years) showed that aging HSCs are characterized not only by myeloid lineage skewing, senescence associated secretory phenotype (SASP), accumulation of reactive oxygen species (ROS), anti-apoptosis, but prominently by elevated glycolysis, glucose uptake, and accumulation of glycogen. This is caused by a subset of HSC that has become more glycolytic than others and not on a per cell basis. Subsequent comparative transcriptome studies of HSCs from human subjects >60 years versus those from <30 years have confirmed this association of elevated glycolysis with aging transcriptome signature.Provided with this background and based on glucose metabolism levels, we have developed a method to isolate human HSCs (CD34+ cells) from bone marrow into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GU high, GU inter, GU low). For human subjects >60 years old (n=9), the proportions of these subsets are: GU high= 5.4+3.5 %, GU inter= 66.4+22.5 %, GU low= 28.2+21.7 %. For subjects <30 years (n=5), the proportions are GU high= 1.7+1.5 %, GU inter= 66.5+36.9 %, GU low= 31.8+36.7. Single-cell RNA-sequencing (scRNA-seq) studies and gene ontology analysis of biological processes revealed that, compared to the GU inter and GU low subsets, the GU high cells showed a significantly higher expression of genes involved in myeloid development, inflammation response (AIF1, CASP2, ANXA1, ZFP36), anti-apoptosis (GSTP1, NME1, BCL2, DMNT1, BAX), cell cycle checkpoint (MCL1, CDK1, CDK4, EIF5A), histone regulation (BCL6, EGR1, KDM1A, MLLT3), b-galactosidase, and significantly lower expressions of genes involved in lymphoid development, and of MDM4, MDM2, FOXP1, SOX4, RB1. Functional studies indicated that the glycolytic enzymes were elevated in elderly HSCs, and the GU low subset corresponded to primitive and more pluripotent HSCs than the GU interand GU high subsets. Pathway analyses have then demonstrated that the GU high subset is associated with up-regulated p53 as well as JAK/STAT signaling pathways, characteristic of senescent HSCs observed in murine models. Applying Gene Set Enrichment Analysis (GSEA) algorithms, we have compared the scRNA-seq data of CD34+ cells derived from young (<30 years) versus older (>60 years) subjects, as well as the scRNA-seq data from GU high subset versus GU inter and GU lowsubsets from each individual subject (n = 6). The results are shown in Figure 1. In analogy to the comparison between old (>60 years) versus young (<30 years) HSCs (CD34+ cells), GSEA of the GU high versus GU inter and GU low subsets shows the same pattern of changes - significant upregulation of gene-set expressions for (a) inflammatory response (b) G2M checkpoint, (c) MTORC1, (d) ROS, (Fig. 1B), (e) allograft rejection; and down-regulation of gene-set expressions for (f) pluripotency, (g) androgen response, (h) UV response (Fig. 1C) as well as (i) interferon-a induction during SARS-CoV2-infection (data not shown in Fig. 1). Thus, our novel findings of elevated glycolysis coupled with significant activation of MTORC1 in the senescent cells of the HSC compartment have provided evidence for the important role of calorie restriction (CR) for healthy aging of HSCs. In numerous animal models, aging has been shown to be driven by the nutrient-sensing MTORC1 network. In animal models of aging, CR has been reported to deactivate the MTOR pathway, thus slowing aging and delaying diseases of aging.Conclusion: In a series of multi-omics studies, we have demonstrated that the GU high subset is identical to the senescent cells (SCs) in human HSC compartment. Studies in animal models have shown that SCs in murine bone marrow are responsible for driving the aging process, and elimination of this subset by inhibitors of anti-apoptotic factors is able to rejuvenate hematopoiesis in mice. Our present results have provided cellular and molecular evidence that SCs in human HSC compartment are also dependent on anti-apoptotic factors, elevated MTORC1 as well as increased glycolysis for survival. Inhibition of MTORC1 or glycolysis, either by specific inhibitors or by CR, may eliminate senescent HSCs and promote rejuvenation of human hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Transcriptomics Study of Human Hematopoietic Progenitors Reveals Alterations Associated with Aging and Myeloid Malignancies

Hematopoietic stem and progenitor cells (HSPCs) comprise a continuum of cells with varying differentiation potential and priming toward specific lineages. During both healthy aging and myeloid malignancies, changes occur in the composition and regulation of HSPCs. In this study, we evaluated human HSPCs obtained from young and elderly healthy donors using single-cell RNA sequencing to identify the transcriptional and regulatory alterations associated with aging at single cell resolution. We then applied this knowledge to the study of specific perturbations associated with the development of myeloid pathologies.We isolated >90,000 bone marrow CD34+ cells from 5 young (18-20 y/o), 3 elderly (>65 y/o) healthy donors, 1 patient with myelodysplastic syndrome (MDS) and 1 patient with acute myeloid leukemia (AML), using fluorescence-activated cell sorting. scRNA libraries were prepared with the 10X chromium platform and sequenced. Finally, bioinformatic analysis was performed using available R and Python algorithms such as Seurat, Palantir and Scenic.First, we characterized HSPC subpopulations in young donors by unsupervised clustering and manual annotation. Taking the previous findings as reference, we then classified the elderly and pathological HSPC using elastic-net regularization prediction models (Figure 1A).Comparison of subpopulations in young and elderly donors confirmed the age-related increase in HSC, as well as reduction of lymphoid progenitors and myelomonocytic compartments. Next, we performed differential expression and pathways analysis to uncover age-associated alterations in the transcriptional profile of cells with the same identity. We found a generalized enrichment in elderly HSPC of pathways activated upon stress and inflammation, such as p53, hypoxia and TNF alpha response. This suggests an age-related increased response to the more inflammatory microenvironment of elderly individuals. On the other hand, young HSPC were enriched for cell cycle activation and proliferation pathways, as well as metabolic processes (Figure 1B).Using trajectory analysis, we recovered 6 differentiation paths present in our young donor's data. When compared to the elderly, the greatest changes occurred along the monocytic trajectory. For some genes, expression differed through the whole trajectory, indicating the existence of original transcriptional alterations already at the HSC compartment. On the other hand, expression of myelomonocytic differentiation markers, such as MPO and CD74, reached lower levels in our elderly HSPC data, pointing towards a loss of capacity for monocytic differentiation in progenitors from elderly individuals.Finally, to identify key transcription factors regulating the progression of differentiation routes, we built gene regulatory networks. Overall, we found lower activation levels for transcriptional programs in the early progenitors from elderly donors. In addition, gene ontology enrichment analysis showed that the active networks in the young were enriched for differentiation-related terms, while networks from the elderly were not. These results also indicate an age-associated loss of differentiation capability.We then applied the same computational tools to analyze aberrant hematopoiesis in samples from 2 patients suffering from myeloid malignancies (MDS and AML). On one hand, we subjected the MDS sample to trajectory analysis, focusing on the erythroid lineage. We observed perturbations in the expression dynamics of genes playing a role in erythropoiesis. In the AML sample, we encountered a significant expansion of the most immature cell compartments (HSC, LMPP and MEP). In addition, GRN reconstruction showed up the specific activity of transcription programs activated by factors deregulated during leukemia, such as ZSCAN18 and GFI1.In conclusion, our work described the transcriptional alterations that occur in early hematopoiesis, both during healthy aging and myeloid pathology. We used multiple approaches, such as the study cellular proportions, differentiation trajectories and GRNs. The inclusion of samples from patients with myeloid pathology provided insights into the potential role of single-cell technologies for understanding and treating hematological malignancies. [Display omitted] DisclosuresSanchez-Guijo: Gilead: Consultancy, Honoraria; Celgene/Bristol-Myers-Squibb,: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Diez-Campelo: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Valcarcel: BMS: Consultancy, Honoraria, Speakers Bureau; CELGENE: Consultancy, Honoraria, Speakers Bureau; ASTELLAS: Consultancy, Honoraria, Speakers Bureau; AMGEN: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Speakers Bureau; JAZZ: Consultancy, Honoraria, Speakers Bureau; SOBI: Consultancy, Honoraria, Speakers Bureau; SANOFI: Consultancy, Honoraria, Speakers Bureau. Romero: 10X Genomics: Current Employment. Prosper: Janssen: Honoraria; Oryzon: Honoraria; BMS-Celgene: Honoraria, Research Funding.

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders.Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs.To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies.Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Display omitted] DisclosuresFrank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

NOTCH1 Aberrant Activation Is a Common Nonmutational Early Event in Chronic Lymphocytic Leukemia Hematopoietic Stem-Cells

Chronic lymphocytic leukemia (CLL) is a B cell disorder characterized by constitutive NOTCH1 activation. NOTCH1 mutations are recurrently associated with CLL providing a new clonal marker to track CLL origin. To investigate CLL-initiating cells, we assessed NOTCH1 mutational status and signaling in hematopoietic stem (HSCs) and progenitor cells from CLL bone marrow. We collected a total of 29 bone marrow (BM) samples including 22 CLL patients and 7 healthy donors (HDs). BM cells were sorted into CD34+CD38- HSCs (purity 94.23% ± 3.04% ) and CD34+CD38+ progenitor fraction (98.12% ± 1.34%). While Sanger sequencing analysis of the NOTCH1 failed to detect alterations in CD34+/CD38- HSCs, AS-PCR and Droplet digital PCR (ddPCR) indicated the presence of small HSCs mutated clones in 57% of cases. Altogether, these data confirm that NOTCH1 mutation is an early event in CLL hematopoiesis. The analysis of CD34+/CD38+ progenitors detected the NOTCH1 mutation in the majority of samples (66%). The NOTCH1 mutational burden progressively increased along specific stages of HSC differentiation (6.4%± 4.7 in CD34+CD38- to 14.9%±11.3 in CD34+CD38+CD10+CD19+ cells, 22.7%±6.5 in CD34-CD38+CD10+CD19+ cells and 40.5%±4.3 in neoplastic CD5+CD19+ cells). This suggests that the NOTCH1 lesion is selected and expands during HSC differentiation toward a B neoplastic cell, thus strengthening the hypothesis that the genetic alteration is an initial event associated with the stepwise malignant transformation of CLL. Conversely, CD34+ cells (1x106) from CLL patients carrying the mutation, when transplanted into NOD/SCID/IL2Rgnull (NSG) mice generated a mature CLL phenotype (the median percentage of CD5+CD19+ cells in hCD45+ cells was 38.5% and 43.8% in BM and spleen respectively) lacking NOTCH1 mutation (as showed by AS-PCR assay performed on enriched hCD45 cells). This data is more in line with the hypothesis that this genetic abnormality is acquired at the mature B cell stage as an additional leukemogenic event to transform into clinical CLL. Thus, we analyzed the NOTCH1 signaling status in HSCs and progenitor cells of NOTCH1-mutated and unmutated CLL samples. The flow-cytometric analysis of the active NOTCH1-ICN protein level showed unmutated and mutated CLL has a significantly higher NOTCH1-ICN level than HDs samples in both CD34+/CD38- HSCs and CD34+CD38+ (73.4%±22.9 and 83%±16.4 vs 33.3%±14.8; 94.4%±7.3 and 92.8%±4.3 vs 47.9%±13.8, p<0.01 and p<0.001, respectively). c-MYC expression was found significantly higher in HSCs cells from NOTCH1 mutated and unmutated CLL samples compared to HD (3.5±0.7 and 2.6±0.08 vs 1.3±0.1). Western blot analysis of the expression levels of the NOTCH1-TM subunit revealed that HSCs from mutated and unmutated CLL patients always expressed the NOTCH1-TM protein, compared to HDs where NOTCH1-TM was either absent or expressed at lower levels. The present study indicated that the pool of CD34+ cells, including HSC and progenitor compartments, have NOTCH1 aberrantly expressed and activated in CLL patients compared to HDs demonstrating a common nonmutational NOTCH1 activation occurring early in CLL hematopoiesis. This selective pressure might contribute to the onset of specific NOTCH1 mutations in a DNA context that is prone to spontaneous microdeletion. These data represent a rationale for the use of therapies targeting the NOTCH1 signaling in CLL aimed to inhibit the survival of CLL-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

Loss of De Novo DNA Methyltransferase DNMT3A Impacts Alternative Splicing in Hematopoietic Stem Cells

DNMT3A is a de novo DNA methyltransferase that is crucial to the maintenance of normal hematopoiesis. DNMT3A is frequently mutated in hematopoietic diseases, with mutations reported in over 20% of adult acute myeloid leukemia (AML), and 10-15% of myelodysplastic syndrome (MDS) patients. Our lab has shown that loss of Dnmt3a in mice leads to enhanced self-renewal of the HSC compartment and a block in differentiation, contributing to its role in clonal hematopoiesis (Challen et al. 2011). We have also shown that loss of DNMT3A leads to a spectrum of hematologic malignancies; however, changes in DNA methylation alone are insufficient to explain the leukemic transformation, suggesting that DNMT3A could also regulate stem cells independent of methylation. The mechanisms through which DNMT3A exerts its specific effects are unclear, in part due to our relatively poor understanding of its interactome.To address this gap in knowledge, we generated a doxycycline-inducible biotinylated-DNMT3A transgene in mouse ES cells. Using co-immunoprecipitation and mass spectrometry analysis of biotinylated-DNMT3A in murine ESCs, we identified several candidate interacting proteins. Along with known partners such as DNMT3L and DNMTB, factors associated with chromatin regulation (HDACs, NuRD complex), splicing factors (SF proteins, Hnrnps, and DDXs), and ribosomes (RPSs and RPLs) were pulled down. We validated these interactions by over-expressing myc-tagged DNMT3A in HEK293T cells. Since epigenetic regulators and splicing factors are recurrently co-mutated in hematologic malignancies, we validated its interaction with splicing factors such as DDX39B, SF3B3, and SF3B1 (among others) through co-IP of DNMT3A in CD34+ HSPCs. Furthermore, we found that these interactions are RNA and DNA-independent. N-terminal deletions of DNMT3A domains revealed that the catalytic domain is crucial for its interaction with splicing factors, and AML mutant, Arg882His, disrupts this interaction. These data suggest that through its interaction with splicing factors, DNMT3A may have a crucial role in RNA splicing efficacy.To determine how the putative interaction between DNMT3A and splicing factors affects hematopoiesis, we examined whether loss of Dnmt3a impacted RNA splicing in purified HSCs after competitive transplant using our Mx1-cre-inducible Dnmt3a knockout mouse model. Using intron retention as a measure of splicing efficacy, we performed deep RNA sequencing of poly-A-enriched mRNA from HSCs (LSK CD150+ CD48-). When comparing exon-intron junctions in Dnmt3a KO versus WT HSCs, Dnmt3a KO HSCs exhibited a greater degree of intron retention. Aberrant alternative splicing was observed in genes involved in HSC differentiation and self-renewal, splicing, ribosomal biogenesis, and cell cycle regulation, suggesting that its role in RNA splicing may be an important mechanism through which DNMT3A influences hematopoiesis. In addition, we delineate a key relationship between DNMT3A, intron retention, and HSC differentiation. Inducing differentiation of WT and Dnmt3a KO HSCs with 5-Fluorouracil (5-FU) revealed key differences in alternative splicing. As WT HSCs differentiate, intron retention decreases; however, Dnmt3a KO HSCs are unable to decrease intron retention in key hematopoietic differentiation genes, leading to a block in differentiation. This was also demonstrated after inducing differentiation in WT and Dnmt3a KO mouse ESCs with Retinoic Acid (RA).To determine if loss of DNMT3A also impacts splicing in human HSPCs, we used CRISPR/Cas9 to knock out DNMT3A in human HSPCs and transplanted CD34+ cells into NSG mice. RNA sequencing of CD34+ cells 3 months after transplantation revealed aberrant alternative splicing, specifically, greater intron retention in DNMT3A KO than control cells. Together, these data suggest that DNMT3A plays a crucial role in maintaining proper splicing in HSCs.In summary, this study reveals a novel role for DNMT3A in hematopoiesis. We show that DNMT3A interacts with splicing factors and impacts global alternative splicing patterns in HSCs. In addition, we demonstrate that decreasing intron retention has a specific role in HSC differentiation. The aberrant splicing patterns seen in Dnmt3a KO HSCs may be instrumental in decrypting the crosstalk between DNA methylation and alternative splicing, and their role in HSC differentiation. DisclosuresNo relevant conflicts of interest to declare.

Chromatin Regulator Mutations Are Drivers of Clonal Evolution in the Early Multipotent HSC Compartment of Primary Myelofibrosis

The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients. DisclosuresNo relevant conflicts of interest to declare.

Mutant PPM1D and TP53 populate the hematopoietic compartment after peptide receptor radionuclide therapy (PRRT) exposure.

10605 Background: Mutations in TP53 and PPM1D are putative drivers associated with therapy related-myeloid neoplasm (T-MN) and have been identified in pre-treatment blood samples obtained at the time of primary malignancy, predating clinically evident T-MN. Genomic analysis of patients(pts) who undergo leukemogenic therapies will help understand T-MN biology and devise risk mitigation strategies. PRRT (Lu 177) for neuroendocrine tumors is associated with enhanced risk of T-MNs. The mechanism for T-MN induced by PRRT is largely elusive due to the novelty of this drug. Methods: We analyzed initial (n=13) and serial blood samples (n=4) prior to and following PRRT for clonal mutations in order to elucidate the role of PRRT in exerting selective pressures on HSCs. Genomic DNA was analyzed using a targeted myeloid 100-gene panel and a variant allele frequency (VAF) cutoff 1% was used to call clonal hematopoiesis (CH). Results: Fifty-four percent pts had CH, despite relatively young age of cohort (median age 58 years, range 41-75) and minimal chemo-radiotherapy exposure; baseline characteristics and molecular profile of cohort is published [Singh et al. Blood 2020; 136 (Supplement 1): 35–36]. Serial sample analysis in 4 pts (Table 1) demonstrates that PRRT exposure is associated with clonal evolution and accompanying cytopenias in 75% (3/4) pts. Pt-1 (age 67) with normal baseline hemogram developed persistent cytopenias after PRRT, accompanied by emergence and expansion of mutant- PPM1D (m PPM1D; VAF 20%). These data suggest that cytopenias result from repopulation of the HSC compartment by m PPM1D cells. In Pts 2 and 3 (age 74 and 75), we note expansion of m TP53 and m PPM1D clones respectively, also associated with the development of cytopenias. Pt-4 was younger (age 59) and developed no cytopenias. Exposure to PRRT was associated with loss of m TET2 and m DDX41, possibly due to lack of clonal fitness of m TET2/DDX41 clones and the relatively young HSC microenvironment. Conclusions: We conclude that mutations in PPM1D and TP53 are clinically relevant, contribute to clonal cytopenias and may increase risk of future T-MN. The temporal association of m TP53 and m PPM1D expansion with PRRT exposure in our analysis suggests selection of these clones in response to PRRT-induced stress, outcompeting wild type and less therapy-resistant HSCs. Our study along with others will inform future efforts to strategize methods of surveillance and early detection for clonality assessment and chemoprevention, to reduce adverse effects of leukemogenic therapies.[Table: see text]

Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BackgroundHematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale.ResultsThrough the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state.ConclusionsOur results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia.

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

FOXO activity adaptation safeguards the hematopoietic stem cell compartment in hyperglycemia

AbstractHematopoietic stem cell (HSC) activity is tightly controlled to ensure the integrity of the hematopoietic system during the organism's lifetime. How the HSC compartment maintains its long-term fitness in conditions of chronic stresses associated with systemic metabolic disorders is poorly understood. In this study, we show that obesity functionally affects the long-term function of the most immature engrafting HSC subpopulation. We link this altered regenerative activity to the oxidative stress and the aberrant constitutive activation of the AKT signaling pathway that characterized the obese environment. In contrast, we found minor disruptions of the HSC function in obese mice at steady state, suggesting that active mechanisms could protect the HSC compartment from its disturbed environment. Consistent with this idea, we found that FOXO proteins in HSCs isolated from obese mice become insensitive to their normal upstream regulators such as AKT, even during intense oxidative stress. We established that hyperglycemia, a key condition associated with obesity, is directly responsible for the alteration of the AKT-FOXO axis in HSCs and their abnormal oxidative stress response. As a consequence, we observed that HSCs isolated from a hyperglycemic environment display enhanced resistance to oxidative stress and DNA damage. Altogether, these results indicate that chronic metabolic stresses associated with obesity and/or hyperglycemia affect the wiring of the HSCs and modify their oxidative stress response. These data suggest that the uncoupling of FOXO from its environmental regulators could be a key adaptive strategy that promotes the survival of the HSC compartment in obesity.

A Comprehensive High Resolution Scrna-Seq Analytical Platform for Characterizing Human Hematopoietic Stem/Progenitor Cells States in Ex Vivo Drug Products

Human hematopoietic/stem progenitor CD34+ cells (HSPCs) are the core components of ex vivo lentiviral gene therapy (GT) for the treatment of rare monogenic diseases. Assessment of CD34+ composition before and after genetic modification as well as in vivo upon transplantation in the patient is a critical parameter to establish the efficacy of GT. We recently showed how the currently available immunophenotyping markers for the identification of HSPC subpopulations do not allow a clear-cut identification of the diversity of cell states within the CD34+ compartment. Furthermore, due to the instability of such markers upon in vitro manipulation, immunophenotyping does not offer a reliable assay to measure the HSPC content within the CD34+ Drug Product (DP). We have combined immunophenotyping, FACS sorting, and selective in vitro manipulation conditions with single-cell RNA-Sequencing (scRNA-Seq) capturing a picture of unprecedent clarity of CD34+ cells dynamics before and after expansion and genetic modification. Using the 10X Chromium System we firstly generated a comprehensive baseline map of CD34+ cell states (total of 77,692 single cells) from the 3 main sources of human HSPCs: bone marrow (BM), mobilized peripheral blood (G-CSF+plerixafor mobilization, MPB) and cord blood (CB). The combination of unsupervised clustering and expression of lineage-associated gene signatures allowed the identification of 11 cell states within these bulk CD34+ populations, and unveiled key differences in their composition depending on different cells' origin. We then investigated the dynamic changes of MPB CD34+ cells, the most advanced and clinically relevant HSPC cell source, upon ex vivo manipulation. To this aim we cultured bulk CD34+ cells and 7 FACS-sorted CD34+ subpopulations independently for 40 hours in presence of SCF, IL3, FLT3-L and TPO. Classical immunophenotyping of the CD34+ population before and after culture showed a substantial enrichment of cells with a CD34+CD38- profile suggesting a selective in vitro maintenance and expansion of primitive progenitors. However, immunophenotyping of individually cultured HSPC subsets revealed that the major factor contributing to this observation is instead the progressive loss of CD38 expression by CD38+ committed progenitors. Thus, the currently available CD34+ immunophenotyping characterization is not designed to provide an accurate assessment of the true DP composition before infusion into patients. By means of scRNA-Seq analysis of 81,126 single cells we were able to identify with high granularity the changes in cell state of each HSPC subset during culture. Firstly, the combined scRNA-seq analysis on sorted HSPC subsets at baseline allowed to draw the highest resolution map of MPB CD34+ cell states available to date (total of 24,736 single cells) and allowed the identification of 7 novel transcriptional states which are independent from the original cell fractionation based on the known CD34+ surface markers. Using these transcriptional signatures we interrogated the dynamic changes of each HSPC subset looking for transcriptional divergences/similarities between the baseline and the end of culture cell states. We could identify the trajectories of each subpopulation towards different hematopoietic differentiation stages upon in vitro manipulation. As expected HSCs and MPPs were the only subsets which maintained after culture a fraction of cells retaining the original primitive transcriptional signature. We believe that in this way we could quantify with unprecedented accuracy the putative fraction of cells in the DP that preserved a multipotent long-term potential vs the one that progressed towards myeloid differentiation identifying in the process variable degrees of cell maturation. We are currently exploring specific molecular signatures within the HSC compartment, which remained stable before and after culture, for the identification of novel more reliable markers of human HSC to be validated in vitro and in vivo. Overall our analytical platform provides the basis for unravelling and comparing the impact of multiple conditions of cell cultures and gene modification on the HSPC DP. To our knowledge this constitutes the most advanced suite for the comprehensive characterization of CD34+ cells states, with potential applications spanning manufacturing, pre-clinical and clinical stages. CB and ML: equal contribution Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Loperfido:AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Yan:AVROBIO Inc: Current Employment. Barbarossa:AVROBIO Inc: Other: Trainee. Segura:AVROBIO Inc: Current Employment. Golipour:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company. Mason:AVROBIO Inc: Current Employment, Current equity holder in private company. Biasco:AVROBIO Inc: Current Employment, Current equity holder in publicly-traded company.

Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

Tet2 Deficiency Rejuvenates Hematopoietic Stem and Progenitor Cells during Ageing

Ageing is accompanied by a significant reduction of hematopoietic competence driven by various causes including epigenetic alternations [1-4]. Ten-eleven translocation 2 (TET2) has been well delineated as a critical epigenetic regulator that affects hematopoietic progenitor and stem cells (HSPCs) function. Tet2 deficiency confers advantages in clonal expansion of HSPCs and skews myeloid lineage differentiation, giving rise to increased risk of hematological malignancy transformation [5-8]. TET2 loss-of-function mutations are frequently detected in aged HSPCs [10-11], thereby raising the question of how Tet2 deficiency affects HSPCs self-renewal and lineage specification during ageing. To address this question, we harvested HSPCs from wild-type (WT) or Tet2KO young and aged donor mice, followed by competitive bone marrow transplantations to monitor age-dependent functional alterations. Despite the enlargement of the HSC pool size (the number of cells with regenerative potential) in aged mice, the aged WT HSPCs exhibited lower self-renewal capability and displayed impaired hematopoietic differentiation when competed against young stem cells. However, we found that both aged and young Tet2-deficient HSPCs shared comparable peripheral blood reconstitution, indicating no engraftment defects were caused by age for Tet2-deficient HSPCs. In parallel, scRNA-seq analysis revealed that Tet2 deficiency and age promoted the expansion of HSC compartment in a synergistic manner, leading to the largely augmented pool size of Tet2-deficient aged HSCs. But unlike aged WT stem cells, these expanded aged Tet2-null stem cells retained high self-renewal potential and possessed a competitive advantage of lineage outputs both in vitro and in vivo. Overall, through conducting repopulation assays and single-cell transcriptomes analysis, we have demonstrated that Tet2 ablation alters age-dependent HSC functional decline, revealing a disparate ageing process in the Tet2-deficient haemopoietic system. Disclosures No relevant conflicts of interest to declare.

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.