Early Hematopoietic Progenitors Research Articles

Single-cell transcriptomic analysis of hematopoietic progenitor cells from patients with systemic lupus erythematosus reveals interferon-inducible reprogramming in early progenitors.

Immune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason, we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature. BM samples from four SLE patients, six healthy controls, and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples, and single-cell RNA-sequencing was performed. A total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers, which were characteristic of each cell type, were identified. We identified three HSPC subpopulations, one of which consisted of proliferating cells (MKI67 expressing cells), one T-like, one B-like, and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures, in healthy BM of HSPC clusters 3 and 4 when compared with CB, and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations. This study revealed both quantitative-as evidenced by decreased numbers of non-proliferating early progenitors-and qualitative differences-characterized by an IFN signature in SLE, which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE, which may account for the immune aberrancies and the cytopenias in SLE.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

Novel Fusion Gene Aven-NUTM1 Induces Mice Myeloid Leukemia Vulnerable to HDAC Inhibitors

Chromosomal rearrangements involving the NUTM1 gene produce fusion genes commonly found in NUT carcinoma. While recurring NUTM1 fusions have been reported in other neoplasms, including AML, their roles in leukemogenesis remain unclear. We identified the AVEN-NUTM1 (AN) fusion gene in an AML patient resistant to standard chemotherapy, and went to assess AN's leukemogenicity via bone marrow transduction and transplantation. Mice transplanted with AN-transduced hematopoietic stem/progenitor cells (HSPCs) developed transplantable myeloid leukemia with a median survival of 342 days. RNA sequencing revealed downregulation of genes related to hematopoietic cell lineage commitment and upregulation of genes related to early hematopoietic progenitors and MYC-targeted genes in AN-transformed leukemic cells. Co-immunoprecipitation confirmed that AVEN-NUTM1 can interact with and activate EP300 as previously reported. To determine whether AN's interaction with EP300 is related to its ability to induce malignant transformation, we mutated the interaction site and constructed mutants of other structural domains. Colony-forming and replating assays showed that HSPCs expressing AN with mutations in the EP300 binding site lost their ability to self-renew and form colonies. A CUT&RUN assay followed by sequencing showed that the most enriched motif in overlapping peaks between AN and EP300 were ETS motifs, suggesting the involvement of an ETS transcription factor family member in AN-activated EP300-induced leukemia. We further found that both the patient's primary cells and bone marrow cells from leukemic mice were especially vulnerable to HDAC inhibitors. These results demonstrate that the AVEN-NUTM1 fusion gene can drive the development of myeloid leukemia in mice, AN's ability to bind and activate P300 is closely linked to its leukemogenicity, and that HDAC inhibitors are effective in targeting AN leukemia cells. The study provids valuable insights for the targeted treatment of NUTM1-related leukemia patients.

Elucidation of the Unique Function of GATA1s in the Development of Transient Abnormal Myeloproliferative Disorders

Transient abnormal myeloproliferative disorder (TAM) is a pre-leukemic condition that occurs at birth in children with Down syndrome. The responsible gene of TAM, GATA1, is a hematopoietic transcription factor integral to hematopoiesis. The GATA1 gene has two translation start sites, giving rise to two protein variants: GATA1-fl, a full-length protein, and GATA1s, an isoform lacking the N-terminal domain. Notably, TAM blasts express only the GATA1s due to a mutation in the GATA1 gene, as opposed to normal cells which express both proteins. This implies that the precancerous state is not triggered by the production of an aberrant protein butrather by the absence of the full-length protein and the exclusive expression of the minor isoform protein also present in normal cells. This is in stark contrast with the functions of many other oncogenes. Thus, the prevailing view is that TAM development involves a “loss of function” due to deficiency of GATA1-fl. However, some studies suggest that GATA1s may have unique functions that actively drive the TAM phenotype. Because of these conflicting findings reported, the precise pathomechanism of TAM development remains elusive, despite the identification of the responsible gene. To address this, we analyzed the function of GATA1s using an induced pluripotent stem cell (iPSC) model of TAM. We previously examined the effect of increasing GATA1s expression levels during early hematopoietic differentiation stage by stage using the TAM-iPSC model. We found that, in the absence of GATA1-fl protein, overexpression of GATA1s in early hematopoietic progenitor cells (HPC) shifted megakaryocytic differentiation toward myeloid lineage, intensifying TAM severity by expanding progenitor cells. These findings suggest that GATA1s has a unique function distinct from that of GATA1-fl. In this study, we investigated the function of GATA1s proteins as transcription factors using chromatin immunoprecipitation sequencing (ChIP-seq). Our results demonstrated that genes involved in hematopoiesis or cell proliferation belong to the gene cluster to which overexpressed GATA1s (O/E GATA1s) binds solely in the absence of GATA1-fl at the early HPC stage. Gene Ontology (GO) analysis further validated the significant enrichment of terms relevant to the TAM phenotype, including regulation of myeloid cell differentiation and negative regulation of cell differentiation. Intriguingly, the genes to which O/E GATA1s binds when GATA1-fl is present were unrelated to hematopoiesis or cell proliferation. These observations suggest that GATA1s uniquely binds to genes, instigating TAM onset under GATA1-fl deficient conditions, thus confirming TAM as a disorder hallmarked by the absence of GATA1-fl due to the GATA1 gene mutation. Our RNA-sequencing data indicate that, at the early HPC stage, O/E GATA1s only induces changes in binding to gene regions involved in TAM pathogenesis, without altering gene expression levels. In contrast, gene expression analyses of other later stage HPC fractions (megakaryocyte-erythroid progenitor-like cells: MEP-like cells), revealed increased STAT3 expression and enrichment of the JAK-STAT pathway and myeloid cell differentiation pathway. These results suggest that TAM development may be influenced by shifts in the gene-binding region of GATA1s in the early HPC, which, in turn, induces TAM in more differentiated MEP-like cells by skewing their differentiation towards the myeloid lineage. In conclusion, our study provides important insights into the pathological mechanisms of TAM pathogenesis, particularly with respect to the role of GATA1 in driving the TAM phenotype. the unique function of GATA1s and their specific binding to genes involved in TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis shed light on the complex interplay between GATA1s and TAM pathogenesis. Understanding these mechanisms may pave the way for targeted therapies aimed at modulating GATA1s activity and reducing the severity of TAM.

A Two-Part Study Evaluating the Combination of Tazemetostat and CPX-351 (Part 1) and Palbociclib with CPX-351 (Part 2) for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia

BACKGROUND AND SIGNIFICANCE Acute myeloid leukemia (AML) is an aggressive cancer of early hematopoietic progenitor cells with only 25-30% of patients (pts) surviving 5 years from diagnosis. For fit pts, conventional chemotherapy with DNA-damaging agents remains the mainstay of treatment. Conventional chemotherapeutic agents cause DNA-damage by targeting cells during DNA replication. Chromatin structure is highly influential in the efficacy of DNA-damaging agents, as areas of compacted chromatin are more resistant to DNA-damaging effects. Areas of DNA marked with histone 3 lysine 27 trimethylation (H3K27me3) are transcriptionally repressed and exhibit reduced accessibility to DNA-damaging agents. Enhancer of Zeste Homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repressive complex 2 which methylates H2K27 to produce a di- or trimethylated state (H3K27me2/3). In preclinical studies, treatment of AML cell lines with the combination of an EZH2 inhibitor (EZH2i) and doxorubicin resulted in an increase in DNA damage and apoptosis compared to those treated with doxorubicin alone. Further, a significant decrease in leukemia burden was seen in mice injected with THP-1 (AML) cells and treated with an EZH2i and doxorubicin and cytarabine compared to doxorubicin and cytarabine alone. This effect was further enhanced by pre-treatment of AML cells with palbociclib (PALB), a CDK4/6 inhibitor, by significantly increasing the number of cells in S phase when exposed to the EZH2i and doxorubicin combination, resulting in an increase in DNA damage and apoptosis compared to cells that were not pre-treated with PALB. These findings were reproduced in in vivo studies with mice injected with the primary AML cells (Porazzi P, et al. Cancer Res., 2021). STUDY DESIGN AND METHODS We will perform a 2-part phase I dose escalation study utilizing EZH2i tazemetostat (TAZ) in combination with CPX-351 induction chemotherapy and of PALB pre-treatment followed by CPX-351 in pts with relapsed or refractory (R/R) AML (NCT05627232). All pts will receive CPX-351 at standard dose on days 1, 3 and 5. Part 1 evaluate the addition of TAZ to CPX-351 via traditional 3+3 design. Pts will be treated with TAZ on days -1 to 6 at a starting dose of 600 mg twice daily to open chromatin concurrent with CPX-351 administration. Part 2 evaluate the pre-treatment with PALB via traditional 3+3 design. Pts will be treated with PALB starting dose of 100 mg daily on days -3 to -1 to induce cell cycle G1. PALB will be held on day 1 to allow cells to then enter S phase prior to administration of CPX-351. ( Figure 1). The study will enroll pts with R/R AML treated at Sidney Kimmel Cancer Center. Pts must be ≥18 years of age with R/R AML (non-acute promyelocytic leukemia) and have received at least 1 prior line of therapy and have adequate performance status and organ function. Pts whose participation would result in a total cumulative dose of daunorubicin greater than 550 mg/m 2 (450 mg/m 2 if they previously received mediastinal radiation) and those with active central nervous system involvement of AML, unstable cardiac disease or other severe concurrent disease will be excluded. The primary endpoints of Parts 1 and 2 will be to determine the maximum tolerated dose of TAZ and of PALB in combination with CPX-351, respectively. Secondary endpoints for Parts 1 and 2 include preliminary assessment of efficacy. Correlative studies to evaluate the on-target effect of TAZ on H3K27me3 and DNA-damage and of PALB on cell cycling will be performed at protocol-specified time points. If these two 2-drug combinations prove to be safe, we will amend the protocol to add Part 3 which will evaluate the safety of the 3-drug combination of pre-treatment with PALB followed by co-treatment with TAZ and CPX-351.

A Multimodal Single Cell Atlas of Adult Murine Haematopoiesis Reveals Nine Differentiation Trajectories and Epigenetic Regulatory Programs

Single cell genomics has revolutionised our understanding of differentiating systems and led to a reinterpretation of haematopoiesis, moving from a step-wise hierarchical tree model towards more continous differentiation landscapes. Recent single cell RNA sequencing (scRNA-seq) studies of Lineage- ckit+ (LK) mouse bone marrow progenitors have defined a highly granular landscape of differentiation from multipotent stem cells to committed progenitors in 8 different lineages and have served as a valuable reference landscape for comparisons with perturbation states. While these landscapes have permitted exploration and interrogation of early haematopoietic progenitors, as yet, no map of mouse total bone marrow haematopoiesis exists at single cell resolution, limiting efforts to fully understand the gene programs which specify and the cell surface phenotypes which identify maturing haematopoietic lineages nor the connection between early bifurcation decisions in progenitor cells and mature cellular outputs. To answer these questions, we performed droplet-based scRNA-seq and cell surface proteomics on whole bone marrow from 4 male and 4 female 12-week old C57Bl6 mice, using a panel of 138 antibodies against cell surface antigens. To increase representation of rare progenitor populations, we complemented total bone marrow mononuclear cells with c-kit enrichment and FACS sorted LK and LK sca1-positive (LSK) populations. After quality control filtering the single cell transcriptomes and proteomes, over 198,000 single cells were analysed separately before multimodal integration into a single UMAP embedding. Addition of cell surface proteome information to the scRNA-seq data through multimodal integration produced a representation with greater resolution of haematopoietic cell types, including improved resolution of T-cell subtypes and separation of a rare population of eosinophil progenitors which were previously indistinguishable from basophil progenitors. This integrated landscape of mouse haematopoiesis was then clustered and annotated though an iterative process of label transfer and manual annotation into 41 cell type states (Figure 1). Using cell fate analysis, which utilises transcriptional similarity and pseudotemporal ordering to estimate the probability of a single cell's commitment to defined terminal states, we were able to resolve cell fate probabilities towards nine haematopoietic lineages (erythroid, neutrophil, megakaryocyte, lymphoid, monocyte, dendritic cell (DC), plasmacytoid dendritic cell (pDC), basophil and eosinophil (Figure 2)). From these probabilities we defined nine trajectories from the earliest uncommitted progenitors to terminally differentiated populations, including a pDC trajectory involving both lymphoid and common DC progenitors. Analysis of trajectory-based gene expression trends provides a framework for discovery of lineage drivers as well as shared gene expression programs across multiple lineages. Leveraging cell surface proteome data permits the use of ‘in-silico-FACS’ to describe phenotypes of cell populations which exist at cell fate branch-points, for further in-vitro characterisation. To demonstrate the utility of our atlas as a reference, we projected perturbation datasets from 7 pre-leukaemic mouse models onto the atlas, allowing quantitation of mutation-associated cellular and tissue-scale alterations. Finally, we have leveraged the transcription-start site enrichment of 5' scRNA-seq to compute enhancer RNA (eRNA) expression in our atlas, allowing the identification of dynamic usage of specific enhancer elements during haematopoietic specification and thus providing a novel framework for correlation of eRNA expression (as a proxy for active distal chromatin regions) with gene expression trends along specific haematopoietic lineages.

Analytical Validation of an Automated Semiconductor-Based Next-Generation Sequencing Assay for Detection of DNA and RNA Alterations in Myeloid Neoplasms

Myeloid neoplasms are heterogeneous tumors derived from early hematopoietic progenitors. Most international guidelines, including the European LeukemiaNet 2022 update, recommend testing a comprehensive set of genes, most within a 3- to 5-day period for optimal treatment decisions. Next-generation sequencing gene panels are essential for identifying genetic alterations, risk stratification, and determining targeted therapies for myeloid malignancies. This study describes the analytical validation of the Oncomine Myeloid Assay GX v2 (Myeloid GX v2) in combination with the Ion Torrent Genexus System using commercial controls, 16 variant-negative samples, and 130 clinical samples of myeloid neoplasms. The Myeloid GX v2 panel detected single nucleotide variants (SNVs), insertions/deletions (indels) (allele frequency >5%), and gene fusions (minimum 11 fusion copies/μL) in synthetic controls with a sensitivity of 100%. Specificity for detection of SNVs, indels, or fusions in 16 variant-negative samples was 100%. Sensitivity for detection of SNVs, indels, and gene fusions in 130 clinical samples was 99%, 97%, and 100%, respectively. Overall precision was 100% for SNVs, 96% for indels, and 100% for fusions. The average turnaround time from nucleic acid extraction to results was 2 days. The Myeloid GX v2 panel is highly accurate and reproducible for the detection of SNVs, indels, and gene fusions in myeloid neoplasms. The ability to deliver clinically relevant results in a short time is key to providing personalized treatments.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Senti-202, a Selective, Off-the-Shelf, Preclinical CAR-NK Cell Therapy with CD33 and/or FLT3 Activating CAR, Healthy Cell Protection from Endomucin (EMCN) Inhibitory CAR and Calibrated Release IL-15 for Hematologic Malignancies Including AML

Background: Patients with CD33 and/or FLT3 malignancies, which encompass most myeloid malignancies such as acute myeloid leukemia (AML), have a grim prognosis and a high unmet need. There are no approved cell therapies for patients with AML due to paucity of targets and an immunosuppressive bone marrow milieu. SENTI-202, a preclinical CAR-NK cell is engineered with a logic-gated gene circuit to overcome these challenges. Methods: SENTI-202 is engineered with a FLT3 OR CD33 NOT EMCN logic-gated gene circuit and a proprietary calibrated release IL-15 (crIL-15). These 3 chimeric proteins are engineered onto a natural killer (NK) cell (healthy adult peripheral blood-derived) to create an off-the-shelf chimeric antigen receptor (CAR) NK cell therapy. The bivalent CD33 OR FLT33 (OR GATE) activating CAR (aCAR) enables concurrent targeting of CD33+ and/or FLT3+ AML cells which kills both AML blasts and leukemic stem cells (LSCs) in vitro and in vivo. This concurrent targeting of two tumor antigens could potentially provide longer remission and less chance of relapse. The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) protects healthy EMCN+ hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) from off-tumor toxicity to potentially aid in post-treatment reconstitution of a healthy hematopoietic system. The crIL-15 provides both autocrine and paracrine IL-15 stimulation to the CAR-NK cells (and surrounding immune cells) to promote cell expansion, persistence, and tumor killing. While previous presentations (Garrison et al., 2021) focused on the performance of individual SENTI-202 components in a cell, this presentation focuses on functional data from the complete logic-gated gene circuit and the crIL-15 together in a cell (i.e., the final CAR-NK with all 3 genetic elements). Results: We demonstrated delivery of all circuit components using a single retroviral vector. We then investigated activity of the resulting CAR-NK cells using a traditional in vitro cytotoxicity assay (E:T=1:2; 20hrs) in which SENTI-202 showed >90% killing of leukemia cells (e.g. SEM; P≤0.001), maintained significant serial killing potential (3 rounds; P≤0.0001; P≤0.001; P≤0.001), as well as robust killing against primary AML blasts and LSC-enriched target cell populations. Importantly, SENTI-202 also exhibited significant tumor cell killing and improved survival (P≤0.001) in an MV4-11 xenograft AML mouse model. We investigated the ability of SENTI-202 to reduce off-tumor toxicity toward EMCN+ healthy cells, including primary HSCs. Within in vitro assays, SENTI-202 provided >50% protection to FLT3+ CD33+ model healthy cells that also expressed the EMCN safety antigen, and similar significant protection was maintained through serial protection assays (3 rounds; P≤0.0001; P≤0.001; P≤0.0001). As further evidence of the selectivity imparted using the OR/NOT logic gating approach, in vitro assays with SENTI-202 resulted in killing of leukemia cells but not primary healthy human HSCs and HPCs (~42% protection) (P≤0.01). Finally, in the presence of SENTI-202 in vivo, the NOT GATE protected and enabled significant (P≤0.0001) expansion of EMCN+ FLT3+ CD33+ model healthy cells. Conclusions: In summary, preclinical evaluation of SENTI-202 demonstrated selective killing of CD33 and/or FLT3 expressing AML cell lines while protecting healthy HSCs and HPCs. This selectivity is imparted by the novel logic-gated gene circuit engineered into healthy adult NK cells. Clinical evaluation of SENTI-202 is planned to evaluate the safety and efficacy in patients with hematologic malignancies with high unmet need, including AML.

Single-Cell Transcriptomics Reveals Similarity of Aggressive Infant Acute Lymphoblastic Leukemia Cells to Early Hematopoietic Progenitors

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While the prognosis for children diagnosed with ALL has significantly improved in recent decades, the outlook for infants (under 12 months of age) with ALL remains bleak. In particular, patients with a rearrangement in the KMT2A gene (KMT2A-r) frequently relapse and have low event-free survival. Recent work shows that cancer cells from infant ALL patients with KMT2A-r show a high degree of transcriptional similarity to early lymphocyte precursors (ELPs), suggesting a molecular plasticity that may drive the aggressiveness of this cancer. In this study, we extend these findings to patients with germline KMT2A (KMT2A-g) and assess the predictive value of the ELP signature. Methods: 10x Genomics single-cell Multiome (v1 chemistry) sequencing was performed to generate RNA sequencing (scRNAseq) data for samples collected at diagnosis and remission from 34 patients presenting with ALL at < 12 months of age - 25 KMT2A-r patients and 9 KMT2A-g patients. KMT2A-r patients had either KMT2A-AFF1 (n = 12) or KMT2A-MLLT1 (n = 13) fusions. 19/25 KMT2A-r patients and 0/9 KMT2A-g patients subsequently relapsed. Sequencing data were aligned to GRCh38 and counted using cellranger-6.0.1, with downstream analysis performed using Seurat 4.0.2 in R 4.0.3. A logistic regression model from Khabirova et al., 2022, fit on published scRNAseq data from fetal bone marrow cells (Jardine et al., 2021), was subsequently tested on sequencing data to produce similarity scores for each infant ALL cell to cell types along the hematopoietic developmental trajectory. Results: Our findings confirm that cells from infant ALL patients with KMT2A-r show a high degree of transcriptional similarity to ELPs (Figure A). We find that this holds true at diagnosis (DX) regardless of whether the patients later relapsed or not. Moreover, we observe that in KMT2A-g infant ALL patients, cells show a lower degree of similarity to ELPs than their KMT2A-r counterparts, but nonetheless show a greater similarity to ELPs than other hematopoietic cells. We do not observe a high degree of similarity to ELPs at remission (MD) in any patient group, supporting the idea that it is leukemic blasts that are similar to ELPs. Strikingly, we also observe that among KMT2A-r patients, a higher proportion of cells highly similar to ELPs is not predictive of subsequent relapse (Figure B). Conclusions: Here we confirm that KMT2A-r infant ALL cancer cells are transcriptionally similar to ELPs, highly plastic hematopoietic progenitors. This plasticity may play a significant role in the aggressiveness of this cancer. We show that this similarity does not exist to the same extent in KMT2A-g patients, consistent with their better prognosis. Additionally, we demonstrate that proportion of ELP-like cells is not predictive of relapse among KMT2A-r patients, suggesting that more work is needed to better understand the significance of similarity to ELPs in this disease. Deeper investigation of gene expression in ELP-like leukemic blasts will shed further light on the molecular mechanisms of this cancer and facilitate improved treatment selection and development of novel therapies for infants with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heterogeneous run-and-tumble motion accounts for transient non-Gaussian super-diffusion in haematopoietic multi-potent progenitor cells.

Multi-potent progenitor (MPP) cells act as a key intermediary step between haematopoietic stem cells and the entirety of the mature blood cell system. Their eventual fate determination is thought to be achieved through migration in and out of spatially distinct niches. Here we first analyze statistically MPP cell trajectory data obtained from a series of long time-course 3D in vivo imaging experiments on irradiated mouse calvaria, and report that MPPs display transient super-diffusion with apparent non-Gaussian displacement distributions. Second, we explain these experimental findings using a run-and-tumble model of cell motion which incorporates the observed dynamical heterogeneity of the MPPs. Third, we use our model to extrapolate the dynamics to time-periods currently inaccessible experimentally, which enables us to quantitatively estimate the time and length scales at which super-diffusion transitions to Fickian diffusion. Our work sheds light on the potential importance of motility in early haematopoietic progenitor function.

The hepatic extramedullary hematopoiesis during experimental murine Schistosomiasis mansoni.

Many years ago, our research group has demonstrated extramedullary hematopoiesis in the peripheral zone of murine hepatic schistosomal granulomas. In the present study, we revisit this phenomenon using new technical and conceptual approaches. Therefore, newborn mice were percutaneously infected by Schistosoma mansoni cercariae and euthanized between 35- and 60-days post infection. Liver samples were submitted to histopathology and immunohistochemical analyses. Cells under mitosis and/or expressing Ki67 demonstrated the proliferation of hematopoietic cells both around the parasite’s eggs trapped in the liver and around hepatic vessels. After 50 days post infection, proliferating cells at different levels on differentiation were located preferentially in the peripheral zone of the granulomas, around the vessels and inside the sinusoids. The presence of acidic and sulfated glycoconjugates, reticular fibers and the absence of fibronectin characterized the microenvironment for attraction and maintenance of hematopoiesis. Some neutrophils secreted MMP9 from the earliest points of infection, indicating degradation of the extracellular matrix in regions of histolysis and a possible chemoattraction of hematopoietic stem cells to the liver. Fall-3+ cells and Sca-1+ cells indicated that early hematopoietic progenitors could be mobilized to the liver. Groups of vWF+ megakaryocytes suggest chemoattraction of these cells and/or migration, proliferation, and differentiation of very immature progenitors to this organ. The increase of blood vessels and extramedullary hematopoiesis in this environment, where markers of immature hematopoietic and endothelial cells have been identified, points to the possibility of the presence of progenitors for endothelial and hematopoietic cells in the liver during the infection. There is also the possibility of concomitant migration of more differentiated hematopoietic progenitors, that proliferate and differentiate in the liver, and the occurrence of angiogenesis caused by inflammation or release of ovular antigens that stimulate the activation and proliferation of endothelial cells. Altogether, these data increase knowledge about a murine model that is of interest for investigating the pathology of the schistosomiasis and also the dynamics of hematopoiesis.

Competition between hematopoietic stem and progenitor cells controls hematopoietic stem cell compartment size

Cellular competition for limiting hematopoietic factors is a physiologically regulated but poorly understood process. Here, we studied this phenomenon by hampering hematopoietic progenitor access to Leptin receptor+ mesenchymal stem/progenitor cells (MSPCs) and endothelial cells (ECs). We show that HSC numbers increase by 2-fold when multipotent and lineage-restricted progenitors fail to respond to CXCL12 produced by MSPCs and ECs. HSCs are qualitatively normal, and HSC expansion only occurs when early hematopoietic progenitors but not differentiated hematopoietic cells lack CXCR4. Furthermore, the MSPC and EC transcriptomic heterogeneity is stable, suggesting that it is impervious to major changes in hematopoietic progenitor interactions. Instead, HSC expansion correlates with increased availability of membrane-bound stem cell factor (mSCF) on MSPCs and ECs presumably due to reduced consumption by cKit-expressing hematopoietic progenitors. These studies suggest that an intricate homeostatic balance between HSCs and proximal hematopoietic progenitors is regulated by cell competition for limited amounts of mSCF.

Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

Leukemia-associated transcription factor mllt3 is important for primitive erythroid development in zebrafish embryogenesis.

MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains uncertain. Here, we examine the role of mllt3 in developmental hematopoiesis during embryogenesis using zebrafish. Cloning, sequencing, phylogenetic, and synteny analyses showed high evolutionary conservation between important functional domains of the zebrafish orthologue of mllt3 and MLLT3 in humans. Quantitative reverse transcription-PCR and in situ hybridization analyses revealed that mllt3 is maternally supplied and zygotically expressed throughout embryonic development, and that expression is highest between 10 and 24 hours post-fertilization (hpf) coincident with enrichment in the intermediate cell mass (ICM) and posterior blood island, which are the sites of the primitive and transient definitive hematopoiesis in zebrafish, respectively. Further, we found co-expression of mllt3 with the early hematopoietic progenitor markers tal1, gata2, and gata1a in the posterior ICM. By investigating zebrafish hematopoietic mutants, we discovered that mllt3 is involved in erythroid precursor formation. By 48-72 hpf, mllt3 expression proved to be restricted to non-hematopoietic tissues including head structures, pronephric tubules, and liver primordium. These findings establish a link between mllt3 and primitive erythropoiesis and provide the basis for future functional investigations.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Abstract Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution. Supported by NIH intramural funding

A review of the mutagenic potential of N-ethyl-N-nitrosourea (ENU) to induce hematological malignancies.

This review is intended to summarize the existing literature on the mutagenicity of N-ethyl-N-nitrosourea (ENU) in inducing hematological malignancies, including acute myeloid leukemia (AML) in mice. Blood or hematological malignancies are the most common malignant disorders seen in people of all age groups. Driven by a number of genetic alterations, leukemia rule out the normal proliferation and differentiation of hematopoietic stem cells (HSCs) and their progenitors in the bone marrow (BM) and severely affects blood functions. Out of all hematological malignancies, AML is the most aggressive type, with a high incidence and mortality rate. AML is found as either de novo or secondary therapeutic AML (t-AML). t-AML is a serious adverse consequence of alkylator chemotherapy to the cancer patient and alone constitutes about 10%-20% of all reported AML cases. Cancer patients who received alkylator chemotherapy are at an elevated risk of developing t-AML. ENU has a long history of use as a potent carcinogen that induces blood malignancies in mice and rats that are pathologically similar to human AML and t-AML. ENU, once entered into the body, circulates all over the body tissues and reaches BM. It creates an overall state of suppression within the BM by damaging the marrow cells, alkylating the DNA, and forming DNA adducts within the early and late hematopoietic stem and progenitor cells. The BM holds a weak DNA repair mechanism due to low alkyltransferase, and poly [ADP-ribose] polymerase (PARP) enzyme content often fails to obliterate those adducts, acting as a catalyst to bring genetic abnormalities, including point gene mutations as well as chromosomal alterations, for example, translocation and inversion. Taking advantage of ENU-induced immune-suppressed state and weak immune surveillance, these mutations remain viable and slowly give rise to transformed HSCs. This review also highlights the carcinogenic nature of ENU and the complex relation between the ENU's overall toxicity in the induction of hematological malignancies.

Globin vector regulatory elements are active in early hematopoietic progenitor cells

The globin genes are archetypal tissue-specific genes that are silent in most tissues but for late-stage erythroblasts upon terminal erythroid differentiation. The transcriptional activation of the β-globin gene is under the control of proximal and distal regulatory elements located on chromosome 11p15.4, including the β-globin locus control region (LCR). The incorporation of selected LCR elements in lentiviral vectors encoding β and β-like globin genes has enabled successful genetic treatment of the β-thalassemias and sickle cell disease. However, recent occurrences of benign clonal expansions in thalassemic patients and myelodysplastic syndrome in patients with sickle cell disease call attention to the non-erythroid functions of these powerful vectors. Here we demonstrate that lentivirally encoded LCR elements, in particular HS1 and HS2, can be activated in early hematopoietic cells including hematopoietic stem cells and myeloid progenitors. This activity is position-dependent and results in the transcriptional activation of a nearby reporter gene in these progenitor cell populations. We further show that flanking a globin vector with an insulator can effectively restrain this non-erythroid activity without impairing therapeutic globin expression. Globin lentiviral vectors harboring powerful LCR HS elements may thus expose to the risk of trans-activating cancer-related genes, which can be mitigated by a suitable insulator.

Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.