Differentiation Of Myeloid Progenitors Research Articles

Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients. CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells. To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.

Structural Basis for Activation of the Thrombopoietin Receptor (MPL) By Its Ligand (TPO)

Myeloproliferative leukemia protein (MPL), also known as thrombopoietin receptor, is a class I cytokine receptor expressed on hematopoietic progenitors that is critical for normal platelet production by promoting myeloid progenitor growth and differentiation toward the megakaryocyte lineage. The ligand for MPL, thrombopoietin (TPO), stimulates platelet production through binding MPL via two sequential sites, a high- and then a low-affinity interface that together induce MPL dimerization and result in an active conformation allowing downstream JAK2/STAT5 signaling. Despite intense interest in characterizing the molecular mechanism of the interaction between MPL and TPO over the last several decades, no experimental structural evidence has been reported. Here we present a 3.39 Å cryo-EM structure of the ectodomain of mouse MPL bound to TPO. TPO features both low and high affinity sites that each bind to one MPL monomer in agreement with previous functional studies. Our structure reveals that several patient mutations associated with congenital amegakaryocytic thrombocytopenia (CAMT) and aplastic anemia are found at MPL-TPO interaction sites. The structure further explains additional patient mutations, as well as published experimental mutations that have previously been the basis of structure-function studies. Building on this structure, we generated a molecular dynamics simulation model of the full length human MPL-TPO complex, revealing that TPO binding to full length MPL results in unexpected MPL-MPL intramolecular interactions that stabilize the dimer to allow for efficient signaling. Functional characterization of deleterious mutations predicted by our structure and modelling have revealed additional residues critical to MPL-TPO and MPL-MPL interactions, providing further validation of our proposed structure. Overall, our studies reveal new insight into a receptor-cytokine interaction critical for normal hematopoiesis and platelet production that are frequently mutated in disease, provide the structural basis underlying existing experimental findings, and provide additional insight into the nature of hematopoietic growth factor signaling which could benefit future therapeutic research.

Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates

Current immunotherapeutic targets are often shared between neoplastic and normal hematopoietic stem and progenitor cells (HSPCs), leading to unwanted on-target, off-tumor toxicities. Deletion or modification of such targets to protect normal HSPCs is, therefore, of great interest. Although HSPC modifications commonly aim to mimic naturally occurring phenotypes, the long-term persistence and safety of gene-edited cells need to be evaluated. Here, we deleted the V-set domain of CD33, the immune-dominant domain targeted by most anti-CD33 antibodies used to treat CD33-positive malignancies, including acute myeloid leukemia, in the HSPCs of two rhesus macaques, performed autologous transplantation after myeloablative conditioning, and followed the animals for up to 3 years. CD33-edited HSPCs engrafted without any delay in recovery of neutrophils, the primary cell type expressing CD33. No impact on the blood composition, reconstitution of the bone marrow stem cell compartment, or myeloid differentiation potential was observed. Up to 20% long-term gene editing in HSPCs and blood cell lineages was seen with robust loss of CD33 detection on myeloid lineages. In conclusion, deletion of the V-set domain of CD33 on HSPCs, progenitors, and myeloid lineages did not show any adverse effects on their homing and engraftment potential or the differentiation and functionality of myeloid progenitors and lineages.

Dose-dependent expression of GFI1 alters metabolism in the haematopoietic progenitors and MLL::AF9-induced leukaemic cells.

Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.

1487-P: Inosine Monophosphate Preferentially Regulates Myeloid Progenitor Differentiation toward Neutrophil Production in Diabetes

Previous studies described hyperglycemia-induced myelopoiesis and atherosclerotic progression in mice with type I diabetes. By targeted metabolomics, we reported the increase of inosine monophosphate (IMP) in granulocyte/monocyte progenitors (GMP) in db/db mice. Moreover, IMP treatment stimulated GMP proliferation, resulting in skewed myelopoiesis and enhanced acute pancreatitis progression. However, how IMP modulates granulocyte production is not known. To investigate that, equal amount of FACS-sorted GMP of wild-type mice were cultivated with PBS or 15 mM IMP for 5 days in vitro. FACS analysis revealed that the absolute number of neutrophils was 1.7-fold higher but the number of CD11b+ monocytes was 3.2-fold lower in IMP group compared to control (p<0.05 for both). Thereafter, FACS-sorted GMP treated with or without IMP for 3 days were harvested for single cell RNA sequencing. When classified by marker gene expression, 19 clusters representing 7 cell types were identified including neutrophils, monocytes, GMP, intermediate GMP/neutrophils, basophils, Megakaryocyte erythroid progenitors/megakaryocytes (MEP/Mgk), and platelets. The proportions of neutrophils and MEP/Mgk increased 1.3-, and 1.6-fold in IMP-treated GMP cells compared with controls (p=0.059 for neutrophils; p=0.032 for MEP/Mgk). Nevertheless, the percentage of other cell populations were comparable between two groups (p≥0.08 for all). Furthermore, cycling neutrophils in cluster 3 was 1.4-fold increase in IMP-treated GMP cells compared with controls (p=0.04). Accordingly, GO analysis and Gene set enrichment analysis illustrated that genes involved in cell cycles was enriched in cycling neutrophil clusters. In conclusion, our data demonstrated that IMP preferentially regulated GMP proliferation and differentiation toward neutrophil production, which provides new insights on the crosstalk between skewed metabolites and inflammation in diabetes. Disclosure Y.Dong: None. C.Yan: None. Y.Zhang: None. X.Yang: None. Y.Feng: None. Funding National Natural Science Foundation of China (81670765, 82070841)

Assay optimization for the objective quantification of human multilineage colony-forming units

Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining and validating the identity of progenitor populations isolated by methods such as fluorescence-activated cell sorting (FACS). However, current methods for CFU assays are limited in their ability to robustly assay multipotent progenitors with the ability to differentiate down the myeloid, erythroid, and megakaryocytic lineages because of the lack of specific growth factors necessary for certain lineage outputs. In addition, manual counting of colony types is subjective resulting in user to user variability in assessments of cell types based on colony and cell morphologies. We demonstrate that the addition of granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF into a collagen-based MegaCult medium containing IL-3, IL-6, SCF, EPO, and TPO allows for the differentiation of common myeloid progenitors into expected proportions of colonies containing granulocytic (G), monocytic (M), erythroid (E), and megakaryocytic (Mk) cells. Additionally, we demonstrate an objective method using in situ immunofluorescence (IF) with anti-CD66b, anti-CD14, anti-CD235a, and anti-CD41 to detect G, M, E, and Mk cells, respectively. IF stained colonies can be analyzed individually at a microscope or using high-throughput microscopy. Thus, our improvements to the culture conditions and method for assay readout increase the accuracy, reproducibility, and throughput of the myeloid CFU assay.

Characterization of the pro-inflammatory roles of the goldfish (Carassius auratus L.) M17 protein

The interleukin-6 family of cytokines possesses a diversity of roles with significant redundancy. The roles of these molecules have been relatively well characterized in mammals, with limited attention in other species. Progress has been made in the discovery of homologous molecules in fish. Here we report the characterization of pro-inflammatory properties of recombinant goldfish M17. Recombinant goldfish M17 enhanced phagocytosis, primed production of reactive oxygen intermediates, and was chemotactic to macrophages. Treatment of goldfish macrophages with LPS, heat-killed and live Aeromonas hydrophila resulted in higher M17 mRNA levels. Recombinant M17 (RgM17) induced dose-dependent production of IFNγ and IL-1β1 in goldfish macrophages. Furthermore, treatment of macrophages with rgM17 resulted in upregulation of transcription factors that were important in the differentiation of myeloid progenitors into monocytes/macrophages (Runx1 and GATA2). Our results indicate that goldfish M17 is an essential inflammatory cytokine for proliferation and differentiation of goldfish progenitor cells.

Tumor Necrosis Factor-Alpha/Tumor Necrosis Factor-Alpha Receptor 1 Signaling Pathway Leads to Thymocytes' Cell Death by Necroptosis in a Mouse Model of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is characterized by an increased proliferation and loss of differentiation of hematopoietic myeloid progenitors or precursors. Studies performed in AML-affected patients revealed a T cell deficiency characterized by a reduced thymic output and peripheral functional abnormalities. To assess for the thymus function during AML, we used an AML mouse model and showed a drastic thymic atrophy. We observed a massive loss among double (CD4+CD8+- DP) and single positive (CD4+/8+- SP) thymocytes. We assessed for the expression of different actors of cell death signalling pathways by RT-qPCR or Western blotting. When comparing leukemic to control mice, there was a significant increase in the expression of Mlkl gene, phosphorylated MLKL and RIPK3 proteins, and tumor necrosis factor (TNF)-alpha receptors 1 on DP and SP thymocytes. These findings revealed a necroptosis cell death which was also observed in vitro when using cultured wild-type thymocytes and recombinant TNF-alpha protein. Thus, we demonstrated that TNF-alpha plays a deleterious role in thymic function during AML by contributing to extensive thymocytes' death.

BTYNB, an inhibitor of RNA binding protein IGF2BP1 reduces proliferation and induces differentiation of leukemic cancer cells

Leukemia is a group of diseases characterized by altered growth and differentiation of lymphoid or myeloid progenitors of blood. The existence of specific clusters of cells with stemness-like characteristics like differentiation, self-renewal, detoxification, and resistance to apoptosis in Leukemia makes them difficult to treat. It was recently reported that an oncofetal RNA binding protein, insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), maintains leukemic stem cell properties. BTYNB is an inhibitor of IGF2BP1 that was shown to affect the biological functions of IGF2BP1 however, the effect of BTYNB in Leukemia is not properly established. In this study, we assessed the effect of BTYNB on leukemic cell differentiation and proliferation. We performed cell viability assay to assess the effect of BTYNB in leukemic cells. We then assessed cell morphology of the leukemic cells treated with BTYNB. Further, we conducted an apoptosis assay and cell cycle assay. We found the cell viability of leukemic cells was significantly decreased post treatment with BTYNBs. Further, a noticeable morphological change was observed in BTYNB treated leukemic cells. BTYNB treated leukemic cells showed increased cell death and cell cycle arrest at S-phase. Evidence from the upregulation of BAK and p21 further confirmed apoptosis and cycle arrest. The gene expression of differentiation genes such as CD11B, ZFPM1, and KLF5 were significantly upregulated in BTYNB treated leukemic cells, therefore, confirming cell differentiation. Collectively, our study showed inhibition of IGF2BP1 function using BTYNB promotes differentiation in leukemic cells.

Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.

CD40 is an important costimulatory molecule expressed on antigen-presenting cells (APCs) and plays a critical role for APC activation, offering a promising therapeutic target for preventing allograft rejection. Here, we developed a biodegradable nanoparticle small interfering RNA delivery system (siCD40/NPs) to effectively deliver CD40 siRNA (siCD40) into hematopoietic stem cells (HSCs), myeloid progenitors, and mature dendritic cells (DCs) and macrophages. Injection of siCD40/NPs not only down-regulated CD40 expression in DCs and macrophages but also inhibited the differentiation of HSCs and/or myeloid progenitors into functional DCs and macrophages. Furthermore, siCD40/NPs treatment significantly prolonged allograft survival in mouse models of skin allotransplantation. In addition to reiteration of the role of CD40 in APC activation, our findings highlight a previously unappreciated role of CD40 in DC and macrophage differentiation from their progenitors. Furthermore, our results support the effectiveness of siCD40/NPs in suppressing alloimmune responses, providing a potential means of facilitating tolerance induction and preventing allotransplant rejection.

Inhibiting the biogenesis of myeloid-derived suppressor cells enhances immunotherapy efficacy against mammary tumor progression.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1-based (PD-1-based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8+ T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.

TRAPPC1 is essential for the maintenance and differentiation of common myeloid progenitors inmice.

Myeloid cell development in bone marrow is essential for the maintenance of peripheral immune homeostasis. However, the role of intracellular protein trafficking pathways during myeloid cell differentiation is currently unknown. By mining bioinformatics data, we identify trafficking protein particle complex subunit 1 (TRAPPC1) as continuously upregulated during myeloid cell development. Using inducible ER-TRAPPC1 knockout mice and bone marrow chimeric mouse models, we demonstrate that TRAPPC1 deficiency causes severe monocyte and neutrophil defects, accompanied by a selective decrease in common myeloid progenitors (CMPs) and subsequent cell subsets in bone marrow. TRAPPC1-deleted CMPs differentiate poorly into monocytes and neutrophils invivo and invitro, in addition to exhibiting enhanced endoplasmic reticulum stress and apoptosis via a Ca2+ -mitochondria-dependent pathway. Cell cycle arrest and senescence of TRAPPC1-deleted CMPs are mediated by the activation of pancreatic endoplasmic reticulum kinase and the upregulation of cyclin-dependent kinase inhibitor p21. This study reveals the essential role of TRAPPC1 in the maintenance and differentiation of CMPs and highlights the significance of protein processing and trafficking processes in myeloid cell development.

SHP-2 Regulates Myeloid Cell Differentiation, Anti-Tumor Responses and Innate Immune Memory

PD-1 is a T cell checkpoint inhibitor and the most extensively exploited therapeutic target of cancer immunotherapy. PD-1-mediated T cell inactivation is attributed to the function of SHP-2 phosphatase, which is activated by recruitment to PD-1 cytoplasmic tail. However, T cell-specific SHP-2 deletion did not improve anti-tumor immunity or altered responses to anti-PD-1 immunotherapy. Previously, we determined that PD-1 is also expressed in common myeloid progenitors and granulocyte/macrophage progenitors, which accumulate during cancer-driven emergency myelopoiesis and give rise to immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs). In tumor-bearing mice with myeloid-specific PD-1 ablation, accumulation of MDSCs was prevented, while output of differentiated effector myeloid cells with monocytic lineage dominance was increased. However, the molecular mechanism remains elusive. It is also unknown whether a canonical PD-1:SHP-2 axis is operative in myeloid cells. Temporal activation of SHP-2 is critical for myeloid cell fate. Gain of function SHP-2 mutations, resulting in constitutive phosphatase activation, prevent myeloid differentiation and lead to the accumulation of immature myelocytes and development of leukemia. HOXA proteins, members of the HOX family of transcription factors involved in normal hematopoiesis, are targets of SHP-2, which dephosphorylates HOXA and prevents derepressing of HOXA target genes and myeloid cell differentiation. In addition, SHP-2 regulates the function of IFN regulatory factor (IRF)-8, a transcription factor that has a decisive role in myeloid progenitor differentiation toward monocytes and DCs. IRF8 phosphorylation is mandatory for its nuclear translocation and initiation of gene transcription. The cytoplasmic-nuclear shuttling of IRF8 is regulated by SHP-2, which dephosphorylates IRF8 and prevents its nuclear localization and transcriptional activation. We generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) in T cells (Shp2f/fLckCre) or myeloid cells (Shp2f/fLysMCre), and found that Shp2f/fLysMCre but not Shp2f/fLckCre mice had diminished tumor growth. As determined by RNA-sequencing, this was paralleled by the presence of inflammatory neutrophils and tumor-associated macrophages (TAMs) with molecular signatures of enhanced differentiation, phagocytosis and antigen-processing and presentation. SHP-2 deficient TAMs also had increased monocyte and dendritic cells (DCs) specification transcription factors, chemokine and cytokine production, and expression of immunostimulatory molecules that promote T cell recruitment and activation. Moreover, monocytes from tumor-bearing Shp2f/fLysMCre mice suppressed tumor growth after transfer to naïve recipients indicating development of innate immune memory, named trained immunity. In bone marrow myelocytes, SHP-2 deletion enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8 that promote myeloid differentiation and monocytic/moDC lineage commitment, respectively. In contrast, in WT bone marrow myelocytes GM-CSF induced PD-1 expression, phosphorylation and interaction with SHP-2, the Src family kinase Lyn, and GM-CSF receptor beta chain, indicating that the PD-1:SHP-2 axis targets a key pathway of myelocyte differentiation. Our results reveal a previously unidentified mechanistic role of SHP-2 and the PD-1:SHP-2 axis in regulating myeloid cell differentiation in the context of cancer and provide evidence that SHP-2 poses a signaling restrain to myeloid differentiation and monocyte lineage commitment resulting in a myeloid landscape that suppresses anti-tumor immunity. This process is hijacked by the PD-1/PD-L1 pathway.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.

Immunosenescence, Inflammaging, and Frailty: Role of Myeloid Cells in Age-Related Diseases.

The immune system is the central regulator of tissue homeostasis, ensuring tissue regeneration and protection against both pathogens and the neoformation of cancer cells. Its proper functioning requires homeostatic properties, which are maintained by an adequate balance of myeloid and lymphoid responses. Aging progressively undermines this ability and compromises the correct activation of immune responses, as well as the resolution of the inflammatory response. A subclinical syndrome of “homeostatic frailty” appears as a distinctive trait of the elderly, which predisposes to immune debilitation and chronic low-grade inflammation (inflammaging), causing the uncontrolled development of chronic and degenerative diseases. The innate immune compartment, in particular, undergoes to a sequela of age-dependent functional alterations, encompassing steps of myeloid progenitor differentiation and altered responses to endogenous and exogenous threats. Here, we will review the age-dependent evolution of myeloid populations, as well as their impact on frailty and diseases of the elderly.

Adenosine Signaling Perturbs Erythropoiesis and Promotes Myeloid Differentiation

BackgroundAdenosine is a major signaling nucleoside that activates cellular signaling pathways through a family of four different G protein-coupled adenosine receptors (ARs), A 1, A 2A, A 2B, and A 3. At steady state conditions, extracellular levels of adenosine remain low (10 to 200 nM) either through its rapid cellular uptake by specialized nucleoside transporters, mainly through the equilibrative nucleoside transporter 1 (ENT1), or its degradation by adenosine deaminases. However, the extracellular levels of adenosine can be rapidly elevated up to 100 μM in response to hypoxia, inflammation, or tissue injury. Under pathophysiological conditions, adenosine signaling is involved in modulating inflammation, fibrosis, and ischemic tissue injury. In sickle cell disease (SCD), adenosine signaling is enhanced and contributes to the pathophysiology of the disease. Despite the importance of adenosine signaling in regulating cell proliferation, and stem cell regeneration, as well as in red blood cell functions and adaptation to hypoxia, very little is known about its implication in hematopoiesis, and more specifically during erythropoiesis. Here, we aimed to investigate the effects of high extracellular adenosine on the erythroid commitment and differentiation of hematopoietic progenitors, and to decipher the implication of ARs in these processes.ResultsTo investigate the role of high extracellular adenosine in regulating erythroid commitment and differentiation of hematopoietic progenitors, we performed ex vivo erythropoiesis of healthy CD34 + cells in the presence or absence of increased extracellular adenosine concentrations ranging from 10 to 200 µM. Our results showed that adenosine decreases erythroid proliferation in a dose dependent manner. High adenosine levels (>50μM) inhibited the proliferation of erythroid precursors and increased apoptosis via a cell cycle arrest in G1. Accordingly, western blots revealed the accumulation of p53 and its downstream target p21, a well-known mediator of G1 cell-cycle arrest, in adenosine-treated cells.Moreover, adenosine treatment led to the persistence of a non-erythroid GPA neg subpopulation expressing myeloid markers (CD18, CD11a, CD13, CD33). May-Grünwald Giemsa staining of this subset revealed granular cells at different stages of differentiation. The culture of FACS-sorted CD36 + and CD36 - cells suggested that this adenosine-induced GPA neg population originates from the survival of CD36 - myeloid progenitors even in the presence of erythropoietin. Importantly, these effects were specific to adenosine as neither guanosine, uridine nor cytidine affected the proliferation and differentiation of erythroid precursors. Furthermore, we have recently shown that ENT1-mediated adenosine uptake is essential for optimal erythroid differentiation. Therefore, we suggested that elevated extracellular adenosine perturbs erythropoiesis via its signaling upon ARs activation. To confirm this hypothesis, we assessed the effect of ARs activation during erythropoiesis. Given that A 2B and A 3 are the only known ARs expressed in human hematopoietic progenitors and erythroid precursors, we used BAY60-6583 and CI-IB-MECA, two highly selective agonists for A 2B and A 3 receptors, respectively. Both BAY60-6583 and CI-IB-MECA increased apoptosis and decreased erythroblast maturation and enucleation, while only Cl-IB-MECA led to the upregulation of CD33 and CD11a myeloid markers and promoted the differentiation of a GPA neg myeloid subpopulation.ConclusionOverall, our results place adenosine signaling as a new player in hematopoiesis regulation. Adenosine signaling via A 3 perturbs erythropoiesis and promotes the survival and differentiation of myeloid progenitors even in an erythroid favoring environment. While the activation of A 2B hampers optimal erythropoiesis without impacting the myeloid differentiation. As both ineffective erythropoiesis and increased leucocyte counts are reported in SCD, and given the detrimental role of high adenosine levels in its pathophysiology, further studies are ongoing to address the impact of adenosine signaling on hematopoiesis in this disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PD-1 Mediates Lineage Fate Determination and Function of Myeloid Cells in Response to Tumor-Mediated “Emergency” Myelopoiesis

PD-1 is an inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as “emergency” myelopoiesis. This process is co-opted by tumors to evade the immune system by inducing potent immunosuppression. Cancer-driven granulo-monocytopoiesis modifies the composition of the hematopoietic output by stimulating the expansion of tumor promoting immature myeloid populations, defined as myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Monocytic (M-) and granulocytic (PMN-) MDSCs and TAMs differentiate from granulocyte/macrophage progenitors (GMP) in the bone marrow. We analyzed the myeloid compartment of tumor-bearing mice to determine the expression and function of PD-1 in myeloid cells during cancer-driven emergency myelopoiesis. Using three different mouse tumor models (MC38, B16 and MC17), we determined that PD-1 and its natural ligand PD-L1, were expressed at high levels in all subsets of myeloid cells isolated from the spleen of tumor-bearing mice, including CD11b+Ly6C+ M-MDSC, CD11b+Ly6G+ PMN-MDSC, CD11b+F/480+ macrophages and CD11b+HLA-DR+ dendritic cells (DC). This was in contrast to the significantly lower or undetectable levels of PD-1 expressed in these cellular counterparts isolated from non-tumor bearing mice. High expression of PD-1 and PD-L1 was also identified in these myeloid subsets isolated from the tumor site and at sites of secondary myelopoiesis in the gut. Because these myeloid cell populations differentiate from GMP upon cancer-mediated emergency myelopoiesis, we examined bone marrow myeloid progenitors in tumor bearing and control, non-tumor bearing mice. In non-tumor bearing mice, PD-1 was detected in Lin-/Sca+/Kit+ (LSK) myeloid progenitors, whereas PD-L1 was expressed in Lin-/Sca-/Kit+ (LK), common myeloid progenitors (CMP) and GMP. In tumor-bearing mice, both PD-1 and PD-L1 expression was significantly higher in all myeloid progenitors compared to tumor-free control mice. To determine whether PD-1-mediated signaling in myeloid cells might affect the magnitude and composition of the myeloid output that is altered by cancer-mediated emergency response, we used PD-1 deficient mice. PD-1 ablation, which decreased tumor growth and metastasis, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6Chi monocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocyte/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6Chi monocytes and resolution of inflammation. To examine whether the effects of PD-1 on cancer-mediated emergency myelopoiesis and on myeloid lineage commitment and differentiation were mediated by myeloid cell-intrinsic mechanisms, we generated mice with conditional targeting of the PD-1 gene in the myeloid compartment by crossing PD-1fl/fl with LysM-Cre mice. Myeloid-specific ablation of PD-1 signaling in PD-1fl/fl/LysM-Cre mice impaired tumor growth to a similar extent as global PD-1 ablation in PD1-KO mice and induced a myeloid cell fate switch from MDSCs to effector-like myeloid cells that could be identified in spleen and tumor. Further, abrogation of PD-1 signaling in the myeloid compartment prevented the expansion of tumor-stress associated GMP in PD-1fl/fl/LysM-Cre mice and resulted in increased differentiation of committed monocyte progenitors (CMOP) and monocyte progenitors (MP) in the bone marrow. Moreover, PD-1 deletion significantly diminished NO production by M-MDSC and reversed their ability to suppress antigen-specific T cell responses. Our results reveal a previously unidentified role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors during tumor-mediated emergency myelopoiesis to promote MDSCs and suppress effector monocytes and mo-DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates its anti-tumor function. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Lipid Metabolism and Cell Cycle Progression By PU.1 in Myeloid Progenitor Cells

PU.1 is a transcription factor essential for myeloid development. High PU.1 expression leads to myeloid differentiation and cell cycle arrest while low expression leads to increased proliferation and self-renewal. Reduced PU.1 can lead to the development of acute myeloid leukemia. PU.1 induces microRNAs targeting genes involved in cell cycle and lipid metabolism. Included in these genes are E2f1 and Acly that are associated with lipogenesis. E2F1 is directly involved in cell cycle activation and lipid metabolism. Acly encodes ATP citrate lyase (ACL) that is responsible for the production of acetyl-CoA from mitochondrial citrate. Acetyl-CoA is the first precursor molecule for lipid biosynthesis, and lipogenesis is essential for proliferation. Therefore, we hypothesized that PU.1 activates genes encoding microRNAs to downregulate E2F1 and ACL, leading to cell cycle arrest. To test this hypothesis, we utilized a PU.1 inducible cell line system that is derived from myeloid progenitor cells in the liver of fetal BN mice (Spi1BN/BN) that express PU.1 at 20% of normal levels (iBN cells). Following doxycycline administration, iBN cells can be induced to express high levels of PU.1, causing cell cycle arrest and differentiation of these myeloid progenitor cells into a macrophage like phenotype. MicroRNA (miR)-223 and miR-141 were identified as PU.1 inducible miRs that target E2f1 and Acly, respectively. Supplementation with acetyl-CoA or acetate significantly rescued cell cycle inhibition caused by PU.1 induction. Inhibition of ACL activity by the chemical inhibitor BMS303141 (BMS) was sufficient to reduce cell cycle progression in cultured iBN cells. Supplementation with acetyl-CoA or acetate significantly rescued cell cycle progression in iBN cells inhibited with BMS. To determine if ACL levels play a role during normal macrophage differentiation, we investigated mRNA transcript levels of Acly over the course of myeloid progenitor differentiation promoted by M-CSF or GM-CSF. We found that Acly mRNA transcript levels inversely correlated with Adgre1 (encoding F4/80) or Spi1 (encoding PU.1) during macrophage differentiation. We conclude that during myeloid differentiation, PU.1 regulates the expression of genes involved in lipid metabolism through miRs, and influences cell cycle progression by limiting fatty acid synthesis required for proliferation. Elucidation of the mechanisms by which lipid metabolism may control cell cycle in myeloid progenitor cells is an important issue for further study. DisclosuresNo relevant conflicts of interest to declare.

Modeling leukemia with pediatric acute leukemia patient-derived iPSCs

Objectiveediatric acute leukemia (AL) is the most common hematological malignancy in childhood. However, the limitation of clinical specimens hindered the progress of research. Therefore, new research platforms are urgently needed to establish and clarify the pathogenesis of pediatric AL, and it is necessary to try to find novel targeted therapies for the clinical use. Here, the induced pluripotent stem cells (iPSCs) derived from AL provide a reliable model for basic research. Methodseukemia cells were sorted by flow cytometry and then reprogrammed into iPSCs by Sendai virus. Cell cycle assay was used to analyze cell proliferation. ResultsiPS cell lines from T cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells were successfully established. The reprogramming efficiency of AML cells was much higher than that of ALL cells. Disease iPS cells switched off the expression of the disease marker genes at iPS and HPC stage. When different subtypes of AML-iPSCs were differentiated into hematopoietic progenitor cells, iPS derived from acute megakaryocytic leukemia was more readily differentiated into megakaryocyte-erythroid progenitors. Whereas, the differentiation of multipotent lymphoid progenitor (MLP) and granulocyte macrophage progenitor (GMP) were blocked. The iPS derived from acute monocyte leukemia (AMCL) also showed the differentiation of common myeloid progenitors (CMP), GMP and monocytes significantly increased but MLP differentiation was inhibited. The AML-iPSC could form teratomas and we could obverse three germ layers in vivo, indicating that the AML-iPSCs have full pluripotency. However, there were not enough blood cells in teratoma to identify the leukemia. ConclusionsOur results provide a novel platform for AL research and critical insight into the difference of hematopoietic differentiation between ALL and AML.

CDK2-instigates C/EBPα degradation through SKP2 in Acute myeloid leukemia.

Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10-14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein-protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML. Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopshorylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML.