Regulation Of Hematopoiesis Research Articles

Myelosuppression Alleviation and Hematopoietic Regeneration by Tetrahedral-Framework Nucleic-Acid Nanostructures Functionalized with Osteogenic Growth Peptide.

As major complications of chemoradiotherapy, myelosuppression and hematopoietic‐system damage severely affect immunologic function and can delay or even terminate treatment for cancer patients. Although several specific cytokines have been used for hematopoiesis recovery, their effect is limited, and they may increase the risk of tumor recurrence. In this study, osteogenic growth peptide functionalized tetrahedral framework nucleic‐acid nanostructures (OGP‐tFNAs) are prepared; they combine the positive hematopoiesis stimulating effect of OGP and the drug carrying function of tFNAs. The potential of OGP‐tFNAs for hematopoietic stimulation and microenvironment regulation is investigated. It is shown that OGP‐tFNAs can protect bone marrow stromal cells from 5‐fluorouracil (5‐FU)‐induced DNA damage and apoptosis. OGP‐tFNAs pretreatment activates the extracellularly regulated protein kinase signal and downregulates apoptosis‐related proteins. OGP‐tFNAs also alleviate the chemotherapy‐induced inhibition of hematopoiesis‐related cytokine expression, which is crucial for hematopoiesis reconstitution. In conclusion, OGP‐tFNAs can protect hematopoietic cells and their microenvironment from chemotherapy‐induced injuries and myelosuppression, while promoting hematopoiesis regeneration.

Defective RAB31-mediated megakaryocytic early endosomal trafficking of VWF, EGFR, and M6PR in RUNX1 deficiency

AbstractTranscription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate–treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.

Peeling Back the Layers of Lymph Gland Structure and Regulation.

During the past 60 years, the fruit fly, Drosophila melanogaster, has proven to be an excellent model to study the regulation of hematopoiesis. This is not only due to the evolutionarily conserved signalling pathways and transcription factors contributing to blood cell fate, but also to convergent evolution that led to functional similarities in distinct species. An example of convergence is the compartmentalization of blood cells, which ensures the quiescence of hematopoietic stem cells and allows for the rapid reaction of the immune system upon challenges. The lymph gland, a widely studied hematopoietic organ of the Drosophila larva, represents a microenvironment with similar features and functions to classical hematopoietic stem cell niches of vertebrates. Lymph gland studies were effectively supported by the unparalleled toolkit developed in Drosophila, which enabled the high-resolution investigation of the cellular composition and regulatory interaction networks of the lymph gland. In this review, we summarize how our understanding of lymph gland structure and hematopoietic cell-to-cell communication evolved during the past decades and compare their analogous features to those of the vertebrate hematopoietic stem cell niche.

"FLipping" the Story: FLT3-Mutated Acute Myeloid Leukemia and the Evolving Role of FLT3 Inhibitors.

Simple SummaryPatients with acute myeloid leukemia (AML) may have a number of different mutations. Those with mutations in the FLT3 gene have a higher risk of relapse and death than those lacking these mutations. FLT3 is a key receptor on the surface of AML cells, which drives cell survival and growth. Although activation of this receptor is normally tightly controlled, in AML, FLT3 mutations allow it to activate itself, independent of external control. Over the past 5 years, a number of new drugs have been developed to specifically target these mutations. In this article, we discuss these drugs and their uses, as well as the mechanisms by which AML cells may gain resistance to them and how that resistance can be overcome.The treatment of many types of cancers, including acute myeloid leukemia (AML), has been revolutionized by the development of therapeutics targeted at crucial molecular drivers of oncogenesis. In contrast to broad, relatively indiscriminate conventional chemotherapy, these targeted agents precisely disrupt key pathways within cancer cells. FMS-like tyrosine kinase 3 (FLT3)—encoding a critical regulator of hematopoiesis—is the most frequently mutated gene in patients with AML, and these mutations herald reduced survival and increased relapse in these patients. Approximately 30% of newly diagnosed AML carries an FLT3 mutation; of these, approximately three-quarters are internal tandem duplication (ITD) mutations, and the remainder are tyrosine kinase domain (TKD) mutations. In contrast to its usual, tightly controlled expression, FLT3-ITD mutants allow constitutive, “run-away” activation of a large number of key downstream pathways which promote cellular proliferation and survival. Targeted inhibition of FLT3 is, therefore, a promising therapeutic avenue. In April 2017, midostaurin became both the first FLT3 inhibitor and the first targeted therapy of any kind in AML to be approved by the US FDA. The use of FLT3 inhibitors has continued to grow as clinical trials continue to demonstrate the efficacy of this class of agents, with an expanding number available for use as both experimental standard-of-care usage. This review examines the biology of FLT3 and its downstream pathways, the mechanism of FLT3 inhibition, the development of the FLT3 inhibitors as a class and uses of the agents currently available clinically, and the mechanisms by which resistance to FLT3 inhibition may both develop and be overcome.

GFI1B acts as a metabolic regulator in hematopoiesis and acute myeloid leukemia

Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1b deletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1b deletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1b deletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9 cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.

In This Issue

Current murine models of myeloproliferative neoplasms (MPN) cannot address how MPN can invade hematopoietic organs from a defined single anatomical origin as observed in patients. Murakami et al. developed an immunocompetent wild-type mouse model of Polycythemia Vera (PV) spreading from a single bone marrow site. PV development was abrogated in mice with splenectomy or lacking carbonic anhydrase 1 (CA1) in mutant hematopoietic progenitors. Increased expression of CA1 was found in CD34+ cells from PV patients, and its pharmacologic inhibition decreased variant allele frequency in colony-forming assays, suggesting CA1 as a potential therapeutic target for PV.See article, p. 285.Approximately every fifth patient with myeloproliferative neoplasms (MPN) harbors a CALR mutation of either type I or type II, which differ in clinical features and outcomes. Ibarra et al. show that type I, but not type II, mutant CALR proteins lose Ca2+ binding, causing Ca2+ depletion from the endoplasmic reticulum and activation of the IRE1α/XBP1 pathway. IRE1α inhibition prevented type I-mutant CALR cell survival in culture and thrombocythemia development in mice. These results identify IRE1α/XBP1 as a potentially targetable dependency specific to type I CALR-mutated MPNs.See article, p. 298.By single-cell DNA, RNA and proteogenomic sequencing of matched patient samples, Guess et al. uncovered three clonal evolution patterns associated with clinical progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). The Static pattern, characterized by minimal clonal change, had founding clones with mutations in DNA methylation genes and transitioned to sAML in the absence of further genomic aberrations, suggesting nongenetic evolution. Clonal architecture changed dramatically in Dynamic groups, often involving acquisition of mutations in signaling pathways (Dynamic-S) or progressive structural aberrations along with TP53 mutations (Dynamic-C). Collectively, these findings elucidate the interplay of genetic and nongenetic factors in leukemic transformation.See article, p. 316.To investigate patterns of subclone evolution during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML), Menssen et al. sequenced paired MDS and sAML samples from a cohort of patients. Signaling gene mutations were found to occur universally in subclones and undergo complex patterns of clonal evolution, including acquisition, expansion, persistence, and contraction of mutations during progression, with multiple patterns often coexisting in the same patient. The presence of at least one signaling gene mutation at MDS was associated with future progression to sAML in patients with lower risk MDS.See article, p. 330.Nakauchi et al. report the genome-wide landscape of DNA hydroxymethylation (5hmC) in human hematopoietic lineages. The authors map TET2-dependent 5hmC, epigenetic and transcriptional programs matched to competitive advantage, myeloid skewing, and reduced erythroid output in TET2-deficient hematopoietic stem and progenitor cells (HSPC). Vitamin C and azacitidine restore the 5hmC landscape and phenotypes in TET2-mutant HSPCs. These findings offer a comprehensive resource for TET-dependent transcriptional regulation of human hematopoiesis and shed light on the potential mechanisms by which TET deficiency contributes to clonal hematopoiesis and malignancies.See article, p. 346.

Mesenchymal Stromal Cells (MSCs): An Ally of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells in Disease Maintenance and Progression within the Bone Marrow Hematopoietic Niche.

Simple SummaryB-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. Even though the cure rate actually exceeds 85%, the prognosis of relapsed/refractory patients is dismal. Recent literature data indicate that the bone marrow (BM) microenvironment could play a crucial role in the onset, maintenance and progression of the disease. In particular, mesenchymal stromal cells (MSCs), which are key components of the BM niche, actively crosstalk with leukemic cells providing crucial signals for their survival and resistance to therapy. We hereby review the main mechanisms exploited by MSCs to nurture and protect B-ALL cells that could become appealing targets for innovative microenvironment remodeling therapies to be coupled with classical leukemia-directed strategies.Mesenchymal stromal cells (MSCs) are structural components of the bone marrow (BM) niche, where they functionally interact with hematopoietic stem cells and more differentiated progenitors, contributing to hematopoiesis regulation. A growing body of evidence is nowadays pointing to a further crucial contribution of MSCs to malignant hematopoiesis. In the context of B-cell acute lymphoblastic leukemia (B-ALL), MSCs can play a pivotal role in the definition of a leukemia-supportive microenvironment, impacting on disease pathogenesis at different steps including onset, maintenance and progression. B-ALL cells hijack the BM microenvironment, including MSCs residing in the BM niche, which in turn shelter leukemic cells and protect them from chemotherapeutic agents through different mechanisms. Evidence is now arising that altered MSCs can become precious allies to leukemic cells by providing nutrients, cytokines, pro-survivals signals and exchanging organelles, as hereafter reviewed. The study of the mechanisms exploited by MSCs to nurture and protect B-ALL blasts can be instrumental in finding new druggable candidates to target the leukemic BM microenvironment. Some of these microenvironment-targeting strategies are already in preclinical or clinical experimentation, and if coupled with leukemia-directed therapies, could represent a valuable option to improve the prognosis of relapsed/refractory patients, whose management represents an unmet medical need.

High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe−/− and Apoe−/−Nod1−/− mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe−/− mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe−/−Nod1−/− mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe−/− counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe−/−Nod1−/− mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe−/− mice. However, splenic artery ligation reduced the atherogenic burden in Apoe−/− mice an effect that, unexpectedly was lost in Apoe−/−Nod1−/− mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression.

Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.

Simple SummaryAnticancer nucleoside analogs are promising treatments that often result in damaging toxicities and therefore ineffective treatment. Mechanisms of this are not well-researched, but cellular nucleoside transport research in mice might provide additional insight given transport’s role in mammalian hematopoiesis. Cellular nucleoside transport is a notable component of mammalian hematopoiesis due to how mutations within it relate to hematological abnormities. This review encompasses nucleoside transporters, focusing on their inherent properties, hematopoietic role, and their interplay in nucleoside drug treatment side effects. We then propose potential mechanisms to explain nucleoside transport involvement in blood disorders. Finally, we point out and advocate for future research areas that would improve therapeutic outcomes for patients taking nucleoside analog therapies.Anticancer nucleoside analogs produce adverse, and at times, dose-limiting hematological toxicities that can compromise treatment efficacy, yet the mechanisms of such toxicities are poorly understood. Recently, cellular nucleoside transport has been implicated in normal blood cell formation with studies from nucleoside transporter-deficient mice providing additional insights into the regulation of mammalian hematopoiesis. Furthermore, several idiopathic human genetic disorders have revealed nucleoside transport as an important component of mammalian hematopoiesis because mutations in individual nucleoside transporter genes are linked to various hematological abnormalities, including anemia. Here, we review recent developments in nucleoside transporters, including their transport characteristics, their role in the regulation of hematopoiesis, and their potential involvement in the occurrence of adverse hematological side effects due to nucleoside drug treatment. Furthermore, we discuss the putative mechanisms by which aberrant nucleoside transport may contribute to hematological abnormalities and identify the knowledge gaps where future research may positively impact treatment outcomes for patients undergoing various nucleoside analog therapies.

P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments. Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML. Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways. Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers. Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance.

P723: LOSS OF THE NK MOTIF AND ANKYRIN REPEAT DOMAIN 1 (KANK1) LEADS TO LYMPHOID COMPARTMENT DYSREGUATION IN MICE

Background: We identified a novel germline loss of heterozygosity (LOH) mutation encompassing the NK Motif And AnKyrin Repeat Domain 1 (KANK1) gene in a young Myelodysplastic syndrome (MDS) patient and his healthy father, with no additional MDS-related mutations or chromosomal abnormalities. Clinical investigation suggested development of MDS following severe infection, likely triggered by an autoimmune mechanism. KANK1 protein plays a role in the control of cytoskeleton formation by regulating actin polymerization. A t(5;9) translocation resulting in a fusion of the platelet-derived growth factor receptor beta gene (PDGFRB) and KANK1 was detected in a patient with myeloid neoplasm (MN) characterized with severe thrombocythemia. The protein is thought to have a tumour suppressor function, as its expression is downregulated or missing in several tumor tissues, while over-expressing the protein was reported to inhibit the proliferation of tumor cells in solid cancer models. In addition, defects in cytoskeletal proteins are reported to contribute to immunological dysfunction and disruption of normal immune processes. Aims: To investigate the role of KANK1 in the regulation of normal hematopoiesis and the development of myeloid disorders. Methods: We generated a new genetically modified KANK1 (Kank1-/-) knockout mouse model and analysed the phenotypic and developmental changes in hematopoiesis in young and aged mice. Results: We confirmed the loss of Kank1 mRNA and protein expression by q-PCR and targeted protein mass-spectrometry respectively. The mice are viable and fertile, and reproduced according to mendelian ratio. Over the two years observation period, Kank1-/- mice did not exhibit any signs of disease, and their peripheral blood parameters remained within the normal range. Flow cytometry analysis of bone marrow (BM) cells from 17-20 weeks kank1-/- old mice showed minor increase in CD3+ T-cell population, while the expression of the double positive B220/CD19 B-cells population was reduced in Kank1-/- compared to Kank1+/+. Comprehensive flow cytometry analysis of spleen and thymus cells from young (n=8 each, 6-7 weeks old) and aged (n=8, 68-71 weeks old) Kank1-/- mice showed further dysregulation of the lymphoid compartment when compared to Kank1+/+. In young mice, a slight decrease of Treg cells is noticed in the spleen, coupled with a slight decrease of activated CD4 in the peripheral blood. In the spleen of aged Kank1-/- mice, the proportions of macrophages and Treg cells were decreased, while the central memory CD4+ T-cells were increased. Total protein analysis from BM cells of Kank1-/- (n=3) compared to Kank1+/+ mice (n=3) showed a significant reduction (FC >0.5, adj.p. <0.05) in proteins associated with cytoskeleton formation such as Myosin1 (MYH1), Tropomysin-1 (TDM1) and Annexin (ANXA2), and extracellular matrix components such as Collagena(CO1A1) and Matrix metalloproteinase9 (MMP9). We also detected a significant increase in expression of the DNA licensing factor minichromosome maintenance complex component 7 (MCM7), and the mitochondrial membrane associated transporter ATP binding Cassette Subfamily B Member 10 (ABCBA). Summary/Conclusion: Dysregulation of the immune system has been widely recognized to play a role in the development of MDS. Based on our results, we propose a role for KANK1 in regulating the development of immune cells in the mouse through it invovlement in cytoskeleton regulation. In vivo infectious challenge experiments using our model will futher elucidate the importance of KANK1 in the development of MDS.

PB2204: THE ROLE OF HES6 IN NORMAL HUMAN HEMATOPOIESIS

Background: The Notch signaling pathway is an important regulator of normal hematopoiesis and is often involved in leukemia. Some members of the HES gene family of transcription factors, like HES1 and HES4, are known Notch targets and have important roles in hematopoiesis. HES6, on the other hand, does not seem to be Notch dependent and has been poorly studied in the hematopoietic system. Interestingly, in the context of neurogenesis, it has been shown that HES6 can inhibit HES1 function, raising the hypothesis that HES6 might influence canonical Notch signaling activity. Within the hematopoietic system, HES6 has been described to be overexpressed in BPDCN, a very rare and aggressive hematological malignancy of plasmacytoid dendritic cells (pDCs). Also other studies have suggested the involvement of HES6 in cancer, including glioma, colorectal cancer, prostate cancer and uveal melanoma, and this mostly relates to a possible role for HES6 in cell proliferation. Aims: Given that HES6 is poorly studies within the hematopoeitic system, this study aims to unravel the role of HES6 in normal human hematopoiesis. Methods: We analyzed published datasets to examine the expression pattern of HES6 in different blood cell types. Through shRNA-mediated knockdown, we studied the impact of HES6 knockdown on the differentiation of CD34+ hematopoietic stem cells from cord blood to various hematopoietic lineages using in vitro stromal co-cultures and colony-forming unit assays. These experiments were analyzed using flow cytometry for the evaluation of differentiation, and cell counts for the evaluation of cell proliferation. Results: HES6 is highly expressed in megakaryocyte-erythroid progenitors (MEPs) and to a lesser extent in NK cells, pDCs and T cells. When exploring the expression in developing T cells, we observed that HES6 expression is inversely correlated with the expression of HES1. Considering the high HES6 expression in MEPs, we particularly studied the role of this gene during erythroid/megakaryocyte development. In our stromal cultures, HES6 expression remained high in developing erythroblasts and was low in megakaryocytes. Upon HES6 knockdown in CD34+ cells, a significant reduction in the number of megakaryocytes (50-85%) and erythroblasts (75-85%) was observed. The effect of HES6 knockdown in the was significant, with a 55-80%.reduction of colony formation. The number of cells per colony was also reduced by approximately 50%. HES6 knockdown during the development of other lineages also appears to impact cell proliferation but this effect is less obvious and needs further exploration. Summary/Conclusion: Consistent with the high expression of HES6 in MEPs, we observed significantly reduced cell proliferation upon HES6 knockdown during erythroid/mekaryocyte differentiation, with the most distinct effect on erythroblasts. HES6 knockdown also seems to impact the differentiation potential of precursor cells towards the myeloid lineages, as shown through colony-forming assays. Although the impact of HES6 on other hematopoietic lineages needs to be explored further, our results do provide the first clear insights into the functional role of HES6 in human hematopoiesis.

Combined Approach to Leukemic Differentiation Using Transcription Factor PU.1-Enhancing Agents.

The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.

Mesenchymal stroma/stem cells: Haematologists' friend or foe?

Mesenchymal stromal cells (MSCs) are non-haematopoietic cells found in fetal and adult organs, that play important roles in tissue repair, inflammation and immune modulation. MSCs residing in the bone marrow interact closely with haematopoietic cells and comprise an important component of the microenvironment supporting haematopoiesis, in both health and disease states. Since their identification in 1970, basic scientific and preclinical research efforts have shed light on the role of MSCs in the regulation of haematopoiesis and evoked interest in their clinical application in haematopoietic stem cell transplantation (HSCT) and malignant haematology. Over the last two decades, these research efforts have led to numerous clinical trials, which have established the safety of MSC therapy; however, the optimal mode of administration and the benefit remain inconclusive. In this paper, we will review the clinical experience with use of MSCs in HSCT for enhancement of engraftment, prevention and treatment of graft-versus-host disease and haemorrhagic cystitis. Then, we will discuss the contradictory evidence regarding tumour-promoting versus tumour-suppressing effects of MSCs in haematological malignancies, which may have relevance for future clinical applications.

Recurrent noncoding somatic and germline WT1 variants converge to disrupt MYB binding in acute promyelocytic leukemia

AbstractGenetic alternations can occur at noncoding regions, but how they contribute to cancer pathogenesis is poorly understood. Here, we established a mutational landscape of cis-regulatory regions (CREs) in acute promyelocytic leukemia (APL) based on whole-genome sequencing analysis of paired tumor and germline samples from 24 patients and epigenetic profiling of 16 patients. Mutations occurring in CREs occur preferentially in active enhancers bound by the complex of master transcription factors in APL. Among significantly enriched mutated CREs, we found a recurrently mutated region located within the third intron of WT1, an essential regulator of normal and malignant hematopoiesis. Focusing on noncoding mutations within this WT1 intron, an analysis on 169 APL patients revealed that somatic mutations were clustered into a focal hotspot region, including one site identified as a germline polymorphism contributing to APL risk. Significantly decreased WT1 expression was observed in APL patients bearing somatic and/or germline noncoding WT1 variants. Furthermore, biallelic WT1 inactivation was recurrently found in APL patients with noncoding WT1 variants, which resulted in the complete loss of WT1. The high incidence of biallelic inactivation suggested the tumor suppressor activity of WT1 in APL. Mechanistically, noncoding WT1 variants disrupted MYB binding on chromatin and suppressed the enhancer activity and WT1 expression through destroying the chromatin looping formation. Our study highlights the important role of noncoding variants in the leukemogenesis of APL.

Implication of microRNAs in Carcinogenesis with Emphasis on Hematological Malignancies and Clinical Translation.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, that are involved in the multistep process of carcinogenesis, contributing to all established hallmarks of cancer. In this review, implications of miRNAs in hematological malignancies and their clinical utilization fields are discussed. As components of the complex regulatory network of gene expression, influenced by the tissue microenvironment and epigenetic modifiers, miRNAs are “micromanagers” of all physiological processes including the regulation of hematopoiesis and metabolic pathways. Dysregulated miRNA expression levels contribute to both the initiation and progression of acute leukemias, the metabolic reprogramming of malignantly transformed hematopoietic precursors, and to the development of chemoresistance. Since they are highly stable and can be easily quantified in body fluids and tissue specimens, miRNAs are promising biomarkers for the early detection of hematological malignancies. Besides novel opportunities for differential diagnosis, miRNAs can contribute to advanced chemoresistance prediction and prognostic stratification of acute leukemias. Synthetic oligonucleotides and delivery vehicles aim the therapeutic modulation of miRNA expression levels. However, major challenges such as efficient delivery to specific locations, differences of miRNA expression patterns between pediatric and adult hematological malignancies, and potential side effects of miRNA-based therapies should be considered.

Niche Regulation of Hematopoiesis: The Environment Is "Micro," but the Influence Is Large.

Immune cell production is governed by a process known as hematopoiesis, where hematopoietic stem cells (HSCs) differentiate through progenitor cells and ultimately to the mature blood and immune cells found in circulation. While HSCs are capable of cell-autonomous regulation, they also rely on extrinsic factors to balance their state of quiescence and activation. These cues can, in part, be derived from the niche in which HSCs are found. Under steady-state conditions, HSCs are found in the bone marrow. This niche is designed to support HSCs but also to respond to external factors, which allows hematopoiesis to be a finely tuned and coordinated process. However, the niche, and its regulation, can become dysregulated to potentiate inflammation during disease. This review will highlight the architecture of the bone marrow and key regulators of hematopoiesis within this niche. Emphasis will be placed on how these mechanisms go awry to exacerbate hematopoietic contributions that drive cardiovascular disease.

Deconvolution of the hematopoietic stem cell microenvironment reveals a high degree of specialization and conservation

SummaryUnderstanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggest a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows inferring conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology is a stepping-stone toward a comprehensive cell atlas of the BM microenvironment.

The role of JAK inhibitors in hematopoietic cell transplantation.

The Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) pathway is essential for both the regulation of hematopoiesis and the control of inflammation. Disruption of this pathway can lead to inflammatory and malignant disease processes. JAK inhibitors, designed to control the downstream effects of pro-inflammatory and pro-angiogenic cytokines, have been successfully used in pre-clinical models and clinical studies of patients with autoimmune diseases, hematologic malignancies, and the hematopoietic cell transplantation (HCT) complication graft versus host disease (GVHD). In the last decade, JAK inhibitors Ruxolitinib, Fedratinib, and most recently Pacritinib have been United States Federal Drug Administration (FDA) approved for the treatment of myelofibrosis (MF). Ruxolitinib was also recently approved for the treatment of steroid refractory acute as well as chronic GVHD; JAK inhibitors are currently under evaluation in the pre-HCT setting in MF and for the prevention of GVHD. This review will focus on the role of JAK inhibitors in the treatment of hematologic malignancies, the potential function of pre-HCT JAK inhibitors in patients with MF, and the role of JAK inhibitors in the prevention and treatment of acute and chronic GVHD.

Of mice and men: From hematopoiesis in mouse models to curative gene therapy for sickle cell disease

Through studies in mice and in humans, Stuart Orkin showed that GATA-1 is a master transcriptional regulator of hematopoiesis. He has highlighted the role of BCL11A in the fetal-adult hemoglobin switch. The Gairdner Foundation Award recognizes Orkin's contribution to the development of gene therapy of sickle cell disease.