ABSTRACT
Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Mice , Animals , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Hematopoietic Stem Cells/metabolism , Cell Differentiation , Embryonic Stem Cells/metabolism , Doxycycline/pharmacology , Doxycycline/metabolismABSTRACT
This article highlights the presentations from the 2021 scientific meeting of the Club Hematopoiesis and Oncogenesis. This annual meeting focuses on hematopoiesis and oncogenic mechanisms. Various topics were presented: expansion of hematopoietic stem cells with in vivo and ex vivo strategies, the role of the hematopoietic stem cell niches in aging and leukemic resistance, the crossroad between hematology and immunology, the importance of the metabolism in normal hematopoiesis and hematopoietic defects, solid tumors and oncogenesis, the noncoding genome, inflammation in monocyte differentiation and leukemia, and importantly, the recent advances in myeloid malignancies, lymphoid leukemia and lymphoma.
Subject(s)
Leukemia , Lymphoma , Humans , Hematopoiesis/genetics , Hematopoietic Stem Cells , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathologyABSTRACT
Chronic myeloid leukemia (CML) is a clonal hematopoietic malignancy driven by the BCR-ABL1 fusion oncoprotein. The development of tyrosine kinase inhibitors (TKIs) has deeply increased long-term survival of CML patients. Nonetheless, one patient out of four will switch TKI off owing either to drug intolerance or resistance partly due to amplification or mutations of BCR-ABL1 oncogene and alteration in ATP-binding cassette (ABC) transporters. Increasing evidence suggests the involvement of the microRNA miR-495-3p in cancer-associated chemoresistance through multidrug resistance 1 (MDR1) gene, which encodes an ATP-dependent efflux pump. Our study aimed at investigating the potential role of miR-495-3p in CML TKI chemo-sensitivity and determining the underlying molecular circuitry involved. We first observed that miR-495-3p expression was lower in BCR-ABL1-expressing cellular models in vitro. Notably, loss-of-function experiments showed increased proliferation associated with a decreased number of nondividing cells (G0/G1) and resistance to Imatinib. Conversely, our data showed that miR-495-3p overexpression hindered leukemic cell growth and TKI resistance in Imatinib-resistant T315I-mutant cells, as well as drug efflux activity through MDR1 regulation. Further investigating the role of miR-495-3p in CML patients, we found that predicted miR-495-3p targets were upregulated in patients in blast crisis that were involved in protein phosphorylation and associated with the worst prognosis. Taken together, our results demonstrate that downregulation of miR-495-3p expression is important in the malignant phenotype of CML and TKI resistance mechanisms and could be a useful biomarker and a potential therapeutic target to eradicate CML.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , MicroRNAs , Humans , Imatinib Mesylate/pharmacology , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Drug Resistance, Multiple , Adenosine TriphosphateABSTRACT
Oxaliplatin was nearly twice as hematotoxic, with optimal circadian timing differing by 6 h, in women as compared to men with colorectal cancers. Hence, we investigated sex- and timing-related determinants of oxaliplatin hematopoietic toxicities in mice. Body-weight loss (BWL), blood cell counts, bone marrow cellularity (BMC) and seven flow-cytometry-monitored hematopoietic progenitor populations were evaluated 72 h after oxaliplatin chronotherapy administration (5 mg/kg). In control animals, circadian rhythms of circulating white blood cells showed a peak at ZT5 in both sexes, whereas BMC was maximum at ZT20 in males and ZT13h40 in females. All BM progenitor counts presented robust rhythms with phases around ZT3h30 in females, whereas only three of them rhythmically cycled in males with a ≈ -6 h phase shift. In treated females, chronotoxicity rhythms occurred in BWL, WBC, BMC and all BM progenitors with the best timing at ZT15, ZT21, ZT15h15 and ZT14h45, respectively. In males, almost no endpoints showed circadian rhythms, BWL and WBC toxicity being minimal, albeit with a substantial drop in BM progenitors. Increasing dose (10 mg/kg) in males induced circadian rhythms in BWL and WBC but not in BM endpoints. Our results suggest complex and sex-specific clock-controlled regulation of the hematopoietic system and its response to oxaliplatin.
ABSTRACT
The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.
Subject(s)
ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Colonic Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/genetics , Antigens, CD/metabolism , Cadherins/metabolism , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement/genetics , Cell Survival/drug effects , Colonic Neoplasms/metabolism , Databases, Genetic , Epithelial Cell Adhesion Molecule/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Knockdown Techniques , Humans , Keratin-20/metabolism , Neoplasm Invasiveness/genetics , Oxaliplatin/pharmacology , Protein Transport , Vimentin/metabolism , beta Catenin/metabolismABSTRACT
Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.
Subject(s)
ARNTL Transcription Factors/genetics , Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-akt/genetics , TOR Serine-Threonine Kinases/genetics , Tumor Suppressor Protein p53/genetics , ARNTL Transcription Factors/metabolism , Apoptosis , Cell Line, Tumor , Cellular Senescence , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Humans , Proto-Oncogene Proteins c-akt/biosynthesis , Signal Transduction , TOR Serine-Threonine Kinases/biosynthesis , Tumor Suppressor Protein p53/biosynthesisABSTRACT
Recent experimental data suggest that the heterogeneity of chronic myeloid leukemia (CML) stem cells may be the result of the development of unique molecular events generating functional consequences in terms of the resistance and persistence of leukemic stem cells. To explore this phenomenon, we designed a single-cell transcriptome assay evaluating simultaneously the expression of 87 genes. Highly purified CD34+ cells from three CML patients at diagnosis were immobilized in microfluidic chips, and the expression of 87 genes was evaluated in each cell. This analysis identified a group of 13 highly connected genes including NANOG, POU5F1, LIN28A, and SOX2, representing on average 8.59% of the cell population analyzed. Bioinformatics analysis with the corrected matrix and t-distributed stochastic neighbor embedding (tSNE) algorithm identified four distinct clusters, and the pseudotime analysis confirmed the presence of seven stem cell states in the four clusters identified. ALOX5 expression was associated with the group of cells expressing the pluripotency markers. In in vitro analyses, two genes that were predicted to undergo similar regulation using pseudotime analysis (ALOX5 and FGFR) were found to be similarly inhibited by ponatinib, an FGFR inhibitor. Finally, in an independent cohort of CML patients, we found that pluripotency gene expression is a common feature of CD34+ CML cells at diagnosis. Overall, these experiments allowed identification of individual CD34+ cells expressing high levels of pluripotency genes at diagnosis, in which a continuum of transitional states were identified using pseudotime analysis. These results suggest that leukemic stem cell persistence in CML needs to be targeted simultaneously rather than using a single pathway.
Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplasm Proteins/biosynthesis , Neoplastic Stem Cells/metabolism , Single-Cell Analysis , Transcriptome , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplasm Proteins/genetics , Neoplastic Stem Cells/pathologyABSTRACT
Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.
Subject(s)
Carcinoma, Papillary/etiology , Carcinoma, Renal Cell/etiology , Embryoid Bodies/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mutation , Proto-Oncogene Proteins c-met/genetics , Alleles , Carcinoma, Papillary/diagnosis , Carcinoma, Renal Cell/diagnosis , Embryoid Bodies/metabolism , Embryoid Bodies/ultrastructure , Fluorescent Antibody Technique , Gene Expression , Genotype , Humans , Immunohistochemistry , Magnetic Resonance Imaging/methods , Reproducibility of ResultsABSTRACT
Bone marrow (BM) niche cells help to keep adult hematopoietic stem cells (HSCs) in a quiescent state via secreted factors and induction of cell-cycle inhibitors. Here, we demonstrate that the adapter protein CABLES1 is a key regulator of long-term hematopoietic homeostasis during stress and aging. Young mice lacking Cables1 displayed hyperproliferation of hematopoietic progenitor cells. This defect was cell intrinsic, since it was reproduced in BM transplantation assays using wild-type animals as recipients. Overexpression and short hairpin RNA-mediated depletion of CABLES1 protein resulted in p21Cip/waf up- and downregulation, respectively. Aged mice lacking Cables1 displayed abnormalities in peripheral blood cell counts accompanied by a significant reduction in HSC compartment, concomitant with an increased mobilization of progenitor cells. In addition, Cables1-/- mice displayed increased sensitivity to the chemotherapeutic agent 5-fluorouracil due to an abnormal microenvironment. Altogether, our findings uncover a key role for CABLES1 in HSC homeostasis and stress hematopoiesis.
Subject(s)
Cyclins/genetics , Hematopoietic Stem Cells/cytology , Aging , Animals , Antigens, CD34/metabolism , Bone Marrow Cells/cytology , Cell Cycle Checkpoints/drug effects , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclins/antagonists & inhibitors , Cyclins/metabolism , Fluorouracil/pharmacology , Hematopoiesis/drug effects , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.
Subject(s)
MicroRNAs/metabolism , Pluripotent Stem Cells/cytology , Cell Differentiation/physiology , Cell Line , Cell Lineage , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Pluripotent Stem Cells/metabolismABSTRACT
Over the last decade, the possibility of reprogramming malignant cells to a pluripotent state has been achieved in several hematological malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, and chronic myeloid leukemia (CML). It has been shown that it is readily possible to generate induced pluripotent stem cells (iPSCs) from several types of primary CML cells and to generate progenitors and differentiated cells with variable efficiency. Although these experiments have brought some new insights in the understanding of CML pathophysiology, the ultimate goal of generating induced leukemic stem cells (LSCs) with long-term multilineage potential has not yet been demonstrated. Experiments under way will determine whether additional signaling events are required to induce the emergence of bona fide LSCs. However, iPSC modeling offers the unique possibility to generate pluripotent cells harboring cancer-predisposing mutations using patient-derived noncancerous cells, as has been shown in Li-Fraumeni syndrome, BRCA-1 associated breast carcinomas, or RET-mutated medullary thyroid carcinomas. In these conditions, mutated iPSCs can then be used to study the mutational history that precedes the appearance of the malignant transformation and to develop novel drug-screening strategies. The ability to induce a successful differentiation program toward the tissue in which a given cancer develops or to generate tissue-specific cancer organoids in which the full oncogenic potential can be revealed remains a major challenge in the field. Similarly, in hematological malignancies, a significant hurdle remains due to the lack of adequate technology to induce the emergence of leukemic cells that resemble LSCs, which hinders our ability to study the mechanisms of therapy resistance.
Subject(s)
Cell Transformation, Neoplastic , Disease Susceptibility , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Animals , Biomarkers , Cell Differentiation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplastic Syndromes, Hereditary/etiology , Neoplastic Syndromes, Hereditary/metabolism , Neoplastic Syndromes, Hereditary/pathology , Tumor MicroenvironmentABSTRACT
Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplastic Stem Cells/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Basic Helix-Loop-Helix Transcription Factors/agonists , Basic Helix-Loop-Helix Transcription Factors/genetics , Carbazoles/pharmacology , Case-Control Studies , Cell Line, Tumor , Cell Proliferation/drug effects , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Purines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction/drug effects , Tumor Stem Cell AssayABSTRACT
Hematopoietic stem cells (HSCs) undergo self-renewal to maintain hematopoietic homeostasis for lifetime, which is regulated by the bone marrow (BM) microenvironment. The chemokine receptor CXCR4 and its ligand CXCL12 are critical factors supporting quiescence and BM retention of HSCs. Here, we report an unknown function of CXCR4/CXCL12 axis in the protection of HSCs against oxidative stress. Disruption of CXCR4 receptor in mice leads to increased endogenous production of reactive oxygen species (ROS), resulting in p38 MAPK activation, increased DNA double-strand breaks and apoptosis leading to marked reduction in HSC repopulating potential. Increased ROS levels are directly responsible for exhaustion of the HSC pool and are not linked to loss of quiescence of CXCR4-deficient HSCs. Furthermore, we report that CXCL12 has a direct rescue effect on oxidative stress-induced HSC damage at the mitochondrial level. These data highlight the importance of CXCR4/CXCL12 axis in the regulation of lifespan of HSCs by limiting ROS generation and genotoxic stress.
Subject(s)
Chemokine CXCL12/genetics , Hematopoietic Stem Cells/cytology , Hepatocytes/cytology , Reactive Oxygen Species/metabolism , Receptors, CXCR4/genetics , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chemokine CXCL12/metabolism , DNA Breaks, Double-Stranded , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Hepatocytes/metabolism , Mice , Mitochondria/genetics , Mitochondria/metabolism , Oxidative Stress , Phosphorylation , Receptors, CXCR4/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The development of the hematopoietic system is a dynamic process that is controlled by the interplay between transcriptional and epigenetic networks to determine cellular identity. These networks are critical for lineage specification and are frequently dysregulated in leukemias. Here, we identified histone demethylase KDM2B as a critical regulator of definitive hematopoiesis and lineage commitment of murine hematopoietic stem and progenitor cells (HSPCs). RNA sequencing of Kdm2b-null HSPCs and genome-wide ChIP studies in human leukemias revealed that KDM2B cooperates with polycomb and trithorax complexes to regulate differentiation, lineage choice, cytokine signaling, and cell cycle. Furthermore, we demonstrated that KDM2B exhibits a dichotomous role in hematopoietic malignancies. Specifically, we determined that KDM2B maintains lymphoid leukemias, but restrains RAS-driven myeloid transformation. Our study reveals that KDM2B is an important mediator of hematopoietic cell development and has opposing roles in tumor progression that are dependent on cellular context.
Subject(s)
F-Box Proteins/physiology , Hematopoiesis , Jumonji Domain-Containing Histone Demethylases/physiology , Lymphoma/enzymology , Animals , Cell Differentiation , Cell Line, Tumor , Cell Lineage , Cell Transformation, Neoplastic , Gene Expression Regulation, Leukemic , Humans , Lymphoma/pathology , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b's adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b(-/-) progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b's absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis.
Subject(s)
Blood Platelets/physiology , Bone Marrow/physiology , Erythrocytes/physiology , Gene Expression Regulation/genetics , Hematopoiesis/physiology , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Animals , DNA Primers/genetics , Embryonic Stem Cells/metabolism , Flow Cytometry , Genetic Vectors , Kaplan-Meier Estimate , Mice , Mice, Transgenic , Microarray Analysis , Microscopy, Fluorescence , Mutagenesis , Proto-Oncogene Proteins/genetics , Real-Time Polymerase Chain Reaction , Repressor Proteins/geneticsABSTRACT
The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy.
Subject(s)
Cell Differentiation/physiology , Cellular Reprogramming , Hematopoietic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Animals , Animals, Newborn , Cell Line , Cells, Cultured , Chimera , Fibroblasts/cytology , Gene Expression Regulation, Developmental , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/metabolism , Kruppel-Like Factor 4 , Lentivirus/genetics , Mice , Molecular Sequence Data , Transcription Factors/genetics , Transcription Factors/metabolism , Transduction, GeneticABSTRACT
The initial steps in the pathogenesis of acute leukemia remain incompletely understood. The TEL-AML1 gene fusion, the hallmark translocation in Childhood Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical disease, most often in utero. We have generated mice in which TEL-AML1 expression is driven from the endogenous promoter and can be targeted to specific populations. TEL-AML1 renders mice prone to malignancy after chemical mutagenesis when expressed in hematopoietic stem cells (HSCs), but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly increases the number of HSCs and predominantly maintains them in the quiescent (G(0)) stage of the cell cycle. TEL-AML1(+) HSCs retain self-renewal properties and contribute to hematopoiesis, but fail to out-compete normal HSCs. Our work shows that stem cells are susceptible to subversion by weak oncogenes that can subtly alter their molecular program to provide a latent reservoir for the accumulation of further mutations.
Subject(s)
Bone Marrow Cells/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid, Acute/genetics , Oncogene Proteins, Fusion/genetics , Oncogenes , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factors/metabolism , Embryonic Development , Gene Targeting , Hepatocytes/metabolism , Humans , Interleukin-7/metabolism , Leukemia, Myeloid, Acute/metabolism , Mice , Models, Animal , Oncogene Fusion , Oncogene Proteins, Fusion/metabolismABSTRACT
Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells and have been presumed to retain labels such as the thymidine analog 5-bromodeoxyuridine (BrdU). However, BrdU retention is neither a sensitive nor specific marker for HSCs. Here we show that transient, transgenic expression of a histone 2B (H2B)-green fluorescent protein (GFP) fusion protein in mice has several advantages for label-retention studies over BrdU, including rapid induction of H2B-GFP in virtually all HSCs, higher labeling intensity and the ability to prospectively study label-retaining cells, which together permit a more precise analysis of division history. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a stringent marker combination (L(-)K(+)S(+)CD48(-)CD150(+)), revealed unexpected heterogeneity in their proliferation rates and showed that approximately 20% of HSCs divide at an extremely low rate (< or =0.8-1.8% per day).
Subject(s)
Green Fluorescent Proteins/metabolism , Histones/chemistry , Animals , Antigens, CD/biosynthesis , Bone Marrow Cells/cytology , Bromodeoxyuridine/pharmacology , CD48 Antigen , Cell Differentiation , Cell Lineage , Flow Cytometry/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Humans , Mice , Models, Theoretical , Receptors, Cell Surface/biosynthesis , Signaling Lymphocytic Activation Molecule Family Member 1ABSTRACT
The pathophysiology of microthrombocytopenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked thrombocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormalities are suggestive of intrinsic disturbances of production. In contrast to human patients, murine models of WASp deficiency exhibit only mild thrombocytopenia, and platelets are of normal size. Here, we have identified a critical role for WASp during murine platelet biogenesis. By electron microscopy, WASp-deficient MKs appeared to have shed platelets ectopically within the bone marrow space. WASp-deficient megakaryocytes (MKs) also displayed defects in response to fibrillar collagen I (CI) in vitro, the major matrix component of bone. These included a loss of normal CI receptor (alpha2beta1 integrin)-mediated inhibition of proplatelet formation, a marked abrogation of SDF-1-induced chemotactic migration of CD41+ MKs adherent to CI, and an almost complete lack of actin-rich podosomes, normally induced by interaction between CI and its receptors GPVI or alpha2beta1 integrin. These findings highlight the central and highly specialized role of WASp in MKs during platelet biogenesis, and may provide a mechanism for the mild thrombocytopenia observed in WASp-deficient mice. In addition, they suggest a novel explanation for some of the platelet abnormalities characteristic of patients with WAS.
Subject(s)
Blood Platelets/metabolism , Bone Marrow/pathology , Bone Marrow/physiopathology , Thrombocytopenia/pathology , Wiskott-Aldrich Syndrome Protein, Neuronal/deficiency , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Animals , Blood Platelets/drug effects , Cell Differentiation/genetics , Collagen Type I/metabolism , Collagen Type I/pharmacology , Disease Models, Animal , Integrin alpha2beta1/metabolism , Megakaryocytes/drug effects , Megakaryocytes/pathology , Mice , Mice, Inbred C57BL , Thrombocytopenia/geneticsABSTRACT
Hematopoietic stem cells (HSCs) arise, self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal, which controls numerous decisions in embryonic and adult hematopoiesis. We report here that Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of Lyl-1 results in a diminution in the frequency of immature progenitors (Lin(-), CD34(-), sca-1(+), c-kit(+) [LSK], and LSK-side population [LSK-SP]) and in S(12) colony-forming unit (CFU-S(12)) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, Lyl-1(-/-) E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of Lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes Lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that Lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl functions.