scholarly journals SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2189-2189
Author(s):  
Mark C Wilkes ◽  
Aya Shibuya ◽  
Vanessa M Scanlon ◽  
Hee-Don Chae ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Cord Blood ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Culture Media ◽  
Hsp70 Expression ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

scholarly journals SATB1 Regulates GATA1 Protein Expression in Early Hematopoiesis and Is Deregulated in Diamond Blackfan Anemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Mark C Wilkes ◽  
Hee-Don Chae ◽  
Ethan Patrick Wentworth ◽  
Toshinobu Nishimura ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Liver ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Ribosomal Gene ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Diamond Blackfan Anemia

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Developmental Control of Polycomb Subunit Composition Mediates a Switch to Non-Canonical Functions during Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 241-241
Author(s):  
Jian Xu ◽  
Zhen Shao ◽  
Dan Li ◽  
Huafeng Xie ◽  
Woojin Kim ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Function Analysis ◽  
Subunit Composition ◽  
Erythroid Cells ◽  
Developmental Control ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Hematopoietic Malignancies

Abstract The epigenetic machinery plays crucial roles in hematopoiesis, and its deregulation drives the pathogenesis of blood disorders. Polycomb Repressive Complex 2 (PRC2) is a major class of epigenetic repressor that catalyzes the di/tri-methylation of histone H3 lysine 27 (or H3K27me2/3). The canonical PRC2 complex consists of EED, SUZ12, and the histone methyltransferase EZH2. The functions of PRC2 in hematopoiesis remain elusive due in large to the existence of two highly related enzymatic subunits EZH1 and EZH2. While amplification or overexpression of PRC2 proteins is common in many cancers, inactivating mutation in PRC2 is frequently found in hematopoietic malignancies, indicating that PRC2 can be oncogenic or tumor suppressive in different cellular contexts. In light of recent efforts to therapeutically target EZH2 enzyme activities or canonical EZH2-PRC2 functions in various hematopoietic malignancies, it will be critical to fully assess the context-specific activity of this epigenetic complex in normal and malignant developmental processes. The molecular mechanisms by which PRC2 regulates normal and neoplastic hematopoiesis is unclear, as are the non-redundant effects of canonical versus non-canonical PRC2 functions, which are mediated by EZH1 or EZH2 independent of H3K27me2/3. In this study, we demonstrate that the PRC2 enzymatic subunits EZH1 and EZH2 undergo an expression switch during hematopoiesis. EZH2 is highly expressed in primary human CD34+ hematopoietic stem/progenitor (HSPC) cells and progressively downregulated during erythroid and lymphoid specification, whereas EZH1 is significantly upregulated during differentiation. We next examined the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and revealed the existence of an EZH1-SUZ12 sub-complex lacking EED subunit in human erythroid cells. Through genome scale chromatin occupancy (by ChIP-seq) and transcriptional profiling (by RNA-seq) analyses, we provide evidence that EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin domains marked by H3K4me3 and H3K27me1, and positively regulate gene expression. Furthermore, loss of EZH2 expression leads to global repositioning of EZH1 chromatin occupancy to EZH2 targets, and EZH1 complements EZH2 loss within canonical PRC2 target genes. To elucidate the regulatory networks underlying the developmental control of EZH1/2 switch, we profiled the histone modifications and chromatin accessibility surrounding the EZH1 gene in both CD34+ HSPCs and committed erythroid cells. We identified and characterized an erythroid-selective enhancer element that is indispensable for the transcriptional activation of EZH1. Loss of function analysis using CRISPR/cas9-mediated enhancer deletion results in markedly decrease in EZH1 expression in human erythroid cells. Moreover, a switch from GATA2 to GATA1 expression controls the developmental EZH1/2 switch by differential association with distinct EZH1 enhancers during erythroid differentiation. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical PRC2 functions. Our study also establishes a molecular link between the switch of master lineage regulators and developmental control of PRC2 composition, providing a means to coordinate linage-specific transcription and accompanying changes in the epigenetic landscape during blood stem cell specification. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 455-455
Author(s):  
Mark C Wilkes ◽  
Jacqueline D Mercado ◽  
Mallika Saxena ◽  
Jun Chen ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fold Increase ◽  
Healthy Controls ◽  
In Vitro Activity ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-2-SCI-2
Author(s):  
Stefan Karlsson ◽  
Johan Flygare ◽  
Pekka Jaako ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2591-2591
Author(s):  
Mark C Wilkes ◽  
Kaoru Takasaki ◽  
Minyoung Youn ◽  
Hee-Don Chae ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Chromatin Organization ◽  
Hsp70 Expression ◽  
Bone Marrow Failure Syndrome ◽  
Enhancer Element ◽  
Protein Levels ◽  
Diamond Blackfan Anemia ◽  
Increased Risk

Abstract Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome. The disease usually presents within the first year of life and is associated with anemia, congenital abnormalities, and an increased risk of developing cancer. The most prevalent mutations are found in the ribosomal protein RPS19, accounting for over 25% of cases but approximately 70% of DBA patients carry mutations in ribosomal genes. Downregulation of GATA1 has been attributed to the disease, but the upstream mechanisms leading to aberrant erythropoiesis is only beginning to be elucidated. SATB1 is highly upregulated in lymphocytes (especially thymocytes) and steadily downregulated in all myeloid ineages during differentiation. However the more modest expression of SATB1 in early progenitors is required for self-renewal of HSCs and globin switching in early erythropoiesis. Using transcriptomics, we identified SATB1 expression is prematurely lost in RPS19-insufficient erythropoiesis and that over a third (16/42) of RPS19-sensitive early erythroid genes were rescued upon SATB1 re-expression. One of the most deregulated transcripts encode the chaperone HSP70. GATA1 is an essential master regulator in erythroid differentiation and high GATA1 protein levels are required for efficient erythropoiesis. In DBA, GATA1 protein expression is drastically diminished. Two mechanisms contribute to this. At the translational level, GATA1 transcripts fail to be translated adequately due to ribosomal inefficiency at binding short, unstructured 5'UTRs. At the protein level,HSP70 loss leads to reduced GATA1 protein stability. During healthy erythropoiesis HSP70 genes are drastically upregulated (3.21, 4.18 and 1.37- fold) and this is lost in RPS19 insufficiency. Here we report that upregulation of HSP70 in healthy erythropoiesis requires SATB1. SATB1 binds to 3 sites within a 40kb region surrounding the HSP70 gene loci and recruits a distal predicted enhancer element to the proximal promoters via formation of two chromatin loops. During RPS19-insufficiency this chromatin organization is lost, but SATB1 re-expression restores chromatin looping and HSP70 expression, stabilizing GATA1 and promoting efficient erythropoiesis. As with HSP70, it is likely that permissive chromatin organization is essential for other RPS19-sensitive erythroid genes rescued by SATB1 re-expression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting Autophagy as a Therapeutic Pathway in Diamond-Blackfan Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-28-SCI-28
Author(s):  
Sergei Doulatov
Keyword(s):  
Oxidative Stress ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Autophagic Flux ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells ◽  
Induced Pluripotent

Abstract Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. We previously used unbiased drug screens in induced pluripotent stem cells (iPSCs) which identified SMER28 as a potential therpauetic for DBA. SMER28 acts by selectively modulating autophagy, but has distinct effects from the mTOR inhibitor rapamycin, highlighting the need for further study. Autophagy and mitophagy are critical metabolic pathways that mediate turnover of damaged organelles and mitochondria. Autophagy has been linked to regulation of hematopoietic stem cell function and terminal erythroid differentiation. However, the mechanisms by which autophagy regulates hematopoiesis are still poorly understood. We utilize primary cord blood and adult human progenitors and iPSCs to circumvent the paucity of primary patient blood stem and progenitor cells. To understand the role of autophagy, we have developed a lentiviral LC3-based reporter which allows real-time quantitation of autophagic flux. Using this reporter, we show that autophagy is dynamically regulated during erythroid differentiation and closely parallels mitochondrial mass and levels of reactive oxygen species. In our model, oxidative stress in erythroid precursors drives a stress response which involves activation of autophagy and mitophagy pathways. The interplay between oxidative stress and autophagy regulates erythropoiesis in normal and multiple disease contexts, including DBA and myelodysplastic syndromes (MDS). SMER28 and mitochondrial uncouplers promote homeostasis by facilitating mitochondrial clearance. In summary, therpauetic modulation of autophagy may be a broadly applicable therpauetic strategy in both inherited and acquired anemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers

Blood ◽  
2020 ◽  
Vol 136 (6) ◽  
pp. 698-714 ◽  
Author(s):  
Alexandre Fagnan ◽  
Frederik Otzen Bagger ◽  
Maria-Riera Piqué-Borràs ◽  
Cathy Ignacimouttou ◽  
Alexis Caulier ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Hematologic Malignancy ◽  
Ectopic Expression ◽  
Leukemic Cells ◽  
Erythroid Progenitors ◽  
Molecular Subgroup ◽  
Aberrant Expression ◽  
In Vivo Models ◽  
Hematopoietic Stem

Abstract Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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