Delay In Erythroid Differentiation Research Articles

Unraveling the Role of PIEZO1 in Stressed Erythropoiesis: Implications for Dyserythropoiesis and Potential Therapeutic Targets in Dehydrated Hereditary Stomatocytosis

PIEZO1 is a mechanosensitive cation channel that plays a crucial role in various physiological processes as a mechanical force sensor. In erythrocytes, it is responsible for regulating cellular volume and hydration. Gain-of-function (GoF) mutations in PIEZO1 lead to dehydrated hereditary stomatocytosis (DHS) by slowing down the channel's inactivation kinetics. DHS patients often exhibit a range of clinical presentations, including mild to severe hemolytic anemia, and iron overload. In addition, studies on erythroid progenitor cells from DHS patients have indicated a mutation-dependent delay in erythroid differentiation. Furthermore, studies on constitutive GoF Piezo1 mice have revealed stressed erythropoiesis characterized by increased erythrocyte turnover. The primary aim of this study is to investigate the role of PIEZO1 during stressed erythropoiesis in DHS and identify potential druggable targets. A total of 80 DHS patients were analysed, and their BMRI (reticulocyte count × patient's Hb/normal Hb), EPO, and ERFE levels were measured. The findings revealed mutation-dependent dyserythropoietic features similar to those observed in CDAII patients (characterized by dyserythropoiesis). The engineered erythroid model of DHS, Hudep2-PIEZO1-KI (Hudep2-KI), was subjected to erythroid differentiation for 12 days and compared to Hudep2-WT. A notable reduction in CD235a expression was detected on the last day of differentiation, indicating that PIEZO1-mediated alteration occurs during the late stages of differentiation. Morphological analysis of differentiating cells indicated that KI cells experienced a significant reduction in proliferation rate (1.5-fold increase vs. day 0) compared to Hudep2-WT (3.1-fold increase vs. day 0) starting from day 7. By day 12 of differentiation, KI cells displayed only half the cell count of WT cells, with a substantial decrease in the percentage of orthochromatic normoblasts (13.7% vs. WT 54.5%). This was accompanied by a relative increase in reticulocytes (Hudep2-WT: 45.5% vs. Hudep2-KI: 86.3%). To identify the altered signalling pathways involved in differentiation deregulation, RNAseq was performed at various time points during differentiation. The gene ontology analysis indicated that the most enriched biological processes were related to apoptosis, cellular response to hypoxia, and glycolysis. During the differentiation process, various apoptosis-related processes were found significantly enriched on different days. Upon conducting a single gene analysis, an imbalance between pro-apoptotic and antiapoptotic signals became evident. Specifically, in Hudep2-KI cells compared to the WT, we observed that 70.8% of proapoptotic genes were upregulated. Conversely, the regulation of apoptosis did not seem to impact antiapoptotic signals, with 57.9% of them being upregulated and 42.1% downregulated. This observation led us to suggest that PIEZO1 GoF induces apoptosis by activating pro-apoptotic signals. The findings of this study demonstrate the role of PIEZO1 GOF mutation in stressed erythropoiesis in both DHS patients and the erythroid cellular system. PIEZO1 GOF variants, through their effect on intracellular calcium concentration, influence late-stage differentiation and disrupt several critical pathways, including apoptosis, response to hypoxia, and glycolysis. Ongoing research involves confirming the data in CD34+ cells from DHS patients and PIEZO1 GOF mice. Additionally, the identified deregulated markers hold promise as potential druggable targets for dyserythropoiesis treatment.

P1404: HDAC6 REGULATES HUMAN ERYTHROID DIFFERENTIATION THROUGH MODULATION OF JAK2 SIGNALING

Background: Histone desacetylases (HDAC), through regulation of their acetylation level, modify the physico-chemical properties of proteins and modulate their activity, stability, localization and affinity for partners. Among them, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of HDAC6 (HDAC6i) that has been shown to be of value in treating multiple myeloma or lymphoma but was associated with secondary off target effects Among those, anemia is one of the most frequent, arguing for a particular importance of HDAC6 during erythropoiesis. So far, HDAC6 function during human erythropoiesis remains unknown. Aims: We present here an extensive study of HDAC6 expression and function during human erythropoiesis. Methods: We used two cell models: CD34+ progenitor cells obtained from apheresis from healthy donor, driven into in vitro erythroid diffenretiation and the leukemic cell line UT7/EPO. HDAC6 inhibition was obtained chemically using ACY-1215 and using an shRNA-based knockdown. Results: We quantified HDAC6 expression during erythroid differentiation and observed a progressive increase in HDAC6 RNA expression raising from Day 11 to D18. Using indirect immunofluorescence and flow imaging, we observed that HDAC6 expression was mainly cytoplasmic arguing for non-histone cytoplasmic targets. We then cultured CD34+-cells into in vitro erythroid differentiation in the presence of ACY-1215. We observed that 0.5µM ACY-1215 gave the best result in terms of HDAC6 inhibition, leading to an increased acetylated α-tubulin without affecting histone H3 acetylation level. ACY-1215 exposure on CD34+-cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. shRNA-mediated HDAC6 knockdown in the erythroleukemic cell line UT7/EPO also decreased GPA expression at cell surface. In primary cells, the delay in erythroid differentiation was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. We observed 1920 differentially expressed genes including 1134 upregulated and 786 downregulated. Rank-based GSEA analysis showed a (i) significant enrichment of downregulated genes related to bone marrow erythroblast associated with a downregulation of genes involved in heme metabolism, (ii) a significant enrichment of downregulated GATA1 targets and (iii) an enrichment of downregulated genes related to cell cycle. In UT7/EPO and in primary cells, HDAC6i decreased JAK2 phosphorylation in response to EPO as well as STAT5 activation, as assessed using immunoblot and phosphoflow. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure. We analyzed LNK/14-3-3ζ and LNK/JAK2 direct interactions using co-immunoprecipitation assays. ACY-1215 decreased the 14-3-3ζ/LNK interaction while increasing LNK/JAK2 interactions. Summary/Conclusion: Here, we show that LNK availability to interact with JAK2 in erythroid cells is regulated by HDAC6-dependent 14-3-3ζ acetylation level. Thus, we identified here a new mechanism of HDAC6-dependent control of human erythropoiesis through 14-3-3ζ acetylation level, LNK availability and JAK2 activation in response to EPO.

Base Editing-Mediated Dissection of the -200 Region of the γ-Globin Promoters to Induce Fetal Hemoglobin and Rescue Sickle Cell Disease and β-Thalassemia

β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option.The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs.In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters.In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs.Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress.We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype.In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice.In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. DisclosuresEl Nemer: Hemanext: Consultancy.

In Vitro Erythropoietic Response to Enzyme Replacement Therapy in Gaucher Type 1 Patients

Introduction. Gaucher disease type 1 (GD, OMIM #230800) is the most common inherited lysosomal storage disorder resulting from lysosomal glucocerebrosidase (GBA, acid-β-glucosidase) deficiency and subsequent accumulation of glucosylceramide in macrophages. This leads to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). Anemia is attributed to bone marrow infiltration by Gaucher cells, to inflammation and to the presence of splenomegaly; however the underlying pathophysiological mechanism hasn't been fully elucidated.Aim. The aim of this study is to evaluate the in vitro erythroid status at baseline (T0) and the response to Enzyme Replacement Therapy (ERT) after 6 months (T1) and 1 year (T2). We evaluated the expansion and differentiation in erythroid cultures of CD34+ cells obtained from peripheral blood of GD type 1 patients at diagnosis before starting ERT (T0), at 6 months (T1) and at 1 year (T2) of ERT.Methods. CD34+ cells were obtained by immunomagnetic separation from peripheral blood mononuclear cells of GD patients (n=3) and controls (n=5). Isolated CD34+ were evaluated by in vitro clonogenic capacity assays using the methylcellulose semisolid culture medium after 14-16 days of culture. Hematopoietic colonies (burst-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage (CFU-M)) were scored with an inverted microscope. CD34+ cells were expanded in StemSpan medium, 1% StemSpan CC100 cytokine cocktail, 2 U/ml Erythropoietin (EPO) and 1 μM dexamethasone. Differentiation was induced in StemSpan medium with 10 ng/ml SCF and 10 U/ml EPO. Proliferation and surface marker expression (CD34, CD45, Glycophorine A, CD71) were examined by flow cytometry. Cell morphology was analyzed on cytocentrifuged smears stained with May-Grunwald-Giemsa.Results. At T0 we observed a higher number of BFU-E in GD samples (126±23 BFU-E/105 cells) compared to controls (49±12 BFU-E/105 cells) (Tab.1). Similarly, higher macrophage CFU-M were observed in GD samples (29±12 CFU-M/105 cells) compared to controls (10±1 CFU-M/105 cells) (Tab.1). The BFU-E and CFU-M morphology showed no difference between GD patients and controls. The increased proliferation of erythroid precursors of GD patients was associated to impaired ability to differentiate in liquid cultures after 14 days (45,9% proerythroblasts and only 4% polichromatophilic erythroblasts) compared to controls (27% and 19%, respectively) (Tab.1). Flow cytometry confirmed an impaired differentiation of GD erythroblasts compared to controls at day 14 of the erythroid liquid cultures, as shown by high percentage of CD71high/GlyAlow early erythroid precursor cells and low percentage of CD71high/GlyAhigh mature erythroblasts (GD: 73,9 and 4,9%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1). In samples from GD patients, after 1-year treatment with ERT (T2), we observed a reduction in the number of BFU-E compared to T0, reaching values similar to controls (GD T0: 126±23 BFU-E/105 cells, T2: 41±11 BFU-E/105 cells; controls: 49±12 BFU-E/105 cells) and an increased number of mature erythroid cells generated in GD liquid cultures (Tab. 1). Also CFU-M were reduced at T2 compared to T0 (GD T0: 29±12 CFU-M/105 cells, T2: 8±5 CFU-M/105 cells; controls: 10±1 CFU-M/105 cells) (Tab.1). An increased percentage of more differentiated erythroid cells at T2 compared to T0 was confirmed by flow cytometry in cultures from GD patients, as shown by low percentage of the CD71high/GlyAlow early erythroid precursor cells and high percentage of CD71high/GlyAhigh mature erythroblasts on day 14 (GD: 44,7 and 45,6%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1).Conclusions. These preliminary data suggest that anemia in GD might partly be related to a delay in erythroid differentiation. ERT treatment seems to correct the dyserythropoiesis by improving erythroid differentiation. Since glucosylceramide accumulates in macrophages, that are key elements in the erythroid niche, impaired erythroid differentiation might be related to the pathological micro-environment, rather than intrinsic hematopoietic stem cell defect. More extensive studies are needed to better understand the pathophysiology of anemia and pancytopenia of this rare disease.This study was partially supported by Sanofi Genzyme. [Display omitted] DisclosuresCappellini:Celgene: Honoraria; Novartis: Speakers Bureau; Vifor: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau.

Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of Nuclear Factor I X (NFIX) As a Novel Fetal Hemoglobin (HbF) Repressor

Human beta-like globin gene expression is developmentally regulated. Erythroblasts (EBs) derived from fetal tissues, such as umbilical cord blood (CB), primarily express gamma globin mRNA (HBG) and HbF, while EBs derived from adult tissues, such as bone marrow (BM), predominantly express beta globin mRNA (HBB) and adult hemoglobin. Human genetics has validated de-repression of HBG in adult EBs as a powerful therapeutic paradigm in diseases involving defective HBB, such as sickle cell anemia. To identify novel factors involved in the switch from HBG to HBB expression, and to better understand the global regulatory networks driving the fetal and adult cell states, we performed transcriptome profiling (RNA-seq) and chromatin accessibility profiling (ATAC-seq) on sorted EB cell populations from CB or BM. This approach improves upon previous studies that used unsorted cells (Huang J, Dev Cell 2016) or that did not measure chromatin accessibility (Yan H, Am J Hematol 2018). CD34+ cells from CB and BM were differentiated using a 3-phase in vitro culture system (Giarratana M, Blood 2011). Fluorescence-activated cell sorting and the cell surface markers CD36 and GYPA were used to isolate 7 discrete populations, with each sorting gate representing increasingly mature, stage-matched EBs from CB or BM (Fig 1A, B). RNA-seq analysis revealed expected expression patterns of the beta-like globins, with total levels increasing during erythroid maturation and primarily composed of HBB or HBG transcripts in BM or CB, respectively (Fig 1C). Erythroid maturation led to progressive increases in chromatin accessibility at the HBB promoter in BM populations. In CB-derived cells, erythroid maturation led to progressive increases in chromatin accessibility at the HBG promoters through the CD36+GYPA+ stage (Pops 1-5). Chromatin accessibility shifted from the HBG promoters to the HBB promoter during the final stages of differentiation (Pops 6-7), suggesting that HBG gene activation is transient in CB EBs (Fig 1D). Hierarchical clustering and principal component analysis of ATAC-seq data revealed that cell populations cluster based on differentiation stage rather than by BM or CB lineage, suggesting most molecular changes are stage-specific, not lineage-specific (Fig 2A, B). To identify transcription factors driving cell state, and potentially beta-like globin expression preference, we searched for DNA binding motifs within regions of differential chromatin accessibility and found NFI factor motifs enriched under peaks that were larger in BM relative to CB (Fig 2C). Transcription factor footprinting analysis showed that both flanking accessibility and footprint depth at NFI motifs were also increased in BM relative to CB (Fig 2D). Increased chromatin accessibility was observed at the NFIX promoter in BM relative to CB populations, and in HUDEP-2 relative to HUDEP-1 cell lines (Fig 2E). Furthermore, accessibility at the NFIX promoter correlated with elevated NFIX mRNA in BM and HUDEP-2 relative to CB and HUDEP-1, respectively. Together these data implicated NFIX in HbF repression, a finding consistent with previous genome-wide association and DNA methylation studies that suggested a possible role for NFIX in regulating beta-like globin gene expression (Fabrice D, Nat Genet 2016; Lessard S, Genome Med 2015). To directly test the hypothesis that NFIX represses HbF, short hairpin RNAs were used to knockdown (KD) NFIX in primary erythroblasts derived from human CD34+ BM cells (Fig 3A). NFIX KD led to a time-dependent induction of HBG mRNA, HbF, and F-cells comparable to KD of the known HbF repressor BCL11A (Fig 3B-D). A similar effect on HbF was observed in HUDEP-2 cells following NFIX KD (Fig 3E). Consistent with HbF induction, NFIX KD also increased chromatin accessibility and decreased DNA methylation at the HBG promoters in primary EBs (Fig 3F, G). NFIX KD led to a delay in erythroid differentiation as measured by CD36 and GYPA expression (Fig 3H). Despite this delay, by day 14 a high proportion of fully enucleated erythroblasts was observed, suggesting NFIX KD cells are capable of terminal differentiation (Fig 3H). Collectively, these data have enabled identification and validation of NFIX as a novel repressor of HbF, a finding that enhances the understanding of beta-like globin gene regulation and has potential implications in the development of therapeutics for sickle cell disease. Disclosures Chaand: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Johnston:Syros Pharmaceuticals: Employment, Equity Ownership. Moon:Syros Pharmaceuticals: Employment, Equity Ownership. Carulli:Syros Pharmaceuticals: Employment, Equity Ownership. Shearstone:Syros Pharmaceuticals: Employment, Equity Ownership.

PIEZO1 activation delays erythroid differentiation of normal and hereditary xerocytosis-derived human progenitor cells.

Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/β-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STAT5 and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia associated with haploinsufficiency of more than 15 ribosomal protein genes. We identified in the French registry a DBA patient exhibiting a large heterozygous deletion in ribosomal protein gene RPSA, making it a new candidate as a DBA-associated gene. The deletion was confirmed by qPCR using specific intron/exon primers that could discriminate the RPSA gene from pseudo-genes. Consistent loss of function of this allele, RPSA mRNAs were underrepresented in erythroid cultures and in lymphoblastoid cells (LCLs) derived from the patient. Additionally, an important decrease in erythroid proliferation was observed in erythroid progenitor and precursor cells derived from the patient's bone marrow CD34+ cells, together with a delay in erythroid differentiation, the activation of the p53 pathway, and a G0/G1 cell cycle arrest, which are regular features of DBA patient cells. Similar to other RP genes linked to DBA, interruption of RPSA expression in zebrafish embryos strongly affected development of the head and erythroid cell differentiation, supporting its relevance as a candidate DBA gene. Northern blot analysis also revealed impaired pre-ribosomal RNA (pre-rRNA) processing in patient LCLs compared to controls. But unexpectedly, the observed pre-rRNA profile was clearly different from that observed in HeLa cells upon RPSA knockdown and suggested a dysfunction of pre-rRNA processing upstream of that produced by a shortage of RPSA. We noticed that the RPSA gene hosts the intron-encoded H/ACA snoRNAs snoRA6 and snoRA62, the latter being included in the deletion. Q-RT-PCR analysis of patient cells showed decreased levels of snoRA62 when compared to controls and to other RP-mutated DBA patients. Strikingly, knocking out the snoRNA62 coding sequence in human haploid HAP1 cells with Crispr/Cas9 resulted in a pre-rRNA maturation defect similar to that observed in the RPSA+/mut DBA patient, thereby revealing a role for snoRA62 in human pre-rRNA processing. These data strongly suggest that, in addition to RPSA haploinsufficiency, the primum movens of DBA in this particular patient includes the deletion of the intronic snoRA62 hosted in the RPSA gene. To our knowledge, this is the first time that a snoRNA is linked to DBA. We will discuss this ongoing work in light of our most recent results in cells and zebrafish models. DisclosuresNo relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoetic Stem and Progenitor Cells

In recent clinical studies, it has been demonstrated that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). Mutation of ASXL1 leads to truncation of the protein and thereby to a loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in hematopoietic system in vitro mimics the changes observed in MDS-patients.We down regulated ASXL1 in CD34+ cord blood (CB) cells using a lentiviral approach and obtained a 40-50% reduction of ASXL1 expression. Colony forming (CFC) assays revealed that erythroid colony formation was significantly impaired (p=0.01) and, to some extent, granulocytic and macrophage colony formation (p=0.09, p=0.05 respectively). As MDS can affect all hematopoietic lineages, we first stimulated cell differentiation along the myeloid or erythroid lineage in liquid culture. Upon culturing shASXL1 CB CD34+ cells in suspension, we observed a modest reduction in expansion (two-fold at week1) under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p=0.02). The increase of cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. This phenotype is similar to what has been observed in patients, where increased cell death of progenitors occurs, and suggests that ASXL1 loss may reflect an MDS-like phenotype in this culture setting. Furthermore, as MDS is considered a hematopoietic stem cell (HSC)-driven disorder, we tested whether HSCs were affected by ASXL1 loss. Long-term culture initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, we performed cultures on stromal layers with or without cytokines. shASXL1 CB CD34+ cells cultured on MS5 stromal layer showed a modest, two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. In patients, mutations in ASXL1 are frequently accompanied by a loss of p53. Possibly, loss of p53 is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our first data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies compared to shASXL1/shSCR cells, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, when using erythroid liquid conditions, a decrease in apoptosis compared to the ASXL1 single mutation could be observed. Nevertheless, no transformation occurred and ASXL1 mutated cells were eventually lost in the double hit model despite reduced apoptosis, suggesting that the p53 axis might not be sufficient as the second hit for full transformation.In conclusion, our data indicate that mutations in ASXL1 may lead to an increase in cell death and reduced progenitor output in vitro, which may reflect disease development and progression as seen in patients. Unexpectedly, MS5 stromal did not alter the negative phenotype caused by ASXL1 knock down. Therefore, studies are ongoing to investigate whether an already established MDS microenvironment will influence ASXL1 mutation positively. To this end, we are using healthy human mesenchymal stem cells (MSC) and patient derived MDS MSCs. DisclosuresNo relevant conflicts of interest to declare.

Pomalidomide Modulates Transcription Networks Regulating Human Erythropoiesis and Globin Switching: Implications for Treatment of Hemoglobinopathies

Sickle cell disease (SCD) represents a major challenge in hematology, with approximately 100,000 Americans afflicted and the annual number of newborns with SCD set to rise over the next 40 years worldwide. Current treatment approaches rely on increasing levels of fetal hemoglobin (HbF) to prevent painful vaso-occlusive crises and hemolysis secondary to red cell sickling. Hydroxyurea remains the only pharmacologic intervention approved for SCD; however, it has limited efficacy and carries significant side effects such as myelosuppression. Thus, there is a critical need to develop drugs that enhance HbF production without similar dose limiting side effects. Second generation immunomodulatory drugs, such as pomalidomide, are a class of emerging HbF inducers both in vitro and in vivo. Recent work from our laboratory revealed that hydroxyurea and pomalidomide differentially regulate HbF production in CD34+ cells undergoing erythroid differentiation using a 3-phase culture system. Pomalidomide, but not hydroxyurea, was found to decrease BCL11A expression through a yet to be defined mechanism. In the present study, we sought to characterize erythropoiesis and the expression of key transcription factor networks in this 3-phase culture system to determine the mechanisms underlying pomalidomide’s effect. Following a four day expansion period, isolated CD34+ cells from the peripheral blood of SCD or normal individuals were differentiated along erythroid lineage in the presence of pomalidomide (1μM) or DMSO (vehicle) for 14 days. As an additional control, CD34+ cells were also treated with hydroxyurea (10μM). Proliferation and erythroid differentiation were assessed at 7, 11 and 14 days of culture. Although a 50% decrease in cell growth was noted in cells treated with hydroxyurea, no such decrement was found in control, DMSO and pomalidomide-treated cells. Moreover, pomalidomide produced a transient delay in erythroid differentiation between days 6 and 11 of culture phenotypically documented by flow cytometric analysis using glycophorin-A, α-4 integrin and band 3 as surface markers monitoring erythroid differentiation as well as morphologically by May-Grunwald Giemsa staining. In contrast, cells treated with hydroxyurea demonstrated accelerated differentiation, compared to the control cultures. However, by day 14 of culture, no significant difference was observed under any condition, suggesting that the delayed cells eventually finished terminal differentiation. In terms of HbF induction, we confirmed elevated production in the cultures with pomalidomide by measuring the number of F-cells by flow cytometry. We also evaluated the production of γ-globin chains by qRT-PCR and western blot at D4 and D11 and found a dramatic increase in the production of γ-globin, in both SCD and normal samples treated with pomalidomide.We posited that pomalidomide might foster changes in transcription factors known to play a role in both erythropoiesis and globin switching. To this end, we evaluated the expression kinetics of BCL11A, SOX6, KLF1, MI2β, GATA1 and FOG1 via qRT-PCR and western blot analyses. In DMSO-treated cultures the above transcription factors were maximally expressed between days 6-8, and their levels diminished during the remainder of the culture. Conversely, pomalidomide markedly decreased BCL11A, SOX6, KLF1 and MI2β between days 4 and 6 in cultures of both SCD and normal samples. In line with our results, MI2β acts as a positive regulator of BCL11A and KLF1, and previous studies have shown that its knock down in CD34 cells leads to decreased levels of BCL11A and KLF1. Western blot analyses confirmed the qRT-PCR data. Further, the divergent expression patterns correlated temporally with the differentiation delay suggesting that pomalidomide modulates expression of members of the BCL11Atranscription factor complex, thereby augmenting γ-globin production.Taken together, these data provide evidence that pomalidomide influences erythropoiesis by modulating transcription factor expression in CD34+ cells differentiated in the 3-phase culture system, leading to a decrease in BCL11A and activation of γ-globin production. Importantly, further exploration of these pathways that function to regulate erythropoiesis and promote HbF silencing, may help elucidate the mechanism of action of pomalidomide as well as identify additional druggable molecules. DisclosuresNo relevant conflicts of interest to declare.

Ribavirin suppresses erythroid differentiation and proliferation in chronic hepatitis C patients.

Combination therapy with pegylated interferon (pegIFN) plus ribavirin (RBV) is the standard of care for chronic hepatitis C. One of the major treatment-related side effects is anaemia, attributed to RBV-induced haemolysis. However, haemolysis biomarkers are not present in all patients supporting the existence of other pathogenetic mechanisms. We studied the role of RBV in inducing haemolysis and its effects on erythropoiesis. In 18 hepatitis C virus (HCV) genotype 2 patients treated with pegIFN-alpha-2a (180 mcg/week) plus RBV (800 mg/day) for 24 weeks and in 10 hepatitis B virus (HBV) patients treated with pegIFN-alpha-2a (180 mcg/week) for 48 weeks, haemolysis was assessed by serum LDH, haptoglobin and reticulocyte count. Erythropoiesis was evaluated both ex vivo, analysing the clonogenic activity of patients' erythroid progenitors, as well as in vitro adding pegIFN and RBV to liquid cultures obtained from CD34+ cells of healthy volunteers. The majority of patients developed anaemia; the week 4 mean haemoglobin decrease was greater in HCV than in HBV patients (1.7 vs 0.47 g/dL, P = 0.01). Only three HCV patients (17%) and no HBV patients showed signs of haemolysis. The 15 nonhaemolytic HCV patients and all HBV patients showed a delay in erythroid differentiation, with a reduction in colony number and a relative increase in undifferentiated colony percentage. Haemolytic HCV patients had an increase in colony number at week 4 of therapy. In vitro, erythroid cell proliferation and differentiation were inhibited by both pegIFN and RBV. Both pegIFN and RBV have an inhibitory effect on erythroid proliferation and differentiation.

Identification of Phospholipase C Gamma 1 As a Key Regulator of Erythropoiesis

Abstract 4744Erythropoiesis is a complex multistage process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors. Regulation of survival, expansion and differentiation of erythroid progenitors is dependent on a well-coordinated cohort of transcription factors and an intricate network of finely tuned regulatory signalling pathways. In vivo and in vitro studies have highlighted erythropoietin receptor (EpoR) signaling through JAK2 tyrosine kinase as a crucial regulator of erythropoiesis. This leads to the subsequent activation of downstream effectors such as STAT5, MAPK, and PI-3K/Akt pathways. However, detailed knowledge about signalling pathways involved in EPO/EpoR induced differentiation of erythroid progenitors remain elusive.Phosphatidylinositol-specific phospholipase C gamma1 (PLCg1) is known to act as key mediator of calcium-signalling that can substitute for PI-3K/AKT signalling in oncogenic models. Moreover, its loss is associated with lack of erythropoiesis in a straight knockout mouse model. As it is tempting to speculate on the role of Plcg1/Ca-signalling downstream of EpoR/JAK in regulation of erythroid development we aimed to investigate its influence on differentiation and proliferation of hematopoietic cells in vitro and in vivo.Using different cellular models (Ba/F3, 32D) stably transfected with EpoR and wildtype JAK2 we could provide evidence that PLCg1 is a downstream target of EpoR/JAK2 signalling. Knockdown of PLCg1 led to a decreased proliferation of PLCg1-deficient cells compared to control cells whereas survival of these cells was not affected. In contrast, other downstream targets of EpoR signalling were not affected by PLCg1 knockdown. In order to assess specifically its role in erythroid development, we used the murine pro-erythroblast cell line I-11 as well as primary fetal liver cells (FLC). The I-11 cell line was isolated from p53-deficient fetal livers and is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation; primary FLC were harvested at E13.5. PLCg1 knockdown by using RNA-interference technology led to a significant delay in erythroid differentiation and accumulation of immature erythroid progenitors (e.g. pro-erythroblasts) as assessed by cytology and flow cytometry technology. In addition, we tested the colony-forming potential of PLCg1-deficient I-11 and fetal liver cells compared to controls. Colony formation was significantly impaired in both - I-11 and primary FLC - when compared to control cells (shRNA-scr). We performed gene-expression analysis by Q-RT-PCR on sorted hematopoietic stem and progenitor cells and found a higher expression in MEP compared to GMP or CMP. To clarify, whether the effects of Plcg1 knockdown are restricted to erythroid development at the stage of MEP or erythroid progenitors, we aimed to investigate adult hematopoietic stem cells in erythroid development. We infected lineage-depleted/erythroid-enriched (Gr1-, B220-, CD3/4/8, CD19-/ IL7Ra- negative) bone marrow cells with either PLCg1 or control shRNA. Using flow cytometry analysis to examine differentiation we could observe a reduction of megakaryocyte/erythroid progenitor cells (MEP) in PLCg1 knockdown cells compared to control cells while development of other lineages (e.g. GMP) remained unaffected. Currently, competitive repopulation assays investigating the repopulation and differentiation capacity of hematopoietic stem cells after Plcg1 knockdown (or scr controls) are under way to explore the role of Plcg1 signalling in hematopoietic and erythroid development in vivo.Taken together, our findings presume PLCg1 to be a key regulator in erythroid development and understanding of its relevance in development and maintenance of normal hematopoiesis will be a crucial prerequisite for targeting this important pathway in myeloproliferative disease. Disclosures:No relevant conflicts of interest to declare.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Abstract 4829The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our datareveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures:No relevant conflicts of interest to declare.

Activation of p53 Pathway In Erythroid Precursor Cells of Diamond-Blackfan Anemia Leads to Two Distinct Phenotypes

AbstractAbstract 2225Identification of mutations in a number of ribosomal genes has established DBA as a disease of aberrant biogenesis of ribosomes. We have previously documented that knockdown of RPS19, RPL5 and RPL11 mRNA by shRNAs in CD34+ cells from cord blood leads to two distinct invitro erythroid phenotypes. The haploinsufficient RPS19 precursors exhibited decreased erythroid proliferation, normal erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 and no apoptosis. In contrast, RPL5 or RPL11 haploinsufficient precursors showed a larger decrease in erythroid proliferation, a delay in erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 in association with increased apoptosis. To validate these findings, we studied in vitro erythroid differentiation of primary CD34+ cells isolated from peripheral blood of DBA patients. CD34+ cells from 12 DBA individuals carrying mutations in RPS19 gene, 7 DBA individuals with no mutations in RPS19 and 19 normal individuals. CD34+ cells isolated from peripheral blood were cultured in two-phases, first in methyl-cellulose for 7 days and then in liquid culture for additional 5 days. Cells were harvested at Day 7 (D7), D10 and D12 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay or flow cytometry), and mRNA and protein expression levels. From D0 (CD34+ cells) to D7 (CFU-e/proerythroblasts), to D10 (basophilic erythroblasts) and D12 (orthophilic erythroblasts), we observed a decrease in the extent of cell amplification of CD34+ cells from all DBA individuals. Interestingly, this decrease was more pronounced for CD34+ cells not carrying a mutation of RPS19 gene than for cells harbouring a mutation of RPS19. While a 9.5 fold amplification was noted between D0 to D7, for normal CD34+ cells, it was reduced to 6.1 fold for DBA cells with mutant RPS19 and further reduced to 3.8 fold for DBA cells not carrying RPS19 mutations. While we did not find a difference in cell amplification between D10 and D12 for DBA cells with RPS19 mutations compared to normal cells, we noted a large decrease in cell amplification for DBA cells without RPS19 mutations. We also studied 6 additional DBA patients (one with a mutant RPS19 gene, 2 with mutations in RPL11 gene and 3 in whom no RP gene mutations could be identified to date) using the two phase liquid culture system. Once again we noted that CD34+ cells with RPL11 mutations or with yet to be defined mutations exhibited a larger decrease in cell amplification from D0 to D7 and from D10 to D12 compared to CD34+ cells harbouring the RPS19 mutation. While erythroid differentiation was normal on both MGG and by flow cytometry in RPS19 mutant erythroid precursors, we observed a delay in erythroid differentiation in non RPS19 mutated precursors, in particular at D7 or D10 with increased number of CD34+ cells. Importantly, we noted increased apoptosis of cells not carrying RPS19 mutations compared to cells with RPS19 mutations. Strikingly, in all 6 DBA patients, p53 pathway (p53, p21 at the mRNA level and bax) was activated in the erythroid precursor cells. We confirmed an increase in apoptosis in the DBA patient erythroid precursors with non-mutant RPS19 as evidenced by decreases in Hsp70 and procaspase 3, and an increase in cleaved caspase 3. We confirmed cell cycle arrest in G0/G1 in cells from both DBA individuals studied. Reinforcing these findings, phosphoRb was found to be decreased in cells from all 6 DBA patients studied. In summary, using primary CD34+ cells from DBA individuals, we confirmed our earlier finding of the two distinct DBA phenotypes with cell arrest in conjunction with either presence or absence of apoptosis depending on whether the cells carry mutations in RPS19 or not. Strikingly, p53 pathway was activated in primary cells of all DBA patients studied. Our results suggest that erythroblastopenia, the main characteristics of DBA is likely the result of p53 activation leading to cell cycle arrest and apoptosis. Disclosures:No relevant conflicts of interest to declare.

NF-E2 Overexpression Delays Erythroid Differentiation and Increases Erythrocyte Production

The transcription factor Nuclear-Factor-Erythroid2 (NF-E2) is overexpressed in the vast majority of patients with polycythemia vera (PV). PV affects precisely those lineages expressing NF-E2: hematopoietic precursors, erythroid, megakaryocytic and granulocytic cells. In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of EPO. EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic symptom of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression lead to a delay in erythroid differentiation, manifested by a belated appearance of GlycophorinA-positive mature erythroid cells. Differentiation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted differentiation lead to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in BFU-E formation. These data argue that NF-E2 overexpression delays the early phase of erythroid differentiation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV.

Identification of K-ras as the major regulator for cytokine-dependent Akt activation in erythroid progenitors in vivo

Despite intensive investigation, controversial results have been obtained concerning the precise signaling pathway(s) regulated by K-ras in different cell types. We show that in primary fetal liver erythroid progenitors, erythropoietin activates all three Ras isoforms, but preferentially N- and K-ras. In K-ras(-/-) fetal liver cells (FLC), erythropoietin- or stem cell factor-dependent Akt activation is greatly reduced, whereas other pathways including Stat5 and p44/p42 MAP kinase are activated normally. We further studied the effects of reduced cytokine-dependent Akt activation in erythroid differentiation. We find that freshly isolated K-ras(-/-) FLC show an approximately 7-fold increase of apoptosis and delayed erythroid differentiation, but only at the stage of erythroid progenitors and very early erythroblasts. When K-ras(-/-) erythroid progenitors are cultured in vitro, there is a significant delay in erythroid differentiation but little increase in apoptosis. Furthermore, we show that partial pharmacologic inhibition of the phosphatidylinositol 3-kinase/Akt pathway in wild-type erythroid progenitors leads to a delay in erythroid differentiation similar to that observed in K-ras(-/-) FLC. Taken together, our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo.