Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

Abstract LIN28 proteins bind to RNA and regulate developmental timing events in multicellular organisms, in part, by reducing cellular levels of the let-7 family of microRNAs. High-level LIN28 expression in stem cells promotes their self-renewal. Over-expression of the LIN28 proteins causes suppression of let-7 in hematopoietic stem and early progenitor cell populations (CD34+) from adult donors and manifests a more fetal-like phenotype in the erythroid lineage. Here we explore LIN28expression that is restricted to erythroid cells, rather than stem or multi-potential progenitor cells. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes, as well as an internal ribosomal entry site for puromycin selection (vectors: KLF1-LIN28A-OE and SPTA1-LIN28A-OE). Viral supernatants from these constructs were compared with empty-vector controls in matched transductions of CD34+ cells from three adult human volunteers. The cells were transduced and cultured using a three-phase, serum-free model for ex vivo erythropoiesis. Erythroblast proliferation and differentiation were comparable between control and LIN28-transduced cells assessed by cell counting and flow cytometry with staining for CD71, glycophorin A and thiazole orange. To validate restricted expression of LIN28 in the erythroid lineage, colony formation assays were performed in semisolid methylcellulose containing 1.0 ug/ml puromycin. BFU-E, CFU-GM, CFU-G, CFU-M and GEMM colonies were enumerated 14 days after plating. Puromycin addition to KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions resulted in selection of the erythroid colonies (BFU-E as a percentage of total colonies: Control: 44.6 ± 6.1%; KLF1-LIN28A-OE: 98.4 ± 0.7%, p=0.003; SPTA1-LIN28A-OE: 95.2 ± 1.1%, p=0.005). LIN28A over-expression was confirmed by RT-QPCR (KLF1-LIN28A-OE: 2.1E+05 ± 7.0E+04 copies/ng; SPTA1-LIN28A-OE: 2.2E+05 ± 8.3E+04 copies/ng; Controls: below detection limits) and Western analyses after transduction. Suppression of all let-7 miRNA family members to less than 30% control levels were detected for both vectors resulting in a reduction in total let-7 miRNA (RT-QPCR: Control: 2.0E+07 ± 9.7E+05 copies/ng; KLF1-LIN28A-OE: 5.6E+06 ± 5.6E+05 copies/ng, p=0.003; SPTA1-LIN28A-OE: 4.6E+06 ± 6.2E+05 copies/ng, p=0.003). BCL11A expression levels were also measured by RT-QPCR and Western analyses. While BCL11A showed no significant change at the mRNA level (Control: 1.2E+03 ± 4.5E+02 copies/ng; KLF1-LIN28A-OE: 2.9E+02 ± 7.4E+01 copies/ng, p=0.07; SPTA1-LIN28A-OE: 4.2E+02 ± 3.3E+02 copies/ng, p=0.07), protein analyses of nuclear BCL11A showed moderately reduced levels after KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions. Globin mRNA and protein levels were investigated and compared with controls. Gamma-globin mRNA was significantly increased in LIN28A-OE samples (Control: 3.6E+06 ± 8.2E+05 copies/ng; KLF1-LIN28A-OE: 1.9E+07 ± 1.7E+06 copies/ng, p=0.007; SPTA1-LIN28A-OE: 1.7E+07 ± 8.9E+05 copies/ng, p=0.003). Fetal hemoglobin (HbF) production was measured at the end of the culture period using High Performance Liquid Chromatography, and was increased in the KLF1-LIN28A-OE and SPTA1-LIN28A-OE samples compared to the control (Control: 7.0 ± 1.4%; KLF1-LIN28A-OE: 31.9 ± 2.7%, p=0.004; SPTA1-LIN28A-OE: 43.0 ± 6.2%, p=0.004). Flow cytometry analyses demonstrated a pan-cellular HbF distribution. In contrast to promoting self-renewal in stem cells, these data suggest that adult erythroblast-restricted LIN28 functions to partially reverse the fetal-to-adult developmental transition in hemoglobin expression. Disclosures No relevant conflicts of interest to declare.

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Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽

10.1182/blood.v126.23.2161.2161 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2161-2161

Author(s):

Jaira F. de Vasconcellos ◽

Y. Terry Lee ◽

Colleen Byrnes ◽

Laxminath Tumburu ◽

Antoinette Rabel ◽

...

Keyword(s):

Protein Expression ◽

Fetal Hemoglobin ◽

Cell Counting ◽

Mrna Levels ◽

Rt Pcr ◽

Globin Mrna ◽

Expression Levels ◽

Gamma Globin ◽

Empty Vector ◽

Gene And Protein Expression

Abstract HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

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The Role of Truncated c-kit Receptor (tr-kit) in Maintenance and Differentiation of Hematopoietic Stem Cells and Multipotent Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v106.11.4193.4193 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4193-4193 ◽

Cited By ~ 1

Author(s):

Jie Yang ◽

Candice I. Saltiel ◽

Ronald G. Nachtman ◽

Xin Jing ◽

Roland Jurecic

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Signaling Pathways ◽

Rna Binding ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Hematopoietic Stem ◽

Over Expression ◽

Multipotent Progenitors ◽

Kit Receptor

Abstract Intrinsic mechanisms that regulate self-renewal of mammalian stem cells are slowly being elucidated. Self-renewal of stem cells in Drosophila and C. elegans is regulated by members of the conserved Pumilio family of RNA-binding proteins. Previously, we have cloned and characterized two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the function of mammalian Pum proteins in HSC and multipotent progenitors, the RNA-binding domain of Pum2 was over-expressed in a stem cell factor (SCF)-dependent HSC-like cell line EML. In the presence of SCF EML cells undergo SCF-dependent self-renewal and remain undifferentiated. In the presence of various cytokines (IL-3, GM-CSF, G-CSF, Epo, Tpo, IL-7, Flt3L) EML cells differentiate into erythroid, granulocytic, megakaryocytic and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and suppresses their mutilineage differentiation in the absence of SCF. This uncoupling of the maintenance and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, (b) cell intrinsic, SCF-independent activation of c-kit, and (c) constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. These results indicate that Pum2 could be supporting maintenance of multipotent hematopoietic cells through regulation of SCF/c-kit signaling pathway. An in depth analysis of the pattern of tr-kit expression in murine fetal liver and bone marrow-derived HSC, multipotent progenitors, lineage-committed progenitors and immature blood cells has shown that tr-kit expression is restricted to cell populations highly enriched for HSC and multipotent progenitors. This observation and the finding that an increased expression of tr-kit protein correlates with SCF-independent maintenance of EML cells, suggest that tr-kit could play an important role in SCF-independent activation of full-length c-kit receptor, and participate in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. The fact that Pum2 and tr-kit are co-expressed in bone marrow cells enriched for HSC and early multipotent progenitors (e.g. Lin-Sca-1+c-kit+ cells), but not in later progenitors (e.g. Lin-Sca-1−c-kit− cells), suggests an exciting possibility that HSC and early multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent hematopoietic progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. We are currently studying the effects of Pum2 and tr-kit over-expression and attenuation on (a) HSC and progenitor cell maintenance and differentiation, (b) HoxB4 and Notch1 pathways, involved in HSC maintenance and expansion, and (c) maintenance and differentiation of HSC expressing SLAM receptor CD150. Further study of Pum2 and tr-kit function could provide important new insights into the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation.

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Effect Of Deferasirox On Reactive Species Of Oxygen (ROS) Production In Hematopoietic Stem Cells: Up Or Down?

Blood ◽

10.1182/blood.v122.21.1195.1195 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1195-1195

Author(s):

Tiziana Tataranni ◽

Francesca Agriesti ◽

Carmela Mazzoccoli ◽

Vitalba Ruggieri ◽

Fiorella D'Auria ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Hematopoietic Stem Cells ◽

Cell Viability ◽

Western Blotting ◽

Peripheral Blood ◽

Reactive Species ◽

Ros Production ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Deferasirox (DFX) is an iron chelator used to prevent and treat complications related to transfusional iron overload in myelodisplastic syndrome patients (MDS). Intriguingly, DFX treatment induces haematological responses in a consistent percentage of patients whereas other chelators, like deferoxamine (DFO) do not show such an appreciable effect. A body of literature documents a general reduction of oxidative stress in patients treated with DFX but little is known about the direct effect of DFX treatment on hematopoietic stem cells (HSC). Consolidated evidence highlights the importance of redox signalling in the homeostasis of fundamental processes, particularly in controlling the balance between self-renewal and differentiation of stem cells. In this setting, reactive species of oxygen (ROS) would act as secondary messengers, modulating the expression of master transcription factors and regulatory proteins leading or (pre)conditioning stem cells towards differentiation. In the present study we investigated the effect of DFX and DFO on ROS production in hematopoietic stem/progenitor cells (HSPCs) in order to identify a molecular mechanism explaining the differential effect of iron chelators in rescuing altered hematopoiesis. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy subjects and incubated for 24 hours with DFO and DFX in a range of doses between 6.2mM and 200mM. Human HSPCs, isolated upon informed consent from peripheral blood of G-CSF-treated healthy donors by immuno-selection against the specific markers CD133 and CD34, were treated with 100 mM DFX or DFO for 24 hours. To completely abrogate ROS production, cells were co-incubated with diphenil iodide (DPI) 100mM, a known inhibitor of the main proteins able to generate ROS: the flavo-oxidases of the respiratory chain and the NADPH oxidases. Cell viability was determined by trypan blue staining. ROS levels were analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry after the incubation at 37°C for 15 minutes with the intracellular H2O2 specific probe dichlorodihhyrofluorescein-diacetate (H2DCFDA) 10mM. b-catenin and BMI1 protein levels were assessed by western blotting. Data were presented as mean±s.e.m. and were compared by unpaired Student T-Test; a p<0.05 was considered significant. The flow cytometry analysis on PBMCs revealed that DFX, surprisingly, was able to induce ROS production in a dose-dependent manner with a significant increase at 100mM and 200mM compared to CTRL (p=0.02) whereas DFO treatment didn’t show any effect compared to CTRL. Since the cell viability resulted to be not affected by both drugs, the dose of 100mM was chosen for the following experiments performed with HSPCs where the ability of DFX to cause a significant increase of H2O2 was confirmed. A deeper analysis aimed to clarify the localization of ROS released following DFX treatment was performed by LSCM: a significant up-regulation of mitochondrial ROS induced by DFX and not by DFO was clearly observed compared to untreated samples. Importantly, the addition of DPI strongly reduced the entity of the signal detected by DCF (p=0.01 versus DFX). In order to understand a possible link between ROS production and the ability of DFX to restore the hematopoiesis, the expression of b-catenin and BMI1, both of them sensitive to the intracellular redox state and involved in the hematopoietic function, was analysed by western blotting. It was shown that DFX significantly reduced the expression of both b-catenin, strictly linked to hematopoietic function and BMI1 (p=0.02 versus CTRL), a regulatory protein sustaining immaturity and required for the maintaining of adult self-renewing hematopoietic stem cells. Conversely, no significant variation was observed by DFO treatment. Interestingly, the b-catenin and BMI-1expression downregulations, specifically induced by DFX, were completely reversed by ROS abrogation obtained by DPI treatment. Our results show, for the first time, that DFX treatment, independently on its iron-chelating property, is able to induce ROS production that in turn influences key factors involved in self-renewal/differentiation of hematopoietic stem cells. In this scenario, the modulation of ROS, because of their ability to restore the hematopoietic function, could be taken in account as potential further pharmacological target in MDS treatment. Disclosures: No relevant conflicts of interest to declare.

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Relationship Between Self-Renewal and Differentiation Pathways in Stem Cells and Leukemia

Blood ◽

10.1182/blood.v124.21.4789.4789 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4789-4789

Author(s):

Robert S Welner ◽

Danielle E Tenen ◽

Henry Yang ◽

Deepak Bararia ◽

Giovanni Amabile ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Wnt Signaling ◽

Cell Fate ◽

Wnt Pathway ◽

Beta Catenin ◽

Self Renewal ◽

Hematopoietic Stem ◽

Over Expression

Abstract Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate. To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation. Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood. These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. Disclosures No relevant conflicts of interest to declare.

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