LIN28B and ZBTB8B Genes Are Highly Expressed in Vitro in a CD34⁺ Cells Subpopulation of β-Thalassemia Major Patients and May be Involved in Increased HbF Production

Abstract Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. Disclosures Costa: Novartis: Consultancy.

Download Full-text

Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽

10.1182/blood-2019-122770 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2063-2063

Author(s):

Naoya Uchida ◽

Claire Drysdale ◽

Morgan Yapundich ◽

Jackson Gamer ◽

Tina Nassehi ◽

...

Keyword(s):

Rhesus Macaques ◽

Fetal Hemoglobin ◽

Selective Advantage ◽

Erythroid Cells ◽

Post Transplant ◽

Human Cd34 ◽

Cd34 Cells ◽

Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-137477 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Sara El Hoss ◽

Sylvie Cochet ◽

Auria Godard ◽

Hongxia Yan ◽

Michaël Dussiot ◽

...

Keyword(s):

Cell Death ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Ineffective Erythropoiesis ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v124.21.4363.4363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4363-4363

Author(s):

Alexandre Janel ◽

Nathalie Boiret-Dupré ◽

Juliette Berger ◽

Céline Bourgne ◽

Richard Lemal ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Confocal Microscopy ◽

Hematopoietic Stem Cell ◽

Biological Samples ◽

Mesenchymal Cells ◽

Hematopoietic Stem ◽

Bm Niche ◽

Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities

Blood ◽

10.1182/blood.v88.4.1297.bloodjournal8841297 ◽

1996 ◽

Vol 88 (4) ◽

pp. 1297-1305 ◽

Cited By ~ 62

Author(s):

N Uchida ◽

J Combs ◽

S Chen ◽

E Zanjani ◽

R Hoffman ◽

...

Keyword(s):

Stromal Cell ◽

Biological Activities ◽

Single Cells ◽

Hematopoietic Cells ◽

In Utero ◽

Rhodamine 123 ◽

Differentiation Potential ◽

Cd34 Cells

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

Download Full-text

Specific MicroRNA Deregulated in Myleoproliferative Neoplasm Are Regulated by JAK2V617F and May Contribute to Aberrant Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.965.965 ◽

2009 ◽

Vol 114 (22) ◽

pp. 965-965

Author(s):

Xiaoqing Lin ◽

Monica Buzzai ◽

Martin Carroll ◽

Elizabeth Hexner ◽

Fabricio F Costa ◽

...

Keyword(s):

Progenitor Cells ◽

Mirna Expression ◽

Colony Formation ◽

Lentiviral Vector ◽

Myeloproliferative Neoplasms ◽

Rna Extraction ◽

Differentially Expressed ◽

Loss Of Function ◽

Cd34 Cells

Abstract Abstract 965 The myeloproliferative neoplasms (MPN), PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (∼100%, 70% and 50% respectively). Accumulating evidence suggest that JAK2V617F may not be the initiating event in MPN, and other genetic anomalies play an important role in MPN pathogenesis. We hypothesized that miRNA deregulation contributes to the development of MPN. To test this idea, miRNA expression in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls was determined using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. PV (JAK2V617F) and ET cases (JAK2WT) showed 14 and 78 differentially expressed miRNAs, respectively, when compared to controls. 6 miRNAs were commonly deregulated in PV and ET, while the majority were unique to each disease type. When all MPN patients were grouped and compared to controls, 28 miRNAs were significantly deregulated (p<0.05). These miRNAs differ from those previously reported to be differentially expressed in the peripheral blood of PV patients. Among these 28, mir-214 was down-regulated and mir-410, mir-22* and mir-505* were up-regulated most consistently. Several miRNAs, including mir-135b, mir-542-5p, mir-149, mir-133b and mir-134 were undetectable in normal CD34+ cells and activated in MPN patients. We further hypothesized that some miRNAs are regulated through the action of the mutant JAK2V617F kinase. To test this, miRNA levels were assessed by TaqMan array in HEL and UKE-1 cells (harboring JAK2V617F) treated with 2 μM JAK inhibitor I (Calbiochem) for 20h before RNA extraction. In parallel, miRNA expression as determined by TLDA in TF-1 cells rendered cytokine independent by stable expression of JAK2V617F was compared to that of control TF-1 cells, both cultured overnight in the absence of cytokines. A total of 24 miRNAs were significantly deregulated (>2 fold) in at least two cell line systems. To test which deregulated miRNAs in MPN patients were JAK2 responsive, JAK2 activity was manipulated in HEL and TF-1 cells as described above, and the expression of miRNAs was determined by individual Taqman miRNA assays. mir-1, mir-200a, mir-9, mir-133b, mir-22* and mir-155 were responsive to manipulation of JAK2 activity. miR-155 expression was repressed 50% with the inhibition of JAK2 in HEL cells and stimulated almost 2 fold with the overexpression of JAK2V617F in TF-1 cells. By contrast, mir-214 (downregulated in MPN) and mir-134 (upregulated in MPN) were not responsive to manipulation of JAK2V617F activity in either the gain or loss-of-function systems. To further confirm the ability of JAK2V617F to regulate specific miRNAs, lineage negative (lin-) murine marrow progenitor cells were transduced with JAK2V617F or empty vector, allowed to form colonies for 7 days and miRNA levels in the colonies were determined. Again miR-200a, miR-9 and miR-22* and miR-155 were responsive to JAKV617F overexpression, while mir-134 was not. Transduction of lineage negative murine marrow progenitor cells with a lentiviral vector harboring mir-155 yielded a 30% increase in a myeloid colony formation in vitro. The effect is consistent with the reported ability of mir-155 to induce myeloproliferation in mice. Transduction of marrow progenitors with miR-133b, which is activated in MPN patients, responsive to JAK2V617F manipulation and not previously reported to have a role in hematopoiesis, led to an increase in both erythroid and myeloid colony formation. Taken together we conclude that at least 4 miRNAs are deregulated in CD34+ cells of MPN patients as a result of aberrant JAK2 activity. Two of these tested so far have a role in hematopoiesis. Part of the action of JAK2V617F in myeloproliferation may be mediated by specific miRNA, thus representing alternative therapeutic targets in MPN. Disclosures: Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

Download Full-text

Global Transcriptome Profiling Identifies a Key Mir-185-PAK6 Axis That Promotes Survival of Leukemic Stem Cells and Drug-Insensitive Blasts in BCR-ABL+ Human Leukemia

Blood ◽

10.1182/blood-2018-99-113559 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 931-931

Author(s):

Andrew Wu ◽

Lin Hanyang ◽

Katharina Rothe ◽

Min Chen ◽

Jens Ruschmann ◽

...

Keyword(s):

Expression Profiles ◽

Transcriptome Profiling ◽

Differentially Expressed ◽

Leukemic Stem Cells ◽

Cd34 Cells ◽

Differentially Expressed Mirnas ◽

Resistant Cells

Abstract Chronic myeloid leukemia (CML) stem/progenitor cells and BCR-ABL+ acute lymphoblastic leukemia (ALL) blast cells are insensitive to tyrosine kinase inhibitor (TKI) monotherapies. These cells rapidly generate therapy-resistant clones in vitro and in vivo and are often responsible for disease relapse. Therefore, identification of predictive biomarkers and novel treatments that target key molecular events active in leukemic stem cells (LSCs) are needed. MicroRNAs (miRNAs) are small molecules that regulate the gene expression network and are highly deregulated in many cancers. Through global transcriptome profiling, we have recently identified 66 differentially expressed miRNAs in pre-treatment CD34+ stem/progenitor cells from CML patients (n=6) compared to healthy bone marrow (NBM) controls (n=3, adjusted P<0.05); 26 differentially expressed miRNAs were identified between subsequent IM-nonresponders and IM-responders (P<0.05). 21 differentially expressed miRNAs were successfully validated in additional IM-responders (n=11), IM-nonresponders (n=11) and NBM (n=11). Interestingly, miR-185 was discovered to be one of the most highly deregulated miRNAs, with significant reduction in CD34+ cells from IM-nonresponders compared to IM-responders (p=0.0006). This significant change was further demonstrated in CD34+ cells from CML patients (n=60) before and after 3-month TKI nilotinib treatment in a clinical trial (p<0.05). We further demonstrated that miR-185 functions as a tumor suppressor; its restored expression by lentiviral transduction in CD34+ IM-nonresponder cells significantly impaired survival of these cells and sensitized them to TKI treatment in vitro. Restored miR-185 expression in BCR-ABL+ ALL blasts led to a profound decrease in leukemia burden and significantly enhanced survival compared to controls in vivo (median survival 65 vs. 47 days, P=0.0005). Strikingly, mice injected with miR-185-transduced cells and treated with dasatinib (DA) survived much longer than recipients of control cells treated with DA (median survival 83 vs. 60 days, P=0.0018). Moreover, restoration of miR-185 expression combined with DA treatment greatly reduced in vivo long-term regenerative activity of LSCs from IM-nonresponders as compared to control cells treated with DA in NRG mice (<0.2% vs. 5% GFP+ patient cells in the BM, 25 weeks post-transplantation). We observed not only a marked reduction in GFP+CD34+ cells, but also a near elimination of GFP+CD34+CD38- LSCs that were transduced with miR-185 and treated with DA compared to control cells treated with DA, indicating that restored miR-185 expression combined with DA preferentially prevents the growth of patient-derived long-term leukemia-initiating cells in vivo. Several miRNA target genes were further identified by integrating miRNA expression profiles with gene expression profiles from the same patient samples using strand-specific RNA-seq. Based on three out of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar), PAK6, a serine/threonine-protein kinase, was found to be highly expressed in CD34+ IM-nonresponder cells compared to IM-responders (p<0.003), which correlated with reduced expression of miR-185 in these cells (p=0.0002). PAK6 was confirmed as a target gene of miR-185 by a luciferase reporter assay. Western blot analysis showed that restored miR-185 expression caused a marked decrease in protein levels of PAK6 in miR-185-transduced cells and suppression of PAK6 reduced viability of these cells. These results indicate that PAK6 is a critical target of miR-185, and that loss of miR-185 expression in CML may lead to up-regulation of PAK6, which in turn contributes to disease progression and drug resistance. Indeed, the use of a pre-clinically validated pan PAK inhibitor (PF-3758309) significantly inhibited the growth of IM-resistant cells and CD34+ IM-nonresponder cells and these effects could be enhanced by TKIs (p<0.05). Mechanistically, we observed that p-ERK and p-AKT were significantly reduced in PAK6 knockdown or miR185-restored IM-resistant cells in response to IM treatment. Thus, we infer that downregulation of PAK6 may sensitize TKI-resistant cells to TKI therapy through inhibition of the RAS/MAPK pathway. Taken together, PAK6, a novel target of miR-185, emerges as an attractive druggable target for combination therapy of TKI-resistant patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo

Blood ◽

10.1182/blood.2020010020 ◽

2021 ◽

Author(s):

Nikoletta Psatha ◽

Aphrodite Georgakopoulou ◽

Chang Li ◽

Vivek Nandakumar ◽

Grigorios Georgolopoulos ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Therapeutic Window ◽

Pronounced Increase ◽

Human Red Blood Cells ◽

Transfusion Independence ◽

Cd34 Cells ◽

Proliferation And Differentiation ◽

Cis And Trans

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) have an ameliorated clinical phenotype and in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites have been shown to significantly increase expression of endogenous fetal hemoglobin (HbF). To broaden the therapeutic window beyond a single editing approach, we have explored combinations of cis and trans editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of one trans and one cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0 thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe beta globin phenotype.

Download Full-text

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽

10.1182/blood.v110.11.4540.4540 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4540-4540

Author(s):

Frank Neumann ◽

Daniela C. Bruennert ◽

Anne-Marie Koch ◽

Ingmar Bruns ◽

Norbert Gattermann ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Tyrosine Kinase ◽

Cell Line ◽

K562 Cells ◽

Differentially Expressed ◽

Cd34 Cells ◽

Number Of Genes ◽

In Vitro Treatment

Abstract Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.

Download Full-text

Heme-Regulated Inhibitor (HRI) Loss and Pharmacologic Treatments Cooperate to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Blood ◽

10.1182/blood-2018-99-117436 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 721-721

Author(s):

Scott A. Peslak ◽

Jeremy D. Grevet ◽

Xianjiang Lan ◽

Osheiza Abdulmalik ◽

Junwei Shi ◽

...

Keyword(s):

Protein Kinase ◽

Sickle Cell ◽

Cell Formation ◽

Fetal Hemoglobin ◽

Cell Maturation ◽

Erythroid Cell ◽

Cd34 Cells ◽

Hbf Induction ◽

Pharmacologic Treatments

Abstract Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF for SCD patients but is limited in its efficacy. Recent work in our laboratory utilizing a protein kinase-domain focused CRISPR-Cas9 based genetic screen identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 361:6399, 285-290). Depleting HRI in a human erythroid cell line and primary human erythroid cultures significantly raised HbF levels. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. While all these approaches induce HbF in vitro, it remains to be seen whether these would achieve sufficient levels of HbF induction in patients when used as single agents. Although the specific mechanisms by which HRI, HU, pomalidomide and UNC0638 regulate HbF remain to be fully resolved, we hypothesized that they might work in distinct pathways, and that by combining them might improve effect size and diminish potential adverse effects on erythroid cell maturation and viability. We utilized a three-stage in vitro culture system that recapitulates normal erythropoiesis by terminal differentiation of adult CD34+ hematopoietic cells. Primary human CD34+ cells were treated with select pharmacologic inducers of HbF, including hydroxyurea, pomalidomide, or UNC0638, in combination with shRNA lentiviral knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and cation-exchange HPLC. We find that knockdown of HRI leads to significant HbF induction as expected. We further observed an inverse correlation between remaining HRI levels and HbF induction with maximal effect size requiring 85%-90% HRI depletion. HU displayed comparatively little activity and failed to increase the effects of HRI knockdown. However, treatment with pharmacologic levels of pomalidomide or UNC0638 combined with HRI depletion each showed greater than additive effects in HbF with levels reaching 25-30% HbF for UNC0638-treated and 30-40% HbF for pomalidomide-treated combinations, suggesting underlying cooperativity of HbF induction. RT-qPCR and Western blot analyses suggest that the diminished expression of the HbF repressor BCL11A accounts in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which Bcl11A depletion was greater than 90%, while other HbF repressors such as LRF were unchanged. To determine whether HbF induction results in reduced sickle cell formation, we performed combination HRI depletion and HbF pharmacologic induction in primary CD34+ cells derived from sickle cell patients. HRI depletion in primary SCD cells showed a marked increase in HbF from baseline levels; in addition, significant cooperativity with pomalidomide and UNC0638 was observed, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations with no apparent detrimental effects on erythroid differentiation or maturation. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype in vitro. Overall, we find that the combination of HbF pharmacologic induction and shRNA-mediated HRI inhibition results in significant cooperative upregulation of HbF levels in both normal and sickle cell-derived primary human cells without impairing red cell maturation. We are currently exploring additional potential synergies with HbF regulators to determine the optimal modalities to maximize HbF induction. While no effective and specific HRI inhibitors are currently available, our work suggests that future small molecule inhibition of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Long non-coding RNA levels can be modulated by 5-azacytidine in Schistosoma mansoni

Scientific Reports ◽

10.1038/s41598-020-78669-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Murilo S. Amaral ◽

Lucas F. Maciel ◽

Gilbert O. Silveira ◽

Giovanna G. O. Olberg ◽

João V. P. Leite ◽

...

Keyword(s):

Stem Cell ◽

Life Cycle ◽

Schistosoma Mansoni ◽

Drug Targets ◽

Single Cells ◽

Differentially Expressed ◽

Protein Coding ◽

Life Cycle Stages ◽

Network Modules

AbstractSchistosoma mansoni is a flatworm that causes schistosomiasis, a neglected tropical disease that affects more than 200 million people worldwide. There is only one drug indicated for treatment, praziquantel, which may lead to parasite resistance emergence. The ribonucleoside analogue 5-azacytidine (5-AzaC) is an epigenetic drug that inhibits S. mansoni oviposition and ovarian development through interference with parasite transcription, translation and stem cell activities. Therefore, studying the downstream pathways affected by 5-AzaC in S. mansoni may contribute to the discovery of new drug targets. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein coding potential that have been involved in reproduction, stem cell maintenance and drug resistance. We have recently published a catalog of lncRNAs expressed in S. mansoni life-cycle stages, tissues and single cells. However, it remains largely unknown if lncRNAs are responsive to epigenetic drugs in parasites. Here, we show by RNA-Seq re-analyses that hundreds of lncRNAs are differentially expressed after in vitro 5-AzaC treatment of S. mansoni females, including intergenic, antisense and sense lncRNAs. Many of these lncRNAs belong to co-expression network modules related to male metabolism and are also differentially expressed in unpaired compared with paired females and ovaries. Half of these lncRNAs possess histone marks at their genomic loci, indicating regulation by histone modification. Among a selected set of 8 lncRNAs, half of them were validated by RT-qPCR as differentially expressed in females, and some of them also in males. Interestingly, these lncRNAs are also expressed in other life-cycle stages. This study demonstrates that many lncRNAs potentially involved with S. mansoni reproductive biology are modulated by 5-AzaC and sheds light on the relevance of exploring lncRNAs in response to drug treatments in parasites.

Download Full-text