Erythroid K562 Cells Research Articles

A Crucial Role of Proteolysis in the Formation of Intracellular Dinitrosyl Iron Complexes.

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings.

Development of the Novel Cytosine Analog Aza-T-Dcyd (AB1) As a Fetal Hemoglobin Inducer to Treat Sickle Cell Disease.

Introduction: Induction of fetal hemoglobin (HbF) expression in sickle cell disease (SCD) patients using small molecules is considered an effective therapeutic strategy to ameliorate severe clinical phenotypes. With this objective in mind, several drug molecules have been tested for HbF induction. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. Studies have reported that DNA methyltransferase inhibitors such as decitabine stimulate γ-globin gene expression through DNA hypomethylation. However, decitabine has cytotoxic side effects and is most effective by intravenous administration. Thus, it is critical to develop alternative safe and orally active hypomethylating agents for inducing HbF. Our lab in collaboration with AkiraBio, conducted preclinical in vitro studies to assess a novel cytosine analog Aza-T-dCyd (AB1), for its ability to induce HbF in human K562 cells and erythroid progenitor cells derived from SCD patients. Methods: K562 cells were cultured in Iscove's Modified Dulbecco's Eagle medium, 10% fetal bovine serum and penicillin/streptomycin. The human erythroid progenitors were generated from peripheral blood mononuclear cells isolated from two SCD patients under IRB exempt protocol as previously established in our lab (Li et al., Haematologica. 2018). The cultured K562 cells were treated either with AB1 alone at gradually increasing concentrations from 3nm to 200nm, decitabine (0.2µM), hydroxyurea (HU100µM) or combined AB1 with HU for 48 hours. The cultured sickle erythroid progenitors were treated with AB1 on day 8 and then harvested on day 10. The harvested cells were analyzed for cell viability and flow cytometry to measure HbF positive cells (F-cells). The γ-globin and β-globin mRNA levels were quantified by qRT-PCR and the protein levels of HbF and DNMT-1 were detected by Western blot technique. Results: Initial studies to evaluate drug toxicity were completed. We observed higher cell viability of K562 cells treated with AB1 10nM and 30nM compared to untreated and HU 100µM (p=0.005) treated cells. Hence, subsequent experiments were conducted with the lower AB1 concentrations. By flow cytometry analysis, AB1 treatment significantly increased F-cells from 13.95% to 20.11% and 22.46% in 10nM and 30nM treated cells (p=0.0002) respectively. Moreover, mean fluorescence intensity analysis showed significant HbF expression in AB1 treated cells compared to untreated cells (p=0.00001). At the transcription level, AB1 increased γ-globin mRNA levels 1.4-fold compared to untreated cells. K562 cells treated with a combination of AB1 and HU at their maximum response concentrations (AB1 10 and 30nM; HU 100 µM) did not show additive effects on F-cells or γ-globin mRNA expression. Other combination concentrations were not assessed. In subsequent studies we investigated the HbF inducing capacity of AB1 in human erythroid progenitors generated from SCD patients (N=2). Normal cell viability was observed in sickle progenitors at 3nm, 6nM and 10nm AB1 treatment. The F-cell level increased from 16.21% to 27.68% at 10nm AB1 (p=0.02). By qRT-PCR assay, a significant 2-fold increase in γ-globin mRNA level was observed in AB1 treated erythroid cells, when normalized with total γ-globin and β-globin mRNA; β globin mRNA levels were unchanged. Finally, when we analyze the effects of AB1 on protein levels of HbF and DNMT-1 by Western blot analysis, 10nM and 30nM AB1 induced HbF and inhibited DNMT-1 expression the sickle erythroid progenitors. Conclusion: Our study findings demonstrate that AB1 induced γ-globin transcription in K562 cells and human sickle erythroid progenitors. Studies to test the effects of AB1 in the Townes SCD mouse model are in progress.

Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells.

Pannexin 1 (PANX1) was proposed to drive ATP release from red blood cells (RBCs) in response to stress conditions. Stomatin, a membrane protein regulating mechanosensitive channels, has been proposed to modulate PANX1 activity in non-erythroid cells. To determine whether stomatin modulates PANX1 activity in an erythroid context, we have (i) assessed the in situ stomatin-PANX1 interaction in RBCs, (ii) measured PANX1-stimulated activity in RBCs expressing stomatin or from OverHydrated Hereditary Stomatocytosis (OHSt) patients lacking stomatin, and in erythroid K562 cells invalidated for stomatin. Proximity Ligation Assay coupled with flow imaging shows 27.09% and 6.13% positive events in control and OHSt RBCs, respectively. The uptake of dyes 5(6)-Carboxyfluorescein (CF) and TO-PRO-3 was used to evaluate PANX1 activity. RBC permeability for CF is 34% and 11.8% in control and OHSt RBCs, respectively. PANX1 permeability for TO-PRO-3 is 35.72% and 18.42% in K562 stom+ and stom− clones, respectively. These results suggest an interaction between PANX1 and stomatin in human RBCs and show a significant defect in PANX1 activity in the absence of stomatin. Based on these results, we propose that stomatin plays a major role in opening the PANX1 pore by being involved in a caspase-independent lifting of autoinhibition.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation

Iron‑sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron‑sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5′-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

Transcriptional silencing of fetal hemoglobin expression by NonO.

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.

Histone H3K4me1 and H3K27ac play roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

Histone H3K4me1 and H3K27ac are enhancer-specific modifications and are required for enhancers to activate transcription of target genes. However, the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting the SET domains of histone methyltransferases MLL3 and MLL4 and the HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the β-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 eviction at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the β-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac-induced eRNA transcription without affecting H3K4me1 at the β-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

The Role of TCA Cycle Compounds in Erythroid Heme Synthesis and Its Regulation During an Inflammatory Response

One early hallmark event of terminal erythroid differentiation is the induction of heme and globin synthesis. Onset of heme synthesis occurs when the first pathway enzyme, erythroid-specific 5-aminolevulinate synthase (ALAS2), is induced. The steps of heme biosynthesis are identical between non-erythroid (housekeeping) and differentiating erythroid cells, but two genes encode the first enzyme, 5-aminolevulinate synthase (ALAS). The housekeeping gene (ALAS1) is expressed in non-erythroid cells while the erythroid-specific gene (ALAS2) is expressed only in the erythron. Heme synthesis in metazoan cells requires iron, glycine, and succinyl CoA. The supply of iron required for heme synthesis has been well studied and a reasonable view of the components involved in this process exists. Glycine is abundant in plasma (~250 µM) and studies of glycine transporters are consistent with glycine being supplied from extracellular sources. For succinyl CoA the general assumption is that it is produced by the TCA cycle. This may be possible for housekeeping heme synthesis where only modest levels of heme are made at a given time, but this mechanism would clearly place cells under strain during erythroid differentiation when cells make ~109 molecules of heme in a span of 2-3 days.ALAS2, but not ALAS1, physically interacts with the succinyl CoA synthetase (SCS) ATP-dependent subunit SUCLA2. Since ALAS utilizes succinyl CoA to synthesize aminolevulinic acid (ALA), and because SUCLA2 as a subunit of SCS may be involved in the ATP-dependent reverse reaction of SCS to generate succinyl CoA from succinate, one popular hypothesis is that the ALAS2-SUCLA2 interaction exists to provide succinyl CoA for ALAS2. While the evidence for an association between ALAS2 and SUCLA2 is strong, there is no experimental data showing that SUCLA2 has an impact on ALAS2 activity or that there is a role for this interaction in the supply of succinyl CoA for ALAS2.In the current work we have sought to determine if succinyl CoA for heme synthesis during erythropoiesis in MEL cells originates from the TCA cycle or more directly from metabolism of glutamine via transaminases and α-ketoglutarate dehydrogenase (α-KGDH). Employing 13C-labeled glucose, diethyl succinate, or glutamine we determined that succinate is a poor source of carbons for erythroid heme synthesis, providing a maximum of 5 carbons out of a possible 26 (by isotopomer analysis) and labeling only 2.5% of carbons of isolated heme. Glucose contributes up to 18 carbons and a total of 8.3%, and glutamine contributes to 23 carbons and a total of 23%. Enzyme assays of TCA cycle enzymes revealed that during erythropoiesis only aconitase, isocitrate dehydrogenase, and α-KGDH are induced, which is consistent with glutamine, and not succinate, being the major source of carbon for heme.We also examined the impact of itaconate, a compound produced from the TCA cycle in LPS-induced macrophages, on erythroid differentiation. Itaconate is known to inhibit succinate dehydrogenase, and so might be expected to induce heme synthesis if succinyl CoA is being supplied from accumulating succinate via ATP-dependent SCS. However, dimethyl itaconate (a cell permeable form of itaconate) inhibits heme synthesis in differentiating erythroid K562 cells and this inhibition is reversed by ALA. It has long been assumed that SCS has broad specificity and is capable of making itaconyl CoA (Adler J. et al. 1957, JBC 229:865). However, we found that purified human and porcine SCS do not synthesize itaconyl CoA, but that the enzyme succinyl CoA:glutarate CoA transferase (SUGCT) generates itaconyl CoA from itaconate and succinyl CoA. Additionally we demonstrated that itaconyl CoA inhibits purified ALAS2 at µM concentrations. Previously published data along with our studies on K562 cells in the presence of LPS-induced RAW cells suggest that during the inflammatory response macrophages resident in erythroblastic islands produce itaconate as well as cytokines that induce proximal developing red cells to express a plasmalemmal transporter (possibly SLC13A5) required for itaconate import. Intracellular itaconate is then converted to itaconyl CoA via SUGCT, resulting in ALAS2 inhibition and a concomitant decrease in heme synthesis. This process restricts the amount of protoporphyrin synthesized during infection and, thereby, complements mechanisms that restrict the availability of iron to microbial pathogens. DisclosuresNo relevant conflicts of interest to declare.

Cabozantinib promotes erythroid differentiation in K562 erythroleukemia cells through global changes in gene expression and JNK activation.

Cabozantinib is a potent tyrosine kinase inhibitor with multiple targets including MET, VEGFR2, RET, KIT, and FLT3. Cabozantinib is widely used for the treatment of medullary thyroid cancer and renal cell carcinoma. We recently suggested cabozantinib as a potential therapeutic alternative for acute myeloid leukemia (AML) patients with FLT3-internal tandem duplication (FLT3-ITD). Here, we report that cabozantinib can promote differentiation in erythroid leukemia cells. We found that K562 erythroid leukemia cells treated with 1 μM cabozantinib for 72 h underwent erythroid lineage differentiation. Transcriptomic analysis revealed that various pathways associated with heme biosynthesis, hemoglobin production, and GATA1 targets were upregulated, whereas cell survival pathways were downregulated. Further examination revealed that cabozantinib-induced erythroid differentiation is at least in part regulated by JNK activation and phosphorylation. Levels of phosphorylated BCR-ABL, AKT, STAT5, ERK, and p38 also decreased following cabozantinib treatment. Therefore, we indicate that cabozantinib has dual functions. First, it induces K562 cell differentiation toward the erythroid lineage by upregulating heme biosynthesis, globin synthesis, and erythroid-associated reactions. Second, cabozantinib inhibits K562 cell proliferation by inhibiting the phosphorylation of BCR-ABL and the downstream MAPK, PI3K-AKT, and JAK-STAT signaling pathways.

GATA-1-dependent histone H3K27 acetylation mediates erythroid cell-specific chromatin interaction between CTCF sites.

CCCTC-binding factor (CTCF) sites interact with each other in the chromatin environment, establishing chromatin domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the β-globin locus and is dependent on erythroid-specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the β-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the β-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by Trichostatin A treatment or CBP/p300 KD affected the interactions between CTCF sites around the β-globin locus without changes in CTCF binding. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short interactions than long interactions. GATA-1 was identified as a representative transcription factor that relates with genes present inside the short interactions in erythroid K562 cells. Depletion of GATA-1-reduced H3K27ac at CTCF sites near erythroid-specific enhancers. These results indicate that H3K27ac at CTCF sites is required for cell type-specific chromatin interactions between them. Tissue-specific activator GATA-1 appears to play a role in H3K27ac at CTCF sites in erythroid cells.

HERG K+ Channels Promote Survival of Irradiated Leukemia Cells

Many tumor cells express highly elevated activities of voltage-gated K+ channels in the plasma membrane which are indispensable for tumor growth. To test for K+ channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0–8 Gy, 6 MV photons) and determined K+ channel activity, K+ channel-dependent Ca2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K+ channels which became apparent 1–2 h post-irradiation. This increase in K+ channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G2/M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K+ channel function by applying the hERG1 channel inhibitor E4031 modulated Ca2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K+ channels for the Ca2+ signaling and the cell cycle control during DNA damage response.

Proteomic Analysis of Mitochondrial Membrane Protein FAM210B in Erythroid Cells

(Background) In the context of erythroid differentiation, the importance of transcription factor GATA-1 has been unequivocally demonstrated through cell-based ex vivoassays, knockout mouse models, and rare patients with anemia. GATA-1regulates the expression of erythroid-related genes such as globins, and those involved in heme biosynthesis and an unrecognized function. A novel FAM210B (C20orf108) gene was recently identified as a novel GATA-1 target gene (Kondo et al. Int J Hematol. 2016). FAM210B gene is abundantly expressed in the later stage of erythroid differentiation and encodes a protein containing an N-terminal mitochondrial-targeting sequence, which was considered as a mitochondrial membrane protein. Although the FAM210B protein has been suggested to regulate mitochondrial metabolism (Sun et al. Cell Death Dis. 2017), its detailed function remains to be elucidated. (Method) Endogenous FAM210B protein was deleted by CRISPR/Cas9 based on human iPS-derivederythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2103). To induce erythroid differentiation, HiDEP cells were co-cultured with OP9 stromal cells (ATCC) with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Suzuki et al. MCB2019). Affinity purification of the FAM210B complex was conducted in K562 erythroid cell line (ATCC) stably expressing His/Biotin-tagged FAM210B. MitoXpress Xtra (Agilent) and Human Oligo chip 25K (Toray) were used to evaluate oxygen consumption rate and transcription profiling, respectively. (Results) Clonal lines with HiDEP cells were established, which harbored deletion within coding exon 3 of FAM210B. Quantitative real-time polymerase chain reaction (RT-PCR) analysis confirmed a strong decrease in FAM210B expression. Microarray analysis revealed >1.5-fold up- and down-regulation of 104 and 53 genes caused by the FAM210B knockout, respectively. The down-regulated gene ensemble included TTC19, which is involved in mitochondrial respiratory chain complex III assembly, and genes encoding mitochondrial proteins (AARS2, YWHAE, CLIC1), whereas, the expressions for ALAS2, HBG, HBA, and HMOX1 were not significantly affected by FAM210B depletion. Intriguingly, when wild-type and FAM210B-depleted HiDEP cells were further induced to undergo erythroid differentiation, erythroid differentiation was more pronounced by FAM210B depletion. This was reflected by enhanced nuclear condensation and hemoglobinization, as well as upregulations for ALAS2, HBG, HBA, and HMOX1 in the FAM210B-depleted erythroblasts. Recent report suggests that FAM210B functions as an adaptor protein in the mitochondria (Yien et al. JBC 2018). Thus, FAM210B-interacting protein was purified from K562 cells stably expressing His/Biotin-tagged FAM210B.Mass spectrometry analysis based on the His/Biotin-purified material showed that FAM210B may interact with multiple mitochondrial proteins, including multiple subunits of mitochondrial adenosine triphosphate synthases and mitochondrial heat shock proteins. Further functional analysis showed that oxygen consumption rates might be decreased in FAM210B-depleted HiDEP cells, indicating that FAM210B was possibly involved in mitochondrial energy metabolism in erythroblasts. At present, we are conducting detailed biological analyses to characterize the role of FAM210B during erythroid differentiation. (Conclusion) Further characterization of FAM210B provides new insights in the study of erythroid differentiation, and this may lead to better understanding of the pathophysiology of certain anemias. Disclosures Fukuhara: Mundi: Honoraria; Janssen Pharma: Honoraria; Mochida: Honoraria; Gilead: Research Funding; Bayer: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; AbbVie: Research Funding; Solasia Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Onishi:MSD: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Celgene: Honoraria; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Long noncoding RNA PCED1B-AS1 promotes erythroid differentiation coordinating with GATA1 and chromatin remodeling.

Erythropoiesis is a complex and sophisticated multi-stage process regulated by a variety of factors, including the transcription factor GATA1 and non-coding RNA. GATA1 is regarded as an essential transcriptional regulator promoting transcription of erythroid-specific genes—such as long non-coding RNAs (lncRNA). Here, we comprehensively screened lncRNAs that were potentially regulated by GATA1 in erythroid cells. We identified a novel lncRNA—PCED1B-AS1—and verified its role in promoting erythroid differentiation of K562 erythroid cells. We also predicted a model in which PCED1B-AS1 participates in erythroid differentiation via dynamic chromatin remodeling involving GATA1. The relationship between lncRNA and chromatin in the process of erythroid differentiation remains to be revealed, and in our study we have carried out preliminary explorations.

A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

LRF acts as an activator and repressor of the human β-like globin gene transcription in a developmental stage dependent manner.

Leukemia/lymphoma-related factor (LRF; a hematopoietic transcription factor) has been suggested to repress fetal γ-globin genes in the human adult stage β-globin locus. Here, to study the role of LRF in the fetal stage β-globin locus, we knocked out its expression in erythroid K562 cells, in which the γ-globin genes are mainly transcribed. The γ-globin transcription was reduced in LRF knock-out cells, and transcription factor binding to the β-globin locus control region hypersensitive sites (LCR HSs) and active histone organization in the LCR HSs were disrupted by the depletion of LRF. In contrast, LRF loss in the adult stage β-globin locus did not affect active chromatin structure in the LCR HSs and induced the fetal γ-globin transcription. These results indicate that LRF may act as an activator and repressor of the human β-like globin gene transcription in a manner dependent on developmental stage.

Lymphocyte Phosphatase-Associated Phosphoprotein Is a Substrate of Protein Kinase CK2.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a molecular partner of CD45 phosphatase that plays a key role in the regulation of antigen-specific activation of lymphocytes. The functions of LPAP still remain unknown. We believe that studying LPAP phosphorylation pathways could shed light on its functions. In this work, we studied the phosphorylation of LPAP ectopically expressed in non-lymphoid cells in order to determine the effect of LPAP interaction partners on its phosphorylation. We found that phosphorylation at Ser153 and Ser163 in non-hematopoietic HEK293 cells was conserved, while phosphorylation at Ser99 and Ser172 was almost absent. The pattern of LPAP phosphorylation in K562 erythroid and U937 myeloid cells expressing endogenous CD45 protein was similar to that observed in T and B lymphocytes. We demonstrated for the first time that LPAP is a substrate for protein kinase CK2 that phosphorylates it at Ser153, presumably ensuring LPAP resistance to degradation.

Histone variants H3.3 and H2A.Z are incorporated into the β-globin locus during transcription activation via different mechanisms

Histone variants H3.3 and H2A.Z are often enriched in enhancers and transcriptionally active genes. However, the incorporation dynamics of these variants and the mechanisms of their incorporation are unclear. Here, we examined the distribution of H3.3 and H2A.Z in the human β-globin locus and analyzed their incorporation dynamics during transcription activation. Locus control region hypersensitive sites (LCR HSs), acting as enhancers, and active globin genes were enriched by H3.3 and H2A.Z in erythroid K562 cells, but inactive globin genes were not. Both variants were dynamically incorporated into the β-globin locus after transcription induction in MEL/ch11 cells, and prior to gene transcription the LCR HSs became enriched with the variants. In the activated β-globin gene, H3.3 was highly incorporated during transcription, whereas H2A.Z incorporation appeared to precede it. To further explore the relationship between gene transcription and variant incorporation, we deleted the LCR HS3 enhancer or the β-globin proximal promoter from the β-globin locus using the CRISPR-Cas9 genome editing system. H2A.Z was incorporated into the β-globin gene in the locus lacking promoter, even though the β-globin gene transcription was abolished by these deletions. However, H3.3 incorporation was reduced in the untranscribed β-globin gene. These results suggest that H3.3 and H2A.Z are systematically incorporated into the LCR enhancer and β-globin gene as part of transcription activation, but that their incorporation is carried out via different mechanisms.

Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6−/− red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

An enhancer haplotype may influence BCL11A expression levels and the response to hydroxyurea in β-thalassemia patients.

To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in β-thalassemia patients and the functional significance of this region. Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing. A haplotype harboring three tag SNPs correlated significantly with the HU-response and BCL11A transcript levels in the patients' reticulocytes. Two deletions encompassing this LD block significantly reduced BCL11A transcript levels in K562 cells. Our data suggest an essential role for this LD block in BCL11A expression levels and the response to hydroxyurea in β-thalassemia patients.

Nrf-2-driven long noncoding RNA ODRUL contributes to modulating silver nanoparticle-induced effects on erythroid cells.

The biosafety and biological effects of silver nanoparticles (AgNPs) on human health attract increasing concern. Although considerable studies have been performed to reveal the molecular mechanisms responsible for AgNP-induced effects, the current understanding mainly focuses on oxidative stress-associated signaling pathways activated by Ag particles and/or Ag ions. However, the molecular bases underlying the activation of these stress signaling pathways have not been thoroughly elucidated yet. In the current study, we aimed to shed light on the molecular bases of AgNP-induced effects on erythroid cells from the perspective of long noncoding RNAs. We identified a long-noncoding RNA molecule, ODRUL, which was substantially enhanced in K562 erythroid cells responding to AgNPs, coupled to accelerated cell death. Further, we uncovered oxidative stress-driven Nrf2 transcriptionally promoted ODRUL expression in K562cells. Downstream of Nrf2-ODRUL activation by AgNPs, ODRUL was recognized to interact with PI4Kα protein to modulate the activities of its targets AKT and JNK. As a result, the Bcl-2 level was negatively regulated by PI4K-AKT/JNK signaling under AgNP-induced stress, leading to enhanced cell death. Together, our findings unearthed that Nrf2-mediated lncRNA ODRUL was indispensable for AgNP-induced toxicity in erythroid cells through regulation of AKT/JNK-Bcl-2 signaling dependent on a physical interaction with PI4Kα. Thus, this study would open a new path to depict the molecular bases of AgNP-induced effects on erythroid cells.