Differentiation Of Erythroleukemia Cells Research Articles

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Abstract 5630: Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML

Abstract Acute Myeloid Leukemia (AML) represents a diverse category of blood cancers marked by the clonal proliferation of atypical myeloid precursor cells within the bone marrow. Lysine Specific Demethylase 1 (LSD1), an epigenetic regulator, exhibits elevated expression in AML. Consequently, inhibiting LSD1 has emerged as a promising therapeutic approach for addressing blood and bone marrow cancers. While initial emphasis centered on LSD1's enzymatic inhibition, compelling evidence highlights its pivotal role as a scaffolding protein, orchestrating transcriptional and chromatin remodeling complexes that control cellular differentiation and proliferation. However, our understanding of the precise mechanisms governing these complexes remains limited, necessitating further investigation. The K562 erythroleukemia cell line has served as a prominent model for investigating the molecular factors influencing cell fate determination. In response to specific cues, these cells exhibit a bi-potential capacity, mirroring the behavior of normal hematopoietic megakaryocyte/erythrocyte progenitors (MEP). Notably, when LSD1 is either depleted or inhibited, the erythroid differentiation of K562 cells is hindered, even when exposed to various pro-differentiating triggers, including the BCR-ABL inhibitor Imatinib. In order to gain deeper insights and identify potential new protein partners involved in the K562 differentiation process that interact with LSD1, we employed the erythroleukemic K562 cell model. Using unbiased, proteome-wide proximity labeling we discovered that LSD1 serves as a scaffold for recruitment of the ChAHP complex, comprised of ADNP, CBX3, and CHD4. The ChAHP complex serves fundamental roles in chromatin remodeling, RNA splicing, and transcriptional control. We confirmed these proximity relationships by immunoblotting biotinylated proteins generated in situ by the promiscuous biotin ligase, (BirA*) when anchored to LSD1. We show that depleting ADNP induces spontaneous erythroid differentiation in K562 erythroleukemia cells, which is augmented by BCR-ABL inhibitor Imatinib treatment. ADNP-deficient K562 cells exhibited accelerated erythroid differentiation, as evidenced by a four-fold increase in the surface expression of erythroid markers (CD71, CD235a) and a significant upregulation of beta-globin gene cluster markers. These findings collectively suggest an antagonistic functional relationship between ADNP and LSD1, potentially counterbalancing LSD1's role in lineage allocation. Furthermore, we demonstrate that the loss of the key ChAHP component, ADNP, substantially hampers proliferation and increases apoptosis in K562 cells. These results underscore the potential of ADNP/ChAHP as an appealing target to modulate the choice between erythroid and megakaryocytic fates and offer promise as a target for pro-differentiation therapies in AML Citation Format: Mehraju Din Lone, Mohd Sayeed, Alaa Habieb, Praveen Kumar, Michael E. Engel. Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5630.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin, out of mitochondria that is necessary for hemoglobinization. In this study, we generated Abcb10 deletion cell lines in both mouse murine erythroleukemia and human erythroid precursor human myelogenous leukemia (K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and mouse murine erythroleukemia cells with reduced heme and intermediate porphyrins and decreased levels of aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analyses revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes argininosuccinate synthetase and argininosuccinate lyase. The reduced arginine levels in Abcb10-null cells gave rise to decreased proliferative capacity. Arginine supplementation improved both Abcb10-null proliferation and hemoglobinization upon differentiation. Abcb10-null cells showed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop), ChaC glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), and arginyl-tRNA synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

The NFIA-ETO2 fusion blocks erythroid maturation and induces pure erythroid leukemia in cooperation with mutant TP53

AbstractThe NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver–derived EBs. However, NFIA-ETO2–expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Involvement of autophagy in diosgenin‑induced megakaryocyte differentiation in human erythroleukemia cells.

Natural agents have been used to restart the process of differentiation that is inhibited during leukemic transformation of hematopoietic stem or progenitor cells. Autophagy is a housekeeping pathway that maintains cell homeostasis against stress by recycling macromolecules and organelles and plays an important role in cell differentiation. In the present study, an experimental model was established to investigate the involvement of autophagy in the megakaryocyte differentiation of human erythroleukemia (HEL) cells induced by diosgenin [also known as (25R)-Spirosten-5-en-3b-ol]. It was demonstrated that Atg7 expression was upregulated from day 1 of diosgenin-induced differentiation and was accompanied by a significant elevation in the conversion of light chain 3 A/B (LC3-A/B)-I to LC3-A/B-II. Autophagy was modulated before or after the induction of megakaryocyte differentiation using 3-methyladenine (3-MA, autophagy inhibitor) and metformin (Met, autophagy initiation activator). 3-MA induced a significant accumulation of the LC3 A/B-II form at day 8 of differentiation. It was revealed that 3-MA had a significant repressive effect on the nuclear (polyploidization) and membrane glycoprotein V [(GpV) expression] maturation. On the other hand, autophagy activation increased GpV genomic expression, but did not change the nuclear maturation profile after HEL cells treatment with Met. It was concluded that autophagy inhibition had a more prominent effect on the diosgenin-differentiated cells than autophagy activation.

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Abstract 1159: Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines

Abstract Induction of alkaline phosphatase in colon cancer cells by inhibitors of histone deacetylase activity has been widely used to monitor the induction of differentiation. We have previously identified three human bladder cancer cell lines in which alkaline phosphatase activity can be induced by inhibitors of histone deacetylase (5637, HT1197 and HT1376 cells). In earlier work it was observed that some bromodomain inhibitors inhibit growth and induction of alkaline phosphatase in these cells. After a recent report that HMBA acts as a bromodomain inhibitor, the effects of HMBA on growth and induction of alkaline phosphatase were investigated in two colon cancer cell lines (Caco-2 and HT29) and in 5637, HT1197 and HT1376 bladder cancer cells. Growth was monitored by staining with sulforhodamine B. Incubation with 2 mM butyrate for 72 hours caused induction of alkaline phosphatase in the five cell lines. In some cell lines there was inhibition of growth and of the induction of alkaline phosphatase activity by 2 mM HMBA. Increasing the HMBA concentration to 5 mM caused significant growth inhibition in all five cell lines and significant inhibition of induction of alkaline phosphatase activity by 2 mM butyrate in the 5637, Caco-2 and HT1376 cells, whether expressed as total activity or activity per unit protein. Although the effect of HMBA on induction of alkaline phosphatase in HT29 cells was not statistically significant there is evidence in the literature for inhibition with longer incubation times. The most marked effects on enzyme induction were seen with the HT1376 bladder cancer cells and this distinction was in line with previous studies on the combined effects of butyrate and the well-established bromodomain inhibitor, JQ1. HMBA has been found by several investigators to be an effective inducer of differentiation in erythroleukemia cells as judged by hemoglobin production. The lack of induction of alkaline phosphatase by HMBA as a single agent in any of the cell lines investigated in the present work suggests that different mechanisms of induction of differentiation operate in erythroleukemia cells than in bladder and colon cancer cells. Citation Format: Michael A. Lea. Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1159.

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.

Lyn Kinase Activity Is Required for Akt Mediated Erythroleukemia Cell Differentiation

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation (Fig-1).

MiR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis.

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.

Induction of an embryonic globin gene promoter by short-chain fatty acids

AbstractShort-chain fatty acids (SCFAs) and dimethyl sulfoxide (DMSO) induce adult erythroid differentiation in murine erythroleukemia (MEL) cells, but only SCFAs concurrently up-regulate expression from the endogenous embryonic globin gene ϵy. The ϵy promoter, linked to a reporter gene and stably transfected into MEL cells, was tested during adult erythroid differentiation. Both the ϵy-CACCC site at -114 bp and enhancer sequences (hypersensitive site 2 [HS2]) from the β-globin locus control region (LCR) were essential to maximal SCFA-mediated induction of expression from these constructs in MEL cells. Gel-shift analyses of binding activity from SCFA-induced MEL cell nuclear extracts showed in vitro binding by specificity proteins 1 and 3 (SP1, SP3) and basic or erythroid Krüppel-like factors (BKLF, EKLF) at the ϵy-CACCC site. In a functional analysis, transient cotransfections in nonerythroid NIH/3T3 cells of SP1, SP3, BKLF, or EKLF and HS2 ϵy promoter-luciferase constructs, with or without coactivators (p300, CREB-binding protein [CBP], or p300/CBP-associated factor [PCAF]) and SCFAs, were performed. SP1, SP3, and EKLF further increased expression from HS2 ϵy promoter constructs following exposure to SCFAs. This effect was variably augmented by coactivators and was diminished in EKLF mutants that were unable to undergo histone/factor-acetyl transferase (H/FAT)-mediated acetylation. In addition, acetylation of SP1 was detectable in NIH/3T3 cells following exposure to SCFAs. In sum, LCR sequence and an embryonic globin gene promoter CACCC site were essential to that promoter's up-regulation during SCFA-mediated induction of adult erythroid differentiation in vitro. Of factors that interact at the CACCC site, SCFA-mediated acetylation is implicated in SP1 and EKLF, and may be a mechanism through which SCFAs induce embryonic/fetal globin gene promoters during adult erythroid differentiation. (Blood. 2003;102:4214-4222)

Lyn Kinase Activity Is Required for Akt Mediated Erythroleukemia Cell Differentiation

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation. Disclosures No relevant conflicts of interest to declare.

FAM122A Inhibits Erythroid Differentiation through GATA1.

SummaryFAM122A is a highly conserved housekeeping gene, but its physiological and pathophysiological roles remain greatly elusive. Based on the fact that FAM122A is highly expressed in human CD71+ early erythroid cells, herein we report that FAM122A is downregulated during erythroid differentiation, while its overexpression significantly inhibits erythrocytic differentiation in primary human hematopoietic progenitor cells and erythroleukemia cells. Mechanistically, FAM122A directly interacts with the C-terminal zinc finger domain of GATA1, a critical transcriptional factor for erythropoiesis, and reduces GATA1 chromatin occupancy on the promoters of its target genes, thus resulting in the decrease of GATA1 transcriptional activity. The public datasets show that FAM122A is abnormally upregulated in patients with β-thalassemia. Collectively, our results demonstrate that FAM122A plays an inhibitory role in the regulation of erythroid differentiation, and it would be a potentially therapeutic target for GATA1-related dyserythropoiesis or an important regulator for amplifying erythroid cells ex vivo.

Role of miR-30a-5p and miR-26a-5p in induction of terminal differentiation in human K562 erythroleukemia cells

Background & Aim Background Plasmodium vivax (Pv) infections affect ∼300 million people every year. However, studies into this parasite species are hampered by the inability to set up a continuous, long-term in vitro blood stage culture for Pv. Establishing an in vitro “red blood cell matrix” that would allow continuous access to reticulocytes would facilitate the establishment of continuous, long-term in vitro Pv blood stage cultures and ultimately the study of parasite vulnerabilities that can be exploited in therapeutic approaches. Our study aims to establish culture conditions that allow for the differentiation of K562 cells into enucleated erythroid cells, serving as a constant source of reticulocytes for continuous, long-term Pv blood stage cultures. Methods, Results & Conclusion Materials and Methods Since the K562 cell line (K562) does not express the Duffy blood group antigen receptor (Fy), essential for Pv invasion, the Duffy FyB receptor was stably introduced into the K562 by lentiviral gene transfer. In addition, we combined different factors, which were known to enhance the terminal erythroid differentiation of K562 (knockdown of miR-30a-5p and -26a-5p, co-cultivation with macrophages, EPO, transferrin, insulin and mithramycin A). Effects were analysed by quantification of total cell numbers, surface markers and Giemsa staining. Hb expression was detected by Benzidin staining and PCR. Results K562 cells transduced with anti-miR-26a-5p and -30a-5p (C26) and stimulated with the induction cocktail showed a significantly increase in the amount of enucleated reticulocytes as compared to cells transduced with the empty vector. This is reflected in a stronger down-regulation of CD45 and up-regulation of CD71 as well as in the number of enucleated reticulocytes and the increase in hemoglobin production. CD235a expression was already higher in clone C26 prior to stimulation with the induction cocktail as compared to unstimulated K562 empty cells. The Duffy receptor expression remained stable in stimulated cells of the clone C26, while it was downregulated in K562 empty vector cells over the stimulation period. Conclusions The data show that knockdown of miR-26a-5p and -30a-5p are necessary to induce terminal differentiation of K562 cells. Further optimisation of culture conditions to enhance the yield of RBC blasts from K562 is necessary, as is the confirmation of functionality and susceptibility to Pv infection of these cells.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensablefor the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

Allosteric inhibitor remotely modulates the conformation of the orthestric pockets in mutant IDH2/R140Q

Neomorphic mutation R140Q in the metabolic enzyme isocitrate dehydrogenase 2 (IDH2) is found to be a driver mutation in cancers. Recent studies revealed that allosteric inhibitors could selectively inhibit IDH2/R140Q and induce differentiation of TF-1 erythroleukemia and primary human AML cells. However, the allosteric inhibition mechanism is not very clear. Here, we report the results from computational studies that AGI-6780 binds tightly with the divalent cation binding helices at the homodimer interface and prevents the transition of IDH2/R140Q homodimer to a closed conformation that is required for catalysis, resulting in the decrease of the binding free energy of NADPHs. If the allosteric inhibitor is removed, the original open catalytic center of IDH2/R140Q will gradually reorganize to a quasi-closed conformation and the enzymatic activity might recover. Unlike IDH2/R140Q, AGI-6780 locks one monomer of the wild-type IDH2 in an inactive open conformation and the other in a half-closed conformation, which can be used to explain the selectivity of AGI-6780. Our results suggest that conformational changes are the primary contributors to the inhibitory potency of the allosteric inhibitor. Our study will also facilitate the understanding of the inhibitory and selective mechanisms of AG-221 (a promising allosteric inhibitor that has been approved by FDA) for mutant IDH2.

Clostridium perfringens \u03b1-toxin impairs erythropoiesis by inhibition of erythroid differentiation

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71–TER119+ cell populations but not the CD71+TER119− cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.