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.

Oxidative DNA Damage, Inflammatory Signature, and Altered Erythrocytes Properties in Diamond-Blackfan Anemia

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients’ samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

Effect of Nicotinamide, 1-Methylnicotinamide, and N'-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Análisis de líneas celulares eritroleucémicas con deleciones de genes implicados en el desarrollo de enfermedades hematológicas severas

La línea celular murina MEL (Murine Erythroleukemia cell line) deriva de progenitores eritroides transformados con el complejo vírico Friend, formado por el “spleen focus forming virus” (SFFV) y el “Friend murine leukemia virus” (F-MuLV). Un atributo extremadamente útil de las células MEL radica en su capacidad para retomar el programa de diferenciación mediante la utilización de inductores químicos tales como el hexametilen-bisacetamida (HMBA). En un intento por identificar posibles dianas del HMBA se establecieron líneas celulares eritroleucémicas, derivadas de MEL, que son resistentes a la acción del agente inductor (MEL-R). En un estudio reciente, se han identificado los genes Was y Btk con mayor expresión diferencial en MEL respecto a MEL-R3. Estos genes comparten dos características comunes importantes: están implicados en la organización del citoesqueleto de actina y se activan específicamente en el linaje hematopoyético. Las mutaciones producidas en Was y Btk provocan el desarrollo de enfermedades hematológicas severas. La pérdida de función de Was desencadena el desarrollo del síndrome de Wiskott-Aldrich, asociado a defectos en una gran cantidad de procesos celulares que inducen inmunodeficiencias, trombocitopenia y alteraciones autoinmunes. Asimismo, la inhibición de Btk afecta a las vías de señalización TLR2 y TLR4 de células mieloides, la función de la interleuquina 10 y STAT3 de células dendríticas.

Regulation of Erythroid Heme Synthesis By the Mitochondrial Clpx Unfoldase

Differentiating erythroid cells synthesize large quantities of heme for hemoglobinization. While the transcriptional regulation and enzymatic mechanisms of the heme synthetic enzymes are well characterized, we lack mechanistic understanding of how their protein stability, cofactor incorporation and functional interactions with mitochondrial housekeeping proteins are regulated. These mechanisms can rapidly alter the rate of heme synthesis in response to external stimuli and metabolic requirements, and are critical for heme regulation within a tissue-specific and developmental context. CLPX, a mitochondrial protein unfoldase best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (the CLPXP ATP-dependent protease) plays a central role in regulation of mitochondrial protein turnover, is one such heme regulatory protein. CLPX activates yeast ALAS, which catalyzes the committed step of the heme synthesis pathway, by facilitating the incorporation of its cofactor, PLP, and is required for erythroid heme synthesis in zebrafish (Kardon et al. Cell 2015). Paradoxically, it regulates the turnover of ALAS1 and ALAS2 protein in vertebrate cell lines and appears to regulate the heme synthesis downstream of ALAS (Kubota et al. JBC 2016, Yien et al. PNAS 2017). However, it is not known if vertebrate ALAS was activated by ALAS, or if the requirement for CLPX in vertebrate heme synthesis was caused its regulation of ALAS activity (Figure A). To dissect the roles of CLPX in erythroid heme synthesis, we knocked out Clpxand Clpp in murine erythroleukemia (MEL) cells and assayed the activity, stability, and steady state levels of the heme synthesis enzymes, ALAS2 and FECH, which colocalize with ALAS in the mitochondrial matrix. Consistent with previous observations, Clpx -/- MEL cells had a heme defect, while Clpp -/-cells did not (Figure B). However, in contrast to previous observations in the yeast model, CLPX is not required for ALAS activation in erythroid cells, but plays a key role in regulating ALAS2 turnover in concert with the CLPP peptidase (Figure C). During erythroid differentiation, CLPP protein levels are decreased, stabilizing ALAS2 protein (Figure D). Although differentiating Clpx -/-and Clpp -/- MEL cells did not demonstrate any changes in ALAS2 turnover, likely because steady-state levels of CLPP protein were already decreased (Figure E), we observed an increase in steady-state ALAS2 protein levels and a dramatic increase in ALAS2 enzyme activity. In vitro mitochondrial iron transport/heme synthesis assays revealed a heme defect in Clpx -/-MEL cells, suggesting that CLPX plays a role in mitochondrial iron metabolism. Collectively, these data suggest a complex, differentiation-stage specific regulation of heme synthesis by the CLPXP proteolytic complex (Figure F). As Clpx -/- mouse embryos die by about E9.5 (mousephenotype.org), we dissected the in vivo role of Clpxin erythropoiesis by analyzing the phenotypes of clpxa and clpxb mutant zebrafish obtained from ZIRC. To accomplish this, we crossed clpxa and clpxb mutant zebrafish into Tg(lcr:GFP) zebrafish line in which erythroid cells are fluorescently labeled with GFP (Ganis et al Dev Biol 2012). We observed that clpxa mutant zebrafish had an early erythropoietic defect at 24 hpf that resolved at 48hpf; this developmental defect was not observed in clpxbmutant zebrafish. Benzidine staining of heme in mutant zebrafish revealed that while clpxa was dispensable for erythroid heme synthesis, clpxb was required for erythroid hemoglobinization (Figure G). Lastly, clpxbzebrafish mutants continued to be developmentally delayed and did not survive past 5 dpf. Collectively, our observations in cell lines and in the zebrafish model demonstrate that Clpx is essential for the maintenance of differentiated erythroid cells, as well as for the differentiation of the erythroid lineage. The control of heme synthesis and erythroid development by CLPX reveals how mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with key steps in heme synthesis. Further, our studies provide important genetic tools for dissecting these regulatory components in isolation as well as within the in vivocontext of erythropoiesis. Figure Disclosures No relevant conflicts of interest to declare.

Development of a MEL Cell-Derived Allograft Mouse Model for Cancer Research

Murine erythroleukemia (MEL) cells are often employed as a model to dissect mechanisms of erythropoiesis and erythroleukemia in vitro. Here, an allograft model using MEL cells resulting in splenomegaly was established to develop a diagnostic model for isolation/quantification of metastatic cells, anti-cancer drug screening, and evaluation of the tumorigenic or metastatic potentials of molecules in vivo. In this animal model, circulating MEL cells from the blood stream were successfully isolated and quantified with an additional in vitro cultivation step. In terms of the molecular-pathological analysis, we were able to successfully evaluate the functional discrimination between methyl-CpG-binding domain 2 (Mbd2) and p66α in erythroid differentiation, and tumorigenic potential in spleen and blood stream of allograft model mice. In addition, we found that the number of circulating MEL cells in anti-cancer drug-treated mice was dose-dependently decreased. Our data demonstrate that the newly established allograft model is useful to dissect erythroleukemia pathologies and non-invasively provides valuable means for isolation of metastatic cells, screening of anti-cancer drugs, and evaluation of the tumorigenic potentials.

Hemoglobin Expression and Function in Human Corneal Epithelium

The corneal epithelium forms the outer layer of the cornea. It provides mechanical protection, prevents fluid loss and forms a barrier to invasive pathogens. Though hemoglobin expression has been extensively studied in erythroid cells, recent work in multiple cell systems has documented hemoglobin expression in cells of non-erythroid origin. However, the function of hemoglobin in non-erythroid cells has yet to be established. The hypothesis that hemoglobin is expressed in corneal epithelium and that it functions to protect against oxidative stress will be examined in the present work. Hemoglobin expression and function was examined by immunocytochemistry and western blot analysis. Native human corneal explants and an immortalized human corneal epithelial cell line were examined. Expression of hemoglobin beta and delta chains was demonstrated at the protein level in both native and cell culture preparations. Hexamethylene bisacetamide is a known inducer of hemoglobin beta chain expression in murine erythroleukemia cells. HMBA did function to increase beta chain expression in cultured corneal epithelium as well. Beta chain immunolocalization was primarily cytoplasmic, while the delta chain localized to both cytoplasmic and membrane domains. Oxidative stress, from hydrogen peroxide exposure, was shown to upregulate delta chain expression. In conclusion, hemoglobin chains are expressed in corneal epithelium and could function to protect against oxidative stress. This is relevant given that exposure to light and high oxygen tensions render the cornea particularly susceptible to oxidative damage.

Hemoglobin Expression and Function in Human Corneal Epithelium

The corneal epithelium forms the outer layer of the cornea. It provides mechanical protection, prevents fluid loss and forms a barrier to invasive pathogens. Though hemoglobin expression has been extensively studied in erythroid cells, recent work in multiple cell systems has documented hemoglobin expression in cells of non-erythroid origin. However, the function of hemoglobin in non-erythroid cells has yet to be established. The hypothesis that hemoglobin is expressed in corneal epithelium and that it functions to protect against oxidative stress will be examined in the present work. Hemoglobin expression and function was examined by immunocytochemistry and western blot analysis. Native human corneal explants and an immortalized human corneal epithelial cell line were examined. Expression of hemoglobin beta and delta chains was demonstrated at the protein level in both native and cell culture preparations. Hexamethylene bisacetamide is a known inducer of hemoglobin beta chain expression in murine erythroleukemia cells. HMBA did function to increase beta chain expression in cultured corneal epithelium as well. Beta chain immunolocalization was primarily cytoplasmic, while the delta chain localized to both cytoplasmic and membrane domains. Oxidative stress, from hydrogen peroxide exposure, was shown to upregulate delta chain expression. In conclusion, hemoglobin chains are expressed in corneal epithelium and could function to protect against oxidative stress. This is relevant given that exposure to light and high oxygen tensions render the cornea particularly susceptible to oxidative damage.