ALBERT

Description: Abstract 4228 Human erythropoiesis is a dynamic complex multistep process that involves differentiation of early erythroid progenitors to enucleated red blood cells. Importantly, erythroid differentiation involves lineage-specific activation and restriction of gene expression. However, the mechanisms that play a role in erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1 (EP1), which is selectively expressed in normal human erythroid lineage cells shares 99.5% identity with the malignant fibrous histiocytoma amplified sequence 1(MFHAS1 or MASL1). MASL1 is an important oncogene that is highly expressed in malignant fibrous histiocytomas (MFH). MASL1 protein has several domains; ras, three leucine zipper, ATP/GTP-binding site and leucine-rich tandem repeat motif which are important structural or functional elements for interactions among proteins related to the cell cycle. However, the function of the MASL1 gene in erythropoiesis has not been studied. We hypothesized that MASL1 gene plays a role in the erythroid differentiation, and used a human liquid erythroid culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34+ cells following EPO treatment. Conversely, small interfering RNA-mediated knock down of MASL1 in CD34+ cells resulted in a reduction of a double positive of transferrin receptor and glycophorin A (GPA) (14.1 ± 4.7%) when compared with mock (77.9 ± 4.4%) or control vector-transfected CD34+ cells (76.7 ± 8.8%) at day 14. May-Grunwald-Giemsa staining confirmed a phenotype of MASL1 knockdown CD34+ cells that most of cells were pro- or basophilic erythroblasts. Western blotting also showed a significant decrease in hemoglobin protein levels in MASL1 knockdown CD34+ cells. In addition, MASL1-knockdown in CD34+ cells demonstrated an interruption of Raf/MEK/ERK signaling pathway. Inhibition assay of SH3 domain of Son of Sevenless (SOS) which is an upstream adapter protein in EPO-induced erythroid differentiation, confirmed that a suppression of MASL1 reduced the phosphorylation of Raf/MEK/ERK kinases and delays erythroid differentiation of Epo-induced CD34+ cells. Taken together, our study provides novel insights into MASL1-regulated erythropoiesis through the Raf/MEK/ERK signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Hemoglobin A2 (HbA2 ), which contains δ-globin as its non–α-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the δ-globin gene in adult erythroid cells, we first compared promoter sequences and found that the δ-globin gene differs from the β-globin gene in the absence of an erythroid Krüppel-like factor (EKLF ) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human δ-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5′ flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the δ promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P 〈 .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type δ promoter. A set of substitutions that create an EKLF binding site centered at −85 bp increased the expression by 26.8-fold and 6.5-fold (P 〈 .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase δ-globin gene expression, with potential future clinical benefit.

Description: Increased fetal hemoglobin has been identified to be associated with stress erythropoiesis. However, the mechanisms underlying γ-globin induction during the rapid expansion of erythroid progenitor cells have not been fully elucidated. Here we examined how intracellular signaling pathway modification of specific transcriptional regulators induced γ-globin expression in vitro cultured erythroid progenitor cells in the presence of erythropoietin and stem cell factor (SCF). We find that γ-globin induced by SCF is through a PKCα-dependent Ras/Raf/Erk1/2 signaling pathway involving activation of the transcription factor NF-Ya and inhibition of the repressor Coup-TFII. Specific inhibition of PKCα with Go6976 blocked both activation of Erk1/2 and p38 MAPK induced by SCF, and abrogated the SCF increased γ-globin gene expression. Activation of Erk1/2 plays a critical role in SCF modulated down-stream transcriptional regulators, involving regulation of γ-globin gene induction. SCF induced nuclear translocation of NF-Ya is required to activate Erk1/2 increased phosphorylation of endogenous Nrf2, which involves up-regulation of thioredoxin, and down-regulation of Coup-TFII. Inhibition of either PKCα or Erk1/2 prevented SCF induced recruitment of NF-Ya, RNA polymerase II and displacement of Coup-TFII repressor from γ-globin-promoter, indicating that the PKCα-Erk1/2 MAPK pathway contributes to SCF induced the γ-globin gene induction in adult erythropoiesis. Furthermore, consistent with this concept, SCF induced the γ-globin gene induction attenuated by inhibition of PKCα or Erk1/2 MAPK. Our data suggest that SCF stimulates the PKCα-Erk1/2 MAPK signaling pathway which regulate the downstream transcriptional activator NF-Ya and repressor Coup-TFII resulting in γ-globin reactivation in adult erythropoiesis. These observations provide the molecular pathways that take part in γ-globin augmentation during “stress erythropoiesis”.

Description: Clinical application of retinoic acids (RAs) and demethylation agents has proven to be effective in treating certain myeloid leukemia patients. However, the target genes that mediate these antileukemia activities are still poorly understood. In this study, we identified olfactomedin 4 (OLFM4), a myeloid-lineage–specific gene from the olfactomedin family, as a novel target gene for RAs and the demethylation agent, 5-aza-2′-deoxycytidine. We demonstrated that the retinoic acid receptor alpha/retinoic X receptor alpha heterodimer binds to a retinoic acid response-element (DR5) site in the OLFM4 promoter and mediates all-trans-retinoic acid (ATRA)–induced transactivation of the OLFM4 gene. OLFM4 overexpression in HL-60 cells led to growth inhibition, differentiation, and apoptosis, and potentiated ATRA induction of these effects. Conversely, down-regulation of endogenous OLFM4 in acute myeloid leukemia-193 cells compromised ATRA-induced growth inhibition, differentiation, and apoptosis. Overexpression of OLFM4 in HL-60 cells inhibited constitutive and ATRA-induced phosphorylation of the eukaryote initiation factor 4E-binding protein 1 (4E-BP1), whereas down-regulation of OLFM4 protein in acute myeloid leukemia-193 cells increased 4E-BP1 phosphorylation, suggesting that OLFM4 is a potent upstream inhibitor of 4E-BP1 phosphorylation/deactivation. Thus, our study demonstrates that OLFM4 plays an important role in myeloid leukemia cellular functions and induction of OLFM4-mediated effects may contribute to the therapeutic value of ATRA.

Description: Abstract 3777 Introduction: Olfactomedin 4 (OLFM4) is a member of olfactomedin-related glycoprotein family, which is specifically expressed in neutrophils and gastrointestinal tract. OLFM4 expression is upregulated in gastrointestinal cancer and inflammatory diseases such as chronic inflammatory bowel disease and Helicobacter pylori infection. It has been shown that OLFM4 is a target gene of NF-kB and Notch pathways and that OLFM4 down-regulates innate immunity to H. pylori infection. However, its potential biological functions in neutrophils still remain to be defined. The goal of this study is to determine whether OLFM4 is involved in the bactericidal activity of neutrophils using an OLFM4 deficient mouse model. Results: 1. OLFM4 expression in neutrophils is upregulated in response to Staphylocococus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. 2. We have shown that neutrophils from OLFM4 deficient mice have increased intracellular killing of S. aureus and E. coli bacteria in vitro. 3. The OLFM4 deficient mice displayed enhanced bacterial clearance in vivo when the mice were challenged with intra-peritoneal injection of S. aureus and E. coli. 4. To elucidate the molecular mechanisms that mediate these effects, we performed a yeast 2-hybrid screen and found that OLFM4 interacts with cathepsin C (dipeptidyl peptidase I or DPPI), a lysosomal cysteine protease that has a degraditive function as an exopeptidase and is essential for activation of neutrophil granule-associated serine proteases including neutrophil elastase, cathepsin G and proteinase 3. The direct association of OLFM4 with cathepsin C was confirmed in human primary neutrophils. 4. We have demonstrated that OLFM4 is a direct substrate of DPPI and inhibits DPPI activity in transfected 293T cells. 5. The cathepsin C activity in neutrophils from OLFM4 deficient mice was significantly higher than that in neutrophils from wild-type littermate mice in the absence or presence of bacterial infection, suggesting that OLFM4 is an endogenous inhibitor of cathepsin C in neutrophils. 6. We have also demonstrated increased activities of neutrophil elastase, cathepsin G and proteinase 3 (whose processing and maturity require cathepsin C activity) in OLFM4 deficient neutrophils compared with wild-type neutrophils. 7. Activation of NADPH oxidase, myeloperoxidase (MPO) activity, and neutrophil phagocytosis were not altered in OLFM4 deficient neutrophils compared with wild-type neutrophils. Conclusion: These results suggest that OLFM4 is an important regulator of neutrophil bacterial killing activity via negative regulation of cathepsin C activity and its down stream granule-associated serine proteases. Disclosures: No relevant conflicts of interest to declare.

Description: Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34+ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34+ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34+ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.