Erythroid CD71 Research Articles

Selinexor Plus Ruxolitinib in JAK Inhibitor (JAKi)-Naïve Patients with Myelofibrosis: Long Term Follow up from XPORT-MF-034 Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by splenomegaly, constitutional symptoms, and inflammation due to unregulated, clonal proliferation of hematopoietic stem cells in the bone marrow. Common genetic drivers include JAK2, CALR, and MPL. Although improvements in splenomegaly and symptoms occur with the current approved therapies, additional assessments are needed to understand the impact of therapies on disease modification. Increased burden of variant allele frequency (VAF) of driver mutations is associated with poor disease outcomes, and increased expression of pro-inflammatory cytokines is implicated in disease pathophysiology and symptoms burden. Selinexor (SEL) is an investigational oral XPO1 inhibitor that may inhibit multiple MF-relevant pathways including STAT, ERK, and AKT. Preclinical studies have shown potential synergy between SEL and ruxolitinib (RUX) treatment in vivo. Safety and efficacy analysis of SEL plus RUX in JAKi treatment-naïve MF in a Phase 1 study supported a 60 mg SEL dose as the recommended dose in a Phase 3 study. Here we include exploratory analyses of a range of biomarkers in the Phase 1 study of SEL and RUX in JAKi-naïve myelofibrosis. Methods: Phase 1 of XPORT-MF-034 (NCT04562389) is an open-label study evaluating the safety and efficacy of SEL at 40mg and 60mg once weekly plus RUX with JAKi-naïve MF. Assessments included safety and rates of spleen volume reduction of ≥35% (SVR35) and total symptom score reduction of ≥50% (TSS50). Platelet and hemoglobin levels were also assessed. Longitudinal clinical biomarker assessments included %VAF change at week 24 for driver genes and plasma cytokine levels at week 4 (Cycle 2 Day 1). Bone marrow (BM) biopsies were obtained at screening and week 24, then evaluated for bone marrow fibrosis (BMF) and other BM cellular markers (CD61, CD71). Results: As of April 10, 2023, a total of 24 patients (pts) received at least one dose of SEL (40 mg: n=10; 60 mg n=14). The most common adverse events (AEs) overall of any grade were nausea (75% majority grade 1-2), fatigue (58%), anemia (54%), and thrombocytopenia (54%). Those receiving a prophylactic anti-emetic reported lower incidence and severity of nausea AEs compared to those that did not; pts experienced a median weight increase of 3.3 kg at week 24. Two pts discontinued treatment due to treatment-related AEs (thrombocytopenia and neuropathy). Generally stable hemoglobin levels were observed in 48% of transfusion-independent pts. Median hemoglobin levels were 10.1 g/dL at baseline, 9.3 g/dL at week 12, and 10.2 g/dL at week 24. Median platelet counts remained generally stable with median values of 258 k/uL at baseline, 164 k/uL at week 12, and 193 k/uL at week 24. SVR35 at week 24 in the intent-to-treat (ITT) population was achieved by 40% of the 40 mg cohort and 79% of the 60 mg cohort and TSS50 was achieved by 10% and 58%, respectively. Mutation analysis showed no pts were triple negative, with 18 harboring JAK2 mutations, 5 with CALR, and 1 with MPL. Of 13 pts with available data at week 24, 5 pts (38%) had a reduction of VAF that was ≥20%, and 3/5 of these pts had high VAF (>50%) driver mutations at baseline and were also characterized as HMR. A panel of 74 cytokines was evaluated in 21 MF pts with baseline and week 2 samples (including 10 with end-of-treatment [EOT] samples), and in 10 healthy donors. Comparing baseline MF to healthy samples, 50% of the analyzed cytokines were elevated. A rapid and durable decrease of pro-inflammatory MF-relevant cytokines including TGF-β1, IFNɣ, TNF-α, IL-7, IL-1Ra, and IL-16 was observed in the majority of pts at week 2 and continued into EOT. Notably, the reduction in IL-18 correlated to SVR and TSS response at week 24, with R 2 values of 0.29 and 0.28, respectively. BM analysis at week 24 showed an increased number of erythroid CD71+ elements in 3/14 pts. Conclusions: SEL plus RUX was generally well tolerated and manageable.Evidence of potential disease modification in pts with JAKi-naïve MF patients was seen via rapid stabilization of platelets and maintaining hemoglobin levels, VAF reduction, and pro-inflammatory cytokine reduction. Promising biomarker and efficacy data suggests that SEL in combination with RUX has the potential to become a novel, first-line treatment for pts with MF. Updated clinical data with longer follow-ups and additional subgroup analyses will be available at the time of presentation.

Regulation of Erythroblast Protein Expression By an RNA Splicing Mechanism, Decoy Exon-Mediated Intron Retention

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells >5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65. Disclosures No relevant conflicts of interest to declare.

NRF2 Mediates Epigenetic Changes in DNA and Chromatin Structure to Regulate γ-Globin Gene Expression in a Human βYAC Transgenic Mouse Model

NRF2 is the master regulator for the cellular anti-oxidative stress response and previously shown to activate γ-globin gene expression in human erythroid progenitor cells. The goal of this study was to expand on these findings by exploring the in vivo function of NRF2 using the human β-globin locus YAC transgenic (βYAC) mouse carrying the entire 248kb human β-globin locus (HBB). We observed that NRF2 activation by chronic dimethyl fumarate treatment of βYAC mice, induced human γ-globin gene expression, but had no effect on the adult β-globin gene. Subsequently in a novel βYAC/NRF2 knockout mouse model established in our laboratory, we demonstrated that NRF2 loss increased mouse erythroid CD71 levels while reducing human γ-globin gene expression during erythropoiesis in mouse embryonic E13.5 and E18.5 day fetal livers and peripheral blood. Furthermore, the ability of dimethyl fumarate to induce γ-globin gene expression was abolished after NRF2 loss. By western blot analysis of nuclear protein, we confirmed that part of the mechanism of globin gene regulation by NRF2 loss involves a decline of global histone H3 lysine 4 trimethylation levels, without changing histone acetylation. Interestingly, NRF2 loss decreased the protein levels of the DNA methylcytosine dioxygenases including TET1, TET2 and TET3. Analysis of DNA methylation/hydroxymethylation levels by DNA dot-blot assay of mouse E13.5 fetal livers isolated from βYAC/NRF2 knockout mice, showed inhibition of genome wide DNA hydroxymethylation, while DNA methylation was not affected. In addition, DNA immunoprecipitation confirmed decreased hydroxymethylation in the HBB locus control region (LCR) enhancer and γ-globin gene region. These data suggest an essential role of NRF2 in modifying chromatin structure and assembling transcription complexes to regulate γ-globin gene expression. ChIP assay to assess in vivo DNA-protein interactions showed decreased associations of histone H3 lysine 4 monomethylation and trimethylation, TATA-binding protein and RNA polymerase II to the LCR and γ-globin promoter after NRF2 loss. Final studies were conducted to evaluate long-range chromatin interactions between the LCR and individual globin genes by chromosome conformation capture assay. We observed decreased interactions between the LCR and γ-globin gene promoter region after NRF2 loss while interactions in the adult β-globin was not affected suggesting NRF2 preferentially mediates γ-globin gene regulation. In conclusion, our data suggest that NRF2 alters γ-globin expression through epigenetic DNA/histone modifications in addition to direct DNA binding. Therefore, activation of NRF2 expression using small chemical compounds is an innovative strategy to induce γ-globin gene transcription for the treatment of β-hemoglobinopathies. This work was supported by funding from the National Heart, Lung, and Blood Institute to XZ through the Hemoglobinopathy Translational Research Skills Core component of U01 grant HL117684 and R01 grant HL069234 to BSP. DisclosuresNo relevant conflicts of interest to declare.

Euroflow-Based Immunophenotypic Characterization of CD34+ Cell Compartment in Juvenile Myelomonocytic Leukemia (JMML): A New Tool for Differential Diagnosis

BackgroundJuvenile myelomonocytic leukemia (JMML) is a lethal myeloproliferative disease (MPD) of young childhood characterized by an overproduction of myelomonocytic cells and an increased in vitro sensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor [GM-CSF] (Emanuel PD et al, Blood 1991). Diagnostic criteria for JMML are currently well-established and based on clinical features and laboratory findings. However, in some patients diagnosis of JMML vs other overlapping disease entities, still remains a challenge, immunophenotyping being not part of the diagnostic work-up of JMML. Here, we aimed at detailed characterization of the CD34+ cell compartment in JMML bone marrow (BM) using the standardized EuroFlow myeloid panel in combination with innovative EuroFlow software maturation tools. Our major goal was to determine the potential utility of immunophenotyping of CD34 cells in the diagnostic work-up of JMML.MethodsOverall, we analyzed BM cells from 10 JMML patients at diagnosis (age range: 0-7 years), 17 control subjects (age range: 0-15 years) and 5 patients (age range: 0-5 years) with a suspected diagnosis of JMML that was subsequently not confirmed following standardized EuroFlow antibody combinations:1) cyCD3/ CD45/ cyMPO/ cyCD79a/ CD34/ CD19 / CD7/smCD3 (for early lineage assignement);2) HLADR/CD45/CD16/CD13/CD34/CD117/CD11b/CD10 (neutrophilic maturation);3) HLADR/CD45/CD35/CD64/CD34/CD117/CD300e (IREM2)/CD14 (monocytic maturation);4) HLADR/CD45/CD36/CD105/CD34/CD117/CD33/CD71 (erythroid vs plasmacytoid dendritic cell maturation).Samples were processed and analyzed according to the Euroflow standard operating protocols (van Dongen JJM et al, Leukemia 2012, Kalina T et al, Leukemia 2012). Data analysis was specifically focused on the immunophenotypic profile of CD34+ gated cells.ResultsWithin the CD34+ BM cell compartment the proportion (mean % ± 1SD) of granulocytic and monocytic precursors were not significantly different in JMML as compared to controls: 33% ± 15% vs 25% ± 12% (p = 0.16) and 14% ± 6.3% vs 12% ± 7.1% (p = 0.68) respectively. Otherwise we observed a slightly decreased in erythroid CD34+ progenitors in JMML vs controls (1.0% ± 1.2% vs 2.8% ± 1.7%, p<0.05). Moreover, a significantly different distribution of lymphoid precursors was observed: B-cell precursors were strongly reduced in JMML vs controls (3.0% ± 3.5% vs 53% ± 16%, p<0.0001), while CD7+ lymphoid precursors resulted significantly enhanced (28% ± 18% vs 2.3% ± 1.2%, p<0.0001). We then investigated the presence of unusual immunophenotypes in JMML CD34+ BM cells, including CD7+/MPO+, CD79a+/CD7+, and CD79a+/MPO+ cells. Interestingly, we consistently found CD7+/MPO+ and/or CD79a+/CD7+ cells in 7/7 JMML patients analyzed (mean 7.9% ± 6.4%), while in control subjects these cells were virtually absent (0.02% ± 0.00%, p<0.0001). In contrast, no CD79a+/MPO+ cells were detected among CD34+ precursors. Those 5 patients suspected of having JMML showed a CD34+ BM cell immunophenotypic profile that was not significantly different from that of normal subjects. These patients were finally diagnosed as not having JMML (two had CMV infection, one a Leukocyte Adhesion Deficiency II, one a Noonan Syndrome, the final diagnosis is the other patient being still pending). Of note, JMML peripheral blood (PB) CD34+ cells from 6 JMML patients (3 paired BM-PB samples and 3 additional PB samples) fully confirmed the aberrant immunophenotypic signature seen in BM-derived samples.ConclusionsCD34+ precursor cells from JMML patients display a unique immunophenotypic profile characterized by an inverted ratio of CD19+ B/CD7+ lymphoid precursors, associated with unusual marker coexpressions, which might contribute to fast and more precise diagnostic work-up of JMML. Further studies in larger patient series are required to confirm our observations. DisclosuresBiondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Cellgene: Other: Advisory Board.

Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia.

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

Identification Of Specific Hematopoietic Populations Present In Peripheral Blood Mononuclear Cells That Increase Erythroid Output Directly Or Through Feeder/Stromal Cell-Like Properties

Hematopoietic development occurs in defined niches that ensure specific interactions and cross-talk with the surrounding stromal cells and different hematopoietic cells themselves. For instance, erythropoiesis occurs on the macrophage island within the bone marrow and the central macrophage is believed to regulate pro-erythroblast differentiation, the final stages of enucleation and reticulocyte maturation. We have observed that the expansion of erythroblasts from total peripheral blood mononuclear cells is increased compared to CD34+ Hematopoietic Stem/Progenitor Cells (HS(P)C) isolated from the same amount of blood [van den Akker, Haematologica, 2010]. This suggests i) the presence of CD34-cells that contribute to erythropoiesis and/or ii) that cell-cell contact or specific secreted growth factors by “helper” cells in these cultures can regulate hematopoiesis/erythropoiesis to increase erythroblast yield. Identifying the specific population(s) underlying the increased erythroid yield and understanding their way of action and regulatory mechanism during HSC differentiation and erythropoiesis is not only important to improve erythroblast culture conditions but may also provide clues to the function of hematopoietic effector cells in the various HS(P)C/erythroblast niches.Using specific lineage depletion (among which CD3 and CD14) we have identified and quantified various human erythroid and non-erythroid CD34+ and CD34- populations on the basis of CD36 co-expression in peripheral blood mononuclear cells (PBMC). Erythroid outgrowth from these CD34- populations and CD34+ populations and their contribution to the total erythroid yield from PBMC was assessed. Interestingly, total erythroid yield from the individual sorted populations did not reach the erythroid yield obtained from total PBMC. We hypothesized that support/feeder cells present in total PBMC are positively influencing in vitro erythropoiesis. In agreement with this, PBMC immuno-depletion of specific hematopoietic cell types identified CD14 cells (monocyte/macrophages) and to a lesser extend CD3 cells (lymphocytes) to be also partly responsible for the increased erythroblast yield. Compared to HS(P)C alone, co-culture of CD14 cells and HS(P)C isolated from PBMC resulted in a 5-10 times increase in CD71high/CD235med erythroblasts. Conditioned medium of CD14 cells as well as transwell experiments reconstituted the effect of the HS(P)C-CD14 co-cultures to 70%-80%, indicating that cell-cell contact plays a minor role. CD14 cells could elicit their effect at different stages during HSPC/HSC differentiation to erythroblasts. Co-culture of CD14 cells with pro-erythroblasts did not increase the cellular yield or proliferation rate. In contrast, two days of CD14 co-culture with CD34+ cells results in a 5 fold increase of total colony forming units without altering the colony lineage dynamics. In agreement with this a 5 fold increase in CD34+ cells was observed. These results indicate that CD14 cells elicit their effect on early hematopoietic progenitors but not on the erythroblast population. The results predict that depletion of CD14+ cells from PBMC should result in a decrease in the total number of CD34+cells. Indeed, we observed a 2 fold decrease of specifically HS(P)Cs and MEPs after two days of culture in PBMCs depleted for CD14 cells.Taken together our data i) identify previously unrecognized erythroid and non erythroid CD34- and CD34+ populations in peripheral blood that contribute to erythroid yield from total PBMC and ii) indicate modulation of HS(P)C outgrowth by specific hematopioietic effector cells present in peripheral blood that can also be found near specific hematopoietic niches in the bone marrow. The involvement of CD3 and CD14 immune cells suggests that HS(P)C and erythropoiesis may be modulated by immune-responses. Disclosures:No relevant conflicts of interest to declare.

Slug Is Required for a Functional Hematopoietic Stem Cell (HSC) Niche

AbstractAbstract 2579Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcription factors found in diverse species ranging from C. elegans to humans. Our previous study has already demonstrated that Slug serves intrinsic role in the regulation of HSC fates (Blood, 2010, 115(9):1709-1717). There is bone defect in Slug-/- mice, while Slug is expressed in osteoblast cells which are niche cells for HSCs, suggesting that Slug might be also extrinsically required for HSC regulation. To address this issue, the following experiments and assays were performed. Firstly, the cell proliferation of wild-type (WT) hematopoietic stem/progenitor cells (HS/PCs) (loaded with carboxyfluorescein succinimidyl ester (CFSE)) placed on bone marrow (BM) stromal layers (prepared from either Slug-/- or WT mice) was analyzed. Co-culture proliferation assay illustrated a clear increasing in the ability of Slug-/-BM stroma to support normal proliferation of WT HS/PCs, suggesting a significant and qualitative change in Slug-/- BM stromal cells so that they were able to support normal proliferation by Slug-competent HS/PCs. Secondly, BM microenvironment altered by Slug deficiency was defined by RT-PCR. Slug-/- BM had altered cell cycle profiles associated with increased stromal Notch1, N-cadherin, Vcam and Angiopoietin-1 expression. These findings indicate that Slug may participate in signaling in BM niche cells and thus is capable of influencing their function. Thirdly, the transplantation analysis was performed to address whether Slug impact was stem cell stroma dependent. WT hematopoietic cells in Slug knockout environment (8 weeks after transplantation) displayed a dramatic increase in LSK and LSK-Flk-2 ratio and in total number in BM, but not in spleen, indicating a stroma-determined effect by Slug on HSCs. It also showed a mobilization phenotype similar to that originally observed in Slug+/+ environment. Moreover, the annexin V apoptosis assay and cell-cycle analysis by the Edu incorporation assay suggested that Slug deficiency microenvironment promoted HSC expansion largely by increasing their proliferation, but not cell survival. Fourthly, each cell lineage in BM and spleen after WT BM transplanted into Slug+/+ and Slug-/- recipients was fully analyzed. The total number and ratio of T cells (CD3e, CD4+CD8-, CD4-CD8+ and CD4+CD8+) was markedly decreased in BM, but not in spleen, while the other lineages (granulocytes CD11b+Gr-1+, macrophages CD11b+, immature B lymphoid IgM-B220+, mature B lymphoid IgM+B220+, mature Erythroid CD71-Ter119+, and immature erythroid CD71+Ter119+) had an equivalent number and ratio in BM and spleen. Overall, Slug-/- BM microenvironment results in T cell loss. Finally, the recent study shows BM adipocytes as negative regulators of haematopoietic microenvironment, while the in vivo and in vitro evidences indicate that Slug is a key regulator of the adipocyte differentiation. Thus, in this study, we try to address whether Slug as a key factor adjusts adipocytes in BM niche. The fat of the whole body and the femurs and tibias were measured by DXA (Dual-energy X-ray Absorptiometry). Our data showed Slug-/- mice at 8 wk of age had lowest total fat, as well as ROI (region of interest) fat compared to Slug+/+ mice. In vitro adipocyte differentiation assay indicated there was less adipocyte formation from Slug-/- MSCs from BM. Furthermore, a dramatic loss of adipocytes in the femur in Slug-/- recipient was observed in 8 weeks after transplanting WT hematopoietic cells into Slug+/+ and Slug-/- recipients. Combining the above-mentioned data from WT hemtopoisis in Slug-/- microenvironment showed a dramatic increase in LSK and LSK-Flk-2 ratio and total numbers, lack of BM adipocytes after irradiation in fatless mice (Slug-/- recipient), suggesting a role of Slug-/- BM microenvironment in enhancing haematopoietic progenitor expansion via inhibition of BM adipocytes and post-transplant recovery. Taken together, our previously and present findings demonstrate that Slug serves intrinsic and extrinsic roles in the regulation of HSC fates. Disclosures:No relevant conflicts of interest to declare.

Phosphorylation status of transcription factor C/EBPα determines cell-surface poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units, called poly-LacNAc chains, characterize the histo-blood group i and I antigens, respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus, which expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue, with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene, consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen, granulocytic CD15, and also erythroid CD71 antigens. Taken together, these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role.

GATA‐1 transcription factor is up‐regulated in bone marrow hematopoietic progenitor CD34+ and erythroid CD71+ cells in myelodysplastic syndromes

GATA-1 is a transcription factor governing the production of erythroid and megakaryocytic cells. Unobstructed GATA-1 expression in early progenitor cells commits them to the myeloid lineage, channeling its differentiation towards erythrocytes and megakaryocytes. Myelodysplastic Syndromes (MDS) are clonal disorders of the hematopoietic stem cell frequently presenting dysplasia in erythroid and/or megakaryocytic lineage. We reasoned that measurement of GATA-1 expression levels in hematopoietic progenitor CD34(+) and the committed erythroid CD71(+) cells, from various MDS subcategories, could demonstrate GATA-1 involvement in the pathogenesis of the syndrome. In this study, MDS patients displayed significantly elevated GATA-1 mRNA expression, in bone marrow mononuclear cells (BMMCs), progenitor CD34(+) and erythroid CD71(+) cells in contrast to the control population (P < 0.001). Additionally, GATA-1 mRNA expression in MDS CD71(+) cells was positively correlated with their apoptotic levels (rho = 0.58, P = 0.03). Furthermore, GATA-1 expression levels were found to correlate with the disease progression. MDS patients in high/INT-2 IPSS risk group expressed significantly higher GATA-1 mRNA levels, in both CD34(+) and CD71(+) cells, as opposed to low/INT-1 patients (P < 0.001). Moreover, the former displayed increased apoptosis in the CD71(+) cells and significantly reduced neutrophil and platelet numbers and hemoglobin levels compared with the latter. We conclude that MDS patients display an increase of GATA-1 mRNA expression in BM cells, with high/INT-2 patients showing significantly higher levels. The higher level of GATA-1 mRNA in erythroid cells was positively correlated with their degree of apoptosis. These findings suggest that the up-regulation of GATA-1 may be responsible for the peripheral cytopenias in MDS.

PKCϵ controls protection against TRAIL in erythroid progenitors

AbstractApoptosis plays a central role in the regulation of the size of the hematopoietic stem cell pool as well as in the processes of cell differentiation along the various hematopoietic lineages. TRAIL is a member of the TNF family of cytokines with a known apoptogenic role against a variety of malignant cells and an emerging role in the modulation of normal hematopoiesis. Here we worked on the hypothesis that PKCϵ could act as a switch of the cellular response to TRAIL during erythropoiesis. We demonstrate that EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL at day 0 due to the lack of specific receptor expression. From day 3 onward, erythroid cells express surface death receptors and become sensitive to TRAIL up to day 7/8 when, notwithstanding death-receptor expression, the EPO-driven up-regulation of PKCϵ intracellular levels renders differentiating erythroid cells resistant to TRAIL likely via Bcl-2 up-regulation. Our conclusion is that in human CD34 cells, EPO promotes a series of events that, being finely regulated in their kinetics, restricts the sensitivity of these cells to TRAIL to a specific period of time, which therefore represents the “TRAIL window” for the negative regulation of erythroid-cell numbers.

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon.

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.

Expression of Cellular Prion Protein (PrPc) on Human Red Blood Cells.

Two recent UK cases of vCJD transmission by blood transfusion emphasize urgent need of donor screening test for prion diseases. Pathological form of prion protein, PrPsc, is currently the only specific marker of prion diseases, but its detection in blood poses significant challenge. Blood contains substantial amount of normal PrPc which supposedly differs from PrPsc only in its conformation. Majority of cell associated PrPc in blood reside in platelets (PLT). PrPc is also expressed on PBMCs, but their contribution to quantity of blood PrPc is small. The situation is less clear with PrPc on red blood cells (RBC). Others and we have previously shown that PrPc is detectable on RBC by flow cytometry (FACS). However, this was contradicted by another study and by reported minimal content of PrPc in RBC measured by ELISA. To confirm our finding, we used quantitative FACS with fluorescein labeled monoclonal antibodies (MAbs) FH11, 3F4 and 6H4 against different parts of PrPc molecule (PrP23–85, PrP109–112 and PrP144–152, respectively) for evaluation of PrPc expression on RBC of healthy blood donors (n=8). Mean (range) of MAb molecules bound /cell was: FH11 - 36 (13–74), 3F4 - 80 (33–137), 6H4 - 258 (113–557). Interestingly, 3F4 and 6H4 recognized PrPc on PLT in the same samples equally well. Decrease accessibility of 3F4 epitope is one of the characteristics of PrPsc, suggesting that PrPc on RBC may adopt PrPsc like conformation. To test if PrPc on RBC is resistant to proteolysis we treated blood samples with increasing concentrations of proteinase K (1-50 mg/ml, 30 min., 0°C). FACS demonstrated gradual and complete cleavage of PrPc on both PLT and RBC. Lower 3F4 binding to RBC could be explained by expression of N-terminally truncated form of PrPc. Western blot (WB) analysis of RBC ghosts with MAbs 6H4 and AG4 (PrP31–51) confirmed the presence of PrPc in RBC membranes. PrPc seems to be mainly in diglycosylated form, detected as a diffuse band with molecular weight (m.w.) slightly higher (35–38 kDa) than brain PrPc. The difference in PrPc mobility diminished after deglycosylation of PrPc with PNGase. No prominent bands with lower m.w. suggesting presence of truncated PrPc in RBC were detected. If conformation was the cause of 3F4 epitope inaccessibility, this should be reversed by denaturation. Interestingly, 3F4 displayed similar deficiency in PrPc detection also on WB after denaturation of RBC samples with SDS and boiling. At the same time 3F4 and 6H4 exhibited similar sensitivity in detection of PrPc on WB of dilutions of brain or PLT lysate. The remaining explanation of 3F4 reactivity is that its epitope MKHM on RBC PrPc is modified. Recently, modification of Lys residues of PrPsc by advanced glycosylation end products (AGEs) has been reported. We modified Lys residues of brain PrPc on blots by treatment with increasing concentrations of paraformaldehyde and confirmed that Lys modification leads to loss of 3F4 binding. In opposite to situation in peripheral blood, PrPc on erythroid CD71+ cells in cord blood was detected equally well with 3F4 and 6H4, suggesting that modification of PrPc occurs after release of RBC into periphery. Taken together, human RBC express ~ 200 molecules of PrPc/cell. Due to high RBC count even such low level of expression suggests significant contribution to pool of cell associated PrPc in blood (~ 50%). Methods utilizing MAbs FH11 and 3F4 may underestimate quantity of PrPc in RBC. Likewise, screening tests for presence of PrPsc in blood may encounter difficulties if modification similar to one reported here is present.