ALBERT

Description: Introduction: Protein 4.1R is a cytoskeletal adaptor protein that is responsible for the control of the mechanical stability of erythrocyte membranes, and for the proper anchoring of transmembrane proteins to the membrane skeletal network. Analysis of 4.1R-deficient human and murine erythrocytes revealed the complex array of membrane proteins that bind 4.1R and link these proteins to the spectrin-based skeletal network. 4.1R is composed of four functional domains: the N-terminal 30 kDa domain referred to as the FERM domain, the 16 kDa domain, the 10 kDa spectrin-actin binding domain, and the C-terminal 24 kDa domain. The Kell glycoprotein (93 kDa) is a type II single-span membrane protein which carry the Kell blood group system including the K1 (Kell) and K2 (cellano) antigens. Kell protein has endothelin-3 converting enzyme activity of type II membrane glycoproteins. In this study we have analyzed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(-) HE) and performed detailed characterization of the interaction between 4.1R and Kell glycoprotein. Furthermore we also investigated the expression of membrane proteins exhibiting blood group antigens and the functional activities of AQP1, Band 3 and RhAG in the 4.1(-) HE erythrocyte membrane. Results: Flow cytometry and western blot analyses revealed a severe reduction of Kell in the absence of 4.1R. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R46 R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(-) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3-/Cl- exchange (band 3-dependent), but a normal water (AQP1-dependent) and ammonia (RhAG-dependent) transport in the absence of 4.1R. Discussion: In this study, we provide evidence for a direct interaction between Kell and 4.1R and we propose an updated model for the 4.1R- multiprotein complex in human erythrocyte (Fig 1). The lobe A in the 4.1R FERM domain binds to protein transporters such as band 3, NHE1 and UT-B. Functional and structural experiments are required to confirm the presence of UT-B in this complex. The transmembrane proteins GPC, XK, Duffy and Kell bind to the lobe B and the binding site of p55 is located in lobe C. The deficiency of blood group antigens carrying proteins in HS and HE erythrocytes can be explained by various molecular mechanisms including perturbed traffickingto the erythroblast membrane, aberrant protein sorting duringerythroblast enucleation, and selective loss during reticulocytemembrane remodelling. Establishing when and where these proteins associate during erythroid differentiation should provide mechanistic insights into membrane multi-protein complex formation in both normal and abnormal erythropoiesis. Conclusion: The findings from the present study using 4.1(-) HE human erythrocytes have enabled us to obtain novel insights into the 4.1R complex organization. Table 1. Antigen and protein expression of human erythrocytes.Specific antibody binding capacity, as determined by indirect immunofluorescence using QIFIKIT calibrated beads. *Mean of fluorescence intensity given in arbitrary units. Proteins Normal 4.1(-) HE GPC 54075 (± 1075) 4000 (± 500) Band 3 415000(±7000) 317000(±6000) Kell 4150 (±66) 1220 (±35) CD47 21300 (±1556) 21000 (± 2350) Rh 135200 (±283) 121000 (±113) RhAG 90450 (±2192) 71950 (±13150) GPA 357500 (±7072) 310500(±12500) DARC 2500 (±283) 1850 (±250) CD44 4150 (±71) 4100 (±200) UT-B* 13125 (± 1968) 2702 (±142) Lu/BCAM 1300 (±141) 1624 (±153) AQP1* 256 (±15) 202 (± 22) GLUT-1 10961 (± 239) 11542 (± 806) Figure 1. Proposed model of the 4.1R-multiprotein complex in human erythrocyte. Figure 1. Proposed model of the 4.1R-multiprotein complex in human erythrocyte. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Dynamic intron retention programs exist in the murine megakaryocyte and erythroid and human erythroid lineages. Intron retention inversely correlates with expression levels of a large set of transcripts.

Description: TET family proteins (TET1, TET2 and TET3) have recently emerged as important epigenetic modifiers by catalyzing the conversion of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). Although they have been documented to play important roles in a variety of biological processes, their function in erythroid differentiation has yet to be defined. In the present study, we show that of the three TET family members, TET2 and TET3 but not TET1 are expressed in erythroid cells and that TET3 is more abundantly expressed than TET2. Using shRNA-mediated knockdown approach we explored the role of TET proteins in erythroid differentiation of CD34+ human cells. We first showed that consistent with their role in the production of 5hmc, knockdown of either TET2 or TET3 led to a decrease in global 5hmc levels as assessed by mass spectrometric analysis. However, knockdown of TET2 or TET3 resulted in distinctly different phenotypic changes during erythropoiesis. Knockdown of TET3 in human CD34+ cells resulted in impaired cell growth which is accompanied by increased apoptosis of late stage erythroblasts. Knockdown of TET3 also led to generation of bi/multinucleated polychromatic/orthochromatic erythroblasts which is accompanied by impaired enucleation. To explore the molecular mechanisms for the observed phenotypic changes, we performed RNA-seq analysis on control and TET3 knockdown erythroblasts at same stages of development. Bioinformatics analysis revealed that the expression levels of several apoptosis-promoting genes such as FOXO1, TNFRSF10B, TGFB1 and BTG1 are increased and that of a mitosis/cytokinesis associated gene KLHDC8B is decreased in polychromatic and orthochromatic erythroblasts following TET3 knockdown. Measurement of 5hmc and 5mc at promoter region of KLHDC8B locus revealed decreased 5hmc level concurrent with increased 5mc level. Importantly, knockdown of KLHDC8B in CD34+ cells as with knockdown on TET3 led to generation of increased numbers of bi/multinucleated polychromatic/orthochromatic erythroblasts and impaired enucleation implying a role for this protein in cytokinesis of late stage but not early stage erythroblasts. These findings demonstrate that TET3 regulates erythropoiesis in a stage-specific manner by targeting different set of genes. Importantly, knockdown of TET2 led to phenotypic changes that were very different from that seen following knockdown of TET3 but the observed changes are similar to the erythroid development defects noted in myelodysplastic syndromes (MDS). These include hyper-proliferation of early stage erythroid cells; delayed terminal erythroid differentiation and increased apoptosis of late stage erythroblasts. Together with the fact that TET2 gene mutation is one of the most common mutations in MDS and dyserythropoiesis is a hallmark of this disorder, our findings suggest that TET2 gene mutations can directly account for dyserythropoiesis of MDS. Our findings demonstrate distinct and important roles for TET2 and TET3 in regulating erythropoiesis and provide significant new and novel insights into epigenetic regulation of erythropoiesis at distinct development stages. The findings are likely to be very useful for furthering our understanding of epigenetic regulation of normal and disordered human erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Total splenectomy is recommended in symptomatic cases of hereditary spherocytosis (HS) to reduce the severity of anemia but exposes patients to long-term infectious or thrombotic complications. Alternative strategies such as subtotal splenectomy (STS) have been developed, principally for children under the age of 6 with severe HS, who are not eligible to total splenectomy because of the high infectious risk and for older patients with mild HS complaining of chronic discomfort. Since our original report, several groups have shown that STS reduced the hemolytic rate, increased the red cell lifespan while maintaining an efficient splenic phagocytic function but the extent of follow-up has been limited. In order to define the long-term benefits of STS, we report here an update of our series that includes 90 patients who underwent STS at the Bicêtre hospital between 1985 and 2013, with a median-follow-up of 9.3 years (range 1-23 years). Two groups were defined on the basis of the disease phenotype: Group A included 42 patients for whom STS was performed because of severe/intermediate HS (transfusion requirement or hemoglobin (Hb) level

Description: Peroxiredoxin-2 (Prx2), a typical 2-cysteine (Cys) peroxiredoxin, is a key anti-oxidant system in both normal and pathological erythropoiesis characterized by oxidative stress such as b-thalassemia. We recently showed that the absence of Prx2 worsens ß-thalassemic erythropoiesis and related iron-overload (Matte A et al. Antioxid Redox Signal, 2015). Here, we studied the effects of iron overload in a mouse model genetically lacking Prx2 (Prx2-/-). Two months old female wild-type (WT) and Prx2-/- pure bred mice were fed with a diet containing 2.5% carbonyl-iron compared to standard diet treated mice. We evaluated hematologic parameters, red cell indices and reticulocyte count in both mouse strains at baseline and at 30, 49, 60 and 90 days of treatment with carbonyl-iron. We observed a rapid drop in Hct and reticulocyte count in Prx2-/- mice compared to wild-type mice between 30-49 days of iron supplementation with the appearance of severe hyporegenerative anemia at 60 days of treatment in Prx2-/- characterized by a significant reduction in CD44+TER119+Fsc high cells. This was associated with marked increases in apoptotic Prx2-/- orthochromatic erythroblast compared to either baseline values or WT treated mice. In sorted erythroid precursors from iron overload WT mice, Prx2 expression was significantly increased compared to WT under standard diet. We observed a modulation of Erythropherrone (Erfe) expression during erythropoiesis with upregulation of Erfe in WT orthochromatic erythroblast compared to Prx2-/- erythroblasts. In liver from Prx2-/- mice exposed to iron-overload, we found liver iron content similar to WT mice but Pearls stain analysis showed differential iron distribution in cellular components of liver. While iron accumulated in hepatocytes and Kuppfer cells in Prx2-/- mice, iron-deposits were present only in hepatocytes of WT liver. Oxyblot analysis and liver MDA levels were significantly higher in Prx2-/- mice indicating that the absence of Prx2 promotes a severe liver oxidative stress. In WT liver, Prx2 expression was marked increased in a time depend way during iron supplementation, indicating that Prx2 is part of an adaptive cellular response to iron overload. This is in agreement with increased levels of ferritin-H in Prx2-/- mice compared to WT mice. Hepcidin (HAMP) expression was markedly increased in iron-overload WT mice compared to untreated control group, while no major changes were observed in Prx2-/- mice. Tfr2 expression was significantly increased only in livers of iron-overload WT mice, whereas phospho smad 1-5 was significantly increased in both mouse strains in response to iron-overload. The activation of the signaling pathway through Erk-1/2 only in iron-overload WT mice but not in Prx2-/- mice is most likely related to severe oxidative stress in Prx2-/- resulting in switching off of the Erks pathway. Importantly, administration of PEP1-Prx2 fusion protein rescue almost completely the hematologic phenotype with modulation of Erk signaling pathway towards Tfr2 and the smad system, validating our hypothesis of a role for Erk signaling for the observed phenoytpes. Our data highlight Prx2 as novel factor involved in iron homestasis through the control of oxidative stress modulating signaling pathway towards hepcidin expression. Disclosures No relevant conflicts of interest to declare.

Description: Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Description: Background We and others have shown that normal human erythroid cell maturation requires a transient activation of caspase-3 at late stages of maturation (Zermati et al, J Exp Med 2001). We further documented that, in human erythroblasts, the chaperone HSP70 is constitutively expressed and, at late stages of maturation, translocates into the nucleus and protects GATA-1, the master transcriptional factor critical for erythropoiesis, from caspase-3 cleavage (Ribeil et al, Nature 2007). During the maturation of human β-TM erythroblasts, HSP70 is sequestrated by excess of α-globin chains in the cytoplasm and as a consequence, GATA-1 is no longer protected from caspase-3 cleavage resulting in end-stage maturation arrest and apoptosis (Arlet et al, Nature 2013). Understanding the molecular mechanisms that regulate the localization of HSP70 during erythroid differentiation may help to find new therapeutic targets to reduce ineffective erythropoiesis in beta-thalassemia. Methods CD34 positive cells from normal and thalassemic peripheral blood were cultured in IMDM/BIT media in the presence of SCF, IL3, IL6 for seven days and subsequently cultured for additional 7 to 9 days in media containing SCF, IL3 and Epo. Erythroblasts differentiation, HSP70 localization were analysed by FACS, AMNIS stream, confocal microscopy and western blot analysis. RNAseq and proteomic analysis of highly purified erythroid cells at all distinct stages of differentiation were used to assess expression levels of various exportins. Duolink and Octet analyses were used to assess protein proximity and affinity of interactions, respectively. Results During erythroid differentiation, Hikeshi, the cognate nuclear importin of HSP70, is constitutively expressed and enables HSP70 nucleus entry as assessed by siRNA experiments. However, its expression was not regulated during erythroid differentiation. In contrast, exportin expression analysis showed marked differences in expression levels of XPO1 and XPO7 during erythroid differentiation. XPO1 expression being reduced at the time of c-kit down-regulation and caspase 3 activation while there was a marked increase in XPO7 expression at the late stages of terminal erythroid differentiation. XPO1 interacted in vivo (Duolink analysis) and in vitro with HSP70 (Octet analysis). Likewise, the previously described HSP70 S400A mutant (in the Leucine-rich Nuclear Export Sequence), which is constitutively located in the nucleus interacted with XPO1 with lower affinity compared to HSP70 WT. Stem Cell Factor (SCF) starvation and Pi3k inhibition led to decreased in vivo HSP70/XPO1 interactions. However, neither phosphorylation of HSP70 nor XPO1 were detected by Nanopro and proteomic analysis, and XPO1 expression was not regulated by Pi3K pathway. Expression of RanGTP Activating Protein (RanGAP), a protein critical for XPO1/cargo interaction, was down-regulated at the moment of caspase 3 activation during erythroid maturation, which may explain the decrease in HSP70/XPO-1 interactions. Inhibitors of XPO1 (leptomycin B and KPT 251) were able to induce HSP70 nuclear localization at early stages of differentiation (proE). In erythroid progenitors from β-TM patients, treatment with the Selective Inhibitor of Nuclear Export compound KPT-251 rescued nuclear HSP70 localization and GATA1 expression, and resulted in improved of erythroid terminal differentiation, without cytotoxicity, of thalassemic erythroid progenitors. Conclusion XPO1 is a major regulator of erythropoiesis through the regulation of HSP70 nuclear localization and is a potential new target to decrease ineffective erythropoiesis of thalassemia. Specific XPO-1 inhibitors currently in clinical development are being tested for potential therapy in thalassemic erythroid progenitors. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Gene therapy for sickle cell disease (SCD) is currently in active trials. Finding a safe and effective method for collection of hematopoietic stem cells (HSC) in SCD remains a challenge. Granulocyte colony stimulating factor (G-CSF), used most commonly for collecting HSC, can cause life-threatening vaso-occlusion in SCD. Bone marrow harvest requires general anesthesia and multiple punctures. Plerixafor is an inhibitor of the CXCR4 chemokine receptor on HSC and interferes with binding to SDF-1 on bone marrow stroma. As pre-clinical data in support of a clinical trial in SCD patients studying plerixafor mobilization (NCT02193191), we administered plerixafor to SCD mice to assess the risk of cell activation and vaso-occlusion. Methods: 3-6 month old SS Berkeley (n=12) or SS Townes mice (n= 18) were used. Littermate mice were randomized to subcutaneous treatment with plerixafor (Genzyme-Sanofi) 10 mg/kg once, G-CSF (Amgen) 250 ug/kg daily for 5 days, or equivalent volume (5 uL/g) normal saline daily for 5 days. Peripheral blood was harvested at 1-2 hr (plerixafor) or 4-5 hr (G-CSF and normal saline) after the last dose for the following studies: CBC (Advia), enumeration of HSC mobilization (Lin-Sca-1+ c-kit+ Flt3- (LSKF) cells by flow cytometry), neutrophil activation (L-selectin shedding by flow cytometry), and endothelial activation (soluble P-selectin by ELISA). Berkeley mice underwent MRI imaging before and after completion of treatment. Results: CBC showed the mean WBC and platelet counts of both plerixafor and G-CSF groups to be significantly different from saline, but the WBC differential was only significantly different (in % neutrophils and lymphocytes only) from saline in the G-CSF group (Table 1). The percentages of HSC subsets were significantly higher in both plerixafor and G-CSF groups compared to saline, with no significant differences between plerixafor and G-CSF (Table 2). L-selectin was low and soluble P-selectin high only in the G-CSF group, with both markers significantly different from both plerixafor and saline (Table 3). MRI imaging showed no significant differences in cerebral blood flow (measures oxygen delivery), mean diffusivity (measures vasogenic swelling), or fractional anisotropy (measures axonal integrity) pre- compared to post-treatment in any group. Tables show mean ± SD and significant p-values compared to saline. Discussion: Plerixafor and G-CSF were effective as evidenced by expected changes in WBC and platelet counts with treatment compared to saline. Both plerixafor and G-CSF significantly mobilized HSC subsets. There was a trend towards higher mobilization with G-CSF of the more primitive LSKF subset, but clinical data indicate that addition of plerixafor to G-CSF mobilizes a higher number of more primitive CD34+CD38- than G-CSF alone (Fruehauf S, Cytotherapy 2009). In support of potential safety of plerixafor in SCD patients, there was no evidence of neutrophil or endothelial activation with plerixafor, in contrast to G-CSF. Despite the evidence of neutrophil and endothelial activation with G-CSF, there was no evidence of perfusion-related organ damage as measured by MRI parameters. These findings suggest that plerixafor canbe safely and effectively used for HSC mobilization from SCD patients for use in gene therapy. Acknowledgments: We are grateful to Farid Boulad, MD and Tsiporah Shore, MD for providing plerixafor. Table 1. Significant CBC and WBC differential parameters Treatment group Saline (n=6) Plerixafor (n=8) G-CSF (n=8) WBC (103/uL) 21.7 ± 5.7 39.1 ± 15.5 (p=0.02) 56.3 ± 25.8 (p=0.006) Platelet (103/uL) 1,118 ± 394 554 ± 255 (p=0.007) 582 ± 280 (p=0.01) % neutrophils 20 ± 7 20 ± 6 51 ± 20 (p=0.003) % lymphocytes 76 ± 8 74 ± 7 40 ± 16 (p=0.0003) Table 2. Percent of hematopoietic stem cell subsets in lineage-negative population Treatment group Saline (n=6) Plerixafor (n=8) G-CSF (n=8) SKF 0.04 ± 0.08 0.72 ± 0.58 (p=0.01) 1.33 ± 1.15 (p=0.02) SK 0.12 ± 0.24 1.89 ± 1.42 (p=0.009) 1.42 ± 1.17 (p=0.003) Table 3. Neutrophil and endothelial activation markers Treatment group Saline (n=6) Plerixafor (n=8) G-CSF (n=8) L-selectin fluorescence intensity 8483 ± 5216 8106 ± 3987 2833 ± 1470 (p=0.04) Soluble P-selectin (ng/mL) 197 ± 61 180 ± 36 277 ± 45 (p=0.005) Disclosures No relevant conflicts of interest to declare.

Description: Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

Description: Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing. Disclosures No relevant conflicts of interest to declare.