Haploinsufficiency Of RPS14 Research Articles

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A

AbstractMetabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1–dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Regulated Expression of GATA1 As a Gene Therapy Cure for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure disorder characterized by impaired red blood cell production leading to severe anemia. The only treatment options are regular transfusions, corticosteroids, or bone marrow transplant, and each has variable effectiveness and considerable morbidity. More effective, safer, and permanent cures for DBA are lacking, but few drugs are in active development. DBA is genetically heterogenous, characterized by loss-of-function mutations in one copy of at least 26 different ribosomal protein (RP) genes that alters the mRNA translation of a select group of genes, most notably the hematopoietic master regulator GATA1. Overexpression of GATA1 in hematopoietic stem and progenitor cells (HSPCs) from patients with DBA rescues the erythroid differentiation defects in vitro, but unregulated GATA1 expression in hematopoietic stem cells (HSCs) promotes premature erythroid differentiation at the expense of long-term (LT-) HSC maintenance. Gene therapy is an attractive strategy to achieve a cure for DBA, but a traditional gene therapy approach that overexpresses a functional copy of a mutated gene is not the ideal option, as it would require the development and validation of dozens of gene therapy vectors, each with a copy of one of the mutated DBA genes. Here, we report a unified gene therapy strategy, using developmentally-regulated and highly restricted expression of GATA1 that would be curative in most, if not all, patients with DBA, regardless of the underlying disease-causing mutation. To be a cure for DBA, a GATA1 gene therapy vector must incorporate into the genome of undifferentiated LT-HSCs but only drive robust GATA1 expression in early erythroid progenitors after lineage commitment. To design a vector with lineage-restricted expression, we identified accessible chromatin regions upstream of GATA1 that only gain accessibility in differentiating erythroid cells, but not in upstream HSPCs. Using these selectively accessible regions concatenated together, we constructed a human GATA1 enhancer (hG1E) element and used it to drive GATA1 and/or GFP expression from a lentiviral backbone. To ensure erythroid-restricted expression of the hG1E-GATA1-GFP vector, we infected CD34+ HSCs from healthy human donors and cultured them in conditions that either allowed HSC maintenance or promoted erythrocyte differentiation. We observed very low GFP expression in bulk HSCs and essentially no expression in the subpopulation of immunophenotypic LT-HSCs. However, more than 80% of committed erythroid cells demonstrated high levels of GFP expression, confirming the fidelity of lineage-restricted expression from the hG1E-GATA1 vector. To examine whether lineage-restricted GATA1 expression confers a functional correction of the erythroid maturation block in DBA, we used CRISPR editing to recapitulate RPS19 haploinsufficiency in human HSPCs. In RPS19 edited samples that were infected with a control GFP virus, we observed a progressive depletion over time of RPS19 edits as the cells underwent erythroid differentiation, consistent with the notion that RPS19 editing leads to impaired erythroid differentiation and are selected against. In contrast, RPS19-edited and hG1E-GATA1 treated samples had a more than 10-fold higher preservation of RPS19 edits in the bulk population and in erythroid colonies derived from single progenitor clones, indicating that regulated GATA1 expression could overcome RPS19 haploinsufficiency. To validate the results from the CRISPR model of DBA, we sought to correct the erythroid maturation block in primary HSCs from a DBA patient with a pathogenic mutation in RPL5. There was skewed differentiation of GFP-infected RPL5+/- HSCs away from the erythroid lineage and toward myeloid and megakaryocyte lineages, but regulated GATA1 expression from the hG1E-GATA1 vector reversed that skewing. There was a 10-fold increase in the ratio of erythroid to non-erythroid cells, and a significant expansion of erythroid cells in the hG1E-GATA1 treated patient cells. Preclinical assessment in additional patient samples both in vitro and in vivo is ongoing. Collectively, our preclinical data demonstrate that lineage-restricted expression of GATA1 is sufficient to rescue the impaired erythroid differentiation in DBA regardless of the underlying genetic lesion. These results will set the stage for the first in-human gene therapy trial for DBA.

Evolution of severe (transfusion-dependent) anaemia in myelodysplastic syndromes with 5q deletion is characterized by a macrophage-associated failure of the eythropoietic niche.

Evolution of erythrocyte transfusion-dependent (RBC-TD) anaemia associated with haploinsufficiency of the ribosomal protein subunit S14 gene (RPS14) is a characteristic complication of myelodysplastic syndromes (MDS) with del(5q) [MDS.del(5q)]. Evaluating 39 patients with MDS.del(5q), <5% of anaemia progression was attributable to RPS14-dependent alterations of normoblasts, pro-erythroblasts, or CD34+ CD71+ precursors. Ninety-three percent of anaemia progression and 70% of the absolute decline in peripheral blood Hb value were attributable to disappearance of erythroblastic islands (Ery-Is). Ery-Is loss occurred independently of blast excess, TP53 mutation, additional chromosome aberrations and RPS14-dependent alterations of normoblasts and pro-erythroblasts. It was associated with RPS14-dependent intrinsic (S100A8+ ) and extrinsic [tumour necrosis factor α (TNF-α)-overproduction] alterations of (CD169+ ) marrow macrophages (p<0.00005). In a mouse model of RPS14 haploinsufficiency, Ery-Is disappeared to a similar degree: approximately 70% of Ery-Is loss was related to RPS14-dependent S100A8 overexpression of marrow macrophages, less than 20% to that of CD71high Ter119- immature precursors, and less than 5% to S100A8/p53 overexpression of normoblasts or pro-erythroblasts. Marked Ery-Is loss predicted reduced efficacy (erythrocyte transfusion independence) of lenalidomide therapy (p=0.0006). Thus, erythroid hypoplasia, a characteristic complication of MDS.del(5q), seems to result primarily from a macrophage-associated failure of the erythropoietic niche markedly reducing the productive capacity of erythropoiesis as the leading factor in anaemia progression and evolution of RBC-TD in MDS.del(5q).

Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell–extrinsic contributions to erythropoiesis

AbstractWe follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

Targeting of Calbindin 1 (CALB1) Rescues Erythropoiesis in a Human Model of Diamond Blackfan Anemia: Implications for Novel Therapies

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. The erythroid defects of DBA include macrocytosis, reticulocytopenia, and a selective decrease or absence of erythroid precursors in the bone marrow. It was the first disease linked to ribosomal dysfunction and &amp;gt;70% of patients have since been shown to have a haploinsufficiency of a ribosomal protein (RP) gene. Among these RP mutations, RPS19 mutations were the first to be identified and are the most common, found in up to 25% of DBA patients. Current therapies for DBA include corticosteroids, chronic transfusions, and/or hematopoietic stem cell transplantation. However, each of these therapies have potential for significant side effects and as such there is an urgent need to develop new therapies for the management of these patients. In addition there is a need for improved understanding of the mechanistic basis for the wide range of phenotypic expression and responses to therapy between patients with the same mutation, which implies a key role for modifier genes in this disease. To identify potential modifiers in the phenotype of patients and for developing potential novel therapeutic targets, we performed a genome wide CRISPR screen using A549 cells with RPS19 haploinsufficiency as a cellular model for DBA, which enabled large-scale screening. As the p21 pathway is known to be central to the pathophysiology of DBA, we identified hits that resulted in higher and lower levels of p21 expression in the DBA model vs. control, respectively. Among these hits, we consistently identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily that includes calmodulin and buffers entry of calcium into cells upon stimulation of glutamate receptors. Given the recent findings that calmodulin inhibition can rescue anemia in DBA models by blocking the p90 ribosomal S6 kinase (RSK)-p70 (p70S6K) pathway and the role for glucose and glutamine metabolism in the regulation of human HSC lineage specification, we decided to pursue CALB1 as a potential target of interest. We used peripheral blood derived-CD34+ cells placed in erythroid stimulating media following knockdown of RPS19, and investigated the effects of CALB1 on cell cycle progression and erythroid differentiation by flow cytometry. We observed that knockdown of CALB1 significantly improved cell viability, increased G0/G1 population and promoted erythroid maturation with increased expression of CD36 and glycophorin A in the context of RPS19 deficiency. Together, these data suggest that the knockdown of CALB1 rescues human erythropoiesis in a model of DBA. We are investigating the effects of CALB1 knockdown on signaling pathways and genes involved in DBA due to RPS19 mutations notably GATA-1/HSP70 and TNF-alpha. We anticipate that the identification of a role for CALB1 in the pathophysiology of DBA will provide a new therapeutic target for DBA either alone or in combination with steroids or calmodulin inhibition. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146adeficiencyrecapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146adeficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146adeficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

Acquired ribosomopathies in leukemia and solid tumors.

A mutation in the gene encoding the small subunit-associated ribosomal protein RPS19, leading to RPS19 haploinsufficiency, is one of the ribosomal protein gene defects responsible for the rare inherited bone marrow failure syndrome Diamond Blackfan anemia (DBA). Additional inherited and acquired defects in ribosomal proteins (RPs) continue to be identified and are the basis for a new class of diseases called the ribosomopathies. Acquired RPS14 haploinsufficiency has been found to be causative of the bone marrow failure found in 5q- myelodysplastic syndromes. Both under- and overexpression of RPs have also been implicated in several malignancies. This review will describe the somatic ribosomopathies that have been found to be associated with a variety of solid tumors as well as leukemia and will review cancers in which over- or underexpression of these proteins seem to be associated with outcome.

Ribosome Biogenesis Controls Erythroid Differentiation Via a p53-Dependent Transcriptional Checkpoint and Its Limitation By RPS14 Haploinsufficiency Results in Selective Defects in Translation

Haploinsufficiency in genes encoding ribosomal proteins (RP) or ribosome-associated proteins either by mutation or deletion leads to a predominant erythroid phenotype. In acquired 5q- myelodysplasic syndrome (MDS), the macrocytic anemia has been linked to the monoallelic deletion of RPS14 gene which results in altered ribosome biogenesis. Because of the defective maturation of the small 40S ribosome subunit, the RPL5/RPL11/5SrARN complex, normally involved in the assembly of the large 60S subunit assembly, accumulates and inhibits E3-ligase-HDM2 leading to the stabilization and activation of p53 resultig in cell cycle arrest, increased apoptosis and defective differentiation of maturing erythroblasts. In the present work, we hypothesized that p53 could play a key role in the control of normal erythroid differentiation by ribosome biogenesis and we further investigated the involvement of the decreased pool of ribosome on erythroid defects in the 5q- syndrome.To investigate the first hypothesis, we studied the kinetics of ribosome biogenesis in human primary erythroblasts by mass spectrometry after pulse-SILAC. We noted that ribosome renewal collapses begining at the polychromatophilic erythroblast stage. We subsequently used the pharmacological agent CX-5461 to inhibit RNA polymerase I. When ribosome biogenesis in proerythroblasts is blocked by CX-5461, p53 is activated and proerythroblasts enter the terminal differentiation by expressing GATA1-erythroid target genes without apoptosis. By ChIP-seq in primary erythroblasts, we demonstrated that p53 binds to 1289 genes including 263 genes specifically activated by CX-5461, 6 of them being upregulated during CX-5461-induced erythroid differentiation and 3 of them being known GATA1 targets. We further used an shRNA strategy to demonstrate that one of these genes is required to permit entry into terminal erythroid differentiation when ribosome biogenesis is abrogated. We thus showed that normal erythroid differentiation is controlled by ribosome biogenesis through a p53-dependent checkpoint.In 5q- syndrome, ribosome biogenesis is continuously decreased along all stages of erythropoiesis and in contrast to the normal conditions, erythroid differentiation is defective with an excess of apoptosis affecting mature erythroblasts. We previously reported that GATA1 is targeted by a caspase-dependent cleavage since GATA1 is not protected by its chaperone HSP70 in the nucleus of MDS erythroblasts. We confirmed that GATA1 protein is decreased in 5q- primary erythroblasts and in RPS14 shRNA-expressing normal erythroblasts. To obtain further insights into the defective erythroid maturation of RPS14-deleted erythroblasts, we developed an inducible shRNA to RPS14 in the UT7/Epo cell line. Polysome profiling confirmed the decrease of 40S subunit and absolute quantification of RP by deep proteomics demonstrated a 50% decrease of RPS in conjunction with 50% reduction of ribosome content in these cells. GATA1 expression was decreased and was only partially rescued by treatment with either caspase inhibitor qVD or proteasome inhibitor, bortezomib. We then tested the hypothesis of a decrease in GATA1 translation, as previously shown in a shRNA RPS19 Diamond-Blackfan model, by analyzing and comparing the global transcriptome and the translatome corresponding to transcripts present in high molecular weight polysomes using Affymetrix HTA 2.0 microarrays. We observed a decoupling between transcriptome and translatome suggesting a selectivity of translational defects. Thermodynamic characteristics i.e. the fold energy, energy per base and length of the 3'UTR and the energy per base of the 5'UTR (Vienna RNA Package, UCSC genome browser) were the determinants of transcript selection on the polysome. The shortest transcripts with a highly structured 3'UTR including GATA1 were the transcripts which were less effectively translated. Consistently, the diminution of GATA1 protein was associated with a decrease of its target genes. Our results suggest that GATA1 is a potentially interesting therapeutic target.In summary, our results show that ribosome biogenesis controls erythroid differentiation via a p53-dependent transcriptional regulation and that a reduction of the ribosome pool leads to a selective translation at the expense of erythroid master gene GATA1. DisclosuresFontenay:Celgene: Research Funding.

Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. DisclosuresNo relevant conflicts of interest to declare.

P53 Protein Expression Correlates with Adverse Outcome in Low-Risk Myelodysplastic Syndromes

BackgroundThe presence of diverse spectrum of mutations in myelodysplastic syndromes (MDS) has largely revolutionized the understanding of the disease. Although certain mutations have doubtful prognostic significance, the adverse impact of TP53 mutation is irrefutable in all publications. We and others have shown the strong correlation between presence of TP53 mutation and overexpression of p53 by immunohistochemistry (IHC). This evidence provides the rationale for the evaluation of p53 overexpression which is available in routine diagnostic workup, as a surrogate marker to predict TP53 mutations. Additionally, we predicted that the overexpression of p53 might be an independent prognosticator, even in the absence of TP53 mutations or RPS14 haploinsufficiency, in low risk MDS.In our study,we analyzed the p53 protein expression in a cohort of 277 'lower risk' MDS patients and compared the clinico-pathological features, overall survival and risk of progression and evolution to AML with respect to p53 IHC status.MethodsThis study enrolled 277 adult low risk MDS patients seen at King’s College Hospital between March 2009 and February 2016. All marrow samples were assessed for p53 expression (intensity and proportion of cells) and scored using a Modified Quick Score (MQS). MQS ≥2 was chosen to define p53-positive staining. The median follow-up was 18.7 months [95% confidence interval (CI), 16.5-21.0 months].ResultsAccording to WHO categories, the predominant group was RCMD (78%). Patients were either IPSS low (n=134, 48%) or int-1 (n=91, 33%). We included 52 (19%) pts for whom the IPSS category was not available, due to the lack or failed cytogenetic data, but these were low-risk WHO categories with no excess of blasts. IPSS-R categories were very low 103 (37%), low 77 (28%), intermediate 38 (14%) and high risk 7 (3%). Overall, 19 (7%) patients progressed to AML and 41 (15%) patients died of which 25 (61%) were disease-related deaths.Of the 277 patients, 148 (53%) showed p53 protein expression with MQS ≥ 2 and were considered "p53-positive" patients (p53+). The p53 stain intensity was negative in 125 (45%), weak in 84 (30%), moderate in 40 (14%) and strong in 28 (11%). Seventy six (28%) patients had >5% p53 staining cells.p53 expression correlated with a higher age at diagnosis (median age 64 vs. 60 years, p=0.01). lower haemoglobin levels (9.8 vs 11 g/dL, p=0.002), but a higher platelets count (139 vs 99 x109/L, p =.003) (Table 1). This significance persisted even on exclusion of patients with 5q- and MDS/MPN. P53+ did not correlate with any cytogenetic risk group or degree of fibrosis.Sequencing data was available in 121 patients with 63 harbouring somatic mutations. Among these patients, only 3 showed the TP53 gene mutation; all of them were classified as positive when assessed for p53 stain (MQS≥2), showing that the nuclear staining reflects underlying TP53 mutations in such cases. Additionally, presence of SF3B1 mutation correlated strongly with p53+ (p=0.02).Patients with p53+ had a significantly shorter OS compared with those with p53- (figure 1a) with 2yr and 4yr survival probability of 87% &68% vs 95% & 90% , respectively (p=0.03 ).Analysis of progression-free survival (progression to AML, cytogenetic evolution, increase of blasts or fibrosis) showed that patients with p53 expression had a significantly shorter PFS compared to patients without p53 expression (Figure 1b): 2yr & 4yr PFS 70% & 54% vs 85% &75% respectively, p=0.01). Multivariate analysis confirmed the independent prognostic value of p53 expression for PFS (p=0.013, HR 2.6, 95% CI 1.2-5.2).ConclusionIn conclusion, our study showed that p53 overexpression by IHC, represented as MQS cutoff ≥ 2, correlates with features of poor prognosis, such as more advanced age at diagnosis, lower haemoglobin levels and shorter overall survival, adversely impacting progression-free survival.p53 IHC in MDS patients may represent an important, easily available, cheap and applicable prognostic tool and it should be considered for auxiliary analysis when determining the therapeutic options for a patient. The p53+ predicts poor outcome, especially disease progression, independent of the TP53 mutation status, indicating an alternate yet undefined pathway for p53 overexpression. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Why Patients with 5q- Syndrome Have Thrombocythemia?

Introduction. Fli1 (Friend leukemia virus integration 1) together with other transcription factors induces the megakaryocytic differentiation of MEP (megakarycytic and erythroid progenitor). Refractory anemia and thrombocythemia is typical for 5q- syndrome. We found increased mRNA level of Fli1 in mononuclear bone marrow cells of 5q- syndrome patients in comparison with healthy controls (Neuwirtova et al., Ann Hematol 2013). The reason of the elevated Fli1 in 5q- syndrome is haploinsufficiency of microRNA-145, which targets Fli1 mRNA (Kumar et al., Blood 2011). Due to haploinsufficiency of RPS14 in 5q- syndrome non-consumed ribosomal proteins cause ribosomal stress and inactivate HDM2 in erythroblasts. E3 ubiquitin ligase HDM2 regulates p53 level by p53 degradation in proteasome. Inactivated HDM2 in erythroid precursors of 5q- syndrome leads to apoptosis of erythroblasts and to anemia. Why ribosomal stress does not cause thrombocytopenia and ineffective megakaryopoiesis as well? Our previous results support significant role of Fli1 in this process. Fli1 binds to promoter of the HDM2 gene and increases its transcription (Truong et al., Oncogene 2005). The increased activity of HDM2 in megakaryocytes inspite of ribosomal stress maintains p53 regulation and its degradation in proteasome. Megakaryopoiesis remains effective. Why it is not the case in erythroid precursors? To answer this question it was necessary to detect Fli1 as the protein and to determine in which cells Fli1 is present.Material and Methods. Twenty-three control representative bone marrow trephine biopsies of patients from controls (8 negative staging biopsies in lymphoma) and of patients with various hematological diagnoses (7 MDS with normal chromosome 5, 4 MPN, 3 AML and 1 RARS-T) and from 15 patients with 5q- syndrome were examined. In 13 patients with 5q- syndrome, samples taken before and 6 months after lenalidomide (Revlimid) therapy were available. The expression of Fli1 protein was investigated by immunohistochemistry (IHC). Expression of Fli1 on erythroid precursors was studied by double staining IHC procedure utilizing antibodies against Fli1 and either glycophorin A or E-cadherin known as reliable markers for erythroid precursors.Results. Nuclear expression of Fli1 was demonstrated in normal as well as in dysplastic megakaryocytes, in most cells of granulocytic series and lymphocytes. No staining for Fli1 was seen in erythroblasts and proerythroblasts visualized by expression of either glycophorin A or E-cadherin both in 5q- syndrome and controls. There were no significant differences in Fli1 expression between samples taken before and after lenalidomide treatment.The used IHC technique does not permit quantitative analysis of Fli1 protein levels. This fact could explain why we did not find any difference in Fli1 protein labeling in megakaryocytes before and after lenalidomide treatment while Fli1 mRNA level was decreased in majority of 5q- syndrome patients after six months of this therapy.Conclusion. Fli1 expression was found in normal as well as in dysplastic megakaryocytes. However, no Fli1 positivity was found in erythroid precursors in both 5q- syndrome and controls. Negativity of Fli1 expression in erythroid precursors in 5q- syndrome support our hypothesis of protective role of Fli1 against apoptosis under ribosomal stress in megakaryocytes in contrast to erythroblasts lacking Fli1. This protective role of Fli1 in megakaryocytes consists in Fli1 potentiation of expression of the E3 ubiquitine ligase HDM2 (Truong et al., Oncogene 2005). The presence of increased Fli1 in megakaryocytes helps to explain effective megakaryopoiesis in 5q- syndrome and is the answer to the question in the title of our abstract.Supported by Ministry of Health, Czech Republic-conceptual development of research organization Institute of Hematology and Blood Transfusion 00023736, RVO-VFN64165 and PRVOUK P-27/LF1/2. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2457: Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma

Abstract Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia and congenital anomalies, notably skeletal defects. DBA is known in the majority of cases to be caused by haploinsufficiency of one of the small or large ribosomal protein (rp) subunits. In addition, there is a cancer predisposition in DBA. Indeed, somatic and germline rp mutations have been associated with a number of malignancies. In DBA patients one of the common malignancies is osteogenic sarcoma (OS). It is proposed that selective pressure caused by faulty translation may lead to clonal expansion of cells with acquired interdicting mutations leading to cancer. Purpose: To quantitate the incidence of cancer in patients with DBA and to examine osteoblast formation in vitro using a mouse embryonic stem (mES) cell model haploinsufficient for ribosomal protein Rps19 to identify putative bone defects that may promote tumorigenesis. Methods: The DBA Registry of North America (DBAR), the largest established patient cohort with prospective follow-up since 1991, was interrogated to ascertain the incidence of cancer in DBA. For in vitro studies, mES cells were differentiated towards osteoblasts using 1,25-dihydroxy-vitamin D3, dexamethasone, ascorbic acid and β-glycerophosphate for 10 days. qRT-PCR and histologic stains (alizarin red, alcian blue and alkaline phosphatase) were used to assess osteoblast production in wild type and Rps19+/- cultures. Results: The DBAR reported the first quantitative assessment of cancer incidence in DBA. The significantly elevated observed to expected ratios were 287, 36, 33 and 28 respectively, for myelodysplastic syndrome (MDS), colon carcinoma, OS and acute myeloid leukemia (AML). As of Nov 2015 (N = 751), with a median patient age of 20 years, 11 patients with gastrointestinal carcinoma, 5 with OS, 2 with AML and 8 with MDS are reported to the DBAR. The incidence of gastrointestinal carcinoma and OS is highly significant in this young population. We elected to study OS due to the skeletal defects and poor growth encountered in DBA patients. Using mES cells induced to differentiation towards osteoblasts, we observed reduced mineralization and therefore, bone formation, by alizarin red staining in Rps19+/- cells. Furthermore, rp haplo-insufficient cultures displayed a significant increase in alcian blue staining, a marker for chondrogenesis; these results suggest that regulation of osteochondrogenic potential was altered. In support of this phenotype, qRT-PCR analyses revealed that Rps19 haploinsufficiency induced abnormal expression of the master transcription factors and p53 involved in osteoblast and chondrocyte development. Conclusions: The most common cancers in DBA are AML, OS and adenocarcinoma of the colon. With regards to OS, we are now creating a conditional Rps19 CRISPR murine model, in order to clarify the contribution of both rp and p53 in the etiology of OS. Citation Format: Lionel Blanc, Brian Dulmovits, Julien Papoin, Jessica Kang, Eva Atsidaftos, Jeffrey M. Lipton, Adrianna Vlachos. Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2457.

Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Cooperating Effect of Rps14, Csnk1a1 and miRNA145/miRNA146a Haploinsufficiency in the Activation of the Innate Immune System in Del(5q) MDS

Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We previously generated a mouse model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence.We now sought to determine the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We found significantly increased S100a8 in the erythroid progenitor populations affected by the differentiation block (RIII-RIV population) and in monocytes and macrophages of Rps14 haploinsufficient bone marrows, all representing cells of the erythroblastic niche. Recombinant S100A8 was sufficient to impair erythropoiesis in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression using CRISPR/Cas-mediated gene inactivation in primary mouse Rps14 haploinsufficient HSPC.We validated the association between induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. To examine whether ribosomal haploinsufficiency also leads to activation of S100A8 in patients with del(5q) MDS, we measured S100A8 expression using immunofluorescence in bone marrow biopsies from MDS patients with and without del(5q). In del(5q) MDS, the frequency of S100A8-positive cells was associated with disease severity, as reflected by transfusion burden.RPS14, CSNK1A1 and miR-145 are universally co-deleted in the 5q- syndrome and each represent different clinical features of del(5q) MDS in murine models. Haploinsufficiency of miR-145 or -146a also induces inappropriate activation of innate immune signaling. To analyze the combinatorial effect of haploinsufficiency Rps14, Csnk1a1 and miRNA-145, we transduced hematopoietic stem and progenitor cells (HSPC) from compound haploinsufficient Rps14 and Csnk1a1 mice and stably knocked down both miR-145/miR-146a by retrovirus-mediated overexpression of respective target sequences. Compound haploinsufficiency of Rps14, Csnk1a1 and miR-145/146a led to a progressive anemia comparable to Rps14 haploinsufficiency with splenomegaly and an erythroid differentiation defect at the RIII/RIV population, indicating that the anemia is mainly driven by Rps14 haploinsufficiency. Bone marrow histology demonstrated the typical 5q-phenoytpe of megakaryocytes, in line with significant thrombocytosis. At 10 months of age, hematopoietic stem and progenitor cells were significantly increased (lineagelow ckit+ Sca1+; LSK), in particular multipotent progenitor cells (MPPs; lineagelow ckit+ Sca1+ CD48- CD150+) to significantly higher extents than in solely Rps14 or Csnk1a1 haploinsufficient cells.We next asked if compound haploinsufficiency of the three 5q-genes has combinatorial or synergistic effects on S100a8 expression. Compound haploinsufficiency of Csnk1a1, Rps14 and miR-145/146a induced the highest expression of S100a8 in monocytes, while haploinsufficiency of Rps14 alone induced the highest expression of S100a8 in the RIII erythroid population, suggesting that cell-type specific induction mediates the phenotype.Our data indicate an unexpected link between haploinsufficiency for a ribosomal gene, Rps14, activation of S100A8, and inhibition of erythropoiesis. We demonstrate that compound haploinsufficiency for Csnk1a1 and miR145/146a with Rps14 haploinsuffciency increases the expression of S100a8, mainly in monocytes, and recapitulates the phenotype of del(5q) MDS by cooperating, cell-type specific effects. DisclosuresPlatzbecker:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Boehringer: Research Funding.

HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

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. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiology of Macrocytic Anemia in Diamond Blackfan Anemia and Del(5q) Myelodysplastic Syndrome

Twenty-five percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of RPS19 and ~30% from haploinsufficiencies of other ribosomal proteins. The macrocytic anemia of del(5q) MDS results from RPS14 haploinsufficiency. When tested, these mutations disrupt ribosome assembly, yet how this causes macrocytic anemia remains uncertain. The dominant hypothesis is that P53 pathway activation leads to cell apoptosis, however, why erythropoiesis is severely impacted, while other hematopoietic as well as non-hematopoietic cells function appropriately, is difficult to explain. These observations led us to predict that DBA and del(5q) MDS share a pathogenesis that reflects the rapid kinetics of red cell and hemoglobin production, and specifically that poor ribosome assembly leads to an inability to quickly translate mRNA to protein, delayed globin synthesis in early erythroid precursors, and excess heme. Since free heme is toxic, cell death ensues. To explore this, we studied marrow from DBA (n=3) and del(5q) MDS (n=6) patients in cultures that optimize erythroid differentiation and showed that (a) cell numbers significantly decrease between stages I (lin-CD71+CD36-GlyA-)(BFU-E/early CFU-E) and II, (lin-CD71+CD36+GlyA-)(CFU-E/early proerythroblasts; Li et al. Blood 2014;124:3636); and (b) the cells that survive this insult differentiate normally to stages III (lin-CD36+GlyA+) and IV (lin-CD36-GlyA+). Using marrow from the index DBA patient, a 28-year old transfusion-dependent female with inherited RPS19 haploinsufficiency, and (as available) marrow from other DBA and del(5q) MDS patients (n=2-7 patients per study), we demonstrated that heme synthesis (assessed by ALAS2mRNA and protein) initiates normally while globin production (α- and β-globin mRNA and protein) is slowed, and that stage I and II cells have excess heme. Next, to discern the cell-specific characteristics that allow a DBA or del(5q) MDS cell to survive and fully mature, we compared those cells able to advance to stages III and IV in patient and concurrent control cultures. Stage III-IV GlyA+ DBA cells expressed 2.4-fold less ALAS2 mRNAand 2-fold more FLVCR mRNA than GlyA+ control cells (p values<0.05). Since the levels of ALAS2 and FLVCR mRNA were similar in stage I DBA and control cells, the time when heme synthesis initiates, these data imply that DBA cells capable of down-regulating their heme synthesis (have less ALAS2) or content (have moreFLVCR and increased heme export) preferentially survive. Data from del(5q) MDS patients are similar - cells progressing to stages III and IV have 2.9-fold less ALAS2 mRNA (p<0.05) and 9.4-fold higher FLVCR mRNA (p<0.01) than control cells. That heme synthesis proceeds normally, while globin production initiates slowly, was confirmed by sequential sampling of marrow cultures at days 3, 7, 10, 13, and 17; and as expected, DBA and del(5q) MDS cells at day 7 had increased reactive oxygen species (ROS). Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS cultures by 68-95% (p values<0.05), while the erythroid cell output of concurrent control cultures decreased by 4-13%. Cell death appears to result both from apoptosis (not shown) and ferroptosis, a ROS-induced process previously characterized in kidney epithelial cells and cancer cell lines (Dixon et al. Cell 2012;149:1060). Recent data showing P53 sensitizes cells to ferroptosis by impeding the transcription of SCL7A11, a cysteine-glutamate antiporter, leading to less cysteine uptake, less GSH synthesis, less GPX4, and less antioxidants (Jiang et al. Nature 2015;520:57) link our observations to work implicating P53 activation in DBA and del(5q) MDS anemia. In support of this connection, DBA marrow cells at culture day 10 have 57% less SLC7A11 mRNA than control cells in preliminary experiments. Together, our studies demonstrate that erythropoiesis fails when heme exceeds globin. The data also suggest that decreasing heme synthesis, either directly or by chelating iron, may improve these anemias. They further argue that lenalidomide (destabilizes P53) and therapies decreasing heme should be synergistic. Since diverse mutations could impede brisk protein transcription or translation, and hence brisk globin production, heme toxicity might also contribute to anemia in MDS patients without del(5q). DisclosuresNo relevant conflicts of interest to declare.

Reduced Protein Synthesis and p53 Activation in Late-Stage Erythroblasts Mediate the Erythroid Differentiation Defect in Mice with Ribosomal Protein S14 Haploinsufficiency

The 5q-syndrome is a subtype of myelodysplastic syndrome (MDS) with a defined clinical phenotype associated with heterozygous deletion of Chromosome 5q. The RPS14 gene was identified as a critical gene for the erythroid phenotype of the 5q- syndrome using an RNA interference screen. We generated a murine model for conditional, heterozygous inactivation of Rps14 in the bone marrow to investigate the biological basis of del(5q) MDS.To explore the role of Rps14 on hematopoietic stem cell (HSC) function and erythroid differentiation, we generated a mouse model in which Rps14 exons 2-4 are flanked by loxP sites. Following crosses to Mx1Cre transgenic mice, we induced Rps14 excision in hematopoietic cells by poly(I:C). Two weeks after induction of the gene excision, mice developed significantly reduced hemoglobin and red blood cell counts with a significantly higher MCV compared to Mx1Cre+ controls. Bone marrow analysis confirmed an erythroid differentiation defect specifically at the transition from the CD71+Ter119+ (RII, basophilic erythroblasts/early polychromatophilic erythroblasts) to the CD71intermediate/lowTer119+ (RIII/IV, poly/orthochromatophilic erythroblasts, enucleated erythrocytes) population, accompanied by significant up-regulation of p21 in the RIII population. Histopathology of spleens demonstrated compensatory extramedullar erythropoiesis. The bone marrow was normocellular with a significant increase in hypolobulated megakaryocytes correlating with high platelet counts and dysplastic platelets in the peripheral blood, reflecting the pathognomonic megakaryocyte phenotype in del(5q) MDS. At 50 weeks of age, Rps14-/+Mx1Cre+ developed a more severe macrocytic anemia in the peripheral blood.We thus tested whether a more severe erythroid phenotype could be induced by treatment with the hemolytic agent phenylhydrazine (PH; 2x single dose 25mg/kg body weight, s.c.). In response to PH treatment, Rps14 haploinsufficient mice developed a more severe anemia than control mice, had a delayed reticulocyte response, and a differentiation defect again specifically at the transition from RII to RIII/IV accompanied by a compensatory increase in the RI (CD71+Ter119-) pro-erythroblast/early basophilic population. Megakaryocyte-erythrocyte progenitors were increased, and pre-CFU-E and CFU-E numbers were normal. To further elucidate the mechanisms by which haploinsufficiency of Rps14 causes ineffective erythropoiesis, we analyzed cell cycle, p53 and apoptosis. We found a significant induction of p53 specifically in the RI population and a dramatic increase of apoptotic cells in RIII, suggesting that increased apoptosis accounts for the erythroid failure. We therefore tested if genetic inactivation of p53 rescues the erythroid phenotype. In Rps14-/+p53-/+Mx1Cre+ mice the erythroid differentiation defect was restored and mice had a comparable response to PH as Mx1Cre+ controls. In PH dose escalation experiments (35mg/kg), Rps14 haploinsufficient mice died from the severe anemia and delayed reticulocyte response, while compound p53 loss rescued the erythroid failure. Accordingly, forced erythroid differentiation in vitro was significantly impaired in Rps14 haploinsufficient hematopoietic stem and progenitor cells.We measured protein synthesis by O-propargyl (OP)-puromycin incorporation. Rps14 haploinsufficient cells had a significantly reduced protein synthesis in CD71low cells (late erythroblasts) that could not be fully restored by p53 inactivation although the protein synthesis in p53 heterozygote cells alone was significantly higher. The decreased protein synthesis in Rps14-/+p53-/-Mx1Cre+ compound cells might also account for the decreased stem cell expansion in repopulation assays compared to cells with only p53 loss.Our data provide in vivo evidence that Rps14 haploinsufficiency contributes to the erythroid differentiation defect in del(5q) MDS by reduced protein synthesis and p53 induction in late-stage erythroblasts. This murine model recapitulates the erythroid und megakaryocytic phenotype of the 5q-sydrome and provides a model for understanding the underlying mechanisms in the pathogenesis of ribosomal-mediated erythroid failure in del(5q) MDS. DisclosuresNo relevant conflicts of interest to declare.