Mutations In Ribosomal Proteins Research Articles

Calmodulin Inhibition Rescues DBA Models with Ribosomal Protein Deficiency through Reduction of RSK Signaling

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations are thought to cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a ribosomal gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we tested them in human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19, the CaM inhibitor trifluoperazine (TFP) relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels and reduced p53 activity in the bone marrow. Of note, the effect of TFP was specific to RP deficiency and had no effect on erythroid differentiation or p53 activity in WT cells or mice. This prompted us to hypothesize that TFP is selectively blocking signaling in the RP deficient state, possibly through inhibition of CaM-dependent kinases. In vitro kinase profiling of over 100 kinases revealed that TFP and other positive hits from our screen inhibited the activity of p70 ribosomal S6 kinase (p70S6K) and multiple members of p90 ribosomal S6 kinase (RSK) family. RSKs are highly conserved serine/threonine kinases that regulate cell growth, migration, and survival. They can activate mTOR and directly phosphorylate RPS6. Strikingly, RSK protein levels are elevated in lysates from rps29-/- embryos and treatment with RSK or p70S6K inhibitors increased Hb in rps29-/- embryos in vivo, mimicking CaM inhibitors. RSK phosphorylation is also increased in human RPS19-deficient peripheral blood-derived CD34+ cells compared to WT cells. Treatment with TFP reduced RSK phosphorylation and decreased signaling downstream of RSK, including p70S6K, but only in the presence of RP deficiency. Similarly, TFP reduced p53 and phosphorylation of p53 at S392, but only in the presence of RP deficiency. An in vitro kinase assay determined that p70S6K directly phosphorylated p53 at S392 but not other commonly modified residues, S15 and S20. Mass spectrometry analysis of posttranslational modifications of p53 isolated from TFP treated cells revealed a significant reduction of peptides that contain phosphorylated S392. We hypothesized that TFP inhibited phosphorylation of S392. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a negative control transactivation domain mutant, p53-S15D, resulted in a 4-fold reduction in p21 mRNA levels, while cells containing p53-S392D had no reduction in p21 mRNA in response to TFP. In conclusion, we have shown that RP deficiency increases RSK phosphorylation and CaM inhibitors decrease signaling downstream of RSK, which leads to a reduction of p53 activity and rescues the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP for 2017. DisclosuresZon:Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Two Novel Mutations in Ribosomal Proteins in the Czech National Diamond-Blackfan Anemia Registry

The Czech National Diamond-Blackfan Anemia (DBA) Registry currently consists of 51 patients; in 36 of them ribosomal protein (RP) mutations have previously been identified. Targeted sequencing of genes encoding RP, exomes and transcriptomic sequencing and/or array CGH were performed in newly diagnosed DBA patients and in patients without a causative mutation. Two novel mutations in RPS7 and RPL11 were discovered in 5 individuals from two families.Aheterozygous mutation g[3580153G>T] in exon 6 of a gene coding for RPS7, leading to V134F substitution, was found in an 18-year-old female who was transfusion dependent during infancy, responded to steroids and is currently in remission. The identical mutation was detected in the patient’s mother and older sister, who both are asymptomatic with borderline hemoglobin (Hb) levels, mild macrocytosis with no active treatment. A functional study using cellular models revealed that RPS7-deficient MRC-5 fibroblasts transiently expressing RPS7-V134F exhibit altered protein synthesis, defective ribosomal RNA processing, increased nucleolar and ribosomal stress. This was evidenced by overexpression and translocation of p53 protein to the nucleus in RPS7-V134F but not RPS7 wild type transfected cells. These data are consistent with previously reported cellular phenotypes in DBA. In addition, increased levels of erythrocyte adenosine deaminase (e-ADA) were detected in all three family members with V134F RPS7 mutation (patient: 4.2±0.8 IU/g Hb; mother: 3.5±0.2 IU/g Hb; sister: 4.6±0.3 IU/g Hb; reference range: 0.8-2.5 IU/g Hb). Recently it was shown that ribosomal insufficiency increases oxidative stress in red blood cells (RBC) of DBA patients. In this family, increased levels of reactive oxygen species (ROS) were detected by flow cytometry only in patient’s RBC, but not in RBC of her asymptomatic mother and sister. Consistently, only the patient showed increased anti-oxidative defense parameters compared to the controls and her mother and sister. Markedly elevated levels of reduced glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD), two essential antioxidants, were detected in patient’s RBC (GSH: 4356±44 mM; G6PD: 10.3±0.6 IU/g Hb) compared to controls (GSH: 2434±454 mM; G6PD: 5.4-7.0 IU/g Hb) and patient’s mother (GSH: 2125 mM; G6PD: 5.3±0.2 IU/g Hb) and sister (GSH: 2450±35 mM; G6PD: 6.5±0.1 IU/g Hb). Concomitant increase (approximately 2.5 times) in the activity of hexokinase (HK) and pyruvate kinase (PK) and in the levels of ATP in patient’s RBC indicates augmented energy metabolism to sustain the integrity of RBC. Flow cytometry of Annexin V binding revealed increased exposure of phosphatidylserines on RBC membrane of the patient compared to controls and her mother and sister suggesting that the tendency towards redox balance restoration and membrane integrity does not completely prevent an enhanced recognition and destruction of patient’s RBC by reticuloendothelial macrophages.The second c.281T>G point mutation in RPL11 was found in a 34-year-old woman with unspecified macrocytic anemia, who developed diffuse large B-cell lymphoma (DLBCL). She was transfusion dependent in infancy, responded to steroids and is currently in the anemia remission and second remission of DLBCL. Identical mutation was detected in her 56-year-old mother, followed for macrocytic anemia and gammopathy.In 10 DBA cases without the causative mutation an array CGH was performed, but no large deletions in RPS19, RPS17, RPS26, RPL5 and RPL11 genes were detected.In summary, we present the first case of a missense mutation in RPS7 as the cause of DBA; all to date published RPS7 mutations clinically associated with DBA were located in splice sites. Similarly, the co-occurrence of DBA and DLBCL has not been previously published. We also emphasize that the phenotype of family members with the same mutations could be different, including silent carriers. All family members should therefore be examined even if asymptomatic. Testing of eADA levels could serve as a sensitive screening method in apparently unaffected family members. Finally we suppose that altered RBC metabolism may affect the lifespan of RBC in DBA and thus contribute to the worsening of the blood condition, especially during infections. The analyses in a large cohort of DBA patients are ongoing.Grant Support: AZV16-32105A, GA15-13732S, MH CZ-DRO FNOL 00098892, NPU LO 1304, IGA UP LF_2016_014. DisclosuresNo relevant conflicts of interest to declare.

Ribosomal Protein L22 Loss Predisposes Stem Cells to Transformation By Altering Metabolism

While mutations in ribosomal proteins (RP) have long been associated with increased cancer risk, the molecular basis for this association remains unclear. The prevailing view is that mutations in RP increase cancer risk either by impairing the biogenesis or function of the ribosome. Here, we report that loss of the RP, Rpl22, is frequently observed in human myelodysplastic syndrome (MDS) and is associated with reduced survival in acute myelogenous leukemia (AML). The link between Rpl22 loss and aggressiveness of myeloid disease is causal, since it is replicated in mice lacking Rpl22. Importantly, the development of an MDS-like syndrome in Rpl22-deficient mice and the accelerated development of myeloid leukemia are not caused by reduced protein synthesis in hematopoietic stem cells (HSC). Instead, whole transcriptome analysis of HSC reveals that Rpl22 loss alters the expression of genes controlling cell metabolism and results in an increase in fatty acid oxidation. Moreover, the increased fatty acid oxidation is required for the increased self-renewal and reduced differentiation displayed by Rpl22-deficient HSC, as well as the survival of Rpl22-deficient myeloid leukemia cells. Together, these findings indicate that RP are capable of suppressing transformation potential in ways that do not involve effects on global protein synthesis. Indeed, Rpl22 suppresses transformation by suppressing lipid oxidation, suggesting that MDS and AML patients with reduced Rpl22 expression might benefit from pharmacologic interventions targeting lipid oxidation. DisclosuresNo relevant conflicts of interest to declare.

Successful Treatment of a Diamond-Blackfan Anemia (DBA) Patient with Eltrombopag

▪DBA is a rare bone marrow failure syndrome clinically defined by onset of hypoproliferative anemia in early childhood accompanied by a grossly normocellular bone marrow. With age, a significant fraction of patients progress to pancytopenia and a hypocellular bone marrow, with some increased risk of progression to leukemia. Autosomal dominant or sporadic heterozygous mutations in genes encoding for ribosomal subunit proteins have been etiologically linked to DBA. How loss of function mutations in ribosomal proteins results in selective loss of erythroid progenitors with variable severity is incompletely understood. Chronic transfusion therapy and corticosteroid treatment can alleviate anemia, but both cause severe long term toxicities. Hematopoietic stem cell transplantation, when available, is the only definitive treatment modality for the hematological manifestations, and the only known treatment for DBA patients once pancytopenia develops.Here we report on a 28 year old female patient diagnosed with DBA at age of one month based on clinical criteria. Recent mutational analysis identified a novel mutation in intron 4(c.356+3>C) of the RPS19 gene affecting a known splice donor site that has previously reported in DBA patients. She did not respond to corticosteroids and was maintained on transfusions. She failed various experimental therapies including danazol, hematopoietic growth factors including IL-3 and GM-CSF, and anti-thymocyte globulin/cyclosporine without sustained clinical response. After treatment with daclizumab in 2006 her transfusion requirements decreased for about 4 years. In 2012 she presented with progressive pancytopenia and hypocellular bone marrow without overt dysplasia, and normal cytogenetic analysis. She required red cell transfusions. The patient was enrolled in a clinical research protocol investigating the efficiency and safety of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or those with bone marrow failure with unilineage cytopenia (ClinicalTrials.gov: NCT01328587). The study is designed as a non-randomized, phase II, dose modification study with the primary endpoint hematological response at 16 weeks. EPAG was administered at 50mg daily and escalated every 2 weeks by 25 mg to the maximal dose of 300mg daily. At response assessment the patient’s hemoglobin (Hgb) improved from 7.0g/dl to 8.9g/dl and platelets from 112K/ul to 171k/ul. She required no further transfusions, and was deemed a responder. EPAG was subsequently continued at the same dose level for an additional 6 months when the peripheral cells counts peaked at Hgb 11.9g/dl, platelets 251K/ul, ANC 3.19 K/ul, and EPAG was discontinued for protocol-defined robust response. After discontinuation of EPAG the patient’s hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated at 300mg per protocol. Peripheral blood cell counts rapidly improved and the EPAG dose was slowly tapered. The patient is currently on EPAG 50mg daily with normal blood cell counts. We have not observed any EPAG related toxicities in our patient, and repeated cytogenetic studies on bone marrow documented a normal karyotype.To the best of our knowledge this is the first report documenting EPAG efficacy in a DBA patient. Trautman et al. (2012) previously reported a DBA patient that failed treatment with low dose EPAG after six months. Our observation warrants further systematic investigation of EPAG treatment for DBA patients within a clinical research study setting. [Display omitted] DisclosuresWinkler:GSK/Novartis: Research Funding. Townsley:Novartis: Research Funding. Desmond:Novartis: Honoraria. Dumitriu:GSK/Novartis: Research Funding. Grasmeder:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding.

Small Molecules Targeting the TLR Pathway Alleviate Anemia in a Zebrafish Model of MDS with Del(5q)

MDS with isolated del(5q) (MDS5q) is a defined entity in the WHO classification for myeloid malignanies owing to its unique morphological and biological phenotype and striking responseslenalidamide. Nonetheless approximately 1/3 of patients with MDS5q fail to achieve tansfusion independance and dose limiting toxicity is common. Furthermore 3-year survival remains poor at around 50%, and this disease remains incurable without bone marrow transplantation. Thus there is an unmet need to identify additional therapies for MDS5q.Ribosomal protein gene 14 (RPS14), located on 5q, has been shown to be the likely genetic determinant of anemia in patients with MDS5q. Importantly, loss of 5q has been shown to be an initiating event in the development of MDS in these patients. We have previously shown that Rps14-deficient zebrafish exhibit a robust anemia during development permitting in vivo assessment of an MDS-like phenotype in these animals. We utilized Rps14 deficient embryos in drug screen to identify compounds that alleviate the anemia in this model. Rps14 was knocked down using antisense oligonucleotides directed against the splice donor site of the 2nd exon (first coding exon). At1 day post fertilization (dpf) embryos were plated in a 96 well plate, 2-3 embryos per well and exposed to compounds from the Spectrum library of known bioactive drugs at 5µM and 20µM in duplicate plates. L-Leucine was utilized as a positive control. At 4dpf embryos were assessed visually in vivo by 2 independent observers for anemia and developmental effects. They were then stained with o-dianisidine to assesshemoglobinization. Hits were defined as compounds that scored in at least 2 wells with a minimum of one from each observer. Our primary hit was validated with fresh compound and dose titration (Figure 1A,B). This compound targets TLR signaling which has recently been implicated in the pathogenesis of MDS5q in murine models. We went on to utilize an additional drug targeting the same receptor and observed similar improvements inhemoglobinization, validating this pathway as a putative therapeutic route for MDS5q.To further confirm our findings we generated a stable mutant line carrying a heritable mutation in zebrafish Rps14 using genome editing with Transcription activator-like effector nuclease (TALENS). These mutants have an 11bp complex indelin Exon 2 (Rps14E8fs). Rps14E8fs/E8fs embryos show profound developmental anomalies including a marked anemia in keeping with other ribosomal protein mutants and are embryonic lethal by 5dpf. The majority of Rps14E8fs/+ heterozygotes were indistinguishable from their WT siblings in early development and no overt abnormality in hemoglobinization could be detected by o-dianisidine staining. By contrast heterozygous adults were smaller than their siblings, weighed less and flow cytometry of whole kidney marrow demonstrated pancytopenia. As expected the features were most marked in the erythroid lineage highlighted using Tg(globin:eGFP);Rps14E8fs/+ animals. Rps14E8fs/+ adults also showed reduced hemoglobin levels in their peripheral blood although this was modest compared to the marrow features (Figure 1C). As all lineages appeared to be affected in Rps14E8fs/+ we went on to address myeloid development during development. Using Sudan Black (SB) staining Rps14E8fs/E8fs and Rps14E8fs/+ showed reduced numbers of mature myeloid cells from 2dpf.To further assess the effects of Rps14E8fs/+ in embryonic erythropoiesis we used Tg(Gata1:dsRed); Rps14E8fs/+ embryos and subjected them to flow cytometry. Rps14E8fs/+ at 3dpf showed reduced numbers of dsRed expressing erythroid cells compared to their siblings. Increased numbers of dsRed positive cells were observed when embryos were treated with L-leucine. Finally we treated embryos with our hit compound. Rps14E8fs/+ embryos showed increased numbers of dsRed expressing cells compared to Rps14+/+ siblings confirming our findings from Rps14morphants.In summary we have identified a putative new therapeutic compound that can increase erythroid cell numbers using an in vivo model of MDS5q anemia. We also describe the features of heterozygous Rps14E8fs/+ mutants that recapitulate features of MDS during developmental and adult hematopoiesis providing a novel platform for drug screen and second hit analyses in future studies. Our ongoing work is validating these compounds in patient cells from individuals with MDS5q. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Erythrocyte Adenosine Deaminase Levels Are Elevated in Classic Diamond Blackfan Anemia but Not in the 5q- Syndrome

Diamond Blackfan anemia (DBA) is congenital bone marrow failure syndrome characterized by red cell aplasia. It was the first disease linked to ribosomal dysfunction with 70% of patients having haploinsufficiency of a ribosomal protein (RP) gene. More recently, several patients with DBA have been shown to have mutations in GATA-1. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is also characterized by a severe anemia that is caused by heterozygous loss of RPS14 on chromosome 5q. The reciprocal relationship between these two diseases, which are now collectively known as the ribosomopathies, has spurred interest in how we may be able to better understand the pathophysiology of these disorders to broaden therapeutic options, and to improve diagnosis.Erythrocyte adenosine deaminase (eADA) levels have been used for the last three decades in the diagnosis of DBA based on the finding in 1983 that eADA enzyme levels are significantly elevated in patients with DBA. It is now known that more than 75% of DBA patients have elevated eADA levels. The goal of the present study is to measure the levels of eADA in patients with the 5q- syndrome and determine whether they are elevated implying that eADA may be specific to ribosomal haploinsufficiency in general or normal suggesting that elevated eADA may be specific to DBA.Under a Stanford Univeristy approved IRB protocol, we measured eADA levels in patients with the 5q- syndrome who were undergoing routine blood draws for clinical purposes. The patients with Diamond Blackfan anemia are either followed at our center in Stanford University or were previously referred to us for eADA testing with accompanying clinical information. Analysis was performed using SAS 9.3 (SAS Institute, Carey, NC). Continuous variables were presented as means and standard deviations (SD) and analyzed using 2-tailed T-Test.Complete results by mutation type are summarized in Tables 1 and 2.In a disorder with the marked genotypic and phenotypic variability such as Diamond Blackfan anemia (DBA), it is critical to have sensitive tests to aid with the diagnosis. Erythrocyte adenosine deaminase (eADA) levels have long been used as supportive diagnostic criteria. The test has been especially useful in distinguishing DBA from other causes of anemia and other inherited bone marrow failure syndromes. However, the reason for the elevation of eADA in DBA remains unknown.Our goal was to study eADA levels in the 5q- syndrome where there is an acquired ribosomal haploinsufficiency (from loss of RPS14 on chromosome 5q), which is in contrast to the congenital haploinsufficiency seen in DBA. Interestingly, we found that the eADA levels were normal in the 5q- syndrome suggesting that eADA is specific for DBA. We then studied eADA levels in patients with DBA that is not the results of a ribosomal protein (RP) mutation but instead due to mutations in GATA-1. Once again, we found that eADA levels were normal in this cohort suggesting that elevated eADA is specific feature of DBA due to RP mutations. It is interesting to note that patients with RPS19 mutations, who tend to have a milder clinical phenotype, and patients without an identified mutation had lower (but still elevated) levels of eADA when compared to other RP mutations, in particular RPL11. A larger number of patient samples in future studies would improve the statistical power to compare eADA levels between individual RP mutations.This is the first report to examine the value of eADA in non-classical DBA and in another disorder of ribosomal haploinsufficiency, namely del(5q) MDS. In summary, we found that an elevated eADA strongly suggests the diagnosis of classic DBA although a normal eADA does not exclude the diagnosis, particularly in the setting of GATA-1 mutations. There is no utility for using eADA in the diagnosis of the 5q- syndrome despite the connection of the disease with impaired ribosome function. The reason for elevated eADA in classic DBA remains to be defined. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modeling Diamond Blackfan Anemia and p53 Activation in the Zebrafish

Marrow dysplasias are pre-leukemic conditions that include ribosomopathies, a group of rare genetic diseases, or myelodysplasia preceded by clonal expansion in older adults with somatic mutations. To discover novel therapies for ribosomopathies, we performed two chemical suppressor screens using a ribosomal protein mutant zebrafish and human induced pluripotent stem cells (iPSCs) derived from ribosomopathy patients. In the zebrafish screen, we found calmodulin inhibitors rescued the anemia in zebrafish and also rescued the erythroid defect of the mouse (in vivo) and human (in vitro) models of marrow failure. The screen in RPS19 mutant human iPSCs identified a compound, SMER28, which enhanced erythropoiesis in anemia models through an autophagy-dependent mechanism. The compounds identified in our chemicals screens have provided new insight into the etiology of marrow failure, and calmodulin inhibitors will soon be studied in a clinical trial with adult Diamond-Blackfan anemia patients. To study the abnormal clonal hematopoiesis associated with myelodysplasia, we have generated a new system using the zebrafish in which single stem cells and their progeny express unique combinations of fluorescent proteins, allowing them to be tracked over time by their specific color. Mosaic mutagenesis induced by clustered regularly interspaced short palindromic repeats /cas9 and/or gene overexpression in this system led to expansion of specifically colored blood, modeling the clonal hematopoiesis of human myelodysplasia. Our studies should provide new insight into the ribosomopathies and myelodysplasia. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Mutation in the L3 Ribosomal Protein Could Be Associated with Risk of Selection of High-Level Linezolid-Resistant Staphylococcus epidermidis Strains.

Linezolid (LZD) has arisen as an alternative treatment in diabetic foot osteitis due to staphylococci. LZD resistance selection is difficult and involved various molecular mechanisms. As a better knowledge of those mechanisms could be beneficial for pathogenic strains' screening, we simulated in vitro the spontaneous mutagenesis process that leads to LZD-resistant strains from two Staphylococcus epidermidis strains responsible for monomicrobial diabetic foot osteitis. LZD high resistance was selected for both strains, with the same timeline of mutation appearance. Mutation in L3 protein (G152D) occurred first and quickly, but did not cause phenotypically detectable resistance or fitness cost. It was later followed by different 23S rRNA mutations (G2505A, G2447T), leading this time to detectable resistance (minimum inhibitory concentration [MIC] ≥8 mg/L). This phenomenon underlies the difficulty of resistance selection in coagulase-negative staphylococci (CoNS). This study is the first description of G2505A mutation in CoNS. Various phenotypical impacts were observed depending on strain and mutation: (i) fitness cost of G2505A and G2447T mutations; (ii) loss of erythromycin resistance concomitantly with L3 mutation selection; (iii) correlation between number of mutated rrl copies and LZD resistance level for G2447T. In conclusion, the risk of selection of high-level LZD-resistant S. epidermidis strains is weak, but does exist. It could probably appear in case of long-term treatment and be favored in the case of a pre-existing mutation in L3 ribosomal protein. Thus, broad screening conditions for pathogenic strains should probably be considered.

Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA-rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis.

Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development.

Role of ribosomal protein mutations in tumor development (Review).

Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research.

Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP. DisclosuresEbert:Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Novel Role for Rpl22 in Controlling Lymphoma Progression and Dissemination

Mutations in ribosomal proteins often cause bone marrow failure syndromes associated with increased risk for cancer development; however, in most cases, the basis by which they do so remains unclear. We have shown that Rpl22 is a haploinsufficient tumor suppressor in T acute lymphoblastic leukemia/lymphoma (T-ALL). Loss one Rpl22 allele accelerates T-cell lymphomagenesis in the murine Akt transgenic mouse model, by inducing the stem cell factor Lin28B in an NFkB-dependent manner. Here we employed two distinct murine T lymphoma models to uncover a new role for Rpl22 in lymphoma progression through effects on migration and angiogenesis. Compared with Rpl22+/+ mice, mice deficient for Rpl22 (Rpl22-/-) exhibited significantly increased thymic tumor size, which was associated with markedly enhanced angiogenesis through the activation of VEGF signaling. Unlike Rpl22 sufficient tumors, Rpl22-deficient lymphomas were retained in the thymus as a large mediastinal mass that did not disseminate to the periphery due to a defect in migration. The migration defect in Rpl22-/- tumors results from attenuation of S1P1 receptor expression through downregulation of the KLF2 transcriptional factor, which is responsible for its expression. Indeed, re-expression of S1P1 receptor in Rpl22 deficient tumor cells rescues the migration defect. Re-expression of Rpl22 into Rpl22 deficient cells rescues expression of both KLF2 and its target, S1P1 receptor, indicating that Rpl22 is regulating the KLF2/S1P1 receptor axis, rather than the changes in expression being an indirect consequence of transformation. Consistent with this idea, human T-ALL lines with in which Rpl22 is mutated, exhibit reduced expression of both KLF2 and S1P1 receptor, relative to that in T-ALL lines with intact RPL22 alleles. Collectively, we have defined a new role of Rpl22 in tumorigenesis through regulation of migration. DisclosuresNo relevant conflicts of interest to declare.

The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis.

DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

Transcriptome Analysis of Erythroid Cells Cultured from Diamond Blackfan Anemia Patients with Ribosomal and GATA1 Mutations Reveals Dysregulation of Inflammatory Response Genes

Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have identifiable heterozygous gene mutations or deletions in ribosomal protein genes. Additionally, mutations in GATA1, a key transcription factor in erythropoiesis, have been demonstrated in DBA patients (Sankaran VG et al. JCI. 122: 2439-43, 2012; Ludwig LS et al. Nat Med 20(7):748-53, 2014; Klar J et al. Br J Haem 166(6): 949-51, 2014). Despite our knowledge of the molecular pathology, the mechanism underlying the erythroid failure in DBA is not well understood, largely due to the inherent difficulties in studying primary erythroid cells from DBA patients. Furthermore, DBA patients with GATA1 mutations have not been well characterized and it remains unclear whether the pathogenic mechanisms of GATA1 DBA are similar to that of ribosomal DBA.We designed a two-step, 14 day in vitro culture system to generate erythroid cells from CD34+ stem/progenitor cells isolated from <10ml of peripheral blood (PB) collected from patients enrolled in the DBA Registry of North America. Patients with RPL5, RPS17(n=2), RPS24 and RPL35a, GATA1 mutations (n=2), and patients without identifiable mutations (n=3) were studied and compared to healthy control PB CD34+ cells. At day 14, we routinely obtain at least 10x fewer CD235+ erythroid cells (proerythroblasts and basophilic erythroblasts) in DBA cultures vs. controls (1x106 vs. 1x107 from 1x104 CD34+ cells). Further, a 2-7 day delay in the acquisition of CD235 was observed.Gene expression analyses was performed by Affymetrix GeneChip Human Gene ST Arrays and RNASeq to analyze protein coding and long non-coding RNA transcripts in cells from day 14 of erythroid cultures. Ingenuity pathway analysis (IPA) of the dysregulated genes between patients with ribosomal mutations, GATA1 mutations and controls revealed that many of the genes dysregulated in DBA were involved in multiple leukocyte migration and inflammatory signaling pathways, including the IL8, IL1R1, CXCR4, ICAM3, MPO, TNFSF10, and TLR4 genes with IL6, TNF, and lipopolysaccharide as top upstream regulators. Notably, the dysregulated genes in GATA1 patient cells largely overlapped that of the DBA patients with ribosomal mutations, including disruption of the leukocyte migration and inflammatory response genes. Patients with GATA1 and ribosomal protein mutations shared a number of dysregulated erythroid genes including AHSP, FAM132B, HEMGN, and TRIM10, however GATA1 patient cells showed GATA1 as the top upstream regulator and additionally showed dysregulation of heme biosynthesis pathway genes, including the ALAS2, FECH, CPOX, PPOX, and UROS.Few studies have looked at the pathogenesis of DBA in patients with GATA1 mutations. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of control erythroid cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In cells from the patients with the GATA1 mutation, only GATA1s protein was expressed and the level of GATA1s exceeded the combined level of both GATA1 isoforms in control cells. Erythroid cells from a patient with an RPL5 mutation showed GATA1 protein levels comparable to controls, indicating the DBA phenotype of the RPL5 patient is not due to altered translation of full length GATA1. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing.In summary, our transcriptome analyses have revealed novel insights into the molecular pathogenesis of ribosomal and GATA1-mutated DBA. Significant dysregulation of inflammatory gene pathways in DBA patients with both ribosomal protein and GATA1 mutations were observed, suggesting a shared pathological mechanism. Furthermore, the heme biosynthesis pathway was uniquely disrupted in patients with GATA1 mutations. Further investigating the inflammatory pathways in DBA may reveal novel targets for therapeutic development. DisclosuresNo relevant conflicts of interest to declare.

Disrupted Expression of the Splicing Regulators Tra2-Beta and SRSF1 and Subsequent Aberrant Splicing of FLVCR1 Transcript As an Underlying Mechanism for the Erythropoietic Failure Observed in Diamond-Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited anemia caused by disruption of erythroid progenitor cell development at the burst-forming and colony-forming units. Approximately 50% of DBA patients have additional non-hematopoietic abnormalities including heart defects and short stature. Sixteen genes encoding specific ribosomal proteins (RPs) and the transcription factor GATA1 have been found mutated in approximately 65% of DBA patients, with the RPS19 gene being mutated in 25% of patients. Because RPs are components of the ribosome that is critical for protein synthesis, the identification of RP mutations suggest a translation deficiency as a mechanism for erythroid progenitor failure. However, erythropoietic failure in DBA may arise by causes other non-translational functions of the DBA genes. We have previously reported that inhibiting the heme exporter protein FLVCR1 in human hematopoietic stem cells and progenitors (HSC/Ps), is sufficient to specifically block erythropoiesis, which mimics the hematological features observed in DBA. Moreover, we reported aberrant splicing of exons (E) 2 and 3 of the FLVCR1 transcript in DBA erythroid cells that resulted in disruption of FLVCR1 protein expression and function. We observed aberrant FLVCR1 splicing in erythroid cells from patients with RPS19 and RPL5 mutations and from typical DBA patients whose genetic background has not been known at that time; thus, suggesting FLVCR1 dysfunction as central for the DBA erythropoietic failure.In the present work, we obtained evidence suggesting that DBA pathogenesis is caused by abnormality in the cellular splicing machinery. We performed In silico analysis of the binding motifs of seven SR proteins on each of the FLVCR1 exons. We found binding motifs for TRA2-β, SRSF1 and 9G8 on E2 and/or E3, the two FLVCR1 exons that are skipped in DBA erythroid cells and in RPS19-disrupted K562 cells. We focused on expression analysis of TRA2B and SRSF1 in RPS19-reduced K562 cells. Expression of both TRA2B and SRSF1 mRNA were significantly disrupted in RPS19-disrupted K562 cells, which correlated with disrupted expression of their respective proteins. To test the specificity of disrupted SR splicing regulators and aberrant FLVCR1 splicing, we analyzed TRA2-β, and SRSF1 protein expression, and FLVCR1 aberrant splicing, in K562 cells disrupted in SBDS gene. The SBDS gene product is critical for ribosome biogenesis and the gene is mutated in 90% of patients with Shwachman-Diamond Syndrome. Interestingly, K562 cells down-regulated in SBDS showed normal levels of TRA2-β and SRSF1 expression, and no FLVCR1 aberrant splicing was observed. Importantly, erythroid differentiation potential of the DBA cell model can be rescued by expression of exogenous Tra2-β cDNA or FLVCR1 cDNA.Altogether our data suggests that mutations in the ribosome protein genes in DBA result in down-regulation of the splicing factors, TRA2-β and SRSF1, which impair normal FLVCR1 splicing and consequently erythropoiesis failure. DisclosuresNo relevant conflicts of interest to declare.

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

Plant-Specific Features of Ribosome Biogenesis.

The biogenesis of eukaryotic ribosomes is a fundamental process involving hundreds of ribosome biogenesis factors (RBFs) in three compartments of the cell, namely the nucleolus, nucleus, and cytoplasm. Many RBFs are involved in the processing of the primary ribosomal (r)RNA transcript, in which three of the four rRNAs are imbedded. While pre-rRNA processing is well described for yeast and mammals, a detailed processing scheme for plants is lacking. Here, we discuss the emerging scheme of pre-rRNA processing in Arabidopsis thaliana in comparison to other eukaryotes, with a focus on plant characteristics. In addition, we highlight the impact of the ribosome and its biogenesis on developmental processes because common phenotypes can be observed for ribosomal protein and RBF mutants.

Susceptibility trends including emergence of linezolid resistance among coagulase-negative staphylococci and meticillin-resistant Staphylococcus aureus from invasive infections

Multiresistance in staphylococci constitutes a major challenge for the antimicrobial chemotherapy of invasive infections such as bacteraemia or bone and joint infections (BJIs). A nationwide prospective study was performed to detect antimicrobial resistance trends among staphylococci causing invasive infections. Between October 2011 and February 2012, 367 meticillin-resistant Staphylococcus aureus (MRSA) and 695 coagulase-negative staphylococci (CoNS) were collected from 37 French hospitals, mainly from bacteraemia (59.9%) and osteoarticular infections (29.0%). Minimum inhibitory concentrations (MICs) were determined by broth microdilution, and specific screening and confirmation tests were performed to detect heterogeneous vancomycin-intermediate S. aureus (hVISA). Staphylococcal isolates exhibiting a linezolid MIC>4mg/L were further characterised to determinate their clonal relationships and the mechanism of resistance. MRSA exhibited additional resistances, including levofloxacin (82% associated resistance), gentamicin (13.6%), fusidic acid (13.6%) and rifampicin (6.5%), compromising oral step-down therapy in BJIs. Only two hVISA strains (0.5%) were identified. Among the CoNS, mainly Staphylococcus epidermidis (506/695; 72.8%), resistance to first- and second-line agents was more common. Linezolid resistance was identified in 10 CoNS (1.4%). The most frequent linezolid resistance mechanism was the G2576T mutation in 23S rDNA (9/10). For the first time in France, the cfr gene was found in five related sequence type 2 (ST2) S. epidermidis from two different hospitals, in association with ribosomal RNA and L3 ribosomal protein mutations. These national data must be considered when selecting empirical treatment for invasive staphylococcal infections. Moreover, the emergence and spread of linezolid-resistant CoNS carrying the cfr gene is of concern.

Glucocorticoids improve erythroid progenitor maintenance and dampen Trp53 response in a mouse model of Diamond-Blackfan anaemia.

SummaryDiamond–Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19‐deficient cells‐ in a disease‐specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease‐specific treatments of DBA.