Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts

Role of HSP70 protein in Blacfan-Diamond anemia

L’anémie de Blackfan-Diamond (ABD) est une érythroblastopénie congénitale rare, secondaire à un blocage de la maturation érythroïde entre les stades BFU-e et CFU-e. L’ABD est le plus souvent la conséquence d’une mutation germinale affectant un gène codant pour une protéine ribosomique (RP) de la petite ou de la grande sous-unité du ribosome. Quatorze gènes distincts ont été identifiés. Les gènes les plus fréquemment mutés sont les gènes RPL5, RPL11 et RPS19 (37% des patients). Plus rarement, l’ABD est la conséquence de mutations dans le gène TSR2 ou dans le gène GATA-1. Ce dernier code pour un facteur de transcription majeur de l’érythropoïèse. Chez les patients ABD, les mutations de GATA-1 induisent une perte quasi-totale de la forme longue de GATA-1 qui est nécessaire à la différenciation de la cellule érythroïde. Notre groupe a identifié deux phénotypes de l’ABD in vitro en fonction du gène muté. En cas d’haploinsuffisance RPS19, la prolifération érythroïde est moins réduite qu’en cas d’haploinsuffisance RPL5 ou de RPL11. Une haploinsuffisance RPS19 n’altère pas la différenciation érythroïde et n’induit pas d’apoptose contrairement à l’haploinsuffisance RPL5 ou RPL11 où il existe un retard de différenciation érythroïde et un excès net d’apoptose responsable au moins en partie de la diminution drastique de la prolifération érythroïde dans ces phénotypes.HSP70 est impliquée dans la survie cellulaire et la différenciation érythroïde en protégeant GATA-1 du clivage par la caspase-3, une protéase activée lors de la différenciation érythroïde terminale. Comme la différence entre les deux phénotypes d’ABD in vitro concernait la différenciation érythroïde et la survie cellulaire, nous avons émis l’hypothèse selon laquelle la mutation de certains gènes RP provoque un défaut d’expression d’HSP70 conduisant au blocage de la différenciation érythroïde et à l’excès d’apoptose retrouvés dans les phénotypes sévères d’ABD.Nous avons étudié différents patients atteints d’ABD, porteurs de mutations dans les gènes RPS19, RPL5 ou RPL11 et généré un modèle in vitro d’ABD en exprimant, dans des cellules CD34+ humaines issues de sang de cordon, des ARN interférents ciblant RPL5, RPL11 ou RPS19. Chez les patients comme dans le modèle reproduisant l’ABD, l’haploinsuffisance RPL5 ou RPL11 diminue drastiquement l’expression protéique de HSP70 et de GATA-1 (Western blot, microscopie confocale et en cytométrie couplée à des techniques d’imagerie, (technologie ImageStream) à la différence de 1’haploinsuffisance RPS19. Dans tous les cas, HSP70 est normalement transcrite et traduite. Les inhibiteurs du protéasome (MG132, lactacystine, bortezomib) restaurent l’expression10de HSP70. La diminution d’expression de HSP70 est donc liée à une dégradation protéasomale. L’invalidation de RPL11 induit une polyubiquitinylation importante de HSP70. La transduction lentivirale de l’ADN complémentaire d’HSP70 dans les cellules primitives invalidées pour RPL11 permet de restaurer l’expression de HSP70 et de GATA-1 à un niveau similaire aux contrôles et de rétablir la prolifération cellulaire et la différenciation érythroïde, confirmant le rôle clé de HSP70 dans le phénotype sévère RPL5+/Mut ou RPL11+/Mut. Les formes les plus sévères de l’ABD sont associées à la dégradation de HSP70 par le protéasome. La perte de la protéine chaperone de GATA-1 induit la perte de GATA-1, facteur de transcription majeur de la différenciation érythroïde. Une augmentation de l’expression de HSP70 pourrait ainsi constituer une nouvelle approche thérapeutique dans l’ABD.Diamond-Blackfan anemia (DBA) is 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 GATA-1 genes have been identified in a few DBA families. The GATA-1 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 eytrhoid differentiation and the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA-1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA-1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation. 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 GATA-1 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 of12HSP70 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 depleted RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation, and reduced apoptosis, confirming a critical role for HSP70 in the erythroid defect in the RPL11+/Mut DBA phenotypes. The loss of HSP70 may explain the loss of GATA-1 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

XPO1 regulates erythroid differentiation and is a new target for the treatment of β-thalassemia.

β-thalassemia major (β-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human β-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of β-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of β-TM

Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in diamond-blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission

S6 kinase phosphorylation and autophagy is induced by reactive oxygen species (ROS).

<p>(<b>A</b>) Confocal microscopy analysis of GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i> and either untreated or treated with 10 mM Trolox overnight. Size bars = 10 µM. (<b>B</b>) Quantification of the average number of GFP-LC3 puncta per cell in (<b>A</b>). (<b>C</b>) Western blot analysis of phosphorylated S6 kinase expression in GFP-LC3 HEK transfected with siScr or si<i>RPS19</i> and either untreated or treated with 10 mM Trolox overnight. (<b>D</b>) Western blot analysis of phosphorylated S6 kinase and phosphorylated AKT substrate expression in 2 dpf zebrafish embryos untreated or treated with 10 mM Trolox overnight. (<b>E</b>) Survival rates of embryos treated overnight with 10 mM Trolox. The results are the compilation of three independent experiments with N = 25 embryos representing each mutation. The statistics shown are compared to wild type. (<b>F</b>) Analysis of p53 stabilization in 2 dpf wild type, rpS7-deficient embryos, <i>p53^M214K/M214K</i> mutant embryos, or double mutants (<i>rpS7;p53</i>) by western blotting. (<b>G</b>) Analysis of p53 stabilization by western blotting of zebrafish mutants treated overnight with 100 µM Trolox.</p

Insulin abolishes autophagy in cells with RPS19 loss.

<p>(<b>A</b>) Confocal microscopy analysis of GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i> and either untreated or stimulated with 350 nM insulin for 6 hours. Size bars = 10 µM. (<b>B</b>) Quantification of the average number of GFP-LC3 puncta per cell in (<b>A</b>). (<b>C</b>) Western blot analysis of LC3 expression in cells from (<b>A</b>). Densitometer analysis used to calculate the ratio of LC3II/actin.</p