Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells

Abstract

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q؊ syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q؊ syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineagespecific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q؊ syndrome, DBA, and perhaps other bone marrow failure syndromes. (Blood. 2011;117(9):2567-2576) IntroductionHeterozygous deletions or mutations of ribosomal protein genes have been implicated in 2 human disorders, Diamond-Blackfan anemia (DBA) 1-3 and the 5qϪ syndrome, a subtype of myelodysplastic syndrome (MDS). 4,5 Both disorders are characterized by a severe macrocytic anemia. 2,6 In DBA, a congenital disorder, approximately 25% of patients have mutations in the RPS19 gene, 1 and mutations or deletions have been identified in at least 9 additional ribosomal protein genes. 3,7 In the 5qϪ syndrome, somatic deletion of one allele of chromosome 5q causes haploinsufficiency for the RPS14 gene. 5 A central outstanding question is how a defect in ribosomes, which are expressed in and are essential to all cells, causes a primarily erythroid phenotype.Multiple animal models have demonstrated the effects of ribosomal dysfunction on erythropoiesis and the role of p53, which is known to monitor ribosome function. 8 Morpholinos targeting RPS19 in zebrafish cause an accumulation of p53 and a block in erythropoiesis that is reversed in the absence of p53. 9 Mice with germline mutations in the RPS19 or RPS20 genes have hyperpigmented foot pads with p53 accumulation in the epidermis, a decreased hematocrit, and an increased erythrocyte mean cell volume. Crossing the RPS19 mutant mice with p53 null mice rescued the skin and hematopoietic phenotypes. 10 Finally, a conditional heterozygous deletion of the Cd74-Nid76 interval syntenic to human chromosome 5q, containing 6 genes, including RPS14, in mouse hematopoietic cells, causes a macrocytic anemia that is rescued by crossing to a p53 null background. 11Although p53 appears to play a critical role in these d...

Effects of L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts

“…Recently, a p53-dependent mechanism was found to underlie the pathogenesis of the 5q-syndrome. 4,5 Consistent with these reports, a significant activation of p53 expression (41.5-fold increase in total p53 protein expression measured by intracellular staining and flow cytometry) was observed in the cells with reduced RPS14 expression when compared with the scramble control (Figure 1b). A significant increase in apoptosis (4twofold increase in dead-cell percentage) and cell cycle arrest at G 1 phase were observed in the cells with reduced RPS14 expression when compared with the scramble control (Figures 1c and d).…”

“…3 Several lines of converging evidence suggest that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anemia in the 5q-syndrome. 4,5 The bone marrow cells of patients with the 5q-syndrome show a block in the processing of pre-ribosomal RNA 3 and the CD34 þ cells of these patients show deregulation of multiple ribosomal protein genes and genes involved in the control of translation. 6 These data suggest that the 5q-syndrome represents a disorder of aberrant ribosome biogenesis.…”

“…This may explain the particular sensitivity of the erythroid lineage to reduced expression levels of ribosomal proteins. 5 In this study, we have first investigated the effects of RPS14 haploinsufficiency on human erythropoiesis using an RPS14 short Accepted article preview online 20 March 2012; advance online publication, 18 May 2012…”

PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia

“…A cell extrinsic mechanism for apoptosis in MDS has been favored for many years; 5 however, recent evidence suggests a cell-intrinsic trigger, especially in del(5q) MDS for which there is evidence for activation of the tumor suppressor p53 by ribosomal dysfunction. 6,7 More aggressive stages of MDS have less apoptosis than earlier stages, 8 supporting the paradigm that acquired resistance to apoptosis of malignant progenitors is an important step in cancer progression. 9 Among hematological malignancies, there is strong experimental data to support the tumor suppressor function of apoptosis in lymphoid malignancies [10][11][12] but there is a paucity of data addressing this paradigm in myeloid malignancies including MDS.…”

Ribosome biogenesis dysfunction leads to p53-mediated apoptosis and goblet cell differentiation of mouse intestinal stem/progenitor cells

“…9,10 Studies on cellular and animal models suggest that unscheduled upregulation of p53 may account for many clinical symptoms associated with ribosomopathies. [11][12][13][14][15][16] There are now evidences that ribosome biogenesis dysfunction also triggers p53-independent mechanisms. [17][18][19] Because bone marrow defects is a frequent clinical manifestation of ribosomopathies, most studies focused on the hematopoietic tissue and less is known about the impact of ribosome biogenesis dysfunction in other organs.…”