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...

Summary Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.

Background: Myeloid cell lines were generated from Npm1 ϩ/Ϫ mice to understand the role of NPM1 in MDS.