Developmental Stage-Specific Hematopoietic Failure In Vivo in a Novel Model of Diamond Blackfan Anemia

Abstract

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized by failure of erythropoiesis at the progenitor stages, congenital anomalies, and predisposition to cancer. DBA is most frequently due to mutations in genes encoding a ribosomal protein, the most common one being mutations in RPS19. Despite the well-established role of RPS19 as well as other ribosomal proteins in the erythropoietic failure in in vitro culture systems, there is a paucity of in vivo murine models of DBA due to differences between humans and mice. Indeed, stress erythropoiesis occurs in the spleen of mouse, compensating for the erythropoietic defects observed in the bone marrow. Using CRISPR/Cas9 technologies, we generated a conditional knockout of Rps19 by flanking exon 2 with 2 loxP sites and studied hematopoiesis in this model. While Tie2-cre+;Rps19+/- animals die in utero, Vav-iCre+; Rps19+/- mice are born at expected Mendelian ratios with severe macrocytic anemia. Surprisingly, and unlike any other model generated to date, these mice present with reticulocytopenia, a critical characteristic of patients with DBA and die by post-natal day 10 (P10), due to bone marrow failure and decreased splenic stress erythropoiesis. To understand the role of ribosomal proteins during hematopoietic development, we analyzed fetal livers of Vav-iCre+; Rps19+/- embryos and control littermates at E13.5 and E17.5. Using flow cytometry, we observed defective erythropoiesis and gradual hematopoietic stem cell exhaustion at the fetal to neonatal transition. Mechanistically, and consistent with previous studies using in vitro systems, Rps19mutant cells had increased p53 protein expression and reduced mTORC1 activity. Unexpectedly, hematopoietic progenitors had enhanced global protein synthesis. Using BrDU and EdU stainings, we observed a decrease in the cell cycle duration of erythroblasts, consistent with the macrocytosis and reticulocytopenia seen in neonatal Vav-iCre+; Rps19+/- mice. Since p53 is reported to have a central role in the pathophysiology of DBA, we asked whether deletion of one allele of p53 could rescue the defects observed in Vav-iCre+; Rps19+/-. The Vav-iCre+; Rps19+/-, trp53+/-presented with improved blood counts, increased spleen size and cellularity but died by four weeks after birth. Analyses of the hematopoietic compartment in these animals at P21 revealed bone marrow failure and HSC depletion with skewed progenitor populations. The increase in global protein synthesis observed in Vav-iCre+; Rps19+/- remained unchanged in Vav-iCre+; Rps19+/-, trp53+/-, suggesting mechanisms independent of p53. Taken together, we have developed a novel in vivo model for the study of DBA. Our findings could potentially explain why DBA typically manifests after birth and highlight a critical role for Rps19 during hematopoietic differentiation both in utero and after birth.