Reduced Protein Synthesis and p53 Activation in Late-Stage Erythroblasts Mediate the Erythroid Differentiation Defect in Mice with Ribosomal Protein S14 Haploinsufficiency
Authors: Schneider Rebekka K. , McConkey Marie E. , Chu Lisa P. , Ebert Benjamin L. 
Published: October 2019

Abstract
The 5q-syndrome is a subtype of myelodysplastic syndrome (MDS) with a defined clinical phenotype associated with heterozygous deletion of Chromosome 5q. The RPS14 gene was identified as a critical gene for the erythroid phenotype of the 5q- syndrome using an RNA interference screen. We generated a murine model for conditional, heterozygous inactivation of Rps14 in the bone marrow to investigate the biological basis of del(5q) MDS.
To explore the role of Rps14 on hematopoietic stem cell (HSC) function and erythroid differentiation, we generated a mouse model in which Rps14 exons 2-4 are flanked by loxP sites. Following crosses to Mx1Cre transgenic mice, we induced Rps14 excision in hematopoietic cells by poly(I:C). Two weeks after induction of the gene excision, mice developed significantly reduced hemoglobin and red blood cell counts with a significantly higher MCV compared to Mx1Cre+ controls. Bone marrow analysis confirmed an erythroid differentiation defect specifically at the transition from the CD71+Ter119+ (RII, basophilic erythroblasts/early polychromatophilic erythroblasts) to the CD71 intermediate/lowTer119+ (RIII/IV, poly/orthochromatophilic erythroblasts, enucleated erythrocytes) population, accompanied by significant up-regulation of p21 in the RIII population. Histopathology of spleens demonstrated compensatory extramedullar erythropoiesis. The bone marrow was normocellular with a significant increase in hypolobulated megakaryocytes correlating with high platelet counts and dysplastic platelets in the peripheral blood, reflecting the pathognomonic megakaryocyte phenotype in del(5q) MDS. At 50 weeks of age, Rps14-/+Mx1Cre+ developed a more severe macrocytic anemia in the peripheral blood.
We thus tested whether a more severe erythroid phenotype could be induced by treatment with the hemolytic agent phenylhydrazine (PH; 2x single dose 25mg/kg body weight, s.c.). In response to PH treatment, Rps14 haploinsufficient mice developed a more severe anemia than control mice, had a delayed reticulocyte response, and a differentiation defect again specifically at the transition from RII to RIII/IV accompanied by a compensatory increase in the RI (CD71+Ter119-) pro-erythroblast/early basophilic population. Megakaryocyte-erythrocyte progenitors were increased, and pre-CFU-E and CFU-E numbers were normal. To further elucidate the mechanisms by which haploinsufficiency of Rps14 causes ineffective erythropoiesis, we analyzed cell cycle, p53 and apoptosis. We found a significant induction of p53 specifically in the RI population and a dramatic increase of apoptotic cells in RIII, suggesting that increased apoptosis accounts for the erythroid failure. We therefore tested if genetic inactivation of p53 rescues the erythroid phenotype. In Rps14-/+p53-/+Mx1Cre+ mice the erythroid differentiation defect was restored and mice had a comparable response to PH as Mx1Cre+ controls. In PH dose escalation experiments (35mg/kg), Rps14 haploinsufficient mice died from the severe anemia and delayed reticulocyte response, while compound p53 loss rescued the erythroid failure. Accordingly, forced erythroid differentiation in vitro was significantly impaired in Rps14 haploinsufficient hematopoietic stem and progenitor cells.
We measured protein synthesis by O-propargyl (OP)-puromycin incorporation. Rps14 haploinsufficient cells had a significantly reduced protein synthesis in CD71low cells (late erythroblasts) that could not be fully restored by p53 inactivation although the protein synthesis in p53 heterozygote cells alone was significantly higher. The decreased protein synthesis in Rps14-/+p53-/-Mx1Cre+ compound cells might also account for the decreased stem cell expansion in repopulation assays compared to cells with only p53 loss.
Our data provide in vivo evidence that Rps14 haploinsufficiency contributes to the erythroid differentiation defect in del(5q) MDS by reduced protein synthesis and p53 induction in late-stage erythroblasts. This murine model recapitulates the erythroid und megakaryocytic phenotype of the 5q-sydrome and provides a model for understanding the underlying mechanisms in the pathogenesis of ribosomal-mediated erythroid failure in del(5q) MDS.

Disclosures
No relevant conflicts of interest to declare.