Hematopoietic Stem Cells Supporting Fetal Erythropoiesis Are Differentially Regulated By Small and Large Ribosomal Subunits

Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome commonly associated with mutations or deletions of ribosomal genes, leading to protein haploinsufficiency and resulting in nucleolar stress and p53 activation. Clinically, DBA usually manifests after birth and is characterized by macrocytosis, reticulocytopenia and a paucity of erythroid precursors in the bone marrow. Patients can also present with additional defects in other hematopoietic lineages, suggesting a defect in hematopoietic stem and/or progenitor cells. Notably though, it is still unclear whether these defects are common to all ribosomal protein (RP) mutations or specific to large vs.small ribosomal subunits. Furthermore, the role of RPs in fetal hematopoiesis is poorly understood due to the lack of clinically relevant mouse models for ribosomopathies. To address these questions, we generated Rps19^fl/fl and Rpl5^fl/fl conditional mouse models - representing the most mutated genotypes in DBA - using CRISPR/Cas9 technology and crossed them to a Vav-iCre mouse. Vav-iCre;Rps19^fl/+ mice recapitulated clinical features of DBA, including macrocytic anemia and reticulocytopenia, and died on post-natal day 10 (P10) due to bone marrow failure without stress erythropoiesis in the spleen. These mice exhibited a progressive exhaustion of the hematopoietic stem and progenitor cell (HSPC) compartment by E17.5, resulting in a 40% reduction in the number of erythroid cells at the BFU-E stage. Flow cytometry analyses of terminal erythroid differentiation further demonstrated a 30% reduction in the number of basophilic erythroblasts at E17.5. In contrast with Vav-iCre;Rps19^fl/+ mice, Vav-iCre;Rpl5^fl/+ mice died perinatally from severe anemia. They also differed markedly from Rps19 haplo-insufficient mice as they exhibited a progressive expansion of the HSPC compartment. Rather, the defect in Rpl5 haplo-insufficient mice was restricted to the erythropoietic compartment, with an accumulation of BFU-E and CFU-E erythroid progenitors and a 70% decrease in the number of basophilic erythroblasts by E17.5.

To elucidate the mechanisms underlying the specific HSPC phenotypes in Rps19 and Rpl5 haplo-insufficient mice, we performed scRNAseq analyses of fetal liver cells at E13.5. While Vav-iCre;Rps19^fl/+ mice presented global alterations of the transcriptome at each stage of differentiation, Vav-iCre;Rpl5^fl/+ mice only presented defects in erythroid lineage cells, beginning at the proerythroblast stage. Comparative analyses revealed Uba52, encoding the core RPL40 ribosomal protein, as a differentially regulated gene and western blot analyses confirmed decreased expression of RPL40 in both models, albeit to different extents. RPL40 is associated with translation elongation, and accordingly, we observed acceleration of translation by polysome profiling in the Rps19 model, along with decreases in the phosphorylation of the elongation factor eEF2 in the ckit⁺ population, corroborating the polysome profiling results. By E17.5, the phosphorylation of both the translation initiation factor eIF2a and elongation factor eEF2 were reduced, indicative of increased translation in the mutant mice. Accordingly, we observed reduced mTOR signaling in both these models.

Furthermore, Vav-iCre;Rps19^fl/+ fetal livers demonstrated activation of the p53 pathway, consistent with clinical findings. However, to our surprise, expression of Runx1, a transcription factor that mediates ribosome biogenesis, was significantly increased. Importantly, activation of both p53 and Runx1 pathways played critical roles in the pathological hematopoiesis occurring in Rps19 haplo-insufficient mice as erythroid defects were rescued by conditional deletion of either p53 or Runx1. Deletion of one or both Runx1 alleles significantly rescued fetal HSPC defects but bi-allelic p53 loss was needed to fully rescue fetal HSC exhaustion and prevent neonatal lethality in Vav-iCre;Rps19^fl/+ mice.

Taken together, our results unravel distinct requirements for Rps19 and Rpl5 during fetal hematopoiesis and provide novel insights into the mechanism(s) behind ribosomal protein haploinsufficiency leading to DBA.

Zon:Scholar Rock: Current holder of stock options in a privately-held company; Fate Therapeutics: Current holder of stock options in a privately-held company; CAMP4 Therapeutics: Current holder of stock options in a privately-held company; Triveni Bio: Current holder of stock options in a privately-held company. Weiss:Cellarity Inc., Novartis, and Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees. Crispino:Syndax: Research Funding; Cellarity: Consultancy; Alethiomics: Membership on an entity's Board of Directors or advisory committees.