Activation of p53 Pathway In Erythroid Precursor Cells of Diamond-Blackfan Anemia Leads to Two Distinct Phenotypes

Field: Medicine (Health Sciences)

Abstract Abstract 2225 Identification of mutations in a number of ribosomal genes has established DBA as a disease of aberrant biogenesis of ribosomes. We have previously documented that knockdown of RPS19, RPL5 and RPL11 mRNA by shRNAs in CD34+ cells from cord blood leads to two distinct invitro erythroid phenotypes. The haploinsufficient RPS19 precursors exhibited decreased erythroid proliferation, normal erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 and no apoptosis. In contrast, RPL5 or RPL11 haploinsufficient precursors showed a larger decrease in erythroid proliferation, a delay in erythroid differentiation, activation of p53 pathway with cell cycle arrest in G0/G1 in association with increased apoptosis. To validate these findings, we studied in vitro erythroid differentiation of primary CD34+ cells isolated from peripheral blood of DBA patients. CD34+ cells from 12 DBA individuals carrying mutations in RPS19 gene, 7 DBA individuals with no mutations in RPS19 and 19 normal individuals. CD34+ cells isolated from peripheral blood were cultured in two-phases, first in methyl-cellulose for 7 days and then in liquid culture for additional 5 days. Cells were harvested at Day 7 (D7), D10 and D12 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay or flow cytometry), and mRNA and protein expression levels. From D0 (CD34+ cells) to D7 (CFU-e/proerythroblasts), to D10 (basophilic erythroblasts) and D12 (orthophilic erythroblasts), we observed a decrease in the extent of cell amplification of CD34+ cells from all DBA individuals. Interestingly, this decrease was more pronounced for CD34+ cells not carrying a mutation of RPS19 gene than for cells harbouring a mutation of RPS19. While a 9.5 fold amplification was noted between D0 to D7, for normal CD34+ cells, it was reduced to 6.1 fold for DBA cells with mutant RPS19 and further reduced to 3.8 fold for DBA cells not carrying RPS19 mutations. While we did not find a difference in cell amplification between D10 and D12 for DBA cells with RPS19 mutations compared to normal cells, we noted a large decrease in cell amplification for DBA cells without RPS19 mutations. We also studied 6 additional DBA patients (one with a mutant RPS19 gene, 2 with mutations in RPL11 gene and 3 in whom no RP gene mutations could be identified to date) using the two phase liquid culture system. Once again we noted that CD34+ cells with RPL11 mutations or with yet to be defined mutations exhibited a larger decrease in cell amplification from D0 to D7 and from D10 to D12 compared to CD34+ cells harbouring the RPS19 mutation. While erythroid differentiation was normal on both MGG and by flow cytometry in RPS19 mutant erythroid precursors, we observed a delay in erythroid differentiation in non RPS19 mutated precursors, in particular at D7 or D10 with increased number of CD34+ cells. Importantly, we noted increased apoptosis of cells not carrying RPS19 mutations compared to cells with RPS19 mutations. Strikingly, in all 6 DBA patients, p53 pathway (p53, p21 at the mRNA level and bax) was activated in the erythroid precursor cells. We confirmed an increase in apoptosis in the DBA patient erythroid precursors with non-mutant RPS19 as evidenced by decreases in Hsp70 and procaspase 3, and an increase in cleaved caspase 3. We confirmed cell cycle arrest in G0/G1 in cells from both DBA individuals studied. Reinforcing these findings, phosphoRb was found to be decreased in cells from all 6 DBA patients studied. In summary, using primary CD34+ cells from DBA individuals, we confirmed our earlier finding of the two distinct DBA phenotypes with cell arrest in conjunction with either presence or absence of apoptosis depending on whether the cells carry mutations in RPS19 or not. Strikingly, p53 pathway was activated in primary cells of all DBA patients studied. Our results suggest that erythroblastopenia, the main characteristics of DBA is likely the result of p53 activation leading to cell cycle arrest and apoptosis. Disclosures: No relevant conflicts of interest to declare.