ALBERT

Description: Abstract 176 Identification of mutations in a number of ribosomal genes including RPS19, RPL5 and RPL11 in Diamond-Blackfan anenia has established it as a disease of aberrant ribosome biogenesis. To date, it has not been possible to determine if a common cellular mechanism accounts for the erythroid defect in DBA in conjunction with various ribosomal gene mutations identified. To address this issue, we infected normal CD34+ cells from cord blood with specific short hairpin (sh) RNA against RPS19 (shRPS19C), RPL5 (shRPL5A, B, C) and RPL11 (shRPL11A, B, C) and determined their ability to undergo erythroid differentiation in a liquid culture system. Efficiency of each shRNA was verified at the RNA level in UT7 cell lines and CD34+. They all decreased dramatically mRNA expression of RPS19, RPL5 and RPL11 from 90% to 50% depending on the shRNA and the cells, except shRPL11B and C, which reduced RPL11 mRNA expression from 40% to 20%. Erythroid precursors after sh RNA infection were harvested at D7, D9, D11, D14 and D16 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay and flow cytometry), and mRNA and protein expression. During in vitro erythroid differentiation, we noted a decrease in the extent of cell amplification, from D0 (CD34+ cells) to D7 (CFU-e/proerythroblasts), to D9 (basophilic erythroblasts), to D11, D14, D16 (orthophilic erythroblasts) for all the shRPS19C, shRPL5 (A, B, C) and shRPL11A infected cells compared to uninfected or scramble infected cells. The decrease in erythroid proliferation was less important (50%) in shRPS19 infected cells compared to shRPL5A,B,C and shRPL11A infected CD34+ cells (90–95%). In association with documented decrease in expression of RPS19 mRNA after shRPS19 CD34+ infection, we did not find either an alteration in erythroid differentiation or increased apoptosis at any stage of cell differentiation. In marked contrast, CD34+ cells infected with either shRPL5A,B,C or shRPL11A exhibited importantly a delayed erythroid differentiation with a marked increase in apoptosis. These findings have enabled us to document that while decreased cell proliferation is a feature of all CD34+ cells with decreased expression of different ribosomal proteins, defective differentiation and increased apoptosis of erythroid cells is a distinguishing feature of only CD34+ cells with decreased expression of RPL5 and RPL11. To obtain mechanistic insights into the observed differences in apoptotic phenotype, we analyzed p53 and apoptosis pathways of CD34+ following infection with shRPS19, shRPL5A-B-C, and shRPL11A. All the proteins tested: p53, p21, caspase 3, Bax, Noxa exhibited a decreased expression levels following infection with shRPL5A,B,C and shRPL11A, while exhibiting an increased expression at the mRNA level, suggesting a general inhibition of protein synthesis following RPL5 and RPL11 mRNA depletion. Strikingly, RPS19 depletion resulted increased levels of p53 and p21 expression at both mRNA and protein levels, while Bax and Noxa (proapoptotic) mRNA were as the same level compared to the controls. These different patterns of alteration in p53 and apoptotic protein levels can account for different apoptotic phenotype observed following depletion of RPS19 and of RPL5 and RPL11. In summary, we identified for the first time distinct erythroid proliferation and differentiation defects in conjunction with different ribosomal protein defects. These findings may have implications for future genotype-phenotype relationships in DBA patients. Disclosures: No relevant conflicts of interest to declare.

Description: 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.