Defective Nucleotide Metabolism Contributes To p53 Activation In Diamond-Blackfan Anemia

Abstract

Diamond-Blackfan Anemia (DBA) is a rare childhood bone marrow failure disorder, characterized by the presence of red cell aplasia, congenital abnormalities, and increased levels of adenosine deaminase (ADA). Haploinsufficiency of ribosomal proteins (RPs) due to mutations in RPS19 and RPL11 occurs approximately 25% and 5% of DBA patients, respectively. The pathogenesis of DBA has been associated with activation of p53, but the mechanism of how this leads to the erythroid defect in DBA patients is not well understood.To understand the molecular pathways leading to DBA, we used previously published zebrafish models of RPS19 and RPL11 deficiency, in addition to primary human fetal liver CD34+ cells transduced with RPS19 shRNA to evaluate signaling pathways upstream of p53. One of the earliest responses to RP deficiency was upregulation of rrm1, a subunit of ribonucleotide reductase (RNR), responsible for de novosynthesis of dNTPs. Since ADA expression is increased in DBA and ADA is involved in nucleotide catabolism, we hypothesized that RP deficient zebrafish and primary human fetal liver CD34+ cells have defects in nucleotide metabolism, which result in replication stress and activation of p53 through the ATR/ATM/Chk1/2 pathways.Several genes involved in de novo nucleotide synthesis, nucleotide catabolism, and purine salvage are upregulated in RPL11 and RPS19 deficient zebrafish, including rrm1 (2-7 fold), ada (2.8 fold), xdh (2.3 fold), and hprt1 (3-fold). In contrast, genes involved in pyrimidine salvage, such as tk1 and dck were downregulated in RP deficient zebrafish (0.4 and 0.8 fold, respectively). RP deficient zebrafish also had imbalanced dNTP pools, with increased dTTP and decreased dCTP levels compared to controls. To test whether these alterations in nucleotide metabolism lead to replication stress, we evaluated DNA damage and stress-induced proteins in primary human fetal liver CD34+ cells transduced with RPS19 shRNA and found increased phosphorylation of p53 (Ser15 and Ser37), ATM (Ser1981), 53BP1 (Ser1778), Chk1 (Ser345), and Chk2 (Thr68), indicating activation of the ATR/ATM/Chk1/2/p53 pathway in RP deficient cells. To rescue the RP-deficient zebrafish, we treated them with a mixture of exogenous nucleosides. Nucleoside treatment decreased p53 expression, restored rrm1, ada, tk1, and dck to normal levels, decreased number of apoptotic cells, and increased the number of Gata1-expressing cells in RP deficient zebrafish. Our data suggest that defective nucleotide metabolism contributes to p53 upregulation in DBA, and that nucleoside supplements alleviate replication stress and may prove beneficial for patients with DBA. Disclosures:No relevant conflicts of interest to declare.