TARGETING P53 AND NUCLEOLAR STRESS IN DIAMOND-BLACKFAN ANAEMIA

Abstract

Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.