Abstract

A functional siRNA screening approach to discover therapeutic candidates for the treatment of Diamond-Blackfan anaemia and other rare bone marrow failure syndromes

Amee George, Research Officer, Peter MacCallum Cancer Centre

Diamond-Blackfan Anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia, characterised by low numbers of red blood cells and in many cases, by abnormalities of the face and head, upper limbs and heart. The disease usually presents in infancy (2-3 months of age) and is caused by deleterious mutations in a number of genes that encode ribosomal proteins (RPs), in particular, RPS19, accounting for 20-25% of known cases. While most DBA patients respond positively to treatment (steroids and/or blood transfusions), a significant proportion of DBA mortalities are due to complications arising from treatment. Recent work by us, and others, has demonstrated that impaired ribosome biogenesis (due to nucleolar stress) results in the sequestration of HDM2 (MDM2) by free RPs, leading to the accumulation of p53, which subsequently induces cell cycle arrest or apoptosis. In the case of DBA, we hypothesise that nucleolar stress (due to RPs and other modifying elements) and activation of p53 results in preferential apoptosis or cell cycle arrest of the erythroid progenitors required for red blood cell development and normal development. To this end, we have completed a high content siRNA screen of the entire human genome using the Dharmacon SMARTpool siRNA library (targeting the expression of ~18,000 different candidates), to identify genes required for the nucleolar stress-induced induction of p53 following RPS19 knockdown. We have identified a number of high confidence novel candidates that reduce p53 induction by 40% or more, and we are currently reassessing these candidates in further siRNA screens and by candidate-based approaches. Our next step is to validate our candidates in in vivo models of DBA and other congenital bone marrow failure disorders. It is hoped that these candidates may provide a novel therapeutic strategy for the treatment of these group of diseases. Amee J. George, et al.