A High-Throughput Screening Approach to Identify Therapeutics for the Treatment of Diamond-Blackfan Anaemia

Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 20-25% of all cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent Nucleolar Surveillance Pathway (NSP). In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In DBA, one of the molecular mechanisms impairing the proliferation and thus reducing the number of erythroid progenitors that can progress to mature red blood cells is an elevation of p53 protein mediating activation of the NSP.

In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened compound libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated both cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. Using an RPS19 depleted A549 cell line as a model system, the screen successfully identified different therapeutic groups. In the DBA context, we were most interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 22 molecules were re-evaluated in vitro, again using RPS19 depleted A549 cells, through the quantification of p53 protein expression and densitometry analysis. From this, 10candidates were evaluated ex vivofor their effects on proliferation using bone marrow obtained from an inducible Rps19 knockdown (DBA) mouse model.

While we are currently testing a number of compounds in vivo using the Rps19 DBA mouse model (as described), one of the compounds tested thus far has demonstrated a partial rescue of the cKit+ population, no changes in erythroid precursors but interestingly a reversal of the defect in the Granulocyte-Monocyte Progenitor (GMP) population. Impairment in lineage progression in the GMP compartment has also been reported to present in bone marrow failure Shwachman-Diamond Syndrome. We are currently evaluating the mechanism by which this drug is rescuing the c-Kit+ and GMP populations in these mice.

In summary, our high-throughput screening approach and follow up studies have identified a suite of novel therapies that may be beneficial for repurposing for the treatment of bone marrow failure by increasing hematopoietic progenitor cells. We plan to evaluate this, and potentially other therapies, in a clinical trial with DBA patients.