A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.