Differential Down-Regulation of RPL35a in Human Bone Marrow Progenitors Demonstrates a p53 Independent Mechanism Mediating the Diamond Blackfan Anemia Phenotype.

Abstract Abstract 1096 Poster Board I-118 We have previously reported the first known mutations in a gene that encodes a large ribosomal subunit protein, Rpl35a that are linked to Diamond Blackfan Anemia (DBA), a congenital pure red cell aplasia. We have demonstrated that down-regulation of RPL35a results in decreased cellular proliferation, increased apoptosis, impaired ribosomal RNA processing and defective ribosome biogenesis. In order to examine the mechanisms by which defects in RPL35a lead to the development of DBA, we have developed a lentiviral system containing shRNA to down-regulate RPL35a and RPS19, a gene encoding a small ribosomal protein known to be defective in about 25% of DBA cases. Defects in these cellular processes have been linked to disruption of cell cycle progression through activation of nucleolar associated regulatory mechanisms. Cell cycle analysis of the normal human bone marrow progenitors transduced with the specific shRNA demonstrates a G1 arrest when compared to cells transduced with a luciferase shRNA control construct. Similar findings were made in the p53-mutant cell line, TF-1. In contrast, cell cycle analysis of the p53-mutant UT-7/Epo cell line demonstrates a primarily G2/M arrest. To further investigate the mechanism of the cell cycle arrest in bone marrow progenitors, p53 expression was analyzed by Western Blot. Only a nearly complete knockdown resulted in increased p53 expression; shRNA affecting a haploinsufficient state had no evidence of increased p53 expression. However, clonogenic studies of human bone marrow progenitors transduced with shRNA constructs that approximate haploinsufficiency demonstrate a selective absence of erythroid colonies compared to myeloid colonies, thus mimicking the phenotype of DBA. In contrast, bone marrow progenitors transduced with shRNA that causes near complete absence of expression of RPL35a have neither myeloid nor erythroid clonogenic potential consistent with a stem cell catastrophe. These data may suggest a p53 independent pathway can mediate the effects of ribosomal protein deficiency in DBA as well as demonstrate a model for further understanding abnormalities in ribosomal proteins that result in an in vitro phenocopy of altered DBA hematopoiesis. Disclosures No relevant conflicts of interest to declare.