Abstract

Diamond-Blackfan Anemia (DBA) is a rare inherited bone marrow failure disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. While several studies have found that the tumor suppressor protein p53 contributes to DBA pathogenesis, and that certain GATA1 mutations can give rise to DBA, the link between ribosomal protein mutations and erythroid defects is not well understood.

To investigate the molecular pathways downstream of RPS19 deficiency, we infected human cord blood CD34+ cells with RPS19 shRNA lentivirus and observed that RPS19 knockdown resulted in decreased GATA1 mRNA and protein expression, resulting in defective erythropoiesis. We also detected increased TNF-α levels in RPS19 knockdown cells, and in zebrafish that have been treated with RPS19 morpholino. TNF-α was primarily upregulated in the non-erythroid population of RPS19 deficient cells, while its receptor, TNFR1, showed increased expression on the surface of erythroid cells.

To understand the relationship between GATA1 and TNF-α, we treated primary hematopoietic CD34+ cells with TNF-α or its inhibitor etanercept. Our results demonstrated that TNF-α treatment reduced GATA1 expression, and that this effect could be rescued by the addition of etanercept. Treatment of RPS19 deficient cells with etanercept improved their erythroid differentiation, and increased GATA1 expression. Additionally, etanercept treatment successfully reversed the anemia phenotype observed in RPS19 deficient zebrafish.

To study pathways downstream of TNF-α, we examined phosphorylation of signaling pathways such as p38 MAPK, NFκB, ERK, STAT1, and STAT5 in RPS19 deficient erythroid cells using phospho-flow cytometry. Among these pathways, we found a significant increase in phosphorylation of p38 MAPK, but not ERK, NFκB, STAT1, or STAT5, suggesting that p38 MAPK activation by TNF-α contributes to decreased GATA1 expression in RPS19 cells. We suggest a novel mechanism for the erythroid defects observed in DBA, in which RPS19 deficiency leads to increased TNF-α production in non-erythroid cells, and activation of p38 MAPK, followed by decreased GATA1 expression, in erythroid cells. Our data also suggest that TNF-α inhibitors, such as etanercept, may be beneficial in treating patients with DBA.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.