Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency by Modulating p53 Activity in Models of Diamond Blackfan Anemia

Abstract 512

Ribosomal protein mutations are common in patients with Diamond Blackfan anemia (DBA), who have red cell aplasia and craniofacial abnormalities, and rps14 deficiency has been linked to 5q- myelodysplastic syndrome. We have characterized a zebrafish mutant in rps29, a ribosomal protein in the small subunit. Rps29^−/− embryos have hematopoietic and endothelial defects, including decreased hematopoietic stem cells, defects in hemoglobinization, and decreased staining of vessel markers. Heads of mutant embryos are also morphologically affected, with increased apoptosis. Consistent with other models of DBA, knockdown of p53 completely rescues the rps29 hematopoietic and apoptotic phenotypes. We wanted to identify chemicals that could rescue the rps29 mutant phenotype. Using 600 compounds from known bioactive libraries, we performed an in vivo chemical screen for rescue of the rps29 apoptotic and endothelial defects. Treatment with one compound, A-3, improved the morphology of the apoptotic embryo head. A-3 is an inhibitor of calmodulin, which interacts with a host of calmodulin-dependent enzymes and can bind to other proteins in the cell, including p21. W-7, another calmodulin inhibitor related to A-3, can rescue the endothelial defect in the rps29 mutant. Calmodulin inhibition in rps29 mutant embryos rescues the hemoglobin defect. Furthermore, structurally unrelated chemicals that inhibit calmodulin rescue the endothelial defect. We hypothesized that calmodulin inhibition was affecting some part of the p53 pathway. We tested whether A-3 had an effect on the phenotype in vitro in a cancer cell line where shRNA knockdown of RPS19 has been shown to stabilize p53 and induce p21. When rps19 is knocked down in A549 cells, treatment with calmodulin inhibitors resulted in a decrease of p21 protein, as measured by flow cytometry, although p53 protein levels were stable. This effect was also observed in primary human cord blood-derived CD34⁺ hematopoietic stem and progenitor cells. In situ immunofluorescence in A549s further revealed that nuclear localization of p53 and p21 upon RPS19 knockdown was disrupted by chemical treatment. Calmodulin inhibitors also rescue ribosomal protein deficiency-mediated erythroid defects both in vitro and in vivo. When RPS19 is knocked down in CD34⁺ cells, there is a defect in erythroid differentiation. Treatment with calmodulin inhibitors rescued production of CD71⁺ cells upon differentiation. In conclusion, we have demonstrated the use of calmodulin inhibitors as a novel approach for mediating p53 activation upon ribosomal protein knockdown, thereby rescuing the erythroid defects of ribosomal deficiency. Our data suggests that small molecules that inhibit calmodulin may be effective therapies for patients with ribosomal deficiency.