Rps29 Mutant Research Articles

Abstract IA2: Developing therapeutics using the zebrafish

Abstract The zebrafish is a wonderful disease model, and it is now possible to develop new therapies to treat human disease. A screen for blood stem cells in the developing aorta led to the discovery of PGE2 as a regulator of stem cell engraftment. After studies with mouse and human hematopoietic cells, a clinical trial proceeded to test PGE2 in cord blood transplantation. In 12 leukemic patients receiving 2 cord blood units, one treated with dmPGE2, 10 patients engrafted with the treated cord blood. The neutrophils and platelets recovered more quickly than the untreated cord blood. Another chemical, leflunomide, was found in zebrafish to block neural crest development. Leflunomide also suppressed neural crest gene transcription in human melanomas, and reduced the rate of human tumor formation in mouse xenografts. The effect was stronger in combination with a BRAF inhibitor known to block cell proliferation. This has led to a clinical trial in which 43 patients will be treated for metastaic melanoma. To date, three patients have been treated. In another example, a chemical suppressor screen was done on the rps29 mutant that resembles Diamond Blackfan anemia. Calmodulin antagonists rescue the defect in hematopoiesis. These antagonists also rescue the hematopoietic block found in human CD34 cells treated with an shRNA to rps19. This suggests that calmodulin antagonists could be used to treat patient with Diamond Blackfan anemia. The zebrafish offers an excellent opportunity to find new therapeutics. Citation Format: Leonard I. Zon. Developing therapeutics using the zebrafish. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA2.

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

Abstract 512Ribosomal 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. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy. Zon:Fate Therapeutics: Founder Other.

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29(-/-) embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.

P53 Dependent and Dose Dependent Effects of Rps29 Mutation In the Zebrafish Embryo.

AbstractAbstract 1170Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by red cell aplasia and craniofacial abnormalities. Ribosomal protein genes are often mutated in patients with this disease, but the mechanism of action is still being investigated. To elucidate the effect of mutations in ribosomal proteins, we are studying a zebrafish rps29 mutant with hematopoietic and endothelial defects. Hematopoietic stem cells (HSCs) in rps29-/- embryos are significantly decreased, as assayed by runx1 and cmyb expression. Although the aorta and posterior cardinal vein form in the mutant, intersomitic vessel formation is affected. To test whether decreased p53 levels can rescue these defects, we crossed fish with mutated p53 into the rps29 background. In rps29-/-;p53-/- embryos, the vascular and HSC phenotypes are rescued, demonstrating that p53 may be required for these effects of rps29 knockdown. We performed a microarray comparing rps29-/- embryos and their siblings to identify genes that are differentially expressed in the mutant. Using gene set enrichment analysis (GSEA), we determined that the list of genes up-regulated in the rps29 mutant is enriched for genes up-regulated by p53 in response to irradiation. Many of the genes identified have known roles in apoptosis and stress response. We have also identified genes whose expression correlates with the number of wildtype copies of rps29. Orthopedia homolog a (otpa), which is specifically expressed in forebrain and hindbrain tissues at 24 hours post fertilization (hpf), is decreased in heterozygous siblings and further decreased in homozygous siblings. In addition, p53 knockdown partially increases otpa levels in the mutant. These data support a model where p53 activation is one of the critical downstream mediators of rps29 knockdown in several tissues, but the mechanism of tissue specificity remains unclear. The otpa phenotype suggests that regulation of some genes is dependent on rps29 levels. The zebrafish rps29 mutant will be a useful model for understanding how a decrease in ribosomal protein levels can cause specific defects in hematopoietic and neural tissues. Disclosures:Zon:FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.