Ribosomal Proteins Rps19 and Rps14 Cooperate As Tumor Suppressor Genes with p53

Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines.

We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53^m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9.

The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53^m214k/m214k uninjected controls (p<0.0001). Preliminary histology of grown tumors has shown melanomas and malignant peripheral nerve sheath tumors. Zebrafish injected with an unrelated TALEN targeting a zinc transporter (SLC30A10), into p53^m214k mutant embryos do not show any increase in tumor formation compared to uninjected controls. Notably several RP’s have been shown to be haploinsufficient tumor suppressor genes in their own right in zebrafish and drosophila models. To determine if the early tumor development in p53^m214k zebrafish was simply additive to a potential tumor suppressor effect of Rps14 or Rps19 alone, we injected WT embryos with the rps14 and rps19 TALENS. High mortality in rps14 and rps19 TALEN injected WT embryos impeded this analysis; however recent published reports on stable Rps19 mutant zebrafish do not report an increase in tumor incidence. Interestingly, embryo survival was not affected when TALENs were injected into p53^m214/+. Analysis of these zebrafish is ongoing.

Our results show that loss of Rps14 or Rps19 accelerates the development of tumors in the p53^m214k/m214k mutant line. This effect is independent of the RP or the method of mutation (TALEN vs CRISPR), indicating that off target effects are unlikely to be responsible for this observation. As these are mosaic F0 fish, it is possible that tumors may arise from cells with homozygous RP mutations. Further molecular analysis will reveal this. We have now identified 2 stable Rps19 mutant lines, and tumor analysis of F1 fish from these lines is ongoing. In conclusion, we have shown that loss of Rps14 or Rps19 cooperates with a loss of function p53 mutation to accelerate tumor formation and death. Our results highlight the importance of caution in using p53 suppressors as a therapeutic option in RP deficient patients.