Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29^-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29^-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29^-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29^-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP.