Investigating the mechanism underlying the nucleolar surveillance pathway in disease

The nucleolus is not only a hub for ribosome biogenesis, but also a sensor of cellular stress. When the nucleolus detects stress (e.g. ribosome biogenesis perturbations), it activates the 'nucleolar surveillance pathway' (NSP) which maybe dependent/independent of p53. Perturbed ribosome biogenesis is also associated with diseases such as cancer and ribosomopathies (diseases of the ribosome). Diamond-Blackfan Anaemia (DBA) is a rare blood cell aplasia that presents clinically at approximately 2-3 months of age and its main characteristic is reduced erythroid precursors in the bone marrow, i.e. anemia. Mutations in different ribosomal protein (RP) genes have been associated with DBA, with mutations in RPS19 accounting for 25% of cases. It has been proposed that RPS19 deficiency causes perturbations in ribosome biogenesis, thus activation of the p53-dependent NSP. In this context free RPs (predominantly L5 and L11) in a complex with 5S rRNA sequester the E3 ubiquitin ligase murine double minute 2 (MDM2), leading to the accumulation of p53 and subsequent activation of its transcriptional targets mediating cell cycle arrest or apoptosis. In order to identify potential therapeutics that could be repurposed to prevent the activation of NSP in DBA patients, we have screened libraries of clinically approved therapeutics to identify pathways implicated in the p53-dependent NSP due to RPS19 deficiency. We quantitated cell number and the level of p53 expression, identifying compounds that can result in low and high expression of p53, the latter for potential use in cancer therapy. In the DBA context, we were interested in the compounds that reduced p53 and had no negative effect on cell number. A selection of 25 molecules were re-evaluated in vitro through the quantification of p53 protein expression and densitometry analysis. Then, 10 candidates were evaluated ex vivo for their effects on proliferation using bone marrow from the inducible shRps19-D mouse model. Our best candidates, Digoxin and Leflunomide were then evaluated in vivo. In this thesis we show the first in vivo evidence that the cardiac glycoside Digoxin may be used as a potential treatment for bone marrow failure, such as DBA. In our DBA mouse model, Digoxin treatment in mice heterozygous for shRps19 (D/+) significantly increased the number of LSK cells, no changes in CFU-Es or proerythroblasts but interestingly a reversal of the defect in orhtoerythroblasts and reticulocytes and strikingly in the GMP population. The improvement in LSK cells was further confirmed with the study of the colony forming unit capacity of progenitor cells (BFU-E, CFU-GM and CFU-GEMM). We also present evidence that the immunosuppressant Leflunomide, increases the number of proerythroblast in anaemic mice. While our compound screen was originally designed to identify molecules that prevent the activation of the NSR and p53, the analysis and in vitro validation revealed compounds that enhanced p53 protein levels in our A549 model. In the cancer context, we were interested in compounds that enhance p53 and reduced cell number. Compounds with those characteristics were the topoisomerase I inhibitor Camptothecin and the CDK9 inhibitor Flavopiridol. We have combined these two molecules with the Pol I transcription inhibitor CX-5461, currently in clinical trials, which also induces the NSR and has demonstrated to be an efficacious therapy to treat malignancies like acute myeloid leukaemia (AML). In this thesis we demonstrate the ability of CX-5461 to act synergistically with first generation (Flavopiridol) and second generation (Dinaciclib) CDK9 inhibitors both in vitro and in vivo. Our results provide evidence that targeting Pol I and Pol II transcription has a potential to treat aggressive AML in patients. In summary, via screening of FDA compounds we have identified a selection of compounds which are promising candidates for the treatment of ribosomopathies and cancer in the clinic