The nucleolus is the site for RNA polymerase I (Pol) transcription of ribosomal RNA genes and ribosome subunit assembly. Inhibitors of Pol I transcription CX-5461, BMH-21 and PMR116 have demonstrated therapeutic benefits in various preclinical cancer models and in Phase I clinical trials. Our work has shown that Pol I transcription inhibition induces nucleolar stress, leading to cell cycle arrest and cell death in ovarian cancer models1,2. Importantly, CX-5461 and PMR116 exhibit unique sensitivity profiles compared with chemotherapeutics1-3. Thus, we propose that activating nucleolar stress represents a promising new therapeutic approach.
To identify novel nucleolar stress pathways, we conducted an innovative arrayed whole-genome CRISPR-Cas9 screen to identify genes whose deletion causes changes in nucleolar morphology as an indicator of nucleolar stress. We identified and classified five distinct types of nucleolar stress phenotypes that are linked to the inhibition of specific biological processes, including ribosome biogenesis, DNA repair, cell cycle, and RNA metabolism. Our data highlight the tight coordination between these processes and nucleolar fidelity and defines inducers of a potent nucleolar stress phenotype linked to cancer cell growth inhibition. Moreover, we have identified and characterised a new role for a protein phosphatase in the regulation of Pol I transcription.
To complement this screen and identify drugs that target the nucleoli, we completed a high-throughput drug screen of 23,000 compounds utilising the same screening approach, to identify compounds that trigger nucleolar stress and inhibit ovarian cancer cell growth. Cyclin dependent kinases (CDKs), topoisomerases and novel compounds were identified as candidate hits that induce potent nucleolar stress. In summary, our work uncovered specific pathways/factors as mediators of nucleolar stress and potential cancer therapeutic targets.