While ribosomal proteins are generally considered to be essential genes, the existence of ribosomopathies suggests that a therapeutic window for exploiting ribosomal proteins as targets for cancer therapy may exist. We report distinct stress responses of tumor cells and normal cells upon knockdown of Rpl15, a ribosomal protein which was identified in a direct in vivo shRNA screen for new therapeutic targets in liver cancer. While normal cells undergo a reversible cell cycle arrest, we found that in tumor cells, the knockdown of Rpl15 triggers a ribosomal stress response followed by induction of cellular senescence, designated ribosomal checkpoint induced senescence (RCIS). Importantly, Rpl15 suppression triggered RCIS independent of any reduction in global protein translation. Using well established therapy resistant murine HCC models, we show that shRNAs targeting Rpl15 potently suppress tumor development in genetically diverse murine HCCs. Using Rpl15 shRNA transgenic mice, allowing for ubiquitous shRNA mediated suppression of Rpl15, we show that systemic Rpl15 suppression can be tolerated for up to 5 days, thus revealing a therapeutic window for metronomic Rpl15 inhibitory therapies. However, molecular modeling analyses revealed that Rpl15 is not druggable by small molecule inhibitors and we thus set out to explore whether RCIS can be induced via interference with other ribosomal proteins or other factors involved in ribosome biogenesis. We generated and screened a focused shRNA library targeting 41 ribosomal proteins and 19 ribosome biogenesis factors and found that apart from Rpl15, only a small subset of shRNAs scored. From a druggability point of view it was interesting, that shRNAs targeting components of the RNA polymerase I complex had scored, as recently a pharmacological RNA polymerase I inhibitor became available. Both genetic and pharmacological inhibition of RNA polymerase I induced RCIS and mediated an excellent prolongation of survival (far superior to the clinically used standard therapy Sorafenib), however in contrast to targeting Rpl15, targeting RNA polymerase I failed to achieve full long term tumor suppression. Mechanistically we found a less efficient immune mediated clearance of senescent cells as a possible explanation. In line with the idea that distinct secretory profiles underlie differences in immune mediated clearance of RCIS cells, cytokine arrays revealed distinct secretory profiles of RCIS induced by Rpl15 suppression and RCIS induced via shRNA or pharmacological RNA polymerase I inhibition.
Citation Format: Katharina Wolter, Marina Pesic, Sabrina Klotz, Nicolas Herranz, Torsten Wuestefeld, Tae-Won Kang, Marco Seehawer, Rishabh Chawla, Stefan Zwirner, Jonathan Cotton, Benyuan Zhou, Marcel Krüger, Frank Klawonn, Thomas Longerich, Bence Sipos, Bernd Pichler, Jesus Gil, Martin Eilers, Prem K. Premsrirut, Antti Poso, Lars Zender. Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-335. doi:10.1158/1538-7445.AM2017-LB-335