Cell-based assay directly monitoring RNA Polymerase I activity for cancer drug discovery

Project Abstract/Summary Cancer is the second leading cause of death in the US, with ~600k cancer related deaths and 1.7 million new cases of cancer diagnosis expected in 2019. There have been significant advances in cancer detection and treatment, including radiotherapy, surgery, and immunotherapy, leading to an overall decrease in cancer mortality. However, the heterogeneity and mutational resistance of cancers has prevented the widespread success of targeted therapies and anti-cancer agents. While a significant number of new cancer therapeutics have been FDA approved, analysis of their benefits found no increase in efficacy or a reduction in toxicity for a majority of them. Current chemotherapeutics are widely known to have negative side-effects due to off-target interactions. DNA-dependent RNA Polymerase I (Pol I) is responsible for transcription of rRNA, which is absolutely essential for ribosome function. Cancer cells are well known to upregulate Pol I transcription in order to significantly increase ribosome biogenesis, responsible for the uncontrolled cell growth and proliferation that are hallmarks of cancer. Despite the ubiquitous requirement for ribosomes by living cells, untransformed cells tolerate Pol I inhibition well due to activation of cellular checkpoints that halt cell cycle progression. Cancer cells lack or deregulate these checkpoints. This suggests that Pol I is not only an excellent target for anti-cancer therapeutics but inhibition of Pol I would be minimally toxic. However, current technology lacks the ability to easily or directly monitor Pol I transcription. Thus, our goal is to develop a new cell-based HTS platform capable of directly monitoring Pol I transcription. The use of internal controls monitoring the other DNA-dependent RNA polymerases will allow for identification of Pol I specific inhibitors in a single assay. We plan to use our proprietary RNA aptamer technology to develop a reporter under control of the Pol I promoter. In addition, we will utilize a fluorescent protein to monitor Pol II transcription and a second fluorescent RNA aptamer to monitor Pol III transcription. Multiplexing will allow identification of RNA Pol I specific inhibitors, ensuring that hits identified are potent and relatively non-toxic. Phase II of this application will involve a comprehensive stable cell line expressing all three RNA polymerase reports in various cancer specific cell lines based off the design knowledge obtained from this Phase I grant.Project Narrative Tens of millions of people in the US will be diagnosed with cancer in 2019 and there will be ~600k cancer related deaths. We propose to develop a cell-based assay that monitors RNA Polymerase I transcriptional activity in order to facilitate identification of efficacious anti-cancer agents with low toxicity. Successful development of the assay will identify therapeutics specific for RNA Pol I by using internal controls, and the modular assay can be used to test for therapeutics in various cancer cell-types.