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.
