Abstract

Frequent Hitters: A Challenge for Phenotypic Screens

Christian Parker, Lab Head, Novartis Institute For Biomedical Research

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription co-activators YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. Recently we reported on an siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription co-activator at high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays. This presentation will review the nature of frequent hitters in biochemical and cell based assay and compare such compounds to gene targets that appear as frequent hitters in genetic screens.