SPOT-009 Identification of novel hippo pathway regulators using a genome wide CRISPR screen

Abstract

Introduction The Hippo pathway regulates tissue growth and organ size and its dysregulation/inactivation leads to a host of cancers such as breast, colon and bladder cancer. YAP/TAZ are transcriptional regulators which among other functions enhance cell growth. The core kinase cassette consisting of LATS1/2, MST1/2, SAV and Mob phosphorylate YAP and TAZ promoting their cytoplasmic localization and degradation. Currently much remains to be learned about the upstream regulators that modulate the pathway. The purpose of this study was to carry out a genome wide positive selection CRISPR screen to uncover novel upstream regulators of the Hippo pathway. For the screen it is necessary to generate stable cell lines with a reporter responsive to YAP/TAZ. This was carried out by expressing a fusion protein where the catalytic domain of Caspase-9 is fused to a modified FKBP (FK506 binding domain) under the transcriptional control of YAP/TAZ. This protein is homodimerized by the addition of a drug AP20187 which triggers an apoptotic pathway leading to cell death. When YAP/TAZ activity is disrupted, cells are expected to survive, despite addition of AP20187. The cell lines are also designed to express Cas9 which will be used to knockout the genes in the human genome using a 90K guide RNA library targeting over 17 500 human genes. Material and methods T24 bladder cancer cells were maintained in McCoy’s 5A medium with 10% FBS. Lentiviral constructs of Cas9 and FKBP-Caspase9 were obtained (courtesy Dr. Angers) and the YAP/TAZ responsive FKBP-Caspase9 was constructed by restriction digestion and ligation. Using lentivirus T24 cells were transduced with both constructs and single clones were isolated expressing both FKBP-Caspase9 and Cas9 to obtain the screen cell line. The dimerizer AP20187 was obtained from Clontech. The lentiviral library TKO-v1 (gift from Dr. Jason Moffat) was applied to the T24 screen cell line using lentiviral transduction. After 3 days of puromycin selection, the pool of surviving cells were split into replicates and AP20187 was added to one set of replicates while the other set was treated with ethanol as control. Once the AP20187 treated plates were confluent, the cells were collected, genomic DNA was extracted and sequenced by next generation sequencing. Results and discussions The top genes enriched in the surviving cells included well known members of the Hippo pathway including YAP and TAZ along with new proteins. Conclusion These new proteins are currently being investigated for their role in the Hippo pathway.