Gain-of-function screens as a discovery tool for regulators of response to ionising radiation and novel drug targets
1The Crick Institute, London, UK,2The UCL Cancer Institute, London, UK,3Cancer Research Technology Technology, London, UK
Radiotherapy, given with concurrent chemotherapy, remains the standard of care for patients with unresectable non-small cell lung cancer (NSCLC). Previous efforts to sensitize tumour cells to ionizing radiation and improve the tumour control probability have focused predominantly on modulation of the DNA damage response.
The primary objective of this study is to use a transposon based system for unbiased gain-of-function screens in NSCLC cell lines for discovery of genes involved radiation resistance. Transposons are mobile DNA elements that allow functional genomic screens. Using a piggyBac transposon system, which is highly efficient in mammalian cell lines, we have developed a system to switch on expression of genes located downstream of the transposable element. Identification of transposon integration sites can be determined with Sanger or Next Generation Sequencing following Splinkerette PCR. We are able to directly validate transposon-mediated activation of genes with RT-PCR using a unique sequence tag in transcripts expressed from the transposon promoter. The current workflow permits collection of surviving colonies following ionising radiation, enabling a resource for subsequent molecular studies on radio-resistance mechanisms. A panel of NSCLC are currently being screened to generate recurrent overlapping hits.
Results from a pilot screen in the A549 cell lines have generated initial hits:
- RNF168 which ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signalling
- SCMH1 which has E3 Ubiquitin ligase activity for Histone H2A and for Geminin which is a regulator of DNA replication and mitotic progression via the APC/C
- KIAA0195 an uncharacterised transmembrane protein
Further data from genome wide screen coverage in multiple cell lines will be presented. Using the results from the TRACERx - Tracking Lung Cancer Evolution Through Therapy (NCT01888601) observational study we will assess the frequency of activating mutations and copy number gains in screen hits and their spatial heterogeneity to assess in-vivo relevance.