BACR 9: Investigating the biology of high-risk medulloblastoma by targeted siRNA and whole genome CRISPR screening.

Matthew Selby1,Peter Banks1,Ruth Cranston1,Martina Finetti1,Matthew Bashton1,Daniel Williamson1,Steven Clifford1

1Newcastle University, Newcastle Upon Tyne, UK

Presenting date: Monday 2 November
Presenting time: 12.20-13.10

Background

Central Nervous System tumours are the second most prevalent cancers of childhood. Despite our deeper understanding of the underlying biology and advances in treatment, high-risk paediatric brain tumours still have a particularly poor prognosis. Medulloblastoma has in recent years been categorised into four distinct subgroups (WNT, SHH, Group 3 and Group 4) with differing molecular and clinical features (Taylor et al., 2012). Group 3 medulloblastoma (MBGRP3) have the poorest prognosis and whilst modern genomic techniques have generated large primary datasets, there have been no defining pathways identified which are therapeutically targetable.

Method

We are employing two functional genomic screening techniques to identify potential therapeutic targets within a panel of MBGRP3 cell lines.


• A targeted siRNA screen to MBGRP3 specific candidate genes (Group 3 specific DNA methylation changes, copy number aberrations, transcriptomic effects and common mutations) using small interfering RNA (siRNA) to identify genes and pathways of functional therapeutic relevance.

• A GeCKO [Genome Scale CRISPR (clustered regularly interspaced short palindromic repeats) Knock-Out] (Sanjana et al., 2014; Shalem et al., 2014) screen identifying how long term gene knock out affects cellular phenotype.

We also outline a bioinformatic pipeline for the handling of whole genomic CRISPR screening data and how this can be integrated back to a more classical gene editing screen.

Results

Here we present the initial findings of two cost effective screening techniques, of multiple types of molecular alterations, using MBGRP3 as an exemplar. We have identified potentially functionally important genes from a panel of MBGRP3 cell lines and are in the process of validating these findings. We also present the development, quality controls and bioinformatics required in performing a whole genome CRISPR screen.

Conclusion

These screens will further understanding of the underlying molecular biology and identify the best candidate genes/pathways for therapeutic targeting of MBGRP3. Using two high-throughput screening techniques allows cross-validation between technologies and the integration of primary data from our extensive cohort.