Atypical Teratoid/Rhabdoid Tumors are rare, highly aggressive, paediatric tumours with a dismal prognosis. Rhabdoid Tumours (RT) have one key genetic defect (biallelic inactivation of SMARCB1 in > 90% of patients) which, when re-expressed in RT cells, causes differentiation and growth arrest. We used whole genome CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 screening; a technique by which mutations are created systematically in each gene in the genome - to identify novel SMARCB1-dependent therapeutic targets and synthetic lethal relationships in RT.
A panel of RT cell lines were transduced with a GeCKO (Genome Scale CRISPR Knock-Out) library which contains 122,441 guide constructs targeting 19,050 genes and 1,864 miRNAs; each cell contained a single CRISPR insert. This panel was further transduced with a SMARCB1 expression lentivirus or an empty vector control, cells were collected at 0, 7 and14 days post infection (300x library coverage, 3.7 x 107 cells per time point).
CRISPR inserts were sequenced, counted, normalized and analyzed to identify those whose abundance was significantly altered, representing candidate gene targets which promote or inhibit RT cell growth/viability in a SMARCB1-dependent manner. CRISPR hits were further cross-validated with primary patient and functional model genomic data (RNA-sequencing/450k DNA methylation array) to identify several high confidence deleterious events with strong SMARCB1 dependency.
Hundreds of significantly enriched (both positively and negatively) genes were identified which alter when SMARCB1 is present or absent including MYC family targets, cell cycle targets, SHH pathway and the Aurora kinase family. TGFβ was identified as a top upstream SMARCB1/methylation dependent regulator and when SMARCB1 re-expression data was overlaid onto the TGFβ canonical pathway showed a convincing enrichment in pathway regulation. Pathway members SMAD 2/3 showed a ‘synthetic lethal’ phenotype and represent a functional SMARCB1-dependent relationship which is also observed in SMARCB1 re-expression and methylation model data and is characteristic of primary RT. This phenotype was validated functionally by expression of a dominant negative TGFBR2.
Combining RNA-sequencing and 450K DNA methylation microarray data from primary tumours and experimental models, coupled with whole-genome functional screening, provides a genome-wide view of key RT tumorigenic genes/pathways and their SMARCB1 dependence. These data suggest synthetic lethal phenotypes and enable prioritisation of true SMARCB1-dependent events and therapeutic targets, in this rare yet lethal paediatric tumour.
Citation Format: Matthew P. Selby, Martina A. Finetti, Matthew Bashton, Ruth E. Cranston, Yura Grabovska, Alicia Del-Carpio-Pons, Amanda Smith, Simon Bailey, Steven C. Clifford, Daniel Williamson. Investigating the biology of atypical teratoid/rhabdoid tumors by whole genome CRISPR/Cas9 screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 408. doi:10.1158/1538-7445.AM2017-408