Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention.

The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells1,2. However, previous studies reported that a gene-independent anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, leading to false positive results in copy number amplified regions3,4. We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect. As part of our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this dataset. We found that CERES reduced false positive results and estimated sgRNA activity for both this dataset and previously published screens performed with different sgRNA libraries. Here, we demonstrate the utility of this collection of screens, upon CERES correction, in revealing cancer-type-specific vulnerabilities.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma. Significance Biomarkers of response to small-molecule inhibitors of BET bromodomains, a new compound class with promising anti-cancer activity, have been lacking. Here, we reveal MYCN amplification as a strong genetic predictor of sensitivity to BET bromodomain inhibitors, demonstrate a mechanistic rationale for this finding, and provide a translational framework for clinical trial development of BET bromodomain inhibitors for pediatric patients with MYCN-amplified neuroblastoma.

The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions.