Person:

Choi, Peter

Genomic rearrangements are frequently observed in cancer cells but have been difficult to generate in a highly specific manner for functional analysis. Here we report the application of CRISPR/Cas technology to successfully generate several types of chromosomal rearrangements implicated as driver events in lung cancer, including the CD74-ROS1 translocation event and the EML4-ALK and KIF5B-RET inversion events. Our results demonstrate that Cas9-induced DNA breaks promote efficient rearrangement between pairs of targeted loci, providing a highly tractable approach for the study of genomic rearrangements.

Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35) have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA). Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML), in which U2AF1 is somatically mutated in 3–4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3′ splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3′ splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency. DOI: http://dx.doi.org/10.7554/eLife.23268.001

Whole genome analysis approaches are revealing recurrent cancer-associated somatic alterations in non-coding DNA regions. We combined somatic copy number analysis of 12 tumor types with tissue-specific epigenetic profiling to identify significant regions of focal amplification harboring super-enhancers. Copy-number gains of non-coding regions harboring super-enhancers near KLF5, USP12, PARD6B and MYC are associated with over-expression of these cancer-related genes. We show that two distinct focal amplifications of super-enhancers 3′ to MYC in lung adenocarcinoma (MYC-LASE) and endometrial carcinoma (MYC-ECSE), are physically associated with the MYC promoter and correlate with MYC over-expression. CRISPR/Cas9-mediated repression or deletion of a constituent enhancer within the MYC-LASE region led to significant reductions in the expression of MYC and its target genes, and to the impairment of anchorage-independent and clonogenic growth, consistent with an oncogenic function. Our results demonstrate that genomic amplification of super-enhancers represents a common mechanism to activate cancer driver genes in multiple cancer types.

High cancer death rates indicate the need for new anti-cancer therapeutic agents. Approaches to discover new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds by phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells while others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggesting a neomorphic activity. Co-expression of SLFN12 with PDE3A correlates with DNMDP sensitivity, while depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.