Peter S. Choi

Targeted genomic rearrangements using CRISPR/Cas technology

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.

Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

Whole-genome analysis approaches are identifying recurrent cancer-associated somatic alterations in noncoding 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 noncoding regions harboring super-enhancers near KLF5, USP12, PARD6B and MYC are associated with overexpression 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 overexpression. 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 suggest that genomic amplification of super-enhancers represents a common mechanism to activate cancer driver genes in multiple cancer types.

A Pan-Cancer Analysis of Transcriptome Changes Associated with Somatic Mutations in U2AF1 Reveals Commonly Altered Splicing Events

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.

Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics

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.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability

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

Somatic Superenhancer Duplications and Hotspot Mutations Lead to Oncogenic Activation of the KLF5 Transcription Factor

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5-FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type-specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108-25. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1.

MET exon 14 mutation encodes an actionable therapeutic target in lung adenocarcinoma

Targeting somatically activated oncogenes has revolutionized the treatment of non-small cell lung cancer (NSCLC). Mutations in the gene mesenchymal-epithelial transition (MET) near the exon 14 splice sites are recurrent in lung adenocarcinoma and cause exon skipping (METΔ14). Here, we analyzed 4,422 samples from 12 different malignancies to estimate the rate of said exon skipping. METΔ14 mutation and transcript were most common in lung adenocarcinoma. Endogenously expressed levels of METΔ14 transformed human epithelial lung cells in a hepatocyte growth factor-dependent manner. In addition, overexpression of the orthologous mouse allele induced lung adenocarcinoma in a novel, immunocompetent mouse model. Met inhibition showed clinical benefit in this model. In addition, we observed a clinical response to crizotinib in a patient with METΔ14-driven NSCLC, only to observe new missense mutations in the MET activation loop, critical for binding to crizotinib, upon clinical progression. These findings support genomically selected clinical trials directed toward METΔ14 in a fraction of NSCLC patients, confirm second-site mutations for further therapeutic targeting prior to and beyond acquired resistance, and provide an in vivo system for the study of METΔ14 in an immunocompetent host. Cancer Res; 77(16); 4498-505. ©2017 AACR.

An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

Genome-scale analysis identifies paralog lethality as a vulnerability of chromosome 1p loss in cancer

Functional redundancy shared by paralog genes may afford protection against genetic perturbations, but it can also result in genetic vulnerabilities due to mutual interdependency1–5. Here, we surveyed genome-scale short hairpin RNA and CRISPR screening data on hundreds of cancer cell lines and identified MAGOH and MAGOHB, core members of the splicing-dependent exon junction complex, as top-ranked paralog dependencies6–8. MAGOHB is the top gene dependency in cells with hemizygous MAGOH deletion, a pervasive genetic event that frequently occurs due to chromosome 1p loss. Inhibition of MAGOHB in a MAGOH-deleted context compromises viability by globally perturbing alternative splicing and RNA surveillance. Dependency on IPO13, an importin-β receptor that mediates nuclear import of the MAGOH/B-Y14 heterodimer9, is highly correlated with dependency on both MAGOH and MAGOHB. Both MAGOHB and IPO13 represent dependencies in murine xenografts with hemizygous MAGOH deletion. Our results identify MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types and suggest a rationale for targeting the MAGOHB-IPO13 axis in cancers with chromosome 1p deletion.Analysis of paralog gene pairs using data from loss-of-function genetic screens in cancer cells identifies MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types.

Abstract A36: Genome-scale ORF screening to identify modulators of the epithelial-to-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is an essential developmental program and is often reactivated during tumor initiation and progression. EMT is a reversible reprogramming of the cells. It not only promotes cell morphology alterations, but also provokes a profound cell state change in multiple regulatory circuits in global cell signaling, transcriptional and post-transcriptional modifications. Among all breast cancer subtypes, basal-like breast cancer displayed a high degree of mesenchymal and stem-like cell properties. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen in human mammary epithelial cells. We used CD44 cell surface protein as a marker for cells reside in mesenchymal and stem-like cell state. CD44 low cells were presorted and induced with a barcoded genome-scale ORF library. After seven days of propagation, cells converted to CD44 high state were sorted by flow cytometry, and the barcodes enriched in the CD44 high population compared to the cells in the CD44 low population were evaluated with next-generation sequencing. Interestingly, 68 genes scored 3 standard deviations above the mean, including transcriptional factors, RNA splicing factors, epigenetic regulators, kinases/phosphatases, spermatogenesis regulators and amide metabolic modifiers. Strikingly, gene ontology and gene set enrichment analysis showed that RNA splicing process was the most significantly enriched biological process. We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. After performing these assays, we discovered the splicing factors/RNA binding proteins, QKI and RBFOX1, are both necessary and sufficient to promote EMT and stem-like states. We subsequently investigated the downstream targets of these splicing factors by RNA-sequencing analysis. Further, using biochemical and genetic approaches, we characterized the functional roles of these splicing factors and their downstream targets. In summary, alternative pre-mRNA splicing plays a key role in the regulation of EMT. The molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. Genome-scale ORF screening to identify modulators of the epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A36.