Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/ SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancerselective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

Epigenetic dysregulation is an emerging hallmark of cancers, and the identification of recurrent somatic mutations in chromatin regulators implies a causal role for altered chromatin states in tumorigenesis. As the majority of epigenetic mutations are inactivating and thus do not present directly druggable targets, we reasoned that these mutations may alter the epigenomic state of cancer cells and thereby expose novel epigenetic vulnerabilities. To systematically search for epigenetic synthetic lethal interactions, we performed a deep coverage pooled shRNA screen across a large collection of cancer cell lines using a library targeting a diverse set of epigenetic regulators. Strikingly, this unbiased screen revealed that silencing of the SWI/SNF ATPase subunit BRM/SMARCA2, selectively inhibits the proliferation of BRG1-deficient cancer cells. The mammalian SWI/SNF complexes (mSWI/SNF) regulate chromatin structure through ATP-dependent nucleosome remodeling. Recent cancer genome studies have revealed a significant frequency of mutations in several components of the mSWI/SNF complexes including loss of the catalytic subunit BRG1 in non-small cell lung cancers. Our studies reveal that BRM knockdown selectively induced cell cycle arrest in BRG1-mutant cancer cells and significantly impaired the growth of BRG1-mutant lung tumor xenografts. BRM is the paralog of BRG1, suggesting a model in which mSWI/SNF mutations lead to a hypomorphic complex that promotes tumorigenesis but cannot tolerate complete inactivation. Therefore, our studies present BRM as an attractive therapeutic target in BRG1-mutant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR06. Citation Format: Zainab Jagani, Gregory Hoffman, Rami Rahal, Frank Buxton, Gregory McAllister, Kay Xiang, Elizabeth Frias, Janina Huber, Alicia Lindeman, Dongshu Chen, Linda Bagdasarian, Rodrigo Romero, Nadire Ramadan, Tanushree Phadke, Kristy Haas, Mariela Jaskelioff, Boris Wilson, Matthew Meyer, Margaret E. McLaughlin, Charles WM Roberts, Vic Myer, Jeff Porter, Nicholas Keen, Craig Mickanin, Frank Stegmeier. A functional screen of the epigenome identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR06.

The first approach is aimed at approximately 26% of patients that have point mutations in DMD gene. Here we used gene editing to correct DMD E2035X, a pathogenic mutation in DMD patient myoblasts by designing short RNA sequences complementary to the locus called guides, paired with the Streptococcus pyogenes Cas9. We were able to generate targeted double stranded DNA breaks that were corrected through homology directed repair using single stranded oligos carrying the corrected DNA sequence. Other pathogenic DMD mutations are duplications, which are found in approximately 13% of DMD patients. Utilizing a modified CRISPR strategy that involves a double nuclease approach we deleted a 145 kb (Exons 18-30) duplication and restored dystrophin expression in patient cells. Furthermore, as an universal, mutation-independent treatment approach, we have also utilized a third modified CRISPR system to upregulate utrophin expression to compensate for the loss of dystrophin. In DMD patient myoblasts, we utilized S.pyogenes Cas9, fused to transcriptional activator VP160 to target utrophin promoter. We demonstrated 2.5-5 fold utrophin upregulation resulting in increased expression of β-dystroglycan, providing evidence for functional significance of this strategy.Lastly, in order to determine if this tool is of potential clinical significance, we have began to interrogate CRISPR/Cas9 system in dystrophin-deficient mdx mouse. We have developed strategies for the delivery of CRISPR machinery via rAAV with the goal to achieve dystrophin editing and utrophin upregulation simultaneously in vivo. Taken together, our proof of concept studies provide insights into the potentially far-reaching impact of the therapeutic benefits of the CRISPR system that can be adapted to target a variety of mutations in DMD.

S230can be expressed in both pluripotent stem cells and differentiated cells from all three major germ layers. This allows for the real-time observation of mutations causing phenotypic differences that can be measured in in vitro tissues and organoids during culture as opposed to defined end points. The live cell and tissue assays on ArrayEdit are compatible with standard screening platforms and a variety of human cell types, thus permitting robust and sensitive detection of differences in proliferation, differentiation and other downstream effects of gene editing. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.