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Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

Supplementary Figure S1. Genome-scale CRISPR-Cas9 screening identifies a strong correlation between copy number and sensitivity to CRISPR-Cas9 genome editing. Supplementary Figure S2. Global summary of the relationship of CRISPR-Cas9 guide scores to genomic copy number for all 33 cell lines screened. Supplementary Figure S3. Amplified genes represent the strongest perceived dependencies in pooled CRISPR-Cas9 screening data. Figure S4. Evaluation of the influence of copy number on CRISPR-Cas9 dependency scores in published data from Hart et al. (25). Figure S5. Representative examples of structural variations leading to copy number amplification underlying the gene-independent anti-proliferative response to CRISPRCas9 targeting of loci within these regions.

Sample Information: Table with cell line identities and relevant information for this manuscript.

Supplementary Methods

Glucose is a universal energy currency for living organisms, however, its non-energetic functions in processes such as differentiation are undefined. In epidermis, differentiating cells exhibit dynamic changes in gene expression1–4driven by specific transcription factors (TFs)5–9. The interplay between such TFs and biomolecules that also change in this process is not understood. Metabolomic analyses revealed that increased intracellular glucose accompanies differentiation of epidermal keratinocytes. This elevation also occurred in differentiating cells from other tissues and was verified in epidermal tissue engineered with glucose sensors, which detected a glucose gradient that peaked in the outermost differentiated layers. Free glucose accumulation, unaccompanied by its increased metabolism, was essential for epidermal differentiation and required GLUT1, GLUT3, and SGLT1 transporters. Glucose affinity chromatography and azido-glucose click chemistry uncovered glucose binding to diverse regulatory proteins, including the IRF6 TF, whose epidermal knockout confirmed its requirement in glucose-dependent differentiation. Direct glucose binding enabled IRF6 dimerization, DNA binding, genomic localization, and induction of IRF6 target genes, including essential pro-differentiation TFsGRHL1, GRHL3, HOPXandPRDM1. The IRF6R84Cmutant found in undifferentiated cancers was unable to bind glucose. These data identify a new role for glucose as a gradient morphogen that modulates protein multimerization in cellular differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

The easyCLIP protocol describes a method for both normal CLIP library construction and the absolute quantification of RNA cross-linking rates, data which could be usefully combined to analyze RNA-protein interactions. Using these cross-linking metrics, significant interactions could be defined relative to a set of random non-RBPs. The original easyCLIP protocol did not use index reads, required custom sequencing primers, and did not have an easily reproducible analysis workflow. This short paper attempts to amend these deficiencies. It also includes some additional technical experiments and investigates the usage of alternative adapters. The results here are intended to allow more options to easily perform and analyze easyCLIP.

A central question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into 'co-essential' pathways, but this approach has been limited by ubiquitous false positives. In the present study, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This atlas recapitulates diverse pathways and protein complexes, and predicts the functions of 108 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, a key enzyme for plasmalogen synthesis. We also show that C15orf57 encodes a protein that binds the AP2 complex, localizes to clathrin-coated pits and enables efficient transferrin uptake. Finally, we provide an interactive webtool for the community to explore our results, which establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of new gene functions.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models., T- and NK-cell lymphomas (TCL) are a group of lymphoid malignancies characterized by poor prognosis, but the absence of appropriate pre-clinical models has hampered the development of effective therapies. Here the authors establish several pre-clinical models and identify vulnerabilities that could be further exploited to treat patients afflicted by these diseases.

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1-infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

SUMMARYA central remaining question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into ‘co-essential’ pathways, but this approach has been limited by ubiquitous false positives. Here, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This almanac recapitulates diverse pathways and protein complexes and predicts the functions of 102 uncharacterized genes. Validating top predictions, we show thatTMEM189encodes plasmanylethanolamine desaturase, the long-sought key enzyme for plasmalogen synthesis. We also show thatC15orf57binds the AP2 complex, localizes to clathrin-coated pits, and enables efficient transferrin uptake. Finally, we provide an interactive web tool for the community to explore the results (coessentiality.net). Our results establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of novel gene functions.

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 antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. Significance: We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914–29. ©2016 AACR. See related commentary by Sheel and Xue, p. 824. See related article by Munoz et al., p. 900. This article is highlighted in the In This Issue feature, p. 803

High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

SummaryRAG initiates antibody V(D)J recombination in developing lymphocytes by generating “on-target” DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG “off-target” breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

Summary: Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with “quiet” genomes. : Using a diverse set of rhabdoid tumor cell lines and both small-molecule and CRISPR-Cas9 gene-knockout screening, Oberlick et al. find high expression and dependency upon a wide range of receptor tyrosine kinases (RTKs) and SHP2 downstream. These RTK inhibitors are also effective against a rhabdoid tumor mouse model. Keywords: rhabdoid tumors, SMARCB1, high-throughput drug screening, genome-wide CRISPR screening, receptor tyrosine kinase, PTPN11

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 cells. However, previous studies reported that an anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, particularly in the setting of copy number gain1-4. We performed genome-scale CRISPR-Cas9 essentiality screens on 342 cancer cell lines and found that this effect is common to all lines, leading to false positive results when targeting genes in copy number amplified regions. 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 well as variable sgRNA activity. We applied CERES to sets of screens performed with different sgRNA libraries and found that it reduces false positive results and provides meaningful estimates of sgRNA activity. As a result, the application of CERES improves confidence in the interpretation of genetic dependency data from CRISPR-Cas9 essentiality screens of cancer cell lines.

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In 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 data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.

Summary To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer., Graphical Abstract, Highlights • Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI • Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges • Distinct ligand-receptor and spatial niche associations for tumor and stromal cells. • Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening, Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.

Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

SummaryActivation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.

Mature IgM(+) B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM(+) B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination-associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas.

Primary B-cell development is thought to be restricted to the bone marrow; here it is shown to occur also in intestinal tissues of postnatal mice, that it peaks at the time of weaning and is increased upon colonization of germ-free mice, and is thus influenced by commensal microbes. Primary B-cell development is thought to be restricted to the bone marrow, but here Frederick Alt and colleagues present the surprising finding that it also occurs in the gut, where it is stimulated by the gut microbes. The authors describe a population of early B-lineage cells developing within the intestinal mucosa — specifically in the lamina propria — of postnatal mice. B-cell production peaks at the time of weaning and is increased upon colonization of germ-free mice. The repertoire of these B cells differs from that of cells derived from the bone marrow, and may be shaped by commensal microbes. The RAG1/RAG2 endonuclease (RAG) initiates the V(D)J recombination reaction that assembles immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B-cell antigen-binding receptor (BCR). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3,4,5. This ‘receptor editing’ process, which can also lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen encounter at the Rag-expressing immature B-cell stage helps shape pre-immune BCR repertoires. As the major site of postnatal B-cell development, the bone marrow is the principal location of primary immunoglobulin repertoire diversification in mice. Here we report that early B-cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP immunoglobulin repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B-lineage cells that harbour intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing bone marrow counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B-cell development occurs in the intestinal mucosa, where it is regulated by extracellular signals from commensal microbes that influence gut immunoglobulin repertoires.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of

During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons1,2. In mice, six additional sets of constant region exons (CHs) lie 100-200 kb downstream in the same transcriptional orientation as V(D)J and Cμ exons2. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH2. Activation-Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region2,3; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors3. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB (Fig. 1a). However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high-throughput genome-wide translocation sequencing (HTGTS)4 into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.

Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features that confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits. Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term noncanonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (I-BRD9, GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains. Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a subset of all mSWI/SNF complex target genes, and that associated GO terms for these genes are involved in epigenetic regulation, hemopoiesis, and differentiation. Finally, we applied a newly generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of ncBAF subunits, substantiating previous small-molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel mSWI/SNF complex composition with distinct function in cancer. This abstract is also being presented as Poster 14. Citation Format: Brittany C. Michel, Andrew R. D'Avino, Josh Pan, Robin M. Meyers, Aviad Tsherniak, Paola Grandi, Phillip Humphreys, Neil Garton, Rab K. Prinja, Cigall Kadoch. BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR15.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on-from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six standard deviations from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression-based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Efforts to define protein complexes and their functional networks are critical for systems-level understanding of the pathways involved in human cancer. Current methods to catalog human protein complexes via physical interaction are often unable to resolve functional differences between complex members or infer relationships governed by sub-stoichiometric interactions. While functional wiring maps in yeast have been generated by measuring epistatic interactions between pairs of genes, efforts to scale this concept in individual human cell lines have been met with challenges and have only been able to characterize limited numbers of genes at a time. We have developed a scalable approach that can measure functional similarity without the constraints of pairwise genetic interaction experiments. Using data from genome-wide RNAi and CRISPR dropout screens performed in hundreds of cancer cell lines, we leveraged the heterogeneity of gene dependencies across cancer types to measure functional similarity between thousands of genes at once, which in turn allowed us to recreate known inter- and intra-complex functional relationships and to uncover tumor suppressive and oncogenic functional modules in cancer-relevant pathways such as proteolysis, metabolism and transcription. Applying these approaches to the mammalian SWI/SNF (BAF) chromatin remodeling complex, which is mutated in over 20% of human cancer, revealed three functional modules that arose separately during metazoan evolution, one of which is entirely novel and uncharacterized. We then performed biochemical experiments that fully support three specialized complex configurations, each with distinct size, subunit composition, and function. These data reorganize the BAF complex into previously unrecognized modules that better explain mutational burden in human cancer. Notably, we observe that that all known BAF-driven, highly penetrant rare cancers and neurodevelopmental disorders involve disruption within a single functional module we defined, underscoring the value of evaluating disease genomics through the lens of functional modularity. Citation Format: Joshua Pan, Robin M. Meyers, Brittany C. Michel, Ann E. Sizemore, Francisca Vazquez, Barbara A. Weir, William C. Hahn, Aviad Tsherniak, Cigall Kadoch. Using cancer dependency data to discover tumor suppressive and oncogenic functional modules [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 5559. doi:10.1158/1538-7445.AM2017-5559

Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features which confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits. Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term non-canonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains. Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a distinct subset of all mSWI/SNF complex target genes and, consistent with previous studies, maintain proliferation of AML cells. Finally, we applied a newly- generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of three ncBAF subunits including BRD9, substantiating previous small molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel BAF complex composition with distinct function in cancer. Citation Format: Brittany C. Michel, Joshua Pan, Robin M. Meyers, Paola Grandi, Phillip G. Humphreys, Neil G. Garton, Rab K. Prinja, Cigall Kadoch. BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML [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 1020. doi:10.1158/1538-7445.AM2017-1020

Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sμ and in a downstream acceptor S region, with a DSB in Sμ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sμ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.

Precision Cancer Medicine requires the identification of vulnerabilities linked to genetic features of tumors. Recent studies utilizing highly annotated small molecule collections to assess dependencies across hundreds of genomically annotated cell lines have demonstrated the potential for such large-scale preclinical “Dependency Map” projects. We have undertaken a complementary approach using genetic perturbation tools (RNAi and CRISPR-Cas9 based loss-of-function viability screens), to systematically catalog preferential genetic dependencies and markers that predict response. These efforts are providing a foundation for the discovery of novel targets poised for early therapeutic discovery projects together with patient populations that may be enriched for responders to such therapies. Here, we present results from our initial Cancer Dependency Map consisting of RNAi loss-of-function screens across 503 cell lines, including both solid and hematopoietic tumors. We discovered 43 genes whose mutation or copy number creates a cancer dependency (oncogene addiction) including a novel dependency on the small GTPase, GNAI2 in Diffuse large B-cell Lymphoma. We discovered 142 genes in which elevated levels of expression create a dependency (gene addiction), a group of genes highly enriched for master regulator transcription factors such as SOX10, SPDEF, PAX8 and HNF1B. We discovered 474 genes for which hemizygous copy number creates a dependency (CYCLOPS genes), a group of genes highly enriched for members of macromolecular protein complexes including the spliceosome and proteasome. Finally, we discovered 171 genes that become a dependency when a redundant functional paralog is lost in cancer cells (redundant essentials). We demonstrate the mechanistic basis behind one such redundant essential dependency relationship in which promoter methylation of the UBB ubiquitin gene eliminates a compensatory mechanism leading to a novel vulnerability on the suppression of the UBC ubiquitin gene. These observations begin to provide an initial census, categorization and prioritization of robust cancer dependencies and support the potential impact for expanding early efforts to develop dependency maps of cancer. Citation Format: Francisca Vazquez, Aviad Tsherniak, Barbara Weir, Phil Montgomery, Glenn Cowley, Stanley Gill, Gregory Kryukov, Sasha Pantel, Will Harrington, Mike Burger, Robin Meyers, Levi Ali, Amy Goodale, Yenarae Lee, Levi Garraway, Jesse Boehm, David Root, Todd Golub, William Hahn. Emerging targets from Cancer Dependency Map v0.1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B44.

The mapping of cancer genomes is rapidly approaching completion. The genomic information encoded by individual patients' tumors should, in principle, provide a guide for predicting acquired cancer dependencies. Unfortunately, while the success of precision cancer genomics hinges on the decoding of such dependencies, we lack the ability to predict dependencies for most individual tumors. The challenge stems from the absence of clinical data relating genotypes with dependencies since most cancer mutations are rare and our arsenal of cancer drugs is incomplete. A comprehensive Cancer Dependency Map comprised of a catalog of genetic and small molecule vulnerabilities across a diverse set of cancers, along with robust statistical models able to predict these vulnerabilities from molecular and genomic features, would provide a roadmap of targets ripe for therapeutic development and would help reveal the mechanisms underlying the emergence of these vulnerabilities. Here, we report progress in creating a Cancer Dependency Map consisting of the following components: 1) Systematic genetic perturbation (RNAi/CRISPR) of over 600 cancer cell models representing a wide range of human cancers and cell lineages using massively parallel genome scale loss-of-function screens. 2) Computational segregation of on- from off-target effects of RNAi enabling the discovery of outlier dependencies. 3) Predictive modeling to discover biomarkers for each dependency. Our results demonstrate that our analytical approach (DEMETER) that models both gene and miRNA-based seed sequence effects effectively segregates on- from off-target effects of shRNAs. We discover 768 preferential dependencies whose suppression decreases viability at a level greater than six standard deviations in at least one of 503 cancer models and 105 such dependencies each present in at least 15 models. We find that 95% of the cancer models screened are strongly sensitive to the suppression of at least one of these dependencies, and that many models have common dependencies so that all models harbor at least one six-sigma dependency out of a set of only 76. Using a custom random forest based predictive modeling framework (ATLANTIS), we discover predictive biomarkers for hundreds of dependencies. These include known and novel vulnerabilities specified by somatic oncogenic alterations, overexpression of genes that specify lineage and differentiation, copy-number driven essentiality, and loss of functionally redundant paralogs. These observations provide a rigorous computational and experimental foundation for the creation of a comprehensive Cancer Dependency Map. Subsampling and projection analyses suggest that over 10,000 genomically characterized cancer cell models will be needed to achieve this important goal. This abstract is also being presented as Poster B43. Citation Format: Aviad Tsherniak, Francisca Vazquez, Barbara Weir, Philip Montgomery, Glenn Cowley, Stanley Gill, Gregory Kryukov, Sasha Pantel, Will Harrington, Mike Burger, Robin Meyers, Levi Ali, Amy Goodale, Yenarae Lee, Levi Garraway, Jesse Boehm, David Root, Todd Golub, William Hahn. Towards a Cancer Dependency Map. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR02.

Recent studies have demonstrated the power of CRISPR-Cas9 screening methods for identifying genetic vulnerabilities in cancer cells. As part of a larger effort to generate a comprehensive catalog of vulnerabilities, we performed CRISPR-Cas9 genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation and survival. We found a strong correlation between gene copy number and cell viability after Cas9-targeting. Copy number alterations are extremely prevalent in human cancers and frequently lead to overexpression of driver oncogenes and potential vulnerabilities. Therefore, we sought to identify such genes by investigating the relationship of genomic copy number with essentiality from our screening data. As expected, known oncogenes scored as essential in cell lines harboring amplifications of these genes. However, the scores of all other genes in these amplified regions were also strongly enriched for apparent essentiality, even among unexpressed genes. Furthermore, the infection of cells with sgRNAs targeting Cas9 to non-coding intergenic sequences within regions of high copy number gain also induced this negative effect on cell viability. We observed this effect across multiple different chromosomal structural alterations, including tandem duplications, breakage-fusion-bridge structures, and arm-level gains. More broadly, we found a striking global correlation between cell viability in response to Cas9-targeting and the genomic copy number of the targeted site, even among low-level copy number gain and loss. For example, Cas9-targeting of genes with two copies resulted in, on average, decreased viability relative to Cas9-targeting of genes with only one copy. By examining sgRNAs that target multiple genomic sites, but not within any amplified loci, we found that this cell response to Cas9-targeting correlated strongly with the total number of target sites. Together, these observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response with a severity proportional to the total number of discrete genomic loci targeted. 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. This result illustrates the sensitivity of cancer cells to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Targeting non-essential genes or non-coding intergenic sequences within regions of copy number amplification may reveal cancer-specific vulnerabilities. Citation Format: Robin M. Meyers, Andrew J. Aguirre, Barbara A. Weir, Francisca Vazquez, Cheng-Zhong Zhang, Uri Ben-David, April Cook, Gavin Ha, William F. Harrington, Mihir Doshi, Stanley Gill, Han Xu, Levi D. Ali, Guozhi Jiang, Sasha Pantel, Yenarae Lee, Amy Goodale, Andrew D. Cherniack, Coyin Oh, Gregory Kryukov, Glenn S. Cowley, Levi A. Garraway, Kimberly Stegmaier, Charles W. Roberts, Todd R. Golub, Matthew Meyerson, David E. Root, Aviad Tsherniak, William C. Hahn. Genomic copy number alterations introduce a gene-independent viability bias in CRISPR-Cas9 knock-out screens of cancer cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B39.

Loss-of-function genetic screens in cancer cell lines permit the systematic interrogation of genes and pathways involved in cell proliferation and viability. The CRISPR-Cas9 system enables effective genome editing for perturbation of gene function. To identify genes that are essential for cancer cell proliferation and survival, we have optimized an approach for efficient genome-scale CRISPR-Cas9 pooled screening and performed negative-selection knock-out screens in 43 human cancer cell lines with diverse genetic and phenotypic features, including 8 pancreatic cancer cell lines. We report that CRISPR-Cas9 efficacy varies depending on the cell context and propose methods to effectively identify differential cancer dependencies across cell lines. Additionally, we identify a strong correlation between genomic copy number and perceived gene dependency, and further elucidate a gene-independent effect of CRISPR-Cas9 cutting. Using these screening data, we performed a detailed interrogation of gene and pathway dependencies in pancreatic cancer. Through integrative analysis of CRISPR-Cas9 screening results with RNA-interference and small molecule screening data, as well as DNA and RNA sequencing, we identified a high-confidence set of recurrently essential genes in pancreatic cancer, including several with associated biomarkers. Moreover, through pathway analyses of CRISPR-Cas9 screening data, we have identified a number of essential biologic processes including key metabolic and cell signaling pathways that may represent potential therapeutic avenues. In particular, we characterize the sterol regulatory element binding protein (SREBP) signaling pathway as a recurrent vulnerability in pancreatic cancer and credential this pathway as a potential therapeutic target. This abstract is also being presented as Poster A13 Citation Format: Andrew J. Aguirre, Wei Shao, Han Xu, Barbara Weir, Francisca Vazquez, Robin Meyers, Cheng-Zhong Zhang, Mihir Doshi, Glenn S. Cowley, Theodore Ewachiw, Zeshaan Rasheed, Todd R. Golub, Kimberly Stegmaier, Charles W. Roberts, Levi A. Garraway, Matthew Meyerson, Aviad Tsherniak, David E. Root, Peter J. Espenshade, William C. Hahn.{Authors}. Genome-scale CRISPR-Cas9 screening to identify essential genes and pathways in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR05.

Recombination activating gene 1/2 endonuclease (RAG) initiates V(D)J recombination in developing B and T lymphocytes via “on-target” activity at bona fide recombination signal sequences (RSSs) to generate diverse antibodies. “Off-target” RAG activity is thought to be rare, but can cause oncogenic interstitial deletions or chromosomal translocations that contribute to immature B and T lymphocyte cancers. Given that potential RAG off-target sites occur frequently in the genome, it has been a mystery how this potentially dangerous activity is suppressed. We generated mice with a pair of bona fide V(D)J RSSs (“V(D)J cassette”) in the first intron of c-myc on mouse chromosome 15. When introduced into an ATM-deficient background, the c-myc V(D)J cassette-containing mice recurrently develop B cell lymphomas with oncogenic c-myc translocations/amplifications involving the Igh locus on chromosome 12. These amplifications are generated by breakage-fusion-bridge (“BFB”) cycles from dicentric (15;12) chromosomes formed through fusion of RAG-generated Igh double-stranded breaks (DSBs) to sequences downstream of c-myc. Notably, the translocations downstream of c-myc occurred exclusively on V(D)J cassette-containing alleles, but did not involve the cassette. Based on this finding, we proposed that the V(D)J cassette mediates DSBs downstream of c-myc by activating RAG cutting at linked cryptic RSSs. We developed a linear amplification-mediated (“LAM”) version of our high-throughput genome-wide translocation sequencing approach (“LAM-HTGTS”) to robustly identify “prey” endogenous DNA DSBs that join to introduced or endogenous “bait” DSBs. We used LAM-HTGTS to reveal genome-wide DSBs that joined to RAG-generated bait DSBs. We found that translocations between RAG-induced DSBs in the c-myc V(D)J cassette to Igh occur only in the absence of ATM. Strikingly, however, we discovered a large number of deletions from the c-myc cassette to sequences just upstream of c-myc (1 kb) and up to 1.7 Mb downstream of c-myc in WT and ATM-deficient cells. Moreover, insertion of a pair of bona fide RSSs into 12 random sites genome-wide induced large numbers of RAG-initiated DSBs leading to cassette involved deletions and inversions spreading upstream and downstream of a given cassette from 200 kb to over 2 Mb, respectively. In each case, regions of RAG off-target activity fell into a well-defined topologically associated domain (TAD) into which the cassette integrated. Such domains generally were flanked by convergent CTCF binding sites (CBEs), such as those our lab implicated in Igh locus V(D)J recombination control, but sometimes also involved sub-domains of a CTCF-defined TAD. We showed these TAD-associated DSBs to enrich at “cryptic RSSs” determined by conserved “CAC” motifs. We also captured these DSBs via LAM-HTGTS from Cas9/gRNA DSBs within a TAD and proved them to be RAG-generated. We found dramatic changes of on-target profiles and expansion of off-target RAG-generated Igh locus DSBs when CBEs acting as boundary of DH-JH recombination domain were deleted. Based on these overall findings, we propose that antigen receptor loci exploit fixed CBE-based TADs to restrict RAG activity within them, but also evolved “regulated” sub-domains to further focus and re-focus RAG activity developmentally. We also used LAM-HTGTS to identify 89 RAG off-targets that participate in chromosomal translocations and show RAG off-targets occur in pairs in highly accessible regions such as enhancers and promoters within a TAD. Beyond providing a mechanisms for the generation of recurrent B cell lymphomas in ATM-deficient mice with a V(D)J cassette in c-myc, our findings help explain the origin of certain recurrent interstitial deletions and translocations in tumors that arise from human developing lymphocytes. Citation Format: Jiazhi Hu, Yu Zhang, Lijuan Zhao, Richard Frock, Zhou Du, Robin M. Meyers, Feilong Meng, David G. Schatz, Frederick W. Alt. Topologically associated domains genome-wide restrict the off-target activity of recombination activating gene 1/2 endonuclease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A180.

Summary In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss the implications of these findings for harnessing antibody diversification mechanisms.