Megha Chandrashekhar

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

Forward genetic screens with CRISPR–Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR–Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP–ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein–DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1–4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.Mutations in all three genes encoding ribonuclease H2 sensitize cells to poly(ADP–ribose) polymerase inhibitors by compromising ribonucleotide excision repair.

Cytokinetic effects of Wee1 disruption in pancreatic cancer

ABSTRACT The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker γH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genome-scale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

Systematic discovery and classification of human cell line essential genes

The study of gene essentiality in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. Technological advances in genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems have set the stage for identifying human cell line core and context-dependent essential genes. However, first generation negative selection screens using CRISPR technology demonstrate extreme variability across different cell lines. To advance the development of the catalogue of human core and context-dependent essential genes, we have developed an optimized, ultracomplex, genome-scale gRNA library of 176,500 guide RNAs targeting 17,661 genes and have applied it to negative and positive selection screens in a human cell line. Using an improved Bayesian analytical approach, we find CRISPR-based screens yield double to triple the number of essential genes than were previously observed using systematic RNA interference, including many genes at moderate expression levels that are largely refractory to RNAi methods. We further characterized four essential genes of unknown significance and found that they all likely exist in protein complexes with other essential genes. For example, RBM48 and ARMC7 are both essential nuclear proteins, strongly interact and are commonly amplified across major cancers. Our findings suggest the CRISPR-Cas9 system fundamentally alters the landscape for systematic reverse genetics in human cells for elucidating gene function, identifying disease genes, and uncovering therapeutic targets.

Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells

CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

A CRISPR screen reveals a WNT7B-FZD5 signaling circuit as a therapeutic opportunity in pancreatic cancer.

CRISPR-Cas9 genome editing enables high-resolution detection of genetic vulnerabilities of cancer cells. We conducted a genome-wide CRISPR-Cas9 screen in RNF43 mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation, and discovered a unique requirement for a WNT7B-FZD5 signaling circuit. Our results highlight an underappreciated level of functional specificity at the ligand-receptor level. We derived a panel of recombinant antibodies that reports the expression of nine out of ten human Frizzled receptors and confirm that WNT7B-FZD5 functional specificity cannot be explained by protein expression patterns. We developed two human antibodies that target FZD5 and robustly inhibited the growth of RNF43 mutant PDAC cells grown in vitro and as xenografts, providing strong orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring a RNF43 variant previously associated with PDAC was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy.

Abstract 233: Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens

The CRISPR-Cas9 system provides an effective way to introduce targeted loss-of-function mutations in mammalian cells. The advance that the CRISPR-Cas9 technology brings to human genetics sets the stage for identifying cellular fitness genes that operate either globally (core fitness genes) or specifically within a particular genetic background or environmental context (context-specific fitness genes). In tumors, this is the foundation for the concept of synthetic lethality as genes required in tumor cells but not in adjacent normal tissues should make ideal therapeutic targets with high effectiveness and minimal side effects. Towards this goal, we have developed a second-generation CRISPR gRNA library of 176,500 guides targeting 17,661 human protein-coding genes. We used the library to screen five human cell lines, representing a cross-section of wild type and cancer tissues, to identify genes whose knockouts induce significant fitness defects. Our screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and identify novel context-specific vulnerabilities. Interestingly, we identified a specific dependency on mitochondrial activity and we validated this using various complex I inhibitors. This strongly supports the idea that oxidative phosphorylation (OXPHOS) dependency - a clear exception to the Warburg effect - is a targetable weakness of some tumors. In order to further understand this metabolic vulnerability, we performed a synthetic lethal screen to identify sensitizers of OXPHOS inhibition. Our screen revealed that inhibition of mitochondrial OXPHOS sensitizes the cells to loss of other metabolic pathways such as glycolysis, pentose phosphate pathway and lipid biosynthesis. Furthermore, loss of the cytosolic aspartate aminotransferase GOT1 was found to be synthetic lethal with perturbation of OXPHOS. This is consistent with the essential role of the electron transport chain in cell proliferation, which is to enable aspartate synthesis. Additionally, we also find novel genes, whose loss of function might alleviate the affect of OXPHOS perturbation. Our findings demonstrate that CRISPR-Cas9 screens enable a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer. Further, synthetic lethal chemical-genetic screens can reveal novel functional drug combinations, which will enhance the efficacy of targeted therapies. Citation Format: Megha Chandrashekhar, Michael Arreger, Ashwin Seetharaman, Traver Hart, Jason Moffat. Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 233.