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Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers Edwin H

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Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers Edwin H Published OnlineFirst September 27, 2017; DOI: 10.1158/0008-5472.CAN-17-2043 Cancer Therapeutics, Targets, and Chemical Biology Research Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers Edwin H. Yau1,2,3, Indrasena Reddy Kummetha1, Gianluigi Lichinchi1, Rachel Tang1, Yunlin Zhang1, and Tariq M. Rana1,3 Abstract Targeting mutant KRAS signaling pathways continues to established synthetic enhancers or synthetic lethals for KRASMUT attract attention as a therapeutic strategy for KRAS-driven colorectal cancer, including targetable metabolic genes. Notably, tumors. In this study, we exploited the power of the CRISPR- genetic disruption or pharmacologic inhibition of the metabolic Cas9 system to identify genes affecting the tumor xenograft enzymes NAD kinase or ketohexokinase was growth inhibitory growth of human mutant KRAS (KRASMUT) colorectal cancers. in vivo. In addition, the chromatin remodeling protein INO80C Using pooled lentiviral single-guide RNA libraries, we con- was identified as a novel tumor suppressor in KRASMUT colo- ducted a genome-wide loss-of-function genetic screen in an rectal and pancreatic tumor xenografts. Our findings define a isogenic pair of human colorectal cancer cell lines harboring novel targetable set of therapeutic targets for KRASMUT tumors. mutant or wild-type KRAS. The screen identified novel and Cancer Res; 77(22); 6330–9. Ó2017 AACR. Introduction specific genome engineering in mammalian cells (3–5). By target- ing the Cas9 nuclease gene using specific single-guide RNAs The RAS family of oncogenes (KRAS, NRAS, and HRAS) is the (sgRNA) and inducing targeted double-strand DNA breaks that target of intense research for two reasons: the crucial role of are repaired by error-prone nonhomologous end joining, inser- mutant RAS proteins in tumorigenesis and the continued unmet 0 tion or deletion mutations can be introduced into 5 exons of need for therapeutic options for RAS-mutated human cancers. coding genes, resulting in loss-of-function mutants. A number of RAS is the most frequently mutated oncogenic driver of human groups have used pooled oligonucleotide array synthesis to cancer, and KRAS is mutated in about 20% of all human cancers, generate and validate large sgRNA libraries for genome-wide including some of the most lethal. Because of the historic diffi- loss-of-function (knockout) studies (6–11). culty in targeting RAS, genomic screening using synthetic lethal/ In cell culture models, genome-wide CRISPR-Cas9 knockout enhancing approaches has become an attractive method to iden- screens have revealed that KRAS wild-type (KRASWT)and tify more tractable therapeutic targets and to further understand mutant (KRASMUT) tumor cells show differential dependencies RAS biology. The approach identifies genetic targets whose dis- on downstream MAPK signaling members and adapters, and ruption does not normally affect survival but becomes lethal/ that KRASMUT tumors are highly dependent on mitochondrial enhancing in the presence of an activating RAS mutation (1, 2). oxidative phosphorylation (7, 10). To extend our knowledge of Functional genomic screens have been revolutionized by appli- the genetic vulnerabilities of KRASMUT tumors to the in vivo cation of the gene-specific editing technology of clustered regu- setting, we performed a pooled CRISPR-Cas9 knockout screen larly interspaced short palindrome repeats (CRISPR) and CRISPR- of tumor xenografts using an isogenic pair of human colorectal associated protein 9 (Cas9) system, which allows efficient and cancer cell lines carrying wild-type or mutant KRAS.Weiden- tified gene knockouts that conferred selective beneficial or detrimental effects in the context of KRAS activation, including 1 Department of Pediatrics and Institute for Genomic Medicine, University of multiple metabolic vulnerabilities, highlighting the therapeutic 2 MUT California San Diego School of Medicine, La Jolla, California. Division of potential of targeting cancer metabolism in KRAS tumors. Hematology-Oncology, Department of Internal Medicine, University of Califor- nia San Diego School of Medicine, La Jolla, California. 3Solid Tumor Therapeutics Using a secondary validation sgRNA library, we additionally fi KRAS Program, Moores Cancer Center, University of California, San Diego, La Jolla, identi ed a novel -dependent tumor-suppressor gene in WT/MUT California. KRAS colorectal cancer and pancreatic adenocarcinoma Note: Supplementary data for this article are available at Cancer Research (PDAC) isogenic xenografts. Online (http://cancerres.aacrjournals.org/). Current addresss of Edwin H. Yau: Department of Cancer Genetics & Genomics, Materials and Methods Roswell Park Cancer Institute, Buffalo, NY. Cell lines and culture Corresponding Author: Tariq M. Rana, University of California San Diego, 9500 The paired isogenic colorectal cancer cell lines HCT116 À À Gilman Drive MC0762, La Jolla, CA 92093. Phone: 8582461100; Fax: KRASWT/ ,HCT116KRASWT/G13D,DLD-1KRASWT/ , and DLD-1 8582461600; E-mail: [email protected] KRASWT/G13D (abbreviated to WT and MUT) were kind gifts from doi: 10.1158/0008-5472.CAN-17-2043 Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). Ó2017 American Association for Cancer Research. Capan-2, SW1990, H348, and H441 cells were from the ATCC. All 6330 Cancer Res; 77(22) November 15, 2017 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 27, 2017; DOI: 10.1158/0008-5472.CAN-17-2043 Genome-Wide CRISPR Screen of KRAS-Mutant Tumor Xenografts cell lines were cultured in DMEM (Invitrogen) with 10% FBS sgRNA constructs were washed with PBS, resuspended in 300 mL (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). of PBS and Matrigel (1:1 ratio), and injected subcutaneously into HCT116 cell lines were validated by PCR amplification and the flanks of nude mice (NU/J; Jackson Labs). For the primary Sanger sequencing to confirm the mutation at the genomic level. GeCKO screen and secondary mini-library screens, 3 Â 107 cells Strict bio-banking procedures were followed, and cells were tested were injected per mouse and groups of 3 mice were injected per for contamination, including mycoplasma. HCT116-dCas9, transduction replicate per cell line. After 14 days, mice were DLD-1-dCas9, H358-dCas9, and Capan-2-dCas9 cell lines were euthanized and the tumors excised and stored in RNAlater generated by lentiviral transduction with lentiCas9-Blast (Qiagen) solution. For individual sgRNA experiments, 1.5 Â (Addgene # 52962) and selection with 10 mg/mL of blasticidin. 106 HCT116, DLD-1, H358, or Capan-2 tumor cells were resus- pended in PBS:Matrigel (1:1) and injected into the flanks of nude Pooled library amplification and viral production mice (3–4 mice per sgRNA, 6–8 mice per gene targeted). Tumor We followed previously published protocols of screens using dimensions were measured with Vernier calipers for 24 days, and the GeCKO library, as developed by the Zhang lab, with slight volumes (mm3) were calculated as (0.5 Â width) Â (height2). modifications. The human GeCKO v2 A library pooled plasmid (lentiCRISPR v2) was obtained from Addgene (cat # Small-molecule inhibitor treatment 1000000048) and amplified according to the recommended Nude mice were injected with HCT116 tumor cells as above. protocols by electroporation in Lucigen Endura electrocompetent After xenografts were palpable (7 days after injection), animals cells. For the secondary focused validation sgRNA library, oligo- were injected intraperitoneally with 100 mL of vehicle, thionico- nucleotide pools (CustomArray) were obtained with variable tinamide (Spectrum; 100 mg/kg body weight) in 1% DMSO, 20 bp sgRNA sequences flanked by universal PCR primers (74 bp or KHK inhibitor (Calbiochem 420640 or synthesized according total synthesized sequence, see Supplementary Experimental to published methods; 25 mg/kg; ref. 12) in PBS. Injections were Procedures for full sequences). Full-length oligonucleotides were repeated every other day for 14 days (7 doses total). Tumor amplified by PCR using Q5 HiFi polymerase (New England dimensions were measured with Vernier calipers, and volumes Biolabs) and size-selected on 2% agarose gels. PCR inserts were (mm3) were calculated as (0.5 Â width) Â (height2). cloned into BsmBI-digested lentiGuide-Puro (Addgene # 52963) using Gibson assembly (New England Biolabs), transformed into sgRNA library quantification by deep sequencing Endura electrocompetent cells, and amplified similarly to the Genomic DNA was extracted from tumors or cells using the GeCKO library. 293FT cells were resuspended in DMEM and previously published salt-precipitation protocol (11). The sgRNA cotransfected with 12 mg of the hGeCKO plasmid library or library representation was determined using a two-step PCR secondary validation mini-pool, 9 mg psPAX2 vector, and 6 mg process in which PCR1 amplifies the lentiviral sequence contain- pMD2.G vector in 10 cm plates using 48 mL of PLUS reagent ing the 20 bp sgRNA cassette and PCR2 attaches Illumina sequenc- (Invitrogen 11514-015) and 60 mL of Lipofectamine in Opti-MEM ing adapters and barcodes. Primer sequences were obtained from medium. The medium was aspirated after 6 hours and replaced the Zhang lab online resource (http://genome-engineering.org/ with fresh DMEM/10% FBS. The supernatant was harvested after gecko/) using v2Adaptor_F and v2Adapter_R for PCR1 and pri- 60 hours, centrifuged at 3,000 rpm at 4 C for 10 minutes, filtered mers F01-F06
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