Genome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma. Its SL partners serve as potential drug targets for the development of targeted cancer therapies. We performed parallel genome-wide CRISPR screens in two pairs of isogenic clear cell renal cell carcinoma cell lines that differ only in the VHL status. Comparative analyses of screening results not only confirmed a well-known role for mTOR signaling in renal carcinoma, but also identified DNA damage response and selenocysteine biosynthesis pathways as novel SL targets in VHL-inactivated cancer cells. Follow-up studies provided cellular and mechanistic insights into SL interactions of these pathway genes with the VHL gene. Our CRISPR and RNA-seq datasets provide a rich resource for future investigation of the function of the VHL tumor suppressor protein. Our work demonstrates the efficiency of CRISPR-based synthetic lethality screening in human isogenic cell pairs. Similar strategies could be employed to unveil SL partners with other oncogenic drivers.
ZnaorA, Lortet-TieulentJ, LaversanneM, et al.International variations and trends in renal cell carcinoma incidence and mortality. Eur Urol. 2015; 67:519–530. DOI: 10.1016/j.eururo.2014.10.002.
3.
YoungAC, CravenRA, CohenD, et al.Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma. Clin Cancer Res. 2009; 15:7582–7592. DOI: 10.1158/1078-0432.CCR-09-2131.
4.
SatoY, YoshizatoT, ShiraishiY, et al.Integrated molecular analysis of clear-cell renal cell carcinoma. Nat Genet. 2013; 45:860–867. DOI: 10.1038/ng.2699.
5.
SchitoL, SemenzaGL. Hypoxia-inducible factors: master regulators of cancer progression. Trends Cancer. 2016; 2:758–770. DOI: 10.1016/j.trecan.2016.10.016.
6.
KurbanG, HudonV, DuplanE, et al.Characterization of a von Hippel Lindau pathway involved in extracellular matrix remodeling, cell invasion, and angiogenesis. Cancer Res. 2006; 66:1313–1319. DOI: 10.1158/0008-5472.CAN-05-2560.
7.
ThomaCR, FrewIJ, KrekW. The VHL tumor suppressor: riding tandem with GSK3beta in primary cilium maintenance. Cell Cycle. 2007; 6:1809–1813. DOI: 10.4161/cc.6.15.4518.
8.
LutzMS, BurkRD. Primary cilium formation requires von hippel-lindau gene function in renal-derived cells. Cancer Res. 2006; 66:6903–6907. DOI: 10.1158/0008-5472.CAN-06-0501.
9.
EstebanMA, HartenSK, TranMG, et al.Formation of primary cilia in the renal epithelium is regulated by the von Hippel-Lindau tumor suppressor protein. J Am Soc Nephrol. 2006; 17:1801–1806. DOI: 10.1681/ASN.2006020181.
10.
SchermerB, GhenoiuC, BartramM, et al.The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth. J Cell Biol. 2006; 175:547–554. DOI: 10.1083/jcb.200605092.
11.
HergovichA, LisztwanJ, BarryR, et al.Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL. Nat Cell Biol. 2003; 5:64–70. DOI: 10.1038/ncb899.
12.
ZhaoWT, ZhouCF, LiXB, et al.The von Hippel-Lindau protein pVHL inhibits ribosome biogenesis and protein synthesis. J Biol Chem. 2013; 288:16588–16597. DOI: 10.1074/jbc.M113.455121.
13.
RoeJS, KimH, LeeSM, et al.p53 stabilization and transactivation by a von Hippel-Lindau protein. Mol Cell. 2006; 22:395–405. DOI: 10.1016/j.molcel.2006.04.006.
14.
ThomaCR, TosoA, GutbrodtKL, et al.VHL loss causes spindle misorientation and chromosome instability. Nat Cell Biol. 2009; 11:994–1001. DOI: 10.1038/ncb1912.
15.
HellMP, ThomaCR, FankhauserN, et al.miR-28-5p promotes chromosomal instability in VHL-associated cancers by inhibiting Mad2 translation. Cancer Res. 2014; 74:2432–2443. DOI: 10.1158/0008-5472.CAN-13-2041.
16.
YoungAP, SchlisioS, MinamishimaYA, et al.VHL loss actuates a HIF-independent senescence programme mediated by Rb and p400. Nat Cell Biol. 2008; 10:361–369. DOI: 10.1038/ncb1699.
17.
WelfordSM, DorieMJ, LiX, et al.Renal oxygenation suppresses VHL loss-induced senescence that is caused by increased sensitivity to oxidative stress. Mol Cell Biol. 2010; 30:4595–4603. DOI: 10.1128/MCB.01618-09.
18.
MetcalfJL, BradshawPS, KomosaM, et al.K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair. Oncogene. 2014; 33:1055–1065. DOI: 10.1038/onc.2013.22.
19.
ChoueiriTK, MotzerRJ. Systemic Therapy for Metastatic Renal-Cell Carcinoma. N Engl J Med. 2017; 376:354–366. DOI: 10.1056/NEJMra1601333.
20.
HartwellLH, SzankasiP, RobertsCJ, et al.Integrating genetic approaches into the discovery of anticancer drugs. Science. 1997; 278:1064–1068.
ChanDA, GiacciaAJ. Harnessing synthetic lethal interactions in anticancer drug discovery. Nat Rev Drug Discov. 2011; 10:351–364. DOI: 10.1038/nrd3374.
23.
WeidleUH, MaiselD, EickD. Synthetic lethality-based targets for discovery of new cancer therapeutics. Cancer Genomics Proteomics. 2011; 8:159–171.
24.
Bommi-ReddyA, AlmecigaI, SawyerJ, et al.Kinase requirements in human cells: III. Altered kinase requirements in VHL-/- cancer cells detected in a pilot synthetic lethal screen. Proc Natl Acad Sci U S A., 2008; 105:16484–16489. DOI: 10.1073/pnas.0806574105.
25.
ChakrabortyAA, NakamuraE, QiJ, et al.HIF activation causes synthetic lethality between the VHL tumor suppressor and the EZH1 histone methyltransferase. Sci Transl Med. 2017; 9:eaal5272. DOI: 10.1126/scitranslmed.aal5272.
26.
ChanDA, SutphinPD, NguyenP, et al.Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality. Sci Transl Med. 2011; 3:94ra70. DOI: 10.1126/scitranslmed.3002394.
27.
GameiroPA, YangJ, MeteloAM, et al.In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation. Cell Metab. 2013; 17:372–385. DOI: 10.1016/j.cmet.2013.02.002.
28.
KondoK, KimWY, LechpammerM, et al.Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 2003; 1:439–444. DOI: 10.1371/journal.pbio.0000083.
29.
KondoK, KlcoJ, NakamuraE, et al.Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell. 2002; 1:237–246.
30.
ShenJP, ZhaoD, SasikR, et al.Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat Methods. 2017; 14:573–576. DOI: 10.1038/nmeth.4225.
31.
ThomasGV, TranC, MellinghoffIK, et al.Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med. 2006; 12:122–127. DOI: 10.1038/nm1337.
32.
ZimmerM, DoucetteD, SiddiquiN, et al.Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL-/- tumors. Mol Cancer Res. 2004; 2:89–95. .
33.
TurcotteS, ChanDA, SutphinPD, et al.A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy. Cancer Cell. 2008; 14:90–102. DOI: 10.1016/j.ccr.2008.06.004.
34.
WolffNC, Pavia-JimenezA, TcheuyapVT, et al.High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma. Oncotarget. 2015; 6:16951–16962. DOI: 10.18632/oncotarget.4773.
35.
TangX, WuJ, DingCK, et al.Cystine deprivation triggers programmed necrosis in VHL-deficient renal cell carcinomas. Cancer Res. 2016; 76:1892–1903. DOI: 10.1158/0008-5472.CAN-15-2328.
36.
WoldemichaelGM, TurbyvilleTJ, VasselliJR, et al.Lack of a functional VHL gene product sensitizes renal cell carcinoma cells to the apoptotic effects of the protein synthesis inhibitor verrucarin A. Neoplasia. 2012; 14:771–777. .
37.
CongL, RanFA, CoxD, et al.Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339:819–823. DOI: 10.1126/science.1231143.
38.
MaliP, YangL, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science. 2013; 339:823–826. DOI: 10.1126/science.1232033.
39.
KomorAC, BadranAH, LiuDR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017; 168:20–36. DOI: 10.1016/j.cell.2016.10.044.
40.
DoudnaJA, CharpentierE. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346:1258096. DOI: 10.1126/science.1258096.
41.
SanderJD, JoungJK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014; 32:347–355. DOI: 10.1038/nbt.2842.
42.
WangH, La RussaM, QiLS. CRISPR/Cas9 in Genome Editing and Beyond. Annu Rev Biochem. 2016; 85:227–264. DOI: 10.1146/annurev-biochem-060815-014607.
43.
KiesslingMK, SchuiererS, StertzS, et al.Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening. BMC Genomics. 2016; 17:723. DOI: 10.1186/s12864-016-3042-2.
44.
AndersonGR, WinterPS, LinKH, et al.A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution. Cell Rep. 2017; 20:999–1015. DOI: 10.1016/j.celrep.2017.07.006.
45.
ToledoCM, DingY, HoellerbauerP, et al.Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells. Cell Rep. 2015; 13:2425–2439. DOI: 10.1016/j.celrep.2015.11.021.
46.
SteinhartZ, PavlovicZ, ChandrashekharM, et al.Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors. Nat Med. 2017; 23:60–68. DOI: 10.1038/nm.4219.
47.
TzelepisK, Koike-YusaH, De BraekeleerE, et al.A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. Cell Rep. 2016; 17:1193–1205. DOI: 10.1016/j.celrep.2016.09.079.
48.
FeiT, ChenY, XiaoT, et al.Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing. Proc Natl Acad Sci U S A. 2017; 114:E5207-E5215. DOI: 10.1073/pnas.1617467114.
49.
HartT, ChandrashekharM, AreggerM, et al.High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell. 2015; 163:1515–1526. DOI: 10.1016/j.cell.2015.11.015.
50.
WangT, BirsoyK, HughesNW, et al.Identification and characterization of essential genes in the human genome. Science. 2015; 350:1096–1101. DOI: 10.1126/science.aac7041.
51.
WangT, YuH, HughesNW, et al.Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell. 2017; 168:890–903. DOI: 10.1016/j.cell.2017.01.013.
52.
WangT, WeiJJ, SabatiniDM, et al.Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014; 343:80–84. DOI: 10.1126/science.1246981.
53.
ShalemO, SanjanaNE, HartenianE, et al.Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014; 343:84–87. DOI: 10.1126/science.1247005.
LiaoY, SmythGK, ShiW. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30:923–930. DOI: 10.1093/bioinformatics/btt656.
56.
LoveMI, HuberW, AndersS. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550. DOI: 10.1186/s13059-014-0550-8.
57.
SubramanianA, TamayoP, MoothaVK, et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102:15545–15550. DOI: 10.1073/pnas.0506580102.
58.
DoenchJG, FusiN, SullenderM, et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34:184–191. DOI: 10.1038/nbt.3437.
LiW, XuH, XiaoT, et al.MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014; 15:554. DOI: 10.1186/s13059-014-0554-4.
61.
SaitoY, YoshidaY, AkazawaT, et al.Cell death caused by selenium deficiency and protective effect of antioxidants. J Biol Chem. 2003; 278:39428–39434. DOI: 10.1074/jbc.M305542200.
GossageL, EisenT, MaherER. VHL, the story of a tumour suppressor gene. Nat Rev Cancer. 2015; 15:55–64. DOI: 10.1038/nrc3844.
64.
FrewIJ, MochH. A Clearer View of the Molecular Complexity of Clear Cell Renal Cell Carcinoma. Annu Rev Pathol. 2015; 10:263–289. DOI: 10.1146/annurev-pathol-012414-040306.
65.
YangH, MinamishimaYA, YanQ, et al.pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2. Mol Cell. 2007; 28:15–27. DOI: 10.1016/j.molcel.2007.09.010.
66.
GattoF, NookaewI, NielsenJ. Chromosome 3p loss of heterozygosity is associated with a unique metabolic network in clear cell renal carcinoma. Proc Natl Acad Sci U S A. 2014; 111:E866–875. DOI: 10.1073/pnas.1319196111.
67.
PauseA, LeeS, LonerganKM, et al.The von Hippel-Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal. Proc Natl Acad Sci U S A. 1998; 95:993–998.
68.
IliopoulosO, KibelA, GrayS, et al.Tumour suppression by the human von Hippel-Lindau gene product. Nat Med. 1995; 1:822–826.
69.
BaileyST, SmithAM, KardosJ, et al.MYC activation cooperates with Vhl and Ink4a/Arf loss to induce clear cell renal cell carcinoma. Nat Commun. 2017; 8:15770. DOI: 10.1038/ncomms15770.
70.
HartT, BrownKR, SircoulombF, et al.Measuring error rates in genomic perturbation screens: gold standards for human functional genomics. Mol Syst Biol. 2014; 10:733. DOI: 10.15252/msb.20145216.
71.
Cancer Genome Atlas Research N. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013; 499:43–49. DOI: 10.1038/nature12222.
72.
MotzerRJ, EscudierB, OudardS, et al.Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet. 2008; 372:449–456. DOI: 10.1016/S0140-6736(08)61039-9.
73.
HudesG, CarducciM, TomczakP, et al.Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. New Engl J Med. 2007; 356:2271–2281. DOI: 10.1056/NEJMoa066838.
74.
SchmidtRL, SimonovicM. Synthesis and decoding of selenocysteine and human health. Croat Med J. 2012; 53:535–550. DOI: 10.3325/cmj.2012.53.535.
75.
HatfieldDL, TsujiPA, CarlsonBA, et al.Selenium and selenocysteine: roles in cancer, health, and development. Trends Biochem Sci. 2014; 39:112–120. DOI: 10.1016/j.tibs.2013.12.007.
76.
KryukovGV, CastellanoS, NovoselovSV, et al.Characterization of mammalian selenoproteomes. Science. 2003; 300:1439–1443. DOI: 10.1126/science.1083516.
77.
AlmondesKG, LealGV, CozzolinoSM, et al.The role of selenoproteins in cancer. Rev Assoc Med Bras (1992). 2010; 56:484–488.
78.
SteinbrennerH, SiesH. Protection against reactive oxygen species by selenoproteins. Biochim Biophys Acta. 2009; 1790:1478–1485. DOI: 10.1016/j.bbagen.2009.02.014.
79.
KaelinWGJr.The VHL Tumor Suppressor Gene: Insights into Oxygen Sensing and Cancer. Trans Am Clin Climatol Assoc. 2017; 128:298–307.
80.
SamantaD, SemenzaGL. Maintenance of redox homeostasis by hypoxia-inducible factors. Redox Biol. 2017; 13:331–335. DOI: 10.1016/j.redox.2017.05.022.
81.
ZhouBB, ElledgeSJ. The DNA damage response: putting checkpoints in perspective. Nature. 2000; 408:433–439. DOI: 10.1038/35044005.
82.
HuenMS, ChenJ. The DNA damage response pathways: at the crossroad of protein modifications. Cell Res. 2008; 18:8–16. DOI: 10.1038/cr.2007.109.
83.
PearlLH, SchierzAC, WardSE, et al.Therapeutic opportunities within the DNA damage response. Nat Rev Cancer. 2015; 15:166–180. DOI: 10.1038/nrc3891.
84.
TsherniakA, VazquezF, MontgomeryPG, et al.Defining a cancer dependency map. Cell. 2017; 170:564–576. DOI: 10.1016/j.cell.2017.06.010.
85.
McDonaldER, 3rd, de WeckA, SchlabachMR, et al.Project DRIVE: a compendium of cancer dependencies and synthetic lethal relationships uncovered by large-scale, deep RNAi screening. Cell. 2017; 170:577–592. DOI: 10.1016/j.cell.2017.07.005.
86.
BehanFM, IorioF, PiccoG, et al.Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens. Nature. 2019; 568:511–516. DOI: 10.1038/s41586-019-1103-9.
87.
MeyersRM, BryanJG, McFarlandJM, et al.Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat Genet. 2017; 49:1779–1784. DOI: 10.1038/ng.3984.
88.
TorranceCJ, AgrawalV, VogelsteinB, et al.Use of isogenic human cancer cells for high-throughput screening and drug discovery. Nat Biotechnol. 2001; 19:940–945. DOI: 10.1038/nbt1001-940.
89.
KaelinWGJr.The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer. 2005; 5:689–698. DOI: 10.1038/nrc1691.
90.
NijmanSM, FriendSH. Cancer. Potential of the synthetic lethality principle. Science. 2013; 342:809–811. DOI: 10.1126/science.1244669.
91.
MeehanRS, ChenAP. New treatment option for ovarian cancer: PARP inhibitors. Gynecol Oncol Res Pract. 2016; 3:3. DOI: 10.1186/s40661-016-0024-7.
92.
RobsonM, ImSA, SenkusE, et al.Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N Engl J Med. 2017; 377:523–533. DOI: 10.1056/NEJMoa1706450.
93.
HanK, JengEE, HessGT, et al.Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nature Biotechnology. 2017; 35:463–474. DOI: 10.1038/nbt.3834.
94.
HorlbeckMA, XuA, WangM, et al.Mapping the genetic landscape of human cells. Cell. 2018; 174:953–967 e922. DOI: 10.1016/j.cell.2018.06.010.
95.
TrachoothamD, AlexandreJ, HuangP. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?. Nat Rev Drug Discov. 2009; 8:579–591. DOI: 10.1038/nrd2803.
96.
ManasanchEE, OrlowskiRZ. Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol. 2017; 14:417–433. DOI: 10.1038/nrclinonc.2016.206.
97.
WetterstenHI, AboudOA, LaraPNJr., et al.Metabolic reprogramming in clear cell renal cell carcinoma. Nat Rev Nephrol. 2017; 13:410–419. DOI: 10.1038/nrneph.2017.59.
98.
KaelinWGJr.Molecular basis of the VHL hereditary cancer syndrome. Nat Rev Cancer. 2002; 2:673–682. DOI: 10.1038/nrc885.
99.
ScarpaA, ChangDK, NonesK, et al.Whole-genome landscape of pancreatic neuroendocrine tumours. Nature. 2017; 543:65–71. DOI: 10.1038/nature21063.
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