- Email: [email protected]
CAS9 Accelerator
EDITORIAL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Cancer Gene Discovery in Hepatocellular Carcinoma: The CRISPR/CAS9 Accelerator See “Genome-wide CRISPR screen identifies regulators of mapk as suppressors of liver tumors in mice,” by Song CQ, Li Y, Mou H, et al, on page 000.
R
ecent advances in gene editing have enabled us to easily manipulate DNA using CRISPR (clustered regularly interspaced short palindromic repeats)/CAS9 (CRISPR-associated protein 9) technology.1 CRISPR/CAS9 is an endonuclease that exploits the DNA double-strand break repair pathway to cleave DNA at specific DNA target sites.1 These properties have been translated into the ability to rapidly and precisely modify DNA in cells, a technological revolution that is accelerating biological research.2 In vivo RNA interference screening has been used previously in mouse models to identify the genes involved in liver tumorogenesis or in resistance to biotherapy.3 CRISPR/ CAS9 genome editing can also be combined with genome scale RNA libraries to perform unbiased genetic screening in cancer research.4–7 In the field of hepatocellular carcinoma (HCC), a comprehensive picture of somatic genetic defects has been extensively described using next-generation sequencing.8,9 The main genetic drivers belong to the following signaling pathways: telomere maintenance (TERT promoter mutations, 60%), cell cycle genes (TP53 14-40%, CDKN2A 2-12%), Wnt/B-catenin (CTNNB1 12-37%, AXIN1 5-15%), epigenetic modifiers (ARID1A 5-17%, ARID2 3-10%), mammalian target of rapamycin pathways (PTEN 1-3%, PIK3CA 1%), and RAS/RAF/MAPK pathways (KRAS 1%, RP6SKA3 2-9%).10–12 However, effects occurring at the cellular level of the most frequently mutated genes need to be explored extensively. Moreover, the functionality of the numerous genes mutated at low frequency (the so-called long tail of infrequently mutated genes) is basically unknown.13 Additional studies are required to determine which genes can be considered drivers and which are merely passengers. In an elegant study published in this issue of Gastroenterology, Song et al7 used the CRISPR/CAS9 system to identify genes involved in liver carcinogenesis and to assess their functionality. The authors transfected liver progenitor cells deleted for P53-null and -overexpressing MYC via a library of single-guide RNA (sgRNA) using the CRIPSR/CAS9 system, and transfected these cells into nude mice. Tumors derived from these experiments are believed to result from clonal selection of cells harboring deletions of genes targeted by sgRNA (Figure 1). Among tumors analyzed, a panel of genes targeted by this system (NF1, TSC2, NF2, BIM, and Plnb1) were identified and potentially
considered to be tumor suppressor genes responsible for hepatocyte transformation. Underlying the robustness of this approach is the fact that TSC2 is a tumor suppressor gene mutated in around of 3% to 5% of HCC, and its role in liver carcinogenesis was already demonstrated in previous studies.14,15 Next, the authors focused on NF1 (neurofibromatosis type 1) analysis, because few data exist concerning its role in liver carcinogenesis. NF1 is a negative regulator of RAS; thus, NF1 inhibition leads to constitutive activation of the RAS/RAF/MAP kinase pathway.16 The authors showed that NF1 knockdown in p53–/–MycþCas9 cells leads to tumor formation in nude mice, with constitutive activation of the RAS/RAF/MAPK pathway. Interestingly, the authors used hydrodynamic tail injection of sgRNA/CAS9 to knock down NF1, together with APC, PTEN, ARID1A, and TET2, in the livers of P53 knockout mice. NF1 inactivation induced tumor formation, suggesting cooperation between RAS/RAF/MAPK pathways and other pathways recurrently mutated in HCC. Interestingly, tumors induced by NF1 inactivation harbored reexpression of some stem cell markers (HMGA2, SOX9) potentially associated with poor prognosis in human HCC. Knockdown of these stem cell markers reversed cell proliferation in NF1-inactivated cell lines and decreased tumor formation in nude mice. This suggests that activation of RAS/RAF/ MAPK could reprogram hepatocytes and reactivate stem cell markers, potentially possessing oncogenic properties (Figure 1). Finally, Song et al7 showed that inhibition of RAS/RAF/MAPK by sorafenib or, more specifically, by the MEK inhibitors AZD6244 and trametinib decreased tumor formation in NF1 knockout cells. Overall, this study showed that CRISPR/CAS9 may help to promote the discovery of new key genes involved in liver carcinogenesis, and the testing of new drugs for targeting these pathways.5 It will also be useful for rapidly validating the functionality (or not) of the “long tail” of genes identified by next-generation sequencing that are rarely mutated.4 However, the genome-wide CRISPR screen proposed by Song et al has several limitations, including the difficulties to shutdown some genes or the absence of exploration of oncogenes. Song et al also pinpointed the role of RAS/RAF/ MAPK pathway activation in liver carcinogenesis.7,17 In human HCC, NF1 is rarely found mutated, with 1% to 2% of mutations.8 However, other mutations known to activate the RAS/RAF/MAPK pathway are recurrently identified in HCC, that is, the KRAS mutation (1%) and RPS6KA3 (2%-9%), and could be targeted by specific inhibitors of the pathway (AZD6244 and trametinib).8,18,19 Moreover, in human HCC, genetic alterations might be cooperative (CTNNB1 with TERT; AXIN1 with TP53) or exclusive (TP53 versus CTNNB1 mutations), suggesting a complex interplay between signaling pathways in liver carcinogenesis.9 This study also Gastroenterology 2017;-:1–3
EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
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EDITORIAL
Figure 1. Role of the MAPK pathway in liver carcinogenesis. The authors performed genomewide CRISPR/CAS 9 screening in liver progenitor cells subcutaneously implanted in nude mice. Genetic analysis of the tumor and subsequent in vivo and in vitro experiments validated the role of neurofibromatosis type 1 (NF1) as a tumor suppressor gene, and the RAS/ RAF/MAPK pathway as a key signaling pathway in hepatocellular carcinoma (HCC) development. Percentages of mutations in human HCC are shown in blue for tumor suppressor genes and red for oncogenes; potential biotherapy is shown in purple.
print & web 4C=FPO
120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
suggests that hydrodynamic tail injection of CAS9 sgRNA in genetically engineered mice will be useful for easy testing of cooperation between signaling pathways.6 In conclusion, the RAS/RAF/MAPK pathway is a key signaling pathway in liver carcinogenesis, potentially
associated with a stem cell phenotype that may be druggable using targeted biotherapy. Overall, CRISPR/CAS9 technology will help to accelerate the discovery of new cancer genes in HCC and to validate their potential role as drivers of liver carcinogenesis.
2 EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
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Q2
JEAN-CHARLES NAULT Liver Unit Hôpital Jean Verdier Hôpitaux universitaires Paris-Seine-Saint-Denis Assistance-publique Hôpitaux de Paris Bondy and Unité mixte de Recherche 1162 Génomique fonctionnelle des Tumeurs solides Institut National de la Santé et de la Recherche médicale Paris and Unité de Formation et de Recherche Santé Médecine et Biologie humaine Université Paris 13 Communauté d’Universités et Etablissements Sorbonne Paris Cité Paris, France
References 1. Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 2015;16:299–311. 2. Wang T, Wei JJ, Sabatini DM, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science 2014;343:80–84. 3. Rudalska R, Dauch D, Longerich T, et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat Med 2014;20:1138–1146. 4. Chen S, Sanjana NE, Zheng K, et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 2015;160:1246–1260. 5. Weber J, Ollinger R, Friedrich M, et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc Natl Acad Sci U S A 2015;112:13982–13987. 6. Sanchez-Rivera FJ, Jacks T. Applications of the CRISPR-Cas9 system in cancer biology. Nat Rev Cancer 2015;15:387–395. 7. Song CQ, Li Y, Mou H, et al. Genome-wide CRISPR screen identifies regulators of mapk as suppressors of liver tumors in mice. Gastroenterology 2016;00. 000-000. 8. Schulze K, Nault JC, Villanueva A. Genetic profiling of hepatocellular carcinoma using next-generation sequencing. J Hepatol 2016;65:1031–1042. 9. Zucman-Rossi J, Villanueva A, Nault JC, et al. Genetic landscape and biomarkers of hepatocellular carcinoma. Gastroenterology 2015;149:1226–1239 e4.
10. Nault JC, Mallet M, Pilati C, et al. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun 2013;4:2218. 11. Schulze K, Imbeaud S, Letouze E, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015;47:505–511. 12. Totoki Y, Tatsuno K, Covington KR, et al. Trans-ancestry mutational landscape of hepatocellular carcinoma genomes. Nat Genet 2014;46:1267–1273. 13. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013;339: 1546–1558. 14. Ho DW, Chan LK, Chiu YT, et al. TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication. Gut 2016 Dec 14 [Epub ahead of print]. 15. Huynh H, Hao HX, Chan SL, et al. Loss of tuberous sclerosis complex 2 (TSC2) is frequent in hepatocellular carcinoma and predicts response to mTORC1 inhibitor everolimus. Mol Cancer Ther 2015; 14:1224–1235. 16. Dhillon AS, Hagan S, Rath O, et al. MAP kinase signalling pathways in cancer. Oncogene 2007;26:3279–3290. 17. Calvisi DF, Ladu S, Gorden A, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006;130:1117–1128. 18. Zhao Y, Adjei AA. The clinical development of MEK inhibitors. Nat Rev Clin Oncol 2014;11:385–400. 19. Guichard C, Amaddeo G, Imbeaud S, et al. Integrated analysis of somatic mutations and focal copynumber changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet 2012;44:694–698.
Reprint requests Address requests for reprints to: Jean-Charles Nault, APHP, Hôpitaux universitaires Paris – Seine Saint-Denis, Site Jean Verdier, Pôle d’Activité Cancérologique spécialisée, Service d’Hépatologie, 93143 Bondy, France. e-mail: [email protected].
Conflicts of interest The author discloses no conflicts.
Q1
© 2017 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2017.02.031
3 EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357
Cancer Gene Discovery in Hepatocellular Carcinoma: The CRISPR/CAS9 Accelerator See “Genome-wide CRISPR screen identifies regulators of mapk as suppressors of liver tumors in mice,” by Song CQ, Li Y, Mou H, et al, on page 000.
R
ecent advances in gene editing have enabled us to easily manipulate DNA using CRISPR (clustered regularly interspaced short palindromic repeats)/CAS9 (CRISPR-associated protein 9) technology.1 CRISPR/CAS9 is an endonuclease that exploits the DNA double-strand break repair pathway to cleave DNA at specific DNA target sites.1 These properties have been translated into the ability to rapidly and precisely modify DNA in cells, a technological revolution that is accelerating biological research.2 In vivo RNA interference screening has been used previously in mouse models to identify the genes involved in liver tumorogenesis or in resistance to biotherapy.3 CRISPR/ CAS9 genome editing can also be combined with genome scale RNA libraries to perform unbiased genetic screening in cancer research.4–7 In the field of hepatocellular carcinoma (HCC), a comprehensive picture of somatic genetic defects has been extensively described using next-generation sequencing.8,9 The main genetic drivers belong to the following signaling pathways: telomere maintenance (TERT promoter mutations, 60%), cell cycle genes (TP53 14-40%, CDKN2A 2-12%), Wnt/B-catenin (CTNNB1 12-37%, AXIN1 5-15%), epigenetic modifiers (ARID1A 5-17%, ARID2 3-10%), mammalian target of rapamycin pathways (PTEN 1-3%, PIK3CA 1%), and RAS/RAF/MAPK pathways (KRAS 1%, RP6SKA3 2-9%).10–12 However, effects occurring at the cellular level of the most frequently mutated genes need to be explored extensively. Moreover, the functionality of the numerous genes mutated at low frequency (the so-called long tail of infrequently mutated genes) is basically unknown.13 Additional studies are required to determine which genes can be considered drivers and which are merely passengers. In an elegant study published in this issue of Gastroenterology, Song et al7 used the CRISPR/CAS9 system to identify genes involved in liver carcinogenesis and to assess their functionality. The authors transfected liver progenitor cells deleted for P53-null and -overexpressing MYC via a library of single-guide RNA (sgRNA) using the CRIPSR/CAS9 system, and transfected these cells into nude mice. Tumors derived from these experiments are believed to result from clonal selection of cells harboring deletions of genes targeted by sgRNA (Figure 1). Among tumors analyzed, a panel of genes targeted by this system (NF1, TSC2, NF2, BIM, and Plnb1) were identified and potentially
considered to be tumor suppressor genes responsible for hepatocyte transformation. Underlying the robustness of this approach is the fact that TSC2 is a tumor suppressor gene mutated in around of 3% to 5% of HCC, and its role in liver carcinogenesis was already demonstrated in previous studies.14,15 Next, the authors focused on NF1 (neurofibromatosis type 1) analysis, because few data exist concerning its role in liver carcinogenesis. NF1 is a negative regulator of RAS; thus, NF1 inhibition leads to constitutive activation of the RAS/RAF/MAP kinase pathway.16 The authors showed that NF1 knockdown in p53–/–MycþCas9 cells leads to tumor formation in nude mice, with constitutive activation of the RAS/RAF/MAPK pathway. Interestingly, the authors used hydrodynamic tail injection of sgRNA/CAS9 to knock down NF1, together with APC, PTEN, ARID1A, and TET2, in the livers of P53 knockout mice. NF1 inactivation induced tumor formation, suggesting cooperation between RAS/RAF/MAPK pathways and other pathways recurrently mutated in HCC. Interestingly, tumors induced by NF1 inactivation harbored reexpression of some stem cell markers (HMGA2, SOX9) potentially associated with poor prognosis in human HCC. Knockdown of these stem cell markers reversed cell proliferation in NF1-inactivated cell lines and decreased tumor formation in nude mice. This suggests that activation of RAS/RAF/ MAPK could reprogram hepatocytes and reactivate stem cell markers, potentially possessing oncogenic properties (Figure 1). Finally, Song et al7 showed that inhibition of RAS/RAF/MAPK by sorafenib or, more specifically, by the MEK inhibitors AZD6244 and trametinib decreased tumor formation in NF1 knockout cells. Overall, this study showed that CRISPR/CAS9 may help to promote the discovery of new key genes involved in liver carcinogenesis, and the testing of new drugs for targeting these pathways.5 It will also be useful for rapidly validating the functionality (or not) of the “long tail” of genes identified by next-generation sequencing that are rarely mutated.4 However, the genome-wide CRISPR screen proposed by Song et al has several limitations, including the difficulties to shutdown some genes or the absence of exploration of oncogenes. Song et al also pinpointed the role of RAS/RAF/ MAPK pathway activation in liver carcinogenesis.7,17 In human HCC, NF1 is rarely found mutated, with 1% to 2% of mutations.8 However, other mutations known to activate the RAS/RAF/MAPK pathway are recurrently identified in HCC, that is, the KRAS mutation (1%) and RPS6KA3 (2%-9%), and could be targeted by specific inhibitors of the pathway (AZD6244 and trametinib).8,18,19 Moreover, in human HCC, genetic alterations might be cooperative (CTNNB1 with TERT; AXIN1 with TP53) or exclusive (TP53 versus CTNNB1 mutations), suggesting a complex interplay between signaling pathways in liver carcinogenesis.9 This study also Gastroenterology 2017;-:1–3
EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
EDITORIAL
Figure 1. Role of the MAPK pathway in liver carcinogenesis. The authors performed genomewide CRISPR/CAS 9 screening in liver progenitor cells subcutaneously implanted in nude mice. Genetic analysis of the tumor and subsequent in vivo and in vitro experiments validated the role of neurofibromatosis type 1 (NF1) as a tumor suppressor gene, and the RAS/ RAF/MAPK pathway as a key signaling pathway in hepatocellular carcinoma (HCC) development. Percentages of mutations in human HCC are shown in blue for tumor suppressor genes and red for oncogenes; potential biotherapy is shown in purple.
print & web 4C=FPO
120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
suggests that hydrodynamic tail injection of CAS9 sgRNA in genetically engineered mice will be useful for easy testing of cooperation between signaling pathways.6 In conclusion, the RAS/RAF/MAPK pathway is a key signaling pathway in liver carcinogenesis, potentially
associated with a stem cell phenotype that may be druggable using targeted biotherapy. Overall, CRISPR/CAS9 technology will help to accelerate the discovery of new cancer genes in HCC and to validate their potential role as drivers of liver carcinogenesis.
2 EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238
EDITORIAL 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297
Q2
JEAN-CHARLES NAULT Liver Unit Hôpital Jean Verdier Hôpitaux universitaires Paris-Seine-Saint-Denis Assistance-publique Hôpitaux de Paris Bondy and Unité mixte de Recherche 1162 Génomique fonctionnelle des Tumeurs solides Institut National de la Santé et de la Recherche médicale Paris and Unité de Formation et de Recherche Santé Médecine et Biologie humaine Université Paris 13 Communauté d’Universités et Etablissements Sorbonne Paris Cité Paris, France
References 1. Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 2015;16:299–311. 2. Wang T, Wei JJ, Sabatini DM, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science 2014;343:80–84. 3. Rudalska R, Dauch D, Longerich T, et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat Med 2014;20:1138–1146. 4. Chen S, Sanjana NE, Zheng K, et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 2015;160:1246–1260. 5. Weber J, Ollinger R, Friedrich M, et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc Natl Acad Sci U S A 2015;112:13982–13987. 6. Sanchez-Rivera FJ, Jacks T. Applications of the CRISPR-Cas9 system in cancer biology. Nat Rev Cancer 2015;15:387–395. 7. Song CQ, Li Y, Mou H, et al. Genome-wide CRISPR screen identifies regulators of mapk as suppressors of liver tumors in mice. Gastroenterology 2016;00. 000-000. 8. Schulze K, Nault JC, Villanueva A. Genetic profiling of hepatocellular carcinoma using next-generation sequencing. J Hepatol 2016;65:1031–1042. 9. Zucman-Rossi J, Villanueva A, Nault JC, et al. Genetic landscape and biomarkers of hepatocellular carcinoma. Gastroenterology 2015;149:1226–1239 e4.
10. Nault JC, Mallet M, Pilati C, et al. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun 2013;4:2218. 11. Schulze K, Imbeaud S, Letouze E, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015;47:505–511. 12. Totoki Y, Tatsuno K, Covington KR, et al. Trans-ancestry mutational landscape of hepatocellular carcinoma genomes. Nat Genet 2014;46:1267–1273. 13. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013;339: 1546–1558. 14. Ho DW, Chan LK, Chiu YT, et al. TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication. Gut 2016 Dec 14 [Epub ahead of print]. 15. Huynh H, Hao HX, Chan SL, et al. Loss of tuberous sclerosis complex 2 (TSC2) is frequent in hepatocellular carcinoma and predicts response to mTORC1 inhibitor everolimus. Mol Cancer Ther 2015; 14:1224–1235. 16. Dhillon AS, Hagan S, Rath O, et al. MAP kinase signalling pathways in cancer. Oncogene 2007;26:3279–3290. 17. Calvisi DF, Ladu S, Gorden A, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006;130:1117–1128. 18. Zhao Y, Adjei AA. The clinical development of MEK inhibitors. Nat Rev Clin Oncol 2014;11:385–400. 19. Guichard C, Amaddeo G, Imbeaud S, et al. Integrated analysis of somatic mutations and focal copynumber changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet 2012;44:694–698.
Reprint requests Address requests for reprints to: Jean-Charles Nault, APHP, Hôpitaux universitaires Paris – Seine Saint-Denis, Site Jean Verdier, Pôle d’Activité Cancérologique spécialisée, Service d’Hépatologie, 93143 Bondy, France. e-mail: [email protected].
Conflicts of interest The author discloses no conflicts.
Q1
© 2017 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2017.02.031
3 EDI 5.4.0 DTD YGAST61014_proof 2 March 2017 12:27 am ce
298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357