이 연구는 국립 신경 질환 및 뇌졸중 연구소(R01 NS048353 및 R01 NS109655 SLC의 보조금으로 지원되었습니다. R01 NS109655-03S1 - D.P.J.), 국립 암 연구소(R01 CA122804 - S.L.C.), 국방부(X81XWH-09-1-0086 및 W81XWH-12-1-0164 - S.L.C.).

종양의 치료 표적을 식별하고 비교하기 위한 게놈 규모 스크리닝

<ol>
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P., Orlow, I., Scheithauer, B. W., Cordon-Cardo, C., Woodruff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletions+of+the+INK4A+gene+occur+in+malignant+peripheral+nerve+sheath+tumors+but+not+in+neurofibromas.">Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas.</a> Am J Pathol. 155 (6), 1855-1860 (1999).</li><li>Nielsen, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+of+neurofibromas+in+neurofibromatosis+1+is+associated+with+CDKN2A/p16+inactivation.">Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation.</a> Am J Pathol. 155 (6), 1879-1884 (1999).</li><li>Gregorian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+dosage+is+essential+for+neurofibroma+development+and+malignant+transformation.">PTEN dosage is essential for neurofibroma development and malignant transformation.</a> Proc Natl Acad Sci U S A. 106 (46), 19479-19484 (2009).</li><li>Lee, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRC2+is+recurrently+inactivated+through+EED+or+SUZ12+loss+in+malignant+peripheral+nerve+sheath+tumors.">PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors.</a> Nat Genet. 46 (11), 1227-1232 (2014).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mutations+of+SUZ12+in+malignant+peripheral+nerve+sheath+tumors.">Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors.</a> Nat Genet. 46 (11), 1170-1172 (2014).</li><li>Varela, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+structural+rearrangements+in+genetically+engineered+mouse+mammary+tumors.">Somatic structural rearrangements in genetically engineered mouse mammary tumors.</a> Genome Biol. 11 (10), 100 (2010).</li><li>Johnson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+genomics+matches+driver+mutations+and+cell+compartments+to+model+ependymoma.">Cross-species genomics matches driver mutations and cell compartments to model ependymoma.</a> Nature. 466 (7306), 632-636 (2010).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+oncogenomics+identifies+NEDD9+as+a+melanoma+metastasis+gene.">Comparative oncogenomics identifies NEDD9 as a melanoma metastasis gene.</a> Cell. 125 (7), 1269-1281 (2006).</li><li>Zender, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+oncogenes+in+liver+cancer+using+an+integrative+oncogenomic+approach.">Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach.</a> Cell. 125 (7), 1253-1267 (2006).</li><li>Uren, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+mutagenesis+in+p19(ARF)-+and+p53-deficient+mice+identifies+cancer+genes+and+their+collaborative+networks.">Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.</a> Cell. 133 (4), 727-741 (2008).</li><li>Starr, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon-based+genetic+screen+in+mice+identifies+genes+altered+in+colorectal+cancer.">A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.</a> Science. 323 (5922), 1747-1750 (2009).</li><li>Dupuy, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+sleeping+beauty+transposon+system+that+can+be+used+to+model+a+wide+variety+of+human+cancers+in+mice.">A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice.</a> Cancer Res. 69 (20), 8150-8156 (2009).</li><li>Carroll, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenge+of+Cancer+Genomics+in+Rare+Nervous+System+Neoplasms:+Malignant+Peripheral+Nerve+Sheath+Tumors+as+a+Paradigm+for+Cross-Species+Comparative+Oncogenomics.">The Challenge of Cancer Genomics in Rare Nervous System Neoplasms: Malignant Peripheral Nerve Sheath Tumors as a Paradigm for Cross-Species Comparative Oncogenomics.</a> Am J Pathol. 186 (3), 464-477 (2016).</li><li>Huijbregts, R. P., Roth, K. A., Schmidt, R. E., Carroll, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+neuropathies+and+malignant+peripheral+nerve+sheath+tumors+in+transgenic+mice+overexpressing+glial+growth+factor+beta3+in+myelinating+Schwann+cells.">Hypertrophic neuropathies and malignant peripheral nerve sheath tumors in transgenic mice overexpressing glial growth factor beta3 in myelinating Schwann cells.</a> J Neurosci. 23 (19), 7269-7280 (2003).</li><li>Kazmi, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+overexpressing+neuregulin-1+model+neurofibroma-malignant+peripheral+nerve+sheath+tumor+progression+and+implicate+specific+chromosomal+copy+number+variations+in+tumorigenesis.">Transgenic mice overexpressing neuregulin-1 model neurofibroma-malignant peripheral nerve sheath tumor progression and implicate specific chromosomal copy number variations in tumorigenesis.</a> Am J Pathol. 182 (3), 646-667 (2013).</li><li>Brosius, S. 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R., Sircoulomb, F., Rottapel, R., Moffat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+error+rates+in+genomic+perturbation+screens:+gold+standards+for+human+functional+genomics.">Measuring error rates in genomic perturbation screens: gold standards for human functional genomics.</a> Mol Syst Biol. 10 (7), 733 (2014).</li><li>Hart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+Design+of+Genome-Wide+CRISPR/SpCas9+Knockout+Screens.">Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.</a> G3. 7 (8), 2719-2727 (2017).</li><li>Longo, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+genomic+characterization+of+a+sporadic+malignant+peripheral+nerve+sheath+tumor+cell+line.">Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line.</a> Sci Rep. 11 (1), 5690 (2021).</li><li>Maser, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447 (7147), 966-971 (2007).</li><li>Rahrmann, E. 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저자는 공개할 이해 상충이 없습니다.

여기에 제시된 자세한 방법은 말초 신경계 종양 및 MPNST 발병 기전을 연구하기 위해 개발되었습니다. 이러한 방법이 효과적인 것으로 확인되었지만 여기에서 설명하는 방법에는 몇 가지 잠재적인 제한 사항이 있음을 인식해야 합니다. 아래에서는 이러한 한계 중 일부와 다른 모델 시스템에서 이를 극복하기 위한 잠재적 전략에 대해 설명합니다.
전체 엑솜 염기서열분석이 P 0-...

악성 말초신경초종양(MPNST)은 종양감수성 증후군인 신경섬유종증 1형(NF1)과 관련하여 발생하는 매우 공격적인 방추세포 신생물로, 일반 인구 집단과 이전 방사선 치료 부위에서 산발적으로 발생합니다 1,2,3. NF1 환자는 NF1 종양 억제 유전자의 야생형 사본과 기능 상실 돌연변이가 있는 두 번째 NF1 대립유전자 를 가지고 태어납니다. 이러한 하플로인기능부전(haploinsufficiency) 상태는 NF1 환자를 야생형 NF1 유전자의 두 번째 기능 상실 돌연변이에 취약하게 만들어 종양형성을 유발합니다. 이 "두 번째 히트" NF1 돌연변이가 슈반(Schwann) 세포 계통의 세포에서 발생하면, 그 결과로 생기는 종양은 피부에서 발생하는 진피 신경섬유종이거나 큰 신경 또는 신경총에서 발생하는 망상형 신경섬유종입니다. 진피 신경섬유종과 망상형 신경섬유종의 병리학은 동일하지만, 생물학적 거동은 상당히 다릅니다 - 진피와 망상신경섬유종은 모두 양성이지만, 망상신경섬유종만이 변형을 겪을 수 있고 MPNST를 일으킬 수 있습니다. NF1 유전자에 의해 암호화된 Ras GTPase 활성화 단백질인 뉴로피브롬의 손실 외에도, MPNST는 TP53 4,5,6,7, CDKN2A 8,9 및 PTEN 10을 포함한 여러 다른 종양 억제 유전자의 돌연변이, 폴리콤 억제 복합체 2 11,12 의 구성 요소를 암호화하는 유전자의 돌연변이(PRC2; SUZ12 및 EED 유전자) 및 수용체 티로신 키나아제 1,2의 비정상적인 발현. NF1 및 상술한 다른 유전자들의 돌연변이는 산발적이고 방사선에 의해 유도된 MPNST에서도 존재한다11,12.
MPNST의 게놈 이상에 대한 이해의 이러한 발전은 발병 기전을 이해하는 데 매우 중요하지만 MPNST에 대한 효과적인 새로운 치료법의 개발로 이어지지는 않았습니다. 새로운 치료법의 개발을 방해하는 주요 장벽은 MPNST가 희귀 암이라는 사실입니다. 이 때문에 TCGA(The Cancer Genome Atlas)에서 수행한 것과 같은 주요 동인 돌연변이를 정의하는 글로벌 분석에 필요한 많은 수의 환자 샘플을 얻기가 어렵습니다. 우리의 경험에 비추어 볼 때, 적당한 수의 인간 MPNST 표본을 축적하는 데에도 몇 년이 걸릴 수 있습니다. 이러한 한계를 극복하기 위해 다른 희귀 암 유형을 연구하는 많은 연구자들은 필수 드라이버 유전자 돌연변이를 식별하고, 관심 종양에서 필수 세포질 신호 전달 경로를 정의하고, 새로운 치료 표적을 식별하기 위해 종 간 비교 종양유전체학을 사용하고 있습니다. 종양형성에 필수적인 신호전달 경로는 인간과 다른 척추동물 종 사이에서 매우 잘 보존되어 있기 때문에 게놈 스케일 shRNA 스크리닝과 같은 기능적 유전체학 접근법을 적용하는 것은 이러한 새로운 드라이버 돌연변이, 신호전달 경로 및 치료 표적을 식별하는 효과적인 수단이 될 수 있습니다 13,14,15,16,17,18,19, 특히 제한된 수로 사용할 수 있는 희귀 인간 종양 유형을 연구할 때20개.
여기에 제시된 방법론에서는 성장 인자 뉴레굴린-1(NRG1)의 슈반(Schwann) 세포 특이적 과발현이 망상형 신경섬유종의 발병 기전과 MPNST로의 후속 진행을 촉진하는 유전자 조작 마우스 모델(GEM)인 P 0-GGFβ3 마우스에서 유래한 인간 MPNST 세포주 및 초기 통과 MPNST 배양에서 게놈 프로파일링을 수행하는 이러한 접근 방식을 설명합니다21, 22,23. 이 접근법의 첫 번째 단계는 P 0-GGFβ3 MPNST, 인간 MPNST 세포주 및 외과적으로 절제된 인간 MPNST에서 후보 드라이버 유전자를 식별하는 것입니다. 이러한 돌연변이의 영향을 받는 신호 전달 경로를 기능적으로 검증하기 위해 게놈 스케일 shRNA 스크리닝을 사용하여 인간 및 마우스 MPNST 세포주에서 증식과 생존에 필요한 유전자를 식별합니다. 증식과 생존에 필요한 유전자를 확인한 후, 약물 유전자 상호작용 데이터베이스(Drug Gene Interaction Database)를 사용하여 "히트(hit)" 컬렉션 내에서 약물 가능한 유전자 산물을 식별합니다. 또한 GEM 모델과 인간 MPNST가 동일한 유전자 및 신호 전달 경로에 대해 유사한 의존성을 보이는지 여부를 결정하기 위해 인간 및 마우스 MPNST 세포의 "적중"을 비교합니다. 증식 및 생존에 필요한 유전자와 영향을 받는 신호 전달 경로의 중복을 식별하는 것은 분자 수준에서 P 0-GGFβ3 마우스 모델을 검증하는 수단이 됩니다. 이 접근법은 또한 인간 스크리닝을 보완하는 역할을 할 수 있는 새로운 치료 표적을 식별하기 위해 인간과 마우스 스크리닝을 결합하는 효과를 강조합니다. 이러한 종간 접근법의 가치는 인간 종양과 세포주를 얻기 어려운 희귀 종양에서 치료 표적을 찾을 때 특히 분명합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioruptor Sonication System</td>
			<td>Diagenode&#160;</td>
			<td>UCD-600</td>
			<td></td>
		</tr>
		<tr>
			<td>CASAVA 1.8.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cbot</td>
			<td>Illumina, San Diego, CA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Celigo Image Cytometer</td>
			<td>Nexcelom</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellecta Barcode Analyzer and Deconvoluter software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrisolve Hybrid</td>
			<td>Decon Laboratories</td>
			<td>5989-27-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-well Black Microplate</td>
			<td>Millipore Sigma</td>
			<td>CLS3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Diagenode Bioruptor 15ml conical tubes</td>
			<td>Diagenode&#160;</td>
			<td>C30010009</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Clontech</td>
			<td>639210</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Thermo Scientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td>Qiagen&#160;</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (200 Proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel&#160;</td>
			<td>Microsoft&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FWDGEX 5&#8217;-CAAGCAGAAGACGGCATACGAGA-3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FWDHTS 5&#8217;-TTCTCTGGCAAGCAAAAGACGGCATA-3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GexSeqS (5&#8217; AGAGGTTCAGAGTTCTACAGTCCGAA-3&#8217;</td>
			<td></td>
			<td></td>
			<td>HPLC purified</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>Dotmatics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Fisherbrand</td>
			<td>245-677</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiScanSQ</td>
			<td>Illumina, San Diego, CA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (4%)</td>
			<td>Thermo Scientific</td>
			<td>J19943-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>PLUS Transfection Reagent</td>
			<td>Thermo Scientific</td>
			<td>11514015</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene Transfection Reagent</td>
			<td>Millipore Sigma</td>
			<td>TR1003G</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick PCR Purification Kit</td>
			<td>Invitrogen</td>
			<td>K310001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen Buffer P1</td>
			<td>Qiagen&#160;</td>
			<td>19051</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>RevGEX 5&#8217;-AATGATACGGCGACCACCGAGA-3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RevHTS1 5&#8217;-TAGCCAACGCATCGCACAAGCCA-3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Taq polymerase</td>
			<td>Clontech</td>
			<td>639210</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimmomatic software</td>
			<td></td>
			<td>www.usadellab.org</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic profiling and genome-scale dropout screening to identify therapeutic targets in mouse models of malignant peripheral nerve sheath tumor

악성 말초 신경초 종양(MPNST)은 슈반(Schwann) 세포 또는 그 전구체에서 유래합니다. 종양 감수성 증후군 신경섬유종증 1형(NF1) 환자에서 MPNST는 가장 흔한 악성 종양이자 주요 사망 원인입니다. 이 희귀하고 공격적인 연조직 육종은 5년 무병 생존율이 34-60%에 달하는 암울한 미래를 제시합니다. MPNST 환자를 위한 치료 옵션은 실망스러울 정도로 제한적이며, 변형 수술이 가장 중요한 치료 옵션입니다. Ras 신호전달 억제제인 티피파르닙(tipifarnib)과 같이 한때 유망했던 많은 치료법이 임상적으로 실패했다. 마찬가지로, 표피 성장 인자(EFGR)를 표적으로 하는 엘로티닙과 혈관 내피 성장 인자 수용체(VEGF), 혈소판 유래 성장 인자 수용체(PDGF)를 표적으로 하는 소라페닙, 그리고 Raf를 표준 화학요법과 병용하는 임상 2상 시험에서도 환자에게 반응을 보이지 못했습니다. 
최근 몇 년 동안 암 세포주의 유전자 프로파일링과 결합된 기능적 게놈 스크리닝 방법은 필수 세포질 신호 전달 경로를 식별하고 표적 특이적 치료법을 개발하는 데 유용한 것으로 입증되었습니다. 희귀 종양 유형의 경우, 종간 비교 종양유전체학(cross-species comparative oncogenomics)으로 알려진 이 접근법의 변형이 새로운 치료 표적을 식별하는 데 점점 더 많이 사용되고 있습니다. 종간 비교 종양유전체학에서 유전자 프로파일링 및 기능적 유전체학은 유전자 조작 마우스(GEM) 모델에서 수행되며 결과는 사용 가능한 희귀 인간 표본 및 세포주에서 검증됩니다. 
본 논문은 전체 엑솜 염기서열분석(whole exome sequencing, WES)을 이용하여 인간 및 마우스 MPNST 세포에서 후보 드라이버 유전자 돌연변이를 식별하는 방법을 설명합니다. 그런 다음 게놈 스케일 shRNA 스크리닝을 수행하여 마우스 및 인간 MPNST 세포에서 중요한 신호 전달 경로를 식별 및 비교하고 이러한 경로에서 약물 가능한 표적을 식별하는 방법을 설명합니다. 이러한 방법론은 다양한 인간 암 유형에서 새로운 치료 표적을 식별하는 효과적인 접근 방식을 제공합니다.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive spindle cell neoplasms that arise in association with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), sporadically in the general population and at sites of previous radiotherapy1,2,3. NF1 patients are born with a wild-type copy of the NF1 tumor suppressor gene and a second NF1 allele with a loss-of-function mutation. This state of haploinsufficiency renders NF1 patients susceptible to a second loss-of-function mutation in their wild-type NF1 gene, which triggers tumorigenesis. When this &#34;second hit&#34; NF1 mutation occurs in a cell in the Schwann cell lineage, the resulting tumor is either a dermal neurofibroma arising in the skin or a plexiform neurofibroma that develops in large nerves or nerve plexuses. Although the pathology of dermal and plexiform neurofibromas is identical, their biologic behavior is quite different-although both dermal and plexiform neurofibromas are benign, only plexiform neurofibromas can undergo transformation and give rise to MPNSTs. In addition to the loss of neurofibromin, the Ras GTPase-activating protein encoded by the NF1 gene, MPNSTs carry mutations of multiple other tumor suppressor genes, including TP534,5,6,7, CDKN2A8,9, and PTEN10, mutations of genes encoding components of&#160;polycomb repressive complex 211,12 (PRC2; the SUZ12 and EED genes) and aberrant expression of receptor tyrosine kinases1,2. Mutations of NF1 and the other genes noted above are also present in sporadic and radiation-induced MPNSTs11,12.
While these advances in our understanding of the genomic abnormalities in MPNSTs have been invaluable for understanding their pathogenesis, they have not yet resulted in the development of effective new therapies for MPNSTs. A major barrier impeding the development of new treatments is the fact that MPNSTs are rare cancers. Because of this, it is difficult to obtain the large number of patient samples that are required for global analyses defining key driver mutations such as those undertaken by The Cancer Genome Atlas (TCGA). In our experience, accumulating even a modest number of human MPNST specimens can take years. To overcome such limitations, many investigators studying other rare cancer types have turned to the use of cross-species comparative oncogenomics to identify essential driver gene mutations, define the essential cytoplasmic signaling pathways in their tumor of interest, and identify new therapeutic targets. Since the signaling pathways that are essential for tumorigenesis are highly conserved between humans and other vertebrate species, applying functional genomics approaches such as genome-scale shRNA screens can be an effective means of identifying these new driver mutations, signaling pathways, and therapeutic targets13,14,15,16,17,18,19, particularly when studying rare human tumor types that are available in limiting numbers20.
In the methodologies presented here, we describe this approach to performing genomic profiling in human MPNST cell lines and early passage MPNST cultures derived from P0-GGF&#946;3 mice, a genetically engineered mouse model (GEM) in which Schwann cell-specific overexpression of the growth factor neuregulin-1 (NRG1) promotes the pathogenesis of plexiform neurofibromas and their subsequent progression to MPNSTs21,22,23. The first step in this approach is to identify candidate driver genes in P0-GGF&#946;3 MPNSTs, human MPNST cell lines, and surgically resected human MPNSTs. To functionally validate the signaling pathways affected by these mutations, we then use genome-scale shRNA screens to identify the genes required for proliferation and survival in human and mouse MPNST cell lines. After identifying the genes required for proliferation and survival, we then identify the druggable gene products within the collection of &#34;hits&#34; using the Drug Gene Interaction Database. We also compare the &#34;hits&#34; in human and mouse MPNST cells, to determine whether the GEM model and human MPNSTs demonstrate similar dependence on the same genes and signaling pathways. Identifying overlaps in the genes required for proliferation and survival and the affected signaling pathways serves as a means of validating the P0-GGF&#946;3 mouse model at a molecular level. This approach also emphasizes the effectiveness of combining human and mouse screens to identify novel therapeutic targets, where the mouse model can serve as a complement to the human screens. The value of this cross-species approach is particularly apparent when looking for therapeutic targets in rare tumors, where human tumors and cell lines are difficult to obtain.

저자 스포트라이트: 게놈 스케일 shRNA 스크리닝을 통한 악성 말초 신경초 종양에 대한 새로운 치료 표적 찾기 

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

We are striving to understand the pathogenesis of sporadic and NF1-associated malignant peripheral nerve sheath tumors. Our goal is to logically develop new treatments for these highly aggressive neoplasms. Technologies we are currently implementing to advance our research include unbiased methods like genome-scale shRNA screens, as well as single-cell sequencing, BaseScope in situ probes, and bioinformatics.Using the various current approaches, we have identified multiple receptors that are essential for malignant peripheral nerve sheath tumors'proliferation and survival, as well as being therapeutically targetable. These receptors include the neuregulin receptor, ErbB3, and lysophosphatidic acid receptor 3. The advantage of using this approach lies in its capacity to examine the proliferative significance of over 15, 000 genes in MPNST cancer cell lines.This is an unbiased approach that identifies unexplored targets in the treatment of MPNSTs. Moving forward, our laboratory's focus will concentrate on validating the targets identified in our screen. This includes using inhibitors on cell lines and culture and ultimately, translating that to animal work.

그림 5 플롯은 스크리닝된 각 인간 세포주에서 TRUE로 표시된 핵심 필수 유전자(CEG)의 결핍 점수를 비 CEG(FALSE로 표시)와 비교하여 보여줍니다. 포인트는 개별 유전자에 대한 접힘 고갈 점수의 log2를 나타내며, 이는 전체 점수 분포의 상자 그림 표현 위에 표시됩니다. 스튜던트 t-검정은 각 세포주에서 두 그룹 간의 공핍 점수 평균의 유의한 차이를 검정하는 데 사용되었?...

Watch this Scientific Journal Video about Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens at JoVE.com

우리는 유전자 조작 마우스 모델에서 발생하는 종양과 해당 인간 종양 유형에서 발생하는 종양의 치료 표적을 식별하고 비교하기 위해 게놈 분석 및 기능적 게놈 스크리닝을 활용하는 종간 비교 종양유전체학 접근 방식을 개발했습니다.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

우리는 산발성 및 NF1 관련 악성 말초 신경초 종양의 발병 기전을 이해하기 위해 노력하고 있습니다. 우리의 목표는 이러한 매우 공격적인 신생물에 대한 새로운 치료법을 논리적으로 개발하는 것입니다. 현재 연구를 발전시키기 위해 구현하고 있는 기술에는 게놈 스케일 shRNA 스크리닝, 단일 세포 염기서열 분석, BaseScope in situ 프로브 및 생물정보학과 같은 편향되지 않은 방법이 포함됩니다.현재의 다양한 접근법을 사용하여 악성 말초 신경초 종양의 증식과 생존에 필수적일 뿐만 아니라 치료적으로 표적이 될 수 있는 여러 수용체를 확인했습니다. 이러한 수용체에는 뉴레굴린 수용체(neuregulin receptor), ErbB3 및 리소포스파티드산 수용체(lysophosphatidic acid receptor) 3가 포함됩니다. 이 접근법 사용의 장점은 MPNST 암 세포주에서 15 이상, 000 유전자의 증식 중요성을 시험하는 능력에 있습니다.이는 MPNST 치료에서 탐색되지 않은 표적을 식별하는 편향되지 않은 접근 방식입니다. 앞으로 우리 실험실의 초점은 화면에서 식별된 표적을 검증하는 데 집중할 것입니다. 여기에는 세포주와 배양에 억제제를 사용하는 것이 포함되며 궁극적으로 이를 동물 실험으로 전환하는 것이 포함됩니다.

genetic-profiling-genome-scale-dropout-screening-to-identify

Research

JoVE Journal

Cancer Research

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

697 조회수.  Medical University of South Carolina. 우리는 산발성 및 NF1 관련 악성 말초 신경초 종양의 발병 기전을 이해하기 위해 노력하고 있습니다. 우리의 목표는 이러한 매우 공격적인 신생물에 대한 새로운 치료법을 논리적으로 개발하는 것입니다. 현재 연구를 발전시키기 위해 구현하고 있는 기술에는 게놈 스케일 shRNA 스크리닝, 단일 세포 염기서열 분석, BaseScope in situ 프로브 및 생물정보학과 같은 편향되지 않은 방?...

비디오: 저자 스포트라이트: 게놈 스케일 shRNA 스크리닝을 통한 악성 말초 신경초 종양에 대한 새로운 치료 표적 찾기 

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

연구

암 연구학

Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens

On day zero plate 10 million 293T cells in 30 milliliters of antibiotic-free DMEM containing 10%FBS in a 15 centimeter dish. Prepare a total of 10 such dishes. On the next day, which is day one, confirm that the cells are 80%confluent and ready for transfection.Then in a 50 milliliter conical tube sequentially, add 600 microliters of 0.5 micrograms per microliter packaging plasmid mix and 60 microliters plasmid barcode library. In a separate 15 milliliter conical tube mix 900 microliters of transfection reagent and 12 milliliters DMEM by vortexing. Add 12.9 milliliters of this mixture to the DNA mix and just flick to combine.Without mixing any further, incubate the mix at room temperature for 15 minutes. Then drop wise add 2.5 milliliters of the incubated mixture to each 15 centimeter dish containing the 293T cells and incubate the cells overnight in a tissue culture incubator. On day three, harvest the virus by passing the media through a 0.2 micron filtration unit.Aliquot the filtrate containing viral particles into 15 milliliter conical tubes. Additionally, prepare five one milliliter aliquots of filtered virus in cryo vials for viral titering. To titer the lentiviral pools, add 65 microliters of Cationic polymer to a sterile glass flask containing 65 milliliters of tumor cell growth media.Pipette one milliliter of the polymer containing media per well into 11 six-well tissue culture plates. Next trypsinize the early passage tumor cells. After counting the cells using a preferred method, transfer the cells into the six-well plates.Thaw the one milliliter aliquots of the 48 hour lentivirus from the freezer in a water bath at 37 degrees Celsius. Follow the table to prepare the infection for each viral module.

게놈 스케일 shRNA 스크리닝을 위한 렌티바이러스 패키징 및 바이러스 적정

Lentiviral Infection of MPNST Cells and Genomic DNA Isolation for Genome-Scale shRNA Screens

For lentiviral infection, plate the trypsinized MPNST target cells after counting them, maintaining 2.5 million cells per 15 centimeter dish. Transduce the cells with the thawed virus at a 0.5 multiplicity of infection, in the presence of five micrograms per milliliter of the cationic polymer. Trypsinize the cells three days after the addition of puromycin.Centrifuge half of the cell population at 200 G for five minutes in a tabletop centrifuge. After re-plating the other half of the cell population, grow them for approximately seven population doublings before harvesting and centrifuging as previously demonstrated. Divide the re-suspended cell pellet corresponding to the desired time into two 15 milliliter polymethylpentene tubes.Add 500 microliters of 10%SDS per tube. Mix and incubate at room temperature for five minutes. Place the tubes in a DNA shearing device to sonicate the DNA.Following the addition of phenol and chloroform. Mix well by vortexing vigorously at the maximum possible speed for 45 to 60 seconds. Transfer three milliliters of the resulting clear upper phase from each tube to another fresh 15 milliliter tube.Add 0.5 milliliters of three molar sodium acetate and four milliliters of isopropanol and mix well. After centrifuging the tubes for 30 minutes at 7, 200 G and 20 degrees Celsius and discarding the supernatant, add 0.5 milliliters of 70%ethanol to the pellet and dislodge it by pipetting up and down. Combine the re-suspended pellets from both tubes into a 1.5 milliliter centrifuge tube.Perform the PCR reactions using the conditions shown on the screen. After the second PCR reaction, combine the four samples into one micro-centrifuge tube. And add 80 microliters of 6X loading dye to it.Visualize the gel and confirm a band at approximately 250 base pairs. Human MPNST cells transduced with a non-target control and lentivirus, expressing four different Bcl-6 short hairpin RNA, revealed that several of the Bcl-6 short hairpin RNAs markedly reduced cell numbers. The immuno blot showed that the degree of decrease in cell numbers correlated with the degree of Bcl-6 knockdown.

게놈 스케일 shRNA 스크리닝을 위한 MPNST 세포의 렌티바이러스 감염 및 게놈 DNA 분리

Cytometer Assays of MPNST Cells Challenged with Therapeutic Agents

To begin, plate the 80%confluent malignant peripheral nerve sheath tumor or MPNST cells at a density of 1200 cells per well in black-walled 96 well plates. After preparing dilutions of the drug to be tested, add each dilution or vehicle to at least three replicate cells. To assess direct cell number, add hoaxed 33342, to a final concentration of one to five micrograms per milliliter to the plates.And incubate the plates for 30 minutes at 37 degrees Celsius. Place the plates on a high throughput imaging cytometer and scan the plates using the direct cell count for total cell number option with 100, 000 to 600, 000 milliseconds exposure times. Analyze the reads using the software, export them into a spreadsheet and use appropriate software for statistical analyses.