This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353 and R01 NS109655 to S.L.C.; R01 NS109655-03S1 to D.P.J.), the National Cancer Institute (R01 CA122804 to S.L.C.), and the Department of Defense (X81XWH-09-1-0086 and W81XWH-12-1-0164 to S.L.C.).

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

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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. 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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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The authors have no conflicts of interest to disclose.

The detailed methods presented here were developed to study peripheral nervous system neoplasia and MPNST pathogenesis. Although we have found these methods to be effective, it should be recognized that there are some potential limitations to the methods we describe here. Below, we discuss some of those limitations and potential strategies for overcoming them in other model systems.
We have found that whole exome sequencing effectively identifies mutations of interest in P0-GGF&#946...

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.

<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

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.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

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

Figure 5&#160;plots display depletion scores of core essential genes (CEGs) labeled as TRUE compared to non-CEGs (labeled as FALSE) in each human cell line that was screened. Points represent log2 of fold depletion scores for individual genes, which are plotted over a boxplot representation of the overall score distribution. Student&#39;s t-test was used to test for a significant difference in the mean of depletion scores between the two groups in each cell line. The resulting p...

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

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.

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

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JoVE Journal

Cancer Research

Lentiviral Packaging and Viral Titering for Genome-Scale shRNA Screens

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

Cytometer Assays of MPNST Cells Challenged with Therapeutic Agents

710 Views.  Medical University of South Carolina.  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.

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

概要