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

Summary

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.

Abstract

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.

Introduction

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 radiotherapy^1,2,3. 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 trigge....

Protocol

Prior to the initiation of the studies, have animal procedures and protocols for handling viral vectors reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). The procedures described here were approved by the Medical University of South Carolina's IACUC and IBC Boards and were performed by properly trained personnel in accordance with the NIH Guide for Care and Use of Laboratory Animals and MUSC's institutional animal care guidelines.

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Discussion

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-GGFβ.......

Materials

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