Published: August 25th, 2023
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 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.
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 trigge....
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........
Figure 5 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'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.......
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.......
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.).....
|Bioruptor Sonication System
|Illumina, San Diego, CA
|Celigo Image Cytometer
|Cellecta Barcode Analyzer and Deconvoluter software
|Corning 96-well Black Microplate
|Diagenode Bioruptor 15ml conical tubes
|Ethanol (200 Proof)
|GexSeqS (5’ AGAGGTTCAGAGTTCTACAGTCCGAA-3’
|Illumina, San Diego, CA
|PLUS Transfection Reagent
|Polybrene Transfection Reagent
|PureLink Quick PCR Purification Kit
|Qiagen Buffer P1
|Qiagen Gel Extraction Kit
|Titanium Taq polymerase
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