Transposon mutagenesis-guided crispr/cas9 screening strongly implicates dysregulation of hippo/yap signaling in malignant peripheral nerve sheath tumor development

Germán L. Vélez-Reyes, Nicholas Koes, Ji Hae Ryu, Gabriel Kaufmann, Mariah Berner, Madison T. Weg, Natalie K. Wolf, Susan K. Rathe, Nancy Ratner, Branden S. Moriarity, David A. Largaespada

Research output: Contribution to journalArticlepeer-review

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive, genomically complex, have soft tissue sarcomas, and are derived from the Schwann cell lineage. Patients with neurofibromatosis type 1 syndrome (NF1), an autosomal dominant tumor predisposition syndrome, are at a high risk for MPNSTs, which usually develop from pre-existing benign Schwann cell tumors called plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the NF1 gene, which encode neurofibromin, a Ras GTPase activating protein (GAP) and negative regulator of RasGTP-dependent signaling. In addition to bi-allelic loss of NF1, other known tumor suppressor genes include TP53, CDKN2A, SUZ12, and EED, all of which are often inactivated in the process of MPNST growth. A sleeping beauty (SB) transposon-based genetic screen for high-grade Schwann cell tumors in mice, and comparative genomics, implicated Wnt/β-catenin, PI3K-AKT-mTOR, and other pathways in MPNST development and progression. We endeavored to more systematically test genes and pathways implicated by our SB screen in mice, i.e., in a human immortalized Schwann cell-based model and a human MPNST cell line, using CRISPR/Cas9 technology. We individually induced loss-of-function mutations in 103 tumor suppressor genes (TSG) and oncogene candidates. We assessed anchorage-independent growth, transwell migration, and for a subset of genes, tumor formation in vivo. When tested in a loss-of-function fashion, about 60% of all TSG candidates resulted in the transformation of immortalized human Schwann cells, whereas 30% of oncogene candidates resulted in growth arrest in a MPNST cell line. Individual loss-of-function mutations in the TAOK1, GDI2, NF1, and APC genes resulted in transformation of immortalized human Schwann cells and tumor formation in a xenograft model. Moreover, the loss of all four of these genes resulted in activation of Hippo/Yes Activated Protein (YAP) signaling. By combining SB transposon mutagenesis and CRISPR/Cas9 screening, we established a useful pipeline for the validation of MPNST pathways and genes. Our results suggest that the functional genetic landscape of human MPNST is complex and implicate the Hippo/YAP pathway in the transformation of neurofibromas. It is thus imperative to functionally validate individual cancer genes and pathways using human cell-based models, to determinate their role in different stages of MPNST development, growth, and/or metastasis.

Original languageEnglish (US)
Article number1584
JournalCancers
Volume13
Issue number7
DOIs
StatePublished - Mar 30 2021

Bibliographical note

Funding Information:
Funding: This work was supported by grants to GVR (T32 GM008244, T32 HL007741), DAL (R01 CA134759, R01 CA113636, R01 NS086219 and American Cancer Society Research Professor Award #123939).

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • Cancer biology
  • Genetic screen
  • Neurofibromatosis Type 1

PubMed: MeSH publication types

  • Journal Article

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