Transposon Mutagenesis-Guided CRISPR/Cas9 Screening Strongly Implicates Dysregulation of Hippo/YAP Signaling in Malignant Peripheral Nerve Sheath Tumor Development

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.

Keywords: cancer biology; genetic screen; neurofibromatosis Type 1.

Figure 1

A streamlined approach to assess tumor suppressor gene candidates and oncogene function in human MPNST cell lines reveals novel MPNST pathways. (A) Diagram depicting summary of candidate selection from sleeping beauty (SB) screen to CRISPR/Cas9 forward genetic screening. (B) Diagram depicting screening method. First, gRNAs were designed to target genes selected in (A) and then stable cell lines were created and assessed for transformation upon genetic knockout. (C,D) show a summary of scores and anchorage-independent growth for predicted and known tumor suppressor gene candidates (1.5× magnification).

Figure 2

TAOK1 is a Schwann cell tumor suppressor gene. (A) Western blot showing TAOK1 decreased levels and increased YAP levels upon gRNA against TAOK1 in a human immortalized Schwann cell line and loss of YAP Western blot in the MPNST cell line S462. (B) Anchorage-independent assay showing cell transformation upon TAOK1 knockout in (C) and anoikis upon treatment with verteporfin (1.5× magnification). (D) Inhibition of YAP/TAZ activity and tyrosine kinase inhibition results in loss of anchorage-independent growth. (E) Tumor formation in NRG mice.

Figure 3

Rho is a MPNST pathway and its negative regulator GDI2 is a novel Schwann cell tumor suppressor gene. (A) Western blot showing knockout of GDI2 in an immortalized Schwann cell line and levels in S462 MPNST cells. (B) Loss of GDI2 results in transformation as seen by increased anchorage-independent growth cell migration and tumor formation in NRGs (1.5× magnification). (C) Loss of GDI2 results in FAK activation and treatment with defactinib, a Rho inhibitor results in decreased anchorage-independent growth (2× magnification). Digital Western blot (Wess by Protein Simple) shows decreased FAK phosphorylation in GDI2-deficient cells upon treatment with defactinib. (D) Loss of GDI2 results in increased YAP levels and nuclear localization.

Figure 4

Wnt pathway negative regulator APC is a Schwann cell tumor suppressor gene and multiple pathways converge downstream resulting in Hippo/YAP activation. (A) Western blot showing loss of APC via CRISPR/Cas9 and increased YAP1 and CTNNB1 levels. (B) Loss of APC results in anchorage-independent growth and cell migration (1.5× magnification). (C) Loss of APC results is tumor formation in NRG xenograft model. (D) Diagram summarizing results showing overall Hippo/YAP activation via the loss of multiple tumor suppressor genes that occur in MPNSTs. Wnt, growth factor activity, cell density and Rho pathway(s) activation results in Hippo/YAP pathway activation in MPNSTs.