Adrienne L. Watson

Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies new genes and pathways driving tumorigenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/β-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance.

Canonical Wnt/β-catenin Signaling Drives Human Schwann Cell Transformation, Progression, and Tumor Maintenance

Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of β-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.

PTEN and NF1 inactivation in Schwann cells produces a severe phenotype in the peripheral nervous system that promotes the development and malignant progression of peripheral nerve sheath tumors.

The genetic evolution from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis type 1 (NF1) syndrome remains unclear. Schwann cells and/or their precursor cells are believed to be the primary pathogenic cell in neurofibromas because they harbor biallelic neurofibromin 1 (NF1) gene mutations. However, the phosphatase and tensin homolog (Pten) and neurofibromatosis 1 (Nf1) genes recently were found to be comutated in high-grade peripheral nerve sheath tumors (PNST) in mice. In this study, we created transgenic mice that lack both Pten and Nf1 in Schwann cells and Schwann cell precursor cells to validate the role of these two genes in PNST formation in vivo. Haploinsufficiency or complete loss of Pten dramatically accelerated neurofibroma development and led to the development of higher grade PNSTs in the context of Nf1 loss. Pten dosage, together with Nf1 loss, was sufficient for the progression from low-grade to high-grade PNSTs. Genetic analysis of human malignant PNSTs (MPNST) also revealed downregulation of PTEN expression, suggesting that Pten-regulated pathways are major tumor-suppressive barriers to neurofibroma progression. Together, our findings establish a novel mouse model that can rapidly recapitulate the onset of human neurofibroma tumorigenesis and the progression to MPNSTs.

Conditional Inactivation of Pten with EGFR Overexpression in Schwann Cells Models Sporadic MPNST

The genetic mechanisms involved in the transformation from a benign neurofibroma to a malignant sarcoma in patients with neurofibromatosis-type-1- (NF1-)associated or sporadic malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. It is hypothesized that many genetic changes are involved in transformation. Recently, it has been shown that both phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) play important roles in the initiation of peripheral nerve sheath tumors (PNSTs). In human MPNSTs, PTEN expression is often reduced, while EGFR expression is often induced. We tested if these two genes cooperate in the evolution of PNSTs. Transgenic mice were generated carrying conditional floxed alleles of Pten, and EGFR was expressed under the control of the 2′,3′-cyclic nucleotide 3′phosphodiesterase (Cnp) promoter and a desert hedgehog (Dhh) regulatory element driving Cre recombinase transgenic mice (Dhh-Cre). Complete loss of Pten and EGFR overexpression in Schwann cells led to the development of high-grade PNSTs. In vitro experiments using immortalized human Schwann cells demonstrated that loss of PTEN and overexpression of EGFR cooperate to increase cellular proliferation and anchorage-independent colony formation. This mouse model can rapidly recapitulate PNST onset and progression to high-grade PNSTs, as seen in sporadic MPNST patients.

Heparan sulfate Ndst1 regulates vascular smooth muscle cell proliferation, vessel size and vascular remodeling

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.

Trp53 Haploinsufficiency Modifies EGFR-Driven Peripheral Nerve Sheath Tumorigenesis

Malignant peripheral nerve sheath tumors (MPNSTs) are genetically diverse, aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 1 syndrome. Reduced TP53 gene expression and amplification/overexpression of the epidermal growth factor receptor (EGFR) gene occur in MPNST formation. We focused on determining the cooperativity between reduced TP53 expression and EGFR overexpression for Schwann cell transformation in vitro (immortalized human Schwann cells) and MPNST formation in vivo (transgenic mice). Human gene copy number alteration data, microarray expression data, and TMA analysis indicate that TP53 haploinsufficiency and increased EGFR expression co-occur in human MPNST samples. Concurrent modulation of EGFR and TP53 expression in HSC1λ cells significantly increased proliferation and anchorage-independent growth in vitro. Transgenic mice heterozygous for a Trp53-null allele and overexpressing EGFR in Schwann cells had a significant increase in neurofibroma and grade 3 PNST (MPNST) formation compared with single transgenic controls. Histological analysis of tumors identified a significant increase in pAkt expression in grade 3 PNSTs compared with neurofibromas. Array comparative genome hybridization analysis of grade 3 PNSTs identified recurrent focal regions of chromosomal gains with significant enrichment in genes involved in extracellular signal-regulated kinase 5 signaling. Collectively, altered p53 expression cooperates with overexpression of EGFR in Schwann cells to enhance in vitro oncogenic properties and tumorigenesis and progression in vivo.

Diet and Energy-Sensing Inputs Affect TorC1-Mediated Axon Misrouting but Not TorC2-Directed Synapse Growth in a Drosophila Model of Tuberous Sclerosis

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, we examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system.

Engineered Swine Models of Cancer.

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

Simple and Efficient Methods for Enrichment and Isolation of Endonuclease Modified Cells

The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KOs in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.

MMuFLR: missense mutation and frameshift location reporter

MOTIVATION Cancer researchers seeking immunotherapy targets in cancer cells need tools to locate highly expressed proteins unique to cancer cells. Missense mutation and frameshift location reporter (MMuFLR), a Galaxy-based workflow, analyzes next-generation sequencing paired read RNA-seq output to reliably identify small frameshift mutations and missense mutations in highly expressed protein-coding genes. MMuFLR ignores known SNPs, low quality reads and poly-A/T sequences. For each frameshift and missense mutation identified, MMuFLR provides the location and sequence of the amino acid substitutions in the novel protein candidates for direct input into epitope evaluation tools. AVAILABILITY http://toolshed.g2.bx.psu.edu/ CONTACT rath0096@umn.edu or johns198@umn.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.