Nicholas Koes

Recombinetics (United States)

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Gabriel Kaufmann

University of Minnesota Medical Center

David A. Largaespada

University of Minnesota

Barbara R. Tschida

University of Minnesota Medical Center

Germán L. Vélez‐Reyes

University of Minnesota Medical Center

Josep M. Llovet

Institució Catalana de Recerca i Estudis Avançats

Vincent W. Keng

Hong Kong Polytechnic University

Branden S. Moriarity

University of Minnesota

Juan E. Abrahante

University of Minnesota

Stanislas Corbiere

Institute for Research in Immunology and Cancer

Ji Hae Ryu

University of Minnesota Medical Center

Cooperative Institutions

University of Minnesota

University of Minnesota Medical Center

Masonic Cancer Center

University of Minnesota System

Hospital Clínic de Barcelona

Twin Cities Orthopedics

Consorci Institut D'Investigacions Biomediques August Pi I Sunyer

Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas

Recombinetics (United States)

Cincinnati Children's Hospital Medical Center

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

Cancers (2021)

Germán L. Vélez‐Reyes Nicholas Koes Ji Hae Ryu Gabriel Kaufmann Mariah J. Berner

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.

Schwann cell

10.3390/cancers13071584

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Citations (9)

Abstract LB-197: Transposon mutagenesis and CRISPR/Cas9 screening reveal pathways driving peripheral nerve sheath tumor development and maintenance

Cancer Research (2018)

Germán Reyes Nicholas Koes Gabriel Kaufmann Esther Ryu David A. Largaespada

Abstract Malignant Peripheral Nerve Sheath tumors(MPNSTs) are highly aggressive soft tissue sarcomas derived Schwann cell tumors. Half of these tumors occur sporadically, while the rest occur in the context of Neurofibromatosis Type 1 Syndrome (NF1) developing from pre-existing plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the gene encoding neurofibromin, a negative regulator of the oncogenic Ras pathway. To better understand genetic factors that give rise to MPNSTs, we performed a Sleeping Beauty (SB) transposon screen in mice. The results implicated Wnt/β-catenin, PI3K-AKT-mTOR, growth factor receptor signaling, and other pathways. It is our goal to understand the role of these pathways in human Schwann cell transformation, tumorigenesis, and tumor maintenance. We next sought to validate SB screen gene hits in a human cellular model using CRISPR/Cas9 technology as a tool to induce loss-of-function mutations in tumor suppressor gene (TSG) and oncogene candidates. A total 103 genes were independently targeted with multiple guideRNAs in immortalized human Schwann and MPNST cell lines and effects on transformation assessed. Transformation was assessed by anchorage-independent colony formation in soft agar, transwell migration, and tumor formation in NRG mice. In these assays, more than 30 genes scored as TSG candidates. Our results revealed a role for the Wnt/β-catenin, Hippo/Yap, PI3K-AKT-mTOR, Rho, and growth factor receptor signaling pathways in human neurofibroma and MPNST development and maintenance. These are generating hypothesis driven pre-clinical treatment studies we're pursuing now. Citation Format: German L. Velez Reyes, Nicholas Koes, Gabriel Kaufmann, Esther Ryu, David A. Largaespada. Transposon mutagenesis and CRISPR/Cas9 screening reveal pathways driving peripheral nerve sheath tumor development and maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-197.

Schwann cell

ErbB

10.1158/1538-7445.am2018-lb-197

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R‐spondin 2 Drives Liver Tumor Development in a Yes‐Associated Protein‐Dependent Manner

Hepatology Communications (2019)

Caitlin B. Conboy Germán L. Vélez‐Reyes Barbara R. Tschida Hsiangyu Hu Gabriel Kaufmann

Each year, more than 25,000 people succumb to liver cancer in the United States, and this neoplasm represents the second cause of cancer-related death globally. R-spondins (RSPOs) are secreted regulators of Wnt signaling that function in development and promote tissue stem cell renewal. In cancer, RSPOs 2 and 3 are oncogenes first identified by insertional mutagenesis screens in tumors induced by mouse mammary tumor virus and by transposon mutagenesis in the colonic epithelium of rodents. RSPO2 has been reported to be activated by chromosomal rearrangements in colorectal cancer and overexpressed in a subset of hepatocellular carcinoma. Using human liver tumor gene expression data, we first discovered that a subset of liver cancers were characterized by high levels of RSPO2 in contrast to low levels in adjacent nontumor tissue. To determine if RSPOs are capable of inducing liver tumors, we used an in vivo model from which we found that overexpression of RSPO2 in the liver promoted Wnt signaling, hepatomegaly, and enhanced liver tumor formation when combined with loss of transformation-related protein 53 (Trp53). Moreover, the Hippo/yes-associated protein (Yap) pathway has been implicated in many human cancers, influencing cell survival. Histologic and gene expression studies showed activation of Wnt/β-catenin and Hippo/Yap pathways following RSPO2 overexpression. We demonstrate that knockdown of Yap1 leads to reduced tumor penetrance following RSPO2 overexpression in the context of loss of Trp53. Conclusion: RSPO2 overexpression leads to tumor formation in the mouse liver in a Hippo/Yap-dependent manner. Overall, our results suggest a role for Yap in the initiation and progression of liver tumors and uncover a novel pathway activated in RSPO2-induced malignancies. We show that RSPO2 promotes liver tumor formation in vivo and in vitro and that RSPO2's oncogenic activity requires Hippo/Yap activation in hepatocytes. Both RSPO2 and YAP1 are suggested to represent novel druggable targets in Wnt-driven tumors of the liver.

Hippo signaling pathway

Liver Cancer

LGR5

Beta-catenin

YAP1

10.1002/hep4.1422

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Citations (21)

MODL-15. BRINGING PRECISION MEDICINE TO GLIOBLASTOMA PATIENTS BY MODELING MOLECULAR TUMOR SUBTYPES IN IMMUNOCOMPETENT SWINE FOR THERAPEUTIC AND BIOMARKER DEVELOPMENT

Neuro-Oncology (2022)

Adrienne L. Watson Derek M. Korpela Nicholas Koes Mandy Taisto Stanislas Corbiere

Abstract Glioblastoma (GBM) is an aggressive primary brain tumor with no effective cure. Standard of care treatment offers dismal prognoses for patients with a median survival time of only 15.6 months, and no new drugs have been approved in 15 years. Contributing factors include tumor heterogeneity, high mutation rates, infiltrative growth, a complex tumor-immune microenvironment, and the blood-brain barrier, all of which are obstacles clinicians and researchers must overcome to improve patient outcomes. Research to identify novel therapeutic strategies are ongoing, but treatments that show promise in preclinical studies often do not pass Phase 2 and 3 clinical trials. These costly failures are attributable to a lack of predictive preclinical models that translate to human application, low patient availability for numerous and robust clinical trials, and an “all-comer” approach to clinical trial design where patients are enrolled based on disease presence and not molecular features of the tumor. To advance treatments for GBM, Recombinetics has pioneered somatic gene-editing technologies to the same genetic changes seen in patient tumors in GBM-initiating cells in an immune-competent swine model. These models rapidly and reproducibly develop GBM tumors that resemble human GBMs genetically, clinically and histologically. In addition to the physiological, immunological, and metabolic parallels between swine and humans, the molecular data collected from our swine mesenchymal and classical subtypes of GBM validate accurate disease modeling and we are pursuing additional GBM subtypes. Using RNA-sequencing, we are characterizing molecular profiles of these tumors, as well as identifying and validating drug targets. Studies are ongoing to characterize the tumor immune microenvironment of our models for improved immune-oncology therapeutic development. These large animal models of GBM will usher in a new era of pre-clinical testing that predicts the efficacy of a variety of therapeutic approaches, using precision medicine to bring novel, safe, and effective therapies to patients.

Drug Development

Brain tumor

10.1093/neuonc/noac209.1143

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Citations (1)