Abstract 5103: Notch is a master regulator of mesenchymal transformation and therapeutic resistance in glioma
Authors: Banu Matei A. , Dovas Athanassios , Cuervo Henar , Pereira Brianna , Mahajan Aayushi , Ding Hongxu , Bansal Mukesh , Pampou Sergey , Karan Charles , Califano Andrea , Sims Peter A. , Kitajewski Jan , Bruce Jeffrey N. , Canoll Peter 
Published: August 2018

Abstract
Introduction: Gliomas are highly infiltrative tumors rendering complete resection impossible. As such, prognosis remains dismal. Recent studies have identified both proneural and mesenchymal cell populations. Crucially, recurring gliomas bear a mesenchymal phenotype. Understanding mechanisms driving mesenchymal transformation is important in developing tailored, more efficient therapeutic approaches.
Methods: Using targeted biopsies from a mouse glioma model and human glioma databases, we applied bioinformatics and identified Notch as an additional master regulator (MR) protein driving mesenchymal transformation. To better understand the role of Notch, we overexpressed active forms of Notch1 (N1IC) or Notch3 (N3IC) in primary cells, orthotopic xenotransplants, and transgenic mouse models. We performed mouse survival studies and examined Notch-induced phenotypic alterations in tumor cells and the microenvironment using molecular and immunohistochemical approaches. Finally, we performed high-throughput drug and radiation screening studies to assess responses to treatment.
Results: In silico analyses on RNAseq of biopsies from spatially distinct tumor regions in a mouse glioma model identified Notch as a highly active pathway in the cores of tumors undergoing mesenchymal transformation. Importantly, MR analysis of TCGA samples and single cell transcriptomic data identified the Notch pathway as a driver of proneural to mesenchymal transformation. Immunophenotyping and RNAseq profiling of human GBMs identified Notch receptors on tumor cells and Notch ligands on astrocytes and microglia suggesting potential microenvironment-mediated plasticity since Notch proteins are not frequently mutated in GBM. Overexpression of N1IC or N3IC in proneural cells induced mesenchymal transformation with slow proliferation and a highly invasive phenotype. Orthotransplantation of N3IC-expressing cells in mice led to slower growing tumors, perivascular invasion, increased recruitment of reactive phagocytes and slightly prolonged survival. A novel transgenic mouse model in which N1IC was overexpressed also led to prolonged survival compared to a control population. Importantly, high-throughput screening studies showed that N1IC and N3IC rendered tumor cells significantly more resistant to standard and targeted therapies, including irradiation and etoposide.
Conclusions: Our study identifies Notch signaling as an important, likely microenvironment-driven pathway driving proneural to mesenchymal transformation in glioma. While Notch was shown to slow tumor growth and prolong survival, cells were more invasive and less sensitive to treatment. These findings suggest that Notch plays a role in the mesenchymal transformation and therapeutic resistance seen in post-treatment recurrence. Targeting Notch may therefore be important in improving therapeutic responses against glioma.
Citation Format: Matei A. Banu, Athanassios Dovas, Henar Cuervo, Brianna Pereira, Aayushi Mahajan, Hongxu Ding, Mukesh Bansal, Sergey Pampou, Charles Karan, Andrea Califano, Peter A. Sims, Jan Kitajewski, Jeffrey N. Bruce, Peter Canoll. Notch is a master regulator of mesenchymal transformation and therapeutic resistance in glioma [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 5103.