Frank Furnari, Ph.D. - US grants

DESCRIPTION (provided by applicant): Minimal improvement in the 12-15 month survival of patients with glioblastoma mutliforme (GBM) has been achieved despite in depth knowledge of pathogenic lesions and decades of advances in neurosurgery, radiation therapy and clinical trials. A central issue that confounds successful treatment is the heterogeneous nature of this aggressive tumor. This heterogeneity presents phenotypically as mixed cytological subtypes, genotypically as mutations and gene amplifications, and transcriptionally as regional differences in gene expression. As a result, multiple and spatially distinct heterotypic populations exist within a GBM, making any lesion- or pathway-specific therapy less effective. While much effort has been placed on understanding the interactions between heterotypic tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within these neoplasms. In GBM, amplification of the epidermal growth factor receptor, a hallmark mutation present in 50% of cases, is often detected in a heterogeneous manner and frequently associated with structural alterations. The most common of these alterations, ?EGFR, (also known as de2-7EGFR, deltaEGFR and EGFR*) results in a constitutively active mutant receptor with tumor enhancing capability. This ability is lacking from amplified wtEGFR despite its highly pervasive tumor expression in comparison to focally occurring ?EGFR. By modeling this type of genetic heterogeneity in vivo, we have determined that an IL-6 paracrine mechanism driven by ?EGFR can recruit wtEGFR-expressing cells into accelerated proliferation and therefore result in the maintenance of heterogeneity. Given the ineffectiveness of EGFR-directed therapeutics, we postulate that ?EGFR/wtEGFR sub-population interactions not only enhance aggressive tumor growth but also play a role in therapeutic resistance. The overall goal of this project is to dissect the mechanisms whereby GBM receptor heterogeneity drives tumor aggressiveness and therapeutic resistance. The following lines of experimentation will be carried out: 1) genetic and biochemical analysis as well as tumor heterogeneity modeling in mice will determine critical effectors intrinsic to ?EGFR- and wtEGFR- signaling that mediate heterogeneity maintenance; 2) mass spectrometry, genetic and biochemical analysis will determine the mechanism of IL-6 receptor-mediated cross- talk activation of wtEGFR; and 3) genetic and pharmacological inhibition of identified effectors will be used to uncouple heterotypic glioma cell interactions to enhance therapeutic efficacy. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM), the most common primary brain tumor in adults is a highly invasive, neurologically destructive tumor with a survival range of 12-15 months, despite maximum treatment efforts. A central issue that contributes to this lack of successful treatment is the tumor's heterogeneous or varied cell type composition. As a result, multiple and spatially distinct tumor cell populations exist within a typical GBM, making any single therapy less effective. This proposal will identify the genes and mechanisms that promote GBM heterogeneity. These insights will lead to new tools for improving the efficacy of known therapeutic agents as well as for the development of new therapeutic approaches.

One major issue confounding successful treatment of glioblastoma multiforme (GBM) is the presence of highly invasive cells disseminating into the brain parenchyma. These cells evade surgical resection and often spread distally in brain parenchyma. Multiple and spatially distinct heterotypic populations exist within a single GBM, giving rise to the disease?s genetic heterogeneity and leading to complex cell intrinsic and extrinsic mechanisms of invasion. Amplification of the epidermal growth factor receptor (EGFR), a hallmark mutation present in 60% of cases, most often occurs in a heterogeneous manner and is frequently associated with deletion of exons 2-7, creating a constitutively active mutant, EGFRvIII. While significant focus has been placed on its kinase activity, comparatively little is known about EGFRvIII?s ability to enhance migration via interaction with adhesion receptors. Our preliminary data supports a dual role for EGFRvIII where it interferes with intrinsic adhesion receptors and also recruits non-transformed counterparts via extrinsic signaling to reduce adhesion of a mixed population. Based on our findings, we hypothesize that this difference in adhesive activity is due to differential signaling associated with EGFRvIII, and that this receptor conveys this phenotype to non-transformed counterparts through cytokine production (Inda, Genes & Dev, 2010; Zanca, Genes & Dev, 2017) to cooperatively invade parenchyma. With this hypothesis, we will use adhesion measurement technologies to dissect cell intrinsic EGFR-mediated invasion mechanisms; given the heterogeneity within tumors, we will also combine newly developed adhesion sorting technologies with high throughput sequencing technologies to identify cell extrinsic mechanisms and targets for subsequent intervention. The following lines of experimentation will be carried out: 1) implementation of biophysical assays and signaling pathway analyses to interrogate how cell intrinsic activity of EGFRvIII leads to labile adhesion and an invasive phenotype; 2) biochemical and functional analysis of the EGFRvIII cell extrinsic, secretome-mediated education of wtEGFR cell adhesive phenotype; 3) expression and epigenetic analyses on adhesion-sorted populations will be used to define a migratome signature, its stability in wtEGFR cells after exposure to the EGFRvIII secretome, and the ability of ?educated? wtEGFR to propagate that epigenetic signature to naïve wtEGFR cells.