CELL BIOLOGY AND SIGNALING

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the co-activation of multiple receptor tyrosine kinases (RTKs), which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Herein we present a novel mechanism by which fibroblast growth factor receptors (FGFRs) and src family kinases (SFKs) impact PI3K signaling in GBM by phosphorylating the PTEN tumor suppressor at a conserved tyrosine residue, Y240. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Experimentally, mutation of PTEN tyrosine 240 to phenylalanine generates an allele of PTEN that potently sensitizes cells expressing mutant EGFR to erlotinib. In contrast, activation of FGFR signaling promotes resistance to erlotinib in glioblastoma cells expressing wild type PTEN, concordant with phosphorylation of PTEN and activation of downstream signaling. Together, our findings identify a novel signaling connection between FGFRs and PTEN and provide a mechanistic link between PTEN regulation and drug resistance, suggesting that blocking PTEN phosphorylation by suppression of src and/or FGFR activity represents a potential strategy to re-sensitize tumors to EGFR inhibitors. INTRODUCTION: The majority of glioblastomas (GBMs) harbor amplification/mutation of a receptor tyrosine kinase (RTK), although clinical trials of RTK inhibitors have shown inconsistent responses. One possible mechanism of resistance is activation of multiple RTKs in a tumor. While co-activation has been documented at the protein level in GBM, its significance in maintaining cell populations is unknown. We investigated whether amplification of two different RTKs can occur independently in GBM tumor cell subpopulations, the mechanism of clonal evolution, and the functional significance of genotype heterogeneity for pharmacologic treatment. METHODS/RESULTS: ACGH profiles of 460 TCGA GBMs and 150 tumors from MSKCC were examined for focal amplifications spanning EGFR, PDGFRA, and MET. Multicolor FISH was performed for EGFR, PDGFRA, and cen7 for 8 coamplified cases. For two cases, derived tumor sphere lines were expanded for study. Coamplification of 2 or more RTKs was observed in 24 specimens. Remarkably, 43% of GBMs with focal PDGFRA amplification also contained either EGFR or MET amplifications. FISH-demonstrated amplicons were primarily in distinct tumor cell subpopulations, interspersed rather than regionally segregated. Expanded cell lines from EGFR/PDGFRA coamplified tumors maintained distinct RTK-driven subpopulations that were subject to selection under EGF or PDGF ligand stimulation in vitro. Simultaneous inhibition of both EGFR and PDGFR by gefitinib and imatinib was necessary to suppress PI3 kinase pathway activation. Analysis of whole exome and whole genome sequencing of coamplified TCGA samples and cell line subpopulations supports a common clonal origin with late divergence of RTK-amplified clades, although sequencing depth is limited. CONCLUSION: This is the first documentation of multiple subpopulations with distinct, high-level RTK amplifications in GBM. These results show certain GBMs commonly harbor subpopulations with other RTK amplifications requiring drugs targeting each subpopulation for effective treatment. This data should be considered for current trials of RTK inhibitors, particularly those targeting PDGFR. The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytoma. We examined the role of RhoG in the invasive behavior of glioblastoma cells. We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG strongly decreases colony formation, demonstrating a role for RhoG in glioblastoma cell survival. Importantly, we found that RhoG is activated by HGF and EGF, 2 factors that are clinically relevant drivers of glioblastoma malignant behavior. In addition, depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. We also observed that in addition to controlling cell invasion, RhoG regulates glioblastoma cell migration and the formation of lamellipodia and invadopodia, all of which are functions that have been shown to be Rac1-dependent. However, unlike Rac1, depletion of RhoG does not significantly inhibit cell proliferation, suggesting that RhoG regulates a subset of Rac1-controlled functions. Importantly, in contrast to Rac1, the functions of RhoG in normal cells appear to be rather restricted, and RhoG knockout mice display no significant phenotypes. Thus, our results suggest that targeting RhoG-mediated signaling presents a novel avenue to limit the malignant behavior of glioblastoma tumors. PURPOSE: Glioblastoma multiforme (GBM) is the highest grade and most aggressive form of glioma. Average patient survival is about 14 months after diagnosis. The relative resistance to conventional therapy displayed by GBM leaves opportunity for vast improvement in survival. Elucidating mechanisms of radiation resistance or uncovering novel targets to sensitize tumors to radiation could significantly benefit patient outcomes. Nicotinamide-N-methyltransferase (NNMT) is an enzyme involved in the nicotinamide pathway as well as in the metabolism of drugs and xenobiotic compounds. While the exact role NNMT plays in the malignant phenotype is poorly understood, it has been investigated for its potential use as a prognostic marker or therapeutic target in other cancers. NNMT is highly expressed in glioblastoma compared to either lower grade astrocytomas or normal brain tissue. Furthermore, Kaplan-Meier plots from publically available clinical data sets have shown that relatively higher NNMT expression is correlated with adverse patient outcome. Gaining a better understanding of NNMT's role in GBM could uncover exciting advances in neuro-oncology. EXPERIMENTAL DESIGN: We explored the expression profile of NNMT in established (ATCC) and in our panel of patient-derived primary GBM cell lines. We generated an isogenic, in vitro model by knocking down as well as overexpressing NNMT in U87 cells. We conducted various functional experiments such as MTS assay, clonogenic survival assay, annexin V, ATP assay, comet assay, and western blotting analysis. In vivo experiments were also used to determine differences in relative tumorigenicity. RESULTS AND CONCLUSION: Intracranial implantation of NNMT knockdown cells in mice showed a delayed tumor growth compared to NNMT overexpression cells. Silencing NNMT leads to an enhanced sensitivity to radiation in vitro, whereas cells overexpressing NNMT are more resistant to radiation. These results merit further investigation into NNMT's role in GBM treatment resistance.