SUMMARY EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document co-expression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in~5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

SUMMARY The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally-stabilized murine N-mycT58A into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem and forebrain. Transplantation of N-mycWT NSCs was insufficient for tumor formation. N-mycT58A cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating SHH-dependence and SHH-independence, respectively. These differences were regulated in-part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal.

Malignant progression is often associated with a mesenchymal phenotype and poor prognosis. To test whether radiotherapy promotes a mesenchymal transition, we irradiated proneural tumors arising in a genetically engineered mouse model for high-grade glioma. Cranial ionizing radiation induced a robust and durable proneural-to-mesenchymal transition in tumors. Radiation of primary proneural high-grade glioma cells derived from mouse and human tumors also induced a sustained cell-intrinsic mesenchymal transition, associated with increased invasiveness and resistance to the alkylating agent temozolomide. The transcription factor STAT3 was activated in response to irradiation, and blockade of STAT3 abrogated the mesenchymal transition and combination treatment of JAK2 inhibitors with radiation extended survival in mice. Our data suggest that clinical JAK2 inhibitors should be tested in conjunction with radiation in patients with proneural high-grade glioma, to block emergence of therapy-resistant mesenchymal tumors at relapse.