Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.

Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

Dominant mutations in the Ret receptor tyrosine kinase lead to the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Mammalian tissue culture studies suggest that Ret MEN2 mutations significantly alter Ret-signaling properties, but the precise mechanisms by which Ret MEN2 promotes tumorigenesis remain poorly understood. To determine the signal transduction pathways required for Ret MEN2 activity, we analyzed analogous mutations in the Drosophila Ret ortholog dRet. Overexpressed dRet MEN2 isoforms targeted to the developing retina led to aberrant cell proliferation, inappropriate cell fate specification, and excessive Ras pathway activation. Genetic analysis indicated that dRet MEN2 acts through the Ras-ERK, Src, and Jun kinase pathways. A genetic screen for mutations that dominantly suppress or enhance dRet MEN2 phenotypes identified new genes that are required for the phenotypic outcomes of dRet MEN2 activity. Finally, we identified human orthologs for many of these genes and examined their status in human tumors. Two of these loci showed loss of heterozygosity (LOH) within both sporadic and MEN2-associated pheochromocytomas, suggesting that they may contribute to Ret-dependent oncogenesis. D URING development, receptor tyrosine kinases (RTKs) integrate extracellular signals to influence cellular processes such as growth and differentiation. Signaling through RTKs requires ligand-induced oligomerization to direct tyrosine autophosphorylation; autophosphorylation both stimulates catalytic activity and creates phospho-tyrosine docking sites for cytoplasmic proteins that activate intracellular signaling pathways. To date, mutations in more than half of all RTKs have been implicated in human cancer (reviewed in Blume-Jensen and Hunter 2001). These mutations commonly function by relieving RTK regulatory constraints, leading to inappropriate kinase activity and hyperactivation of downstream pathways. These events promote oncogenic transformation by driving aberrant cellular growth, proliferation, and survival. Still, tumorigenesis requires mutations in multiple loci: along with dominant mutations in oncogenes such as RTKs, tumorigenesis also requires loss-of-function mutations in tumor suppressors. The relationship between oncogenic tyrosine kinases and tumor suppressors, and the extent to which mutations in each cooperate to direct oncogenic growth, is not well understood.The Ret RTK plays an essential role in both development and oncogenesis. During embryogenesis, Ret is required for development of the sympathetic and enteric nervous systems, the neural crest, and the excretory system (Schuchardt et al. 1994;Durbec et al. 1996;Enomoto et al. 2001). The extracellular portion of Ret contains cysteine repeats and a cadherinlike domain. The intracellular portion of Ret contains a tyrosine kinase catalytic domain and multiple tyrosine autophosphorylation sites. Four activating ligands have been identified: GDNF, Neurturin, Persephin, and Artemin all activate Ret through the GPI-linked co-

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk ؊/؊ overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function.

Purpose: Glioblastomas (GBMs), neoplasms derived from glia and neuroglial progenitor cells, are the most common and lethal malignant primary brain tumors diagnosed in adults, with a median survival of 14 months. GBM tumorigenicity is often driven by genetic aberrations in receptor tyrosine kinases, such as amplification and mutation of EGFR. Experimental Design: Using a Drosophila glioma model and human patient–derived GBM stem cells and xenograft models, we genetically and pharmacologically tested whether the YAP and TAZ transcription coactivators, effectors of the Hippo pathway that promote gene expression via TEA domain (TEAD) cofactors, are key drivers of GBM tumorigenicity downstream of oncogenic EGFR signaling. Results: YAP and TAZ are highly expressed in EGFR-amplified/mutant human GBMs, and their knockdown in EGFR-amplified/mutant GBM cells inhibited proliferation and elicited apoptosis. Our results indicate that YAP/TAZ-TEAD directly regulates transcription of SOX2, C-MYC, and EGFR itself to create a feedforward loop to drive survival and proliferation of human GBM cells. Moreover, the benzoporphyrin derivative verteporfin, a disruptor of YAP/TAZ-TEAD–mediated transcription, preferentially induced apoptosis of cultured patient-derived EGFR-amplified/mutant GBM cells, suppressed expression of YAP/TAZ transcriptional targets, including EGFR, and conferred significant survival benefit in an orthotopic xenograft GBM model. Our efforts led us to design and initiate a phase 0 clinical trial of Visudyne, an FDA-approved liposomal formulation of verteporfin, where we used intraoperative fluorescence to observe verteporfin uptake into tumor cells in GBM tumors in human patients. Conclusions: Together, our data suggest that verteporfin is a promising therapeutic agent for EGFR-amplified and -mutant GBM.