A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma

Abstract

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.

Author Summary

Glioblastomas, the most common primary brain tumor, harbor mutations in receptor tyrosine kinases (RTKs), such as EGFR, and components of the Pi-3 kinase (PI3K) signaling pathway. However, the genes that act downstream of RTK and PI3K signaling to drive glioblastoma remain unclear. To investigate the genetic and molecular basis of this disease, we created a glioblastoma model in the fruit fly Drosophila melanogaster. To identify new genes involved in glioblastoma development, we performed a screen for the genes required for tumor cell proliferation using our Drosophila glioblastoma model and then functionally assessed the activity of human versions of novel genes identified in this screen. Our results revealed that the RIO kinases become overexpressed in human glioblastomas but not in normal human glial or neuronal cells. We found that overexpression of the RIO kinases promotes and maintains signals that drive tumor cell proliferation and survival in RTK- and PI3K-dependent human glioblastoma, and reduction of RIO kinase expression decreased proliferation and prompted cell death and chemosensitivity in glioblastoma cells. Therefore, disruption of the RIO kinases may provide new therapeutic opportunities to target glioblastoma and other RTK- or PI3K-dependent cancers.