Abstract
RATIONALE: Isocitrate dehydrogenase (IDH)-1/2 mutations are known driver mutations for the development of low-grade gliomas (LGGs). However, secondary drivers are needed for tumor growth, and it remains unclear which co-factors and pathways are necessary to develop the LGG tumor phenotype. To identify additional players in IDH mutant-mediated oncogenesis, we investigated the effects of disrupting glioma-associated pathways in a tractable in vitro system of IDH-mutant malignant transformation. METHODS: A doxycycline-inducible IDH(R132H) expressing normal human astrocyte cell line was concomitantly treated with either a Wnt-activator/inhibitor (CHIR99021/IWP-2), TGF-β inhibitor (Repsox) or Notch inhibitor (YO-01027). IDH-associated CD171 marker expression changes were assessed with Flow cytometry, followed by transcriptional and proteomic analysis with RNA-Seq and mass spectrometry. Functional characterization entailed cell viability and clonogenic assays as well as cell cycle analyses. RESULTS: We observed significant IDH-related CD171 expression changes upon treatment with CHIR99021, Repsox and YO-01027. Wnt activation with CHIR99021 profoundly affected the IDH mutant-induced transcriptional landscape, leading to differential usage of LGG and stemness programs including altered expression of SOX4 and SOX11. On the protein level, Wnt activation additionally impacted pathways relevant for cellular architecture, specifically Rho-Rac-CDC42 signaling. This was also reflected by an altered cell morphology with small, rounded up cells, augmented clonal proliferation capacity and increased proportions of cell in S-phase. CONCLUSION: Wnt activation in a IDH(R132H) model system modulated LGG development, leading to differential expression of key LGG and stemness associated profiles, morphologic cell changes and increased replication capacity. While aberrant Wnt signaling is a common feature in glioblastoma, our data point to an effect also on low-grade gliomagenesis, therefore requiring further investigation.