Petra Hamerlik

Abstract Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2–MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2–MYC–IGFBP3 axis as a specific and novel therapeutic target in glioblastoma. Significance: Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type–specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma. This article is highlighted in the In This Issue feature, p. 211

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane Ecadherin low /cytoplasmic E-cadherin high /CD133 high , CD44 high , Tie2 low ) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.