Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity–dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-β receptor 1 (TGF-βR1), which in turn leads to activation of canonical TGF-β signaling in both GSCs and NPCs. TGF-β signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-β pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-βR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies, including cetuximab and panitumumab, are used to treat metastatic colorectal cancer (mCRC). However, this treatment is only effective for a small subset of mCRC patients positive for the wild-type KRAS GTPase. GC1118 is a novel, fully humanized anti-EGFR IgG1 antibody that displays potent inhibitory effects on high-affinity EGFR ligand-induced signaling and enhanced antibody-mediated cytotoxicity. In this study, using 51 CRC patient-derived xenografts (PDXs), we showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. In three KRAS-mutant CRCPDXs, GC1118 was more effective than cetuximab, whereas the two agents demonstrated comparable efficacy against three wild-type KRAS PDXs. Persistent phosphatidylinositol-3-kinase (PI3K)/AKT signaling was thought to underlie resistance to GC1118. In support of these findings, a preliminary improved anti-cancer response was observed in a CRC PDX harboring mutated KRAS with intrinsically high AKT activity using GC1118 combined with the dual PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitor BEZ-235, without observed toxicity. Taken together, the superior antitumor efficacy of GC1118 alone or in combination with PI3K/mTOR/AKT inhibitors shows great therapeutic potential for the treatment of KRAS-mutant mCRC with elevated ratios of high- to low-affinity EGFR ligands and PI3K-AKT pathway activation.

One of the hallmarks of glioblastoma (GBM) is extensive neovascularization. In addition to supplying blood and nutrients, vascular endothelial (VE) cells provide trophic support to GBM cells via paracrine signaling, the precise mechanisms of which are being unraveled. Here, using patient-derived GBM and VE cells as well as orthotopic GBM mouse models, we report that Endocan ( ESM1 ), an endothelial-secreted proteoglycan, confers enhanced proliferative, migratory, and angiogenic properties to GBM cells and regulates their spatial identity. Mechanistically, Endocan exerts at least part of its functions via direct binding and activation of the PDGFRA receptor. Subsequent downstream signaling enhances chromatin accessibility of the Myc promoter and upregulates Myc expression inducing highly stable phenotypic changes in GBM cells. Furthermore, Endocan confers a radioprotection phenotype in GBM cells, both in vitro and in vivo . Inhibition of Endocan-PDGFRA signaling with ponatinib increases survival in the Esm1 wild-type but not in the Esm1 knock-out mouse GBM model. Our findings identify Endocan and its downstream signaling axis as a potential target to subdue the recurrence of GBM and further highlight the importance of vascular to tumor cell signaling for GBM biology. Significance statement: Identification of the Endocan/PDGFRA/Myc axis demonstrates an important role of VE cells in GBM malignancy. The contribution of Endocan to the development of GBM cell populations with different phenotypes reveal an additional pathway underlying the origin of GBM intratumoral heterogeneity. Targeting Endocan-mediated crosstalk may enhance the efficacy of GBM treatment.