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Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions., Stem cell development: hold the sugar Cells that can grow into any type of cell, called embryonic stem cells (ESCs), are signaled to stay in stem cell mode (maintain stemness) by addition of a single sugar molecule to Sox2, a regulatory protein coded for by a developmental gene. Sugar modification of Sox2 was known to be involved in maintaining stemness and sometimes implicated in cancer, but the mechanism was poorly understood. When Hyonchol Jang at the National Cancer Center in South Korea and co-workers prevented sugar modification of Sox2 by changing an amino acid at the sugar-binding site, ESCs showed reduced self-renewal. Rather than repressing genes related to stemness, the modification failed to repress developmental genes, permitting cells to grow into other cell types. These results illuminate both the role of Sox2 in cancer and the importance of sugar modification in stemness.

Glioblastoma (GBM) is the most lethal primary brain tumor with few treatment options. The survival of glioma-initiating cells (GICs) is one of the major factors contributing to treatment failure. GICs frequently produce and respond to their own growth factors that support cell proliferation and survival. In this study, we aimed to identify critical autocrine factors mediating GIC survival and to evaluate the anti-GBM effect of antagonizing these factors. Proteomic analysis was performed using conditioned media from two different patient-derived GBM tumor spheres under a growth factor-depleted status. Then, the antitumor effects of inhibiting an identified autocrine factor were evaluated by bioinformatic analysis and molecular validation. Proteins secreted by sphere-forming GICs promote cell proliferation/survival and detoxify reactive oxygen species (ROS). Among these proteins, we focused on midkine (MDK) as a clinically significant and pathologically relevant autocrine factor. Antagonizing MDK reduced the survival of GBM tumor spheres through the promotion of cell cycle arrest and the consequent apoptotic cell death caused by oxidative stress-induced DNA damage. We also identified PCBP4, a novel molecular predictor of resistance to anti-MDK treatment. Collectively, our results indicate that MDK inhibition is an important therapeutic option by suppressing GIC survival through the induction of ROS-mediated cell cycle arrest and apoptosis., Brain cancer: a key growth factor Targeting the growth factor midkine (MDK) may offer improved treatment for glioblastoma, the most lethal brain cancer. In glioblastoma, the tumor-initiating cells produce their own growth factors, encouraging themselves to keep multiplying, and making glioblastoma very difficult to treat. Do-Hyun Nam and Hyun Ju Kang at the Samsung Medical Center, Seoul, South Korea and coworkers screened the growth factors produced by two glioblastoma samples, then focused on a single cancer-associated factor, MDK. Antagonizing MDK slowed tumor cell growth, and further investigation showed that suppressing MDK restored the susceptibility of tumor cells to DNA damage and the mechanisms that weed out damaged cells. Noting that treatment efficacy varied between tumor samples, the researchers identified a biomarker for sensitivity to MDK treatment. These results point the way to new treatments for this particularly deadly cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Testing new ways to identify untapped opportunities for glioblastoma therapies remains highly significant. Amplification and overexpression of MDM2 gene is frequent in glioblastoma and disrupting the MDM2−p53 interaction is a promising strategy to treat the cancer. RG7112 is the first-in class inhibitor and recently discovered AMG232 is the most potent MDM2 inhibitor known to date. Here, we compared the effects of these two clinical MDM2 inhibitors in six glioblastoma cell lines and ten patient-derived glioblastoma stem cells. Targeted sequencing of the TP53, MDM2 genes and whole transcriptome analysis were conducted to verify genetic status associated with sensitivity and resistance to the drugs. Although TP53 wild-type glioblastoma cell lines are similarly sensitive to AMG232 and RG7112, we found that four TP53 wild-type out of ten patient-derived glioblastoma cells are much more sensitive to AMG232 than RG7112 (average IC50 of 76 nM vs. 720 nM). Among these, 464T stem cells containing MDM2 gene amplification were most sensitive to AMG232 with IC50 of 5.3 nM. Moreover, AMG232 exhibited higher selectivity against p53 wild-type cells over p53 mutant stem cells compared to RG7112 (average selectivity of 512-fold vs. 16.5-fold). Importantly, we also found that AMG232 is highly efficacious in three-dimensional (3D) tumor spheroids growth and effectively inhibits the stemness-related factors, Nestin and ZEB1. Our data provide new evidence that glioblastoma stem cells have high susceptibility to AMG232 suggesting the potential clinical implications of MDM2 inhibition for glioblastoma treatment. These will facilitate additional preclinical and clinical studies evaluating MDM2 inhibitors in glioblastoma and direct further efforts towards developing better MDM2-targeted therapeutics.

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/β-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.

GBM is the most aggressive human primary brain tumor and leads to poor clinical outcomes. According to previous studies, exogenous factors secreted from primary brain tumor cells can change the growth pattern of normal cells. We analyzed the ingredients of cell culture media from two patient glioblastoma cells which can grow without growth factors. As a result, we selected a significant factor for tumor cell growth which is called cytokine A, the Heparin-binding growth factor. Because of the cell viability maintenance ability of cytokine A, we tried to investigate detailed mechanism of cytokine A and applied the result to the combination therapy. Cytokine A is critical for tumorigenesis of GBM by modulating DNA damage signaling and cell cycle through ROS. For that reason, cytokine A is effective therapeutic target and can be used in combination therapy.