Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD + ). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD + synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD + -dependent network. Accordingly, we demonstrate E2F2 is required for GSC selfrenewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD + metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.T he prognosis for glioblastoma, the most common malignant intrinsic brain tumor in adults, remains poor despite aggressive multidisciplinary therapy, including maximal safe surgical resection, radiation therapy, and temozolomide (1, 2). The failure of these interventions to generate a durable response stems in part from inadequate understanding of the metabolic and molecular mechanisms underlying malignant behavior and therapeutic resistance (3, 4). Nicotinamide adenine dinucleotide (NAD + ) has a well-known role in cellular metabolism and is an important cofactor for signaling pathways that regulate aging, inflammation, diabetes mellitus, axonal injury, and cancer (5, 6). Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD + synthesis, produces NAD + precursor nicotinamide mononucleotide (NMN) to drive NAD + -dependent processes. Interestingly, NAMPT expression is extremely low in the mammalian brain compared with other organs (7,8). However, NAMPT is highly expressed in several cancers, and features of cancer cells, including proliferation, invasion, and tumor growth, exhibit a dependence on NAD + (9-11). In noncancer cells, NAD + plays a critical role in transcriptional control, providing a metabolic basis for epigenetic reprogramming (12-16). Enzymes using NAD + as a cofactor, including the sirtuins and poly-ADP ribosyl transferases, regulate transcription factor activity and chromatin structure (13-15). Additionally, NAD + can control transcription by altering DNA methylation in neurons (12). However, in glioblastoma, little is known about NAD + -dependent transcriptional events and whether these even...

SUMMARY Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of novel therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-Anaphase-Promoting Complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma.

The substantial spatial heterogeneity observed in different GBM tumor sectors, especially in spatially restricted hypermutator cases, raises important caveats to our current dependence on single-sector molecular information to guide either targeted or immune-based treatments.

TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94 %) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types.

Cell-cell interactions between tumor cells and constituents of their microenvironment are critical determinants of tumor tissue biology and therapeutic responses. Interactions between glioblastoma (GBM) cells and endothelial cells (ECs) establish a purported cancer stem cell niche. We hypothesized that genes regulated by these interactions would be important, particularly as therapeutic targets. Using a computational approach, we deconvoluted expression data from a mixed physical co-culture of GBM cells and ECs and identified a previously undescribed upregulation of the cAMP specific phosphodiesterase PDE7B in GBM cells in response to direct contact with ECs. We further found that elevated PDE7B expression occurs in most GBM cases and has a negative effect on survival. PDE7B overexpression resulted in the expansion of a stem-like cell subpopulation in vitro and increased tumor growth and aggressiveness in an in vivo intracranial GBM model. Collectively these studies illustrate a novel approach for studying cell-cell interactions and identifying new therapeutic targets like PDE7B in GBM.

Encouraging clinical results using immune checkpoint therapies to target the PD-1 axis in a variety of cancer types have paved the way for new immune therapy trials in brain tumor patients. However, the molecular mechanisms that regulate expression of the PD-1 pathway ligands, PD-L1 and PD-L2, remain poorly understood. To address this, we explored the cell-intrinsic mechanisms of constitutive PD-L1 and PD-L2 expression in brain tumors. PD-L1 and PD-L2 expression was assessed by flow cytometry and qRT-PCR in brain tumor cell lines and patient tumor-derived brain tumor-initiating cells (BTICs). Immunologic effects of PD-L2 overexpression were evaluated by IFN-γ ELISPOT. CD274 and PDCD1LG2 cis-regulatory regions were cloned from genomic DNA and assessed in full or by mutating and/or deleting regulatory elements by luciferase assays. Correlations between clinical responses and PD-L1 and PD-L2 expression status were evaluated in TCGA datasets in LGG and GBM patients. We found that a subset of brain tumor cell lines and BTICs expressed high constitutive levels of PD-L1 and PD-L2 and that PD-L2 overexpression inhibited neoantigen specific T cell IFN-γ production. Characterization of novel cis-regulatory regions in CD274 and PDCD1LG2 lead us to identify that GATA2 is sufficient to drive PD-L1 and PD-L2 expression and is necessary for PD-L2 expression. Importantly, in TCGA datasets, PD-L2 correlated with worse clinical outcomes in glioma patients.. By perturbing GATA2 biology, targeted therapies may be useful to decrease inhibitory effects of PD-L2 in the microenvironment. Glioblastoma (GBM) remains a challenging malignancy to treat. Poor clinical outcomes likely reflect aggressive GBM biology 1 , and growing evidence suggests that there is also signficant immunosuppression in GBM. Several immune resistance mechanisms have been described in GBM patients: increased regulatory T cells, indoleamine 2,3 dioxygenase activation, dysregulated antigen presentation, and STAT3-driven myeloid cell suppression, among others 1-3. Overexpression of inhibitory PD-L1 is another mechanism by which GBM cells may attenuate T cells 4,5 , which is important because PD-1/PD-L1 blocakde is FDA-approved for the treatment of many cancers. In GBM, PD-L1 expression is variable and occurs often without significant infiltrating lymphocytes, suggesting that it may be influenced by tumor intrinsic induction 6 rather than extrinsic stimulation. Several mechanisms can drive cell-intrinsic PD-L1 induction including PTEN loss 7 , aberrant signaling 8 , genomic amplification 9 , and post-translational modifications 10. However, it is unclear which of these mechanisms is most germane to brain tumors.

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays important roles in brain microglial function in neurodegenerative diseases, but the role of TREM2 in the GBM TME has not been examined. Here, we found that TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors and that high TREM2 expression correlates with poor prognosis in patients with GBM. TREM2 loss of function in human macrophages and mouse myeloid cells increased interferon-γ–induced immunoactivation, proinflammatory polarization, and tumoricidal capacity. In orthotopic mouse GBM models, mice with chronic and acute Trem2 loss of function exhibited decreased tumor growth and increased survival. Trem2 inhibition reprogrammed myeloid phenotypes and increased programmed cell death protein 1 (PD-1) + CD8 + T cells in the TME. Last, Trem2 deficiency enhanced the effectiveness of anti–PD-1 treatment, which may represent a therapeutic strategy for patients with GBM.