Emerging Therapies for Glioblastoma.

Glioblastoma is most commonly a primary brain tumor and the utmost malignant one, with a survival rate of approximately 12-18 months. Glioblastoma is highly heterogeneous, demonstrating that different types of cells from the same tumor can manifest distinct gene expression patterns and biological behaviors. Conventional therapies such as temozolomide, radiation, and surgery have limitations. As of now, there is no cure for glioblastoma. Alternative treatment methods to eradicate glioblastoma are discussed in this review, including targeted therapies to PI3K, NFKß, JAK-STAT, CK2, WNT, NOTCH, Hedgehog, and TGFß pathways. The highly novel application of oncolytic viruses and nanomaterials in combating glioblastoma are also discussed. Despite scores of clinical trials for glioblastoma, the prognosis remains poor. Progress in breaching the blood-brain barrier with nanomaterials and novel avenues for targeted and combination treatments hold promise for the future development of efficacious glioblastoma therapies.

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress.

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and ß (IKKα/ß) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK-/- cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines.

BACKGROUND: The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells. METHODS: PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines. RESULTS: We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts. DISCUSSION: We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines. CONCLUSION: Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious.

Correction to: A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

The original article [1] contains three erroneous mentions of usage of a restriction enzyme-BstZ17I-in the Methods section as displayed in the following sentences.

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).