Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid.

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.

Preclinical Efficacy of LP-184, a Tumor Site Activated Synthetic Lethal Therapeutic, in Glioblastoma.

PURPOSE: Glioblastoma (GBM) is the most common brain malignancy with median survival <2 years. Standard-of-care temozolomide has marginal efficacy in approximately 70% of patients due to MGMT expression. LP-184 is an acylfulvene-derived prodrug activated by the oxidoreductase PTGR1 that alkylates at N3-adenine, not reported to be repaired by MGMT. This article examines LP-184 efficacy against preclinical GBM models and identifies molecular predictors of LP-184 efficacy in clinical GBM. EXPERIMENTAL DESIGN: LP-184 effects on GBM cell viability and DNA damage were determined using cell lines, primary PDX-derived cells and patient-derived neurospheres. GBM cell sensitivities to LP-184 relative to temozolomide and MGMT expression were examined. Pharmacokinetics and CNS bioavailability were evaluated in mice with GBM xenografts. LP-184 effects on GBM xenograft growth and animal survival were determined. Machine learning, bioinformatic tools, and clinical databases identified molecular predictors of GBM cells and tumors to LP-184 responsiveness. RESULTS: LP-184 inhibited viability of multiple GBM cell isolates including temozolomide-resistant and MGMT-expressing cells at IC50 = approximately 22-310 nmol/L. Pharmacokinetics showed favorable AUCbrain/plasma and AUCtumor/plasma ratios of 0.11 (brain Cmax = 839 nmol/L) and 0.2 (tumor Cmax = 2,530 nmol/L), respectively. LP-184 induced regression of GBM xenografts and prolonged survival of mice bearing orthotopic xenografts. Bioinformatic analyses identified PTGR1 elevation in clinical GBM subtypes and associated LP-184 sensitivity with EGFR signaling, low nucleotide excision repair (NER), and low ERCC3 expression. Spironolactone, which induces ERCC3 degradation, decreased LP-184 IC50 3 to 6 fold and enhanced GBM xenograft antitumor responses. CONCLUSIONS: These results establish LP-184 as a promising chemotherapeutic for GBM with enhanced efficacy in intrinsic or spironolactone-induced TC-NER-deficient tumors.

Impact of Frontline Ivosidenib on Volumetric Growth Patterns in Isocitrate Dehydrogenase-mutant Astrocytic and Oligodendroglial Tumors.

PURPOSE: Isocitrate dehydrogenase (IDH)-mutant gliomas are usually treated with radiotherapy and chemotherapy, which increases the risk for neurocognitive sequelae during patients' most productive years. We report our experience using off-label first-in-class mutant IDH1 inhibitor ivosidenib and its impact on tumor volume in IDH-mutant gliomas. EXPERIMENTAL DESIGN: We retrospectively analyzed patients ages ≥18 years with radiation/chemotherapy-naïve, mutant IDH1, nonenhancing, radiographically active, grade 2/3 gliomas, and ≥2 pretreatment and ≥2 on-treatment ivosidenib MRIs. T2/FLAIR-based tumor volumes, growth rates, and progression-free survival (PFS) were analyzed. log-linear mixed-effect modeling of growth curves adjusted for grade, histology, and age was performed. RESULTS: We analyzed 116 MRIs of 12 patients [10 males, median age 46 years (range: 26-60)]: 8 astrocytomas (50% grade 3) and 4 grade 2 oligodendrogliomas. Median on-drug follow-up was 13.2 months [interquartile range (IQR): 9.7-22.2]. Tolerability was 100%. A total of 50% of patients experienced ≥20% tumor volume reduction on-treatment and absolute growth rate was lower during treatment (-1.2 ± 10.6 cc/year) than before treatment (8.0 ± 7.7 cc/year; P ≤ 0.05). log-linear models in the Stable group (n = 9) showed significant growth before treatment (53%/year; P = 0.013), and volume reduction (-34%/year; P = 0.037) after 5 months on treatment. After treatment, volume curves were significantly lower than before treatment (after/before treatment ratio 0.5; P < 0.01). Median time-to-best response was 11.2 (IQR: 1.7-33.4) months, and 16.8 (IQR: 2.6-33.5) months in patients on drug for ≥1 year. PFS at 9 months was 75%. CONCLUSIONS: Ivosidenib was well tolerated and induced a high volumetric response rate. Responders had significant reduction in tumor growth rates and volume reductions observed after a 5-month delay. Thus, ivosidenib appears useful to control tumor growth and delay more toxic therapies in IDH-mutant nonenhancing indolently growing gliomas. See related commentary by Lukas and Horbinski, p. 4709.

CEST2022: Amide proton transfer-weighted MRI improves the diagnostic performance of multiparametric non-contrast-enhanced MRI techniques in patients with post-treatment high-grade gliomas.

New or enlarged lesions in malignant gliomas after surgery and chemoradiation can be associated with tumor recurrence or treatment effect. Due to similar radiographic characteristics, conventional-and even some advanced MRI techniques-are limited in distinguishing these two pathologies. Amide proton transfer-weighted (APTw) MRI, a protein-based molecular imaging technique that does not require the administration of any exogenous contrast agent, was recently introduced into the clinical setting. In this study, we evaluated and compared the diagnostic performances of APTw MRI with several non-contrast-enhanced MRI sequences, such as diffusion-weighted imaging, susceptibility-weighted imaging, and pseudo-continuous arterial spin labeling. Thirty-nine scans from 28 glioma patients were obtained on a 3 T MRI scanner. A histogram analysis approach was employed to extract parameters from each tumor area. Statistically significant parameters (P < 0.05) were selected to train multivariate logistic regression models to evaluate the performance of MRI sequences. Multiple histogram parameters, particularly from APTw and pseudo-continuous arterial spin labeling images, demonstrated significant differences between treatment effect and recurrent tumor. The regression model trained on the combination of all significant histogram parameters achieved the best result (area under the curve = 0.89). We found that APTw images added value to other advanced MR images for the differentiation of treatment effect and tumor recurrence.

Radiomics analysis of amide proton transfer-weighted and structural MR images for treatment response assessment in malignant gliomas.

The purpose of this study was to evaluate the value of amide proton transfer-weighted (APTw) MRI radiomic features for the differentiation of tumor recurrence from treatment effect in malignant gliomas. Eighty-six patients who had suspected tumor recurrence after completion of chemoradiation or radiotherapy, and who had APTw-MRI data acquired at 3 T, were retrospectively analyzed. Using a fluid-attenuated inversion recovery (FLAIR) image-based mask, radiomics analysis was applied to the processed APTw and structural MR images. A chi-square automatic interaction detector decision tree was used for classification analysis. Models with and without APTw features were built using the same strategy. Tenfold cross-validation was applied to obtain the overall classification performance of each model. Sixty patients were confirmed as having tumor recurrence, and the remainder were confirmed as having treatment effect, at median time points of 190 and 171 days after therapy, respectively. There were 525 radiomic features extracted from each of the processed APTw and structural MR images. Based on these, the APTw-based model yielded the highest accuracy (86.0%) for the differentiation of tumor recurrence from treatment effect, compared with 74.4%, 76.7%, 83.7%, and 76.7% for T1 w, T2 w, FLAIR, and Gd-T1 w, respectively. Model classification accuracy was 82.6% when using the combined structural MR images (T1 w, T2 w, FLAIR, Gd-T1 w), and increased to 89.5% when using these structural plus APTw images. The corresponding sensitivity and specificity were 85.0% and 76.9% for the combination of structural MR images, and 85.0% and 100% after adding APTw image features. Adding APTw-based radiomic features increased MRI accuracy in the assessment of the treatment response in post-treatment malignant gliomas.

Exploring glioblastoma stem cell heterogeneity: Immune microenvironment modulation and therapeutic opportunities.

Despite its growing use in cancer treatment, immunotherapy has been virtually ineffective in clinical trials for gliomas. The inherently cold tumor immune microenvironment (TIME) in gliomas, characterized by a high ratio of pro-tumor to anti-tumor immune cell infiltrates, acts as a seemingly insurmountable barrier to immunotherapy. Glioma stem cells (GSCs) within these tumors are key contributors to this cold TIME, often functioning indirectly through activation and recruitment of pro-tumor immune cell types. Furthermore, drivers of GSC plasticity and heterogeneity (e.g., reprogramming transcription factors, epigenetic modifications) are associated with induction of immunosuppressive cell states. Recent studies have identified GSC-intrinsic mechanisms, including functional mimicry of immune suppressive cell types, as key determinants of anti-tumor immune escape. In this review, we cover recent advancements in our understanding of GSC-intrinsic mechanisms that modulate GSC-TIME interactions and discuss cutting-edge techniques and bioinformatics platforms available to study immune modulation at high cellular resolution with exploration of both malignant (i.e., GSC) and non-malignant (i.e., immune) cell fractions. Finally, we provide insight into the therapeutic opportunities for targeting immunomodulatory GSC-intrinsic mechanisms to potentiate immunotherapy response in gliomas.

Targeting UDP-α-d-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth.

Glioblastoma (GBM, WHO grade IV glioma) is the most common and lethal malignant brain tumor in adults with a dismal prognosis. The extracellular matrix (ECM) supports GBM progression by promoting tumor cell proliferation, migration, and immune escape. Uridine diphosphate (UDP)-glucose 6-dehydrogenase (UGDH) is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM. We investigated how targeting UGDH in GBM influences the GBM immune microenvironment, including tumor-associated microglia/macrophages (TAMs) and T cells. TAMs are the main immune effector cells in GBM and can directly target tumor cells if properly activated. In co-cultures of GBM cells and human primary macrophages, UGDH knockdown in GBM cells promoted macrophage phagocytosis and M1-like polarization. In orthotropic human GBM xenografts and syngeneic mouse glioma models, targeting UGDH decreased ECM deposition, increased TAM phagocytosis marker expression, reduced M2-like TAMs and inhibited tumor growth. UGDH knockdown in GBM cells also promoted cytotoxic T cell infiltration and activation in orthotopic syngeneic mouse glioma models. The potent and in-human-use small molecule GAG synthesis inhibitor 4-methylumbelliferone (4-MU) was found to inhibit GBM cell proliferation and migration in vitro, mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM. Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvironment.

Review and consensus recommendations on clinical APT-weighted imaging approaches at 3T: Application to brain tumors.

Amide proton transfer-weighted (APTw) MR imaging shows promise as a biomarker of brain tumor status. Currently used APTw MRI pulse sequences and protocols vary substantially among different institutes, and there are no agreed-on standards in the imaging community. Therefore, the results acquired from different research centers are difficult to compare, which hampers uniform clinical application and interpretation. This paper reviews current clinical APTw imaging approaches and provides a rationale for optimized APTw brain tumor imaging at 3 T, including specific recommendations for pulse sequences, acquisition protocols, and data processing methods. We expect that these consensus recommendations will become the first broadly accepted guidelines for APTw imaging of brain tumors on 3 T MRI systems from different vendors. This will allow more medical centers to use the same or comparable APTw MRI techniques for the detection, characterization, and monitoring of brain tumors, enabling multi-center trials in larger patient cohorts and, ultimately, routine clinical use.

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Sox2 induces glioblastoma cell stemness and tumor propagation by repressing TET2 and deregulating 5hmC and 5mC DNA modifications.

DNA methylation is a reversible process catalyzed by the ten-eleven translocation (TET) family of enzymes (TET1, TET2, TET3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Altered patterns of 5hmC and 5mC are widely reported in human cancers and loss of 5hmC correlates with poor prognosis. Understanding the mechanisms leading to 5hmC loss and its role in oncogenesis will advance the development of epigenetic-based therapeutics. We show that TET2 loss associates with glioblastoma (GBM) stem cells and correlates with poor survival of GBM patients. We further identify a SOX2:miR-10b-5p:TET2 axis that represses TET2 expression, represses 5hmC, increases 5mC levels, and induces GBM cell stemness and tumor-propagating potential. In vivo delivery of a miR-10b-5p inhibitor that normalizes TET2 expression and 5hmC levels inhibits tumor growth and prolongs survival of animals bearing pre-established orthotopic GBM xenografts. These findings highlight the importance of TET2 and 5hmC loss in Sox2-driven oncogenesis and their potential for therapeutic targeting.

ATRX loss promotes immunosuppressive mechanisms in IDH1 mutant glioma.

BACKGROUND: ATRX inactivation occurs with IDH1R132H and p53 mutations in over 80% of Grades II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1R132H/p53mut glioma cells and xenografts. METHODS: Isogenic astrocytoma cells (+/-IDH1R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline-inducible IDH1R132H, ATRX shRNA, or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1R132H+/-ATRXloss on cell migration, growth, DNA repair, and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays, and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization, and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1R132H/p53mut/ATRXloss cells. RESULTS: Adding ATRX loss to the IDH1R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune-suppressive transcriptome consisting of up-regulated immune checkpoints (e.g., PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g., IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune-suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition. CONCLUSIONS: ATRX loss activates a BRD-dependent immune-suppressive transcriptome and immune escape mechanism in IDH1R132H/p53mut astrocytoma cells.

Erratum: miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs.

[This corrects the article PMC7470213.].

Very long-chain acyl-CoA synthetase 3 mediates onco-sphingolipid metabolism in malignant glioma.

Gliomas are the largest category of primary malignant brain tumors in adults, and glioblastomas account for nearly half of malignant gliomas. Glioblastomas are notoriously aggressive and drug-resistant, with a very poor 5 year survival rate of about 5%. New approaches to treatment are thus urgently needed. We previously identified an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), as a potential therapeutic target in glioblastoma. Using the glioblastoma cell line U87MG, we created a cell line with genomic deletion of ACSVL3 (U87-KO) and investigated potential mechanisms to explain how this enzyme supports the malignant properties of glioblastoma cells. Compared to U87MG cells, U87-KO cells grew slower and assumed a more normal morphology. They produced fewer, and far smaller, subcutaneous xenografts in nude mice. Acyl-CoA synthetases, including ACSVL3, convert fatty acids to their acyl-CoA derivatives, allowing participation in diverse downstream lipid pathways. We examined the effect of ACSVL3 depletion on several such pathways. Fatty acid degradation for energy production was not affected in U87-KO cells. Fatty acid synthesis, and incorporation of de novo synthesized fatty acids into membrane phospholipids needed for rapid tumor cell growth, was not significantly affected by lack of ACSVL3. In contrast, U87-KO cells exhibited evidence of altered sphingolipid metabolism. Levels of ceramides containing 18-22 carbon fatty acids were significantly lower in U87-KO cells. This paralleled the fatty acid substrate specificity profile of ACSVL3. The rate of incorporation of stearate, an 18-carbon saturated fatty acid, into ceramides was reduced in U87-KO cells, and proteomics revealed lower abundance of ceramide synthesis pathway enzymes. Sphingolipids, including gangliosides, are functional constituents of lipid rafts, membrane microdomains thought to be organizing centers for receptor-mediated signaling. Both raft morphology and ganglioside composition were altered by deficiency of ACSVL3. Finally, levels of sphingosine-1-phosphate, a sphingolipid signaling molecule, were reduced in U87-KO cells. We conclude that ACSVL3 supports the malignant behavior of U87MG cells, at least in part, by altering cellular sphingolipid metabolism.

Monoallelic IDH1 R132H Mutation Mediates Glioma Cell Response to Anticancer Therapies via Induction of Senescence.

Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2-M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non-cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies. IMPLICATIONS: The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant-mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

Mutant IDH1 promotes phagocytic function of microglia/macrophages in gliomas by downregulating ICAM1.

Tumor-associated microglia/macrophages (TAMs) are the main innate immune effector cells in malignant gliomas and have both pro- and anti-tumor functions. The plasticity of TAMs is partially dictated by oncogenic mutations in tumor cells. Heterozygous IDH1 mutation is a cancer driver gene prevalent in grade II/III gliomas, and IDH1 mutant gliomas have relatively favorable clinical outcomes. It is largely unknown how IDH mutation alters TAM phenotypes to influence glioma growth. Here we established clinically relevant isogenic glioma models carrying monoallelic IDH1 R132H mutation (IDH1R132H/WT) and found that IDH1R132H/WT significantly downregulated immune response-related pathways in glioma cells, indicating an immunomodulation role of mutant IDH1. Co-culturing IDH1R132H/WT glioma cells with human macrophages promoted anti-tumor phenotypes of macrophages and increased macrophage migration and phagocytic capacity. In orthotopic xenografts, IDH1R132H/WT decreased tumor growth and prolonged animal survival, accompanied by increased TAM recruitment and upregulated phagocytosis markers, suggesting the induction of anti-tumor TAM functions. Using human cytokine arrays that query 36 proteins, we identified significant downregulation of ICAM-1/CD54 in IDH1R132H/WT gliomas, which was further confirmed by ELISA and immunoblotting analyses. ICAM1 gain-of-function studies revealed that ICAM1 downregulation in IDH1R132H/WT cells played a mechanistic role to mediate the immunomodulation function of IDH1R132H/WT. ICAM-1 silencing in IDH1 wild-type glioma cells decreased tumor growth and increased the anti-tumor function of TAMs. Together, our studies support a new TAM-mediated phagocytic function within IDH1 mutant gliomas, and improved understanding of this process may uncover novel approaches to targeting IDH1 wild type gliomas.

EGFR Activates a TAZ-Driven Oncogenic Program in Glioblastoma.

Hyperactivated EGFR signaling is a driver of various human cancers, including glioblastoma (GBM). Effective EGFR-targeted therapies rely on knowledge of key signaling hubs that transfer and amplify EGFR signaling. Here we focus on the transcription factor TAZ, a potential signaling hub in the EGFR signaling network. TAZ expression was positively associated with EGFR expression in clinical GBM specimens. In patient-derived GBM neurospheres, EGF induced TAZ through EGFR-ERK and EGFR-STAT3 signaling, and the constitutively active EGFRvIII mutation caused EGF-independent hyperactivation of TAZ. Genome-wide analysis showed that the EGFR-TAZ axis activates multiple oncogenic signaling mechanisms, including an EGFR-TAZ-RTK positive feedback loop, as well as upregulating HIF1α and other oncogenic genes. TAZ hyperactivation in GBM stem-like cells induced exogenous mitogen-independent growth and promoted GBM invasion, radioresistance, and tumorigenicity. Screening a panel of brain-penetrating EGFR inhibitors identified osimertinib as the most potent inhibitor of the EGFR-TAZ signaling axis. Systemic osimertinib treatment inhibited the EGFR-TAZ axis and in vivo growth of GBM stem-like cell xenografts. Overall these results show that the therapeutic efficacy of osimertinib relies on effective TAZ inhibition, thus identifying TAZ as a potential biomarker of osimertinib sensitivity. SIGNIFICANCE: This study establishes a genome-wide map of EGFR-TAZ signaling in glioblastoma and finds osimertinib effectively inhibits this signaling, justifying its future clinical evaluation to treat glioblastoma and other cancers with EGFR/TAZ hyperactivation. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3580/F1.large.jpg.

Opinion: miRNAs - The new wave of molecular cancer therapeutics.

Cutting-edge advances in nanomedicine and the recent approval of two siRNA-based therapeutics by the Food and Drug Administration (FDA) has rekindled the interest in RNA interference (RNAi) as vehicles for the development of novel cancer therapeutics. In this perspective, we will briefly discuss how miRNAs are becoming the next-generation RNAi therapeutic, the advances in delivery vehicles for in vivo miRNA delivery, and where miRNA technology stands in terms of clinical translation.

Reprogramming Transcription Factors Oct4 and Sox2 Induce a BRD-Dependent Immunosuppressive Transcriptome in GBM-Propagating Cells.

A subset of stem-like cells in glioblastoma (GBM; GSC) underlies tumor propagation, therapeutic resistance, and tumor recurrence. Immune evasion is critical for GSCs to carry out these functions. However, the molecular mechanisms employed by GSCs to escape antitumor immunity remain largely unknown. The reprogramming transcription factors Oct4 and Sox2 function as core multipotency factors and play an essential role in the formation and maintenance of GSCs, but the roles of these transcription factors in GSC immune escape have not been well explored. Here we examine how Oct4/Sox2 coexpression contributes to the immunosuppressive phenotype of GSCs. Combined transcription profiling and functional studies of Oct4/Sox2 coexpressing GSCs and differentiated GBM cells demonstrated that Oct4 and Sox2 cooperatively induce an immunosuppressive transcriptome consisting of multiple immunosuppressive checkpoints (i.e., PD-L1, CD70, A2aR, TDO) and dysregulation of cytokines and chemokines that are associated with an immunosuppressive tumor microenvironment. Mechanistically, induction and function of BRD/H3k27Ac-dependent immunosuppressive genes played a role in the immunosuppressive phenotype of GSCs. Pan-BET bromodomain inhibitors (e.g., JQ1) and shBRD4 constructs significantly inhibited the immunosuppressive transcriptome and immunosuppressive biological responses induced by Oct4/Sox2. Our findings identify targetable mechanisms by which tumor-propagating GSCs contribute to the immunosuppressive microenvironment in GBM. SIGNIFICANCE: This report identifies mechanisms by which the reprogramming transcription factors Oct4 and Sox2 function to drive the immunomodulatory transcriptome of GSCs and contribute to the immunosuppressive microenvironment in GBM.

Molecular clarification of brainstem astroblastoma with EWSR1-BEND2 fusion in a 38-year-old man.

The majority of astroblastoma occur in a cerebral location in children and young adults. Here we describe the unusual case of a 38-year-old man found to have a rapidly growing cystic enhancing circumscribed brainstem tumor with high grade histopathology classified as astroblastoma, MN1-altered by methylome profiling. He was treated with chemoradiation and temozolomide followed by adjuvant temozolomide without progression to date over one year from treatment initiation. Astroblastoma most frequently contain a MN1-BEND2 fusion, while in this case a rare EWSR1-BEND2 fusion was identified. Only a few such fusions have been reported, mostly in the brainstem and spinal cord, and they suggest that BEND2, rather than MN1, may have a more critical functional role, at least in these regions. This unusual clinical scenario exemplifies the utility of methylome profiling and assessment of gene fusions in tumors of the central nervous system.

miR-425-5p, a SOX2 target, regulates the expression of FOXJ3 and RAB31 and promotes the survival of GSCs.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and prognosis is poor despite maximum therapeutic efforts. GBM is composed of heterogeneous cell populations, among which the glioma stem-like cells (GSCs) play an important role in tumor cell self-renewal and the ability to initiate and drive tumor growth and recurrence. The transcription factor SOX2 is enriched in GSCs where it controls the stem cell phenotype, invasion and maintenance of tumorigenicity. Therefore, understanding the molecular mechanisms governed by SOX2 in GSCs is crucial to developing targeted therapies against this resistant cell population. In this study, we identified and validated a miRNA profile regulated by SOX2 in GSCs. Among these miRNAs, miR-425-5p emerged as a significant robust candidate for further study. The expression of miR-425-5p was significantly enriched in clinical GBM specimens compared with a human brain reference sample and showed a positive correlation with SOX2 expression. Using a combination of in silico analyses and molecular approaches, we show that SOX2 binds to the promoter of miR-425-5p. Loss of function studies show that repressing miR-425-5p expression in multiple GSCs inhibited neurosphere renewal and induced cell death. More importantly, miR-425-5p inhibition extended survival in an orthotopic GBM mouse model. Finally, combining several bioinformatics platforms with biological endpoints in multiple GSC lines, we identified FOXJ3 and RAB31 as high confidence miR-425-5p target genes. Our findings show that miR-425-5p is a GBM stem cell survival factor and that miR-425-5p inhibition function is a potential strategy for treating GBM.