Briana Prager

A Three-Dimensional Organoid Culture System Derived from Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell Heterogeneity of Tumors Found In Vivo.

Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2(+), OLIG2(+), and TLX(+) cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465-77. ©2016 AACR.

Sphingosine 1-phosphate signaling at the blood-brain barrier.

The characterization of molecular pathways that modulate blood-brain barrier (BBB) function and integrity has been fueled by a growing body of literature implicating BBB dysfunction in a wide range of neurologic diseases. Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that has been effectively targeted by the immunomodulatory S1P1 functional antagonist fingolimod in the treatment of multiple sclerosis (MS). Investigation into the pathways modulated by S1P has revealed its important role in regulating BBB integrity via signaling through receptor isoforms on astrocytes and endothelial cells (ECs). Current evidence supports a significant role for S1P signaling as a key determinant of BBB permeability and hence as a potential pathogenic player or therapeutic target in diseases characterized by BBB dysfunction.

Zika virus has oncolytic activity against glioblastoma stem cells

Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.

Targeting glioma stem cells through combined BMI1 and EZH2 inhibition

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile, and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress due to downregulation of the E3 ligase RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1). Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.

Nicotinamide metabolism regulates glioblastoma stem cell maintenance

Metabolic dysregulation promotes cancer growth through not only energy production, but also epigenetic reprogramming. Here, we report that a critical node in methyl donor metabolism, nicotinamide N-methyltransferase (NNMT), ranked among the most consistently overexpressed metabolism genes in glioblastoma relative to normal brain. NNMT was preferentially expressed by mesenchymal glioblastoma stem cells (GSCs). NNMT depletes S-adenosyl methionine (SAM), a methyl donor generated from methionine. GSCs contained lower levels of methionine, SAM, and nicotinamide, but they contained higher levels of oxidized nicotinamide adenine dinucleotide (NAD+) than differentiated tumor cells. In concordance with the poor prognosis associated with DNA hypomethylation in glioblastoma, depletion of methionine, a key upstream methyl group donor, shifted tumors toward a mesenchymal phenotype and accelerated tumor growth. Targeting NNMT expression reduced cellular proliferation, self-renewal, and in vivo tumor growth of mesenchymal GSCs. Supporting a mechanistic link between NNMT and DNA methylation, targeting NNMT reduced methyl donor availability, methionine levels, and unmethylated cytosine, with increased levels of DNA methyltransferases, DNMT1 and DNMT3A. Supporting the clinical significance of these findings, NNMT portended poor prognosis for glioblastoma patients. Collectively, our findings support NNMT as a GSC-specific therapeutic target in glioblastoma by disrupting oncogenic DNA hypomethylation.

MYC-regulated mevalonate metabolism maintains brain tumor–initiating cells

Metabolic dysregulation drives tumor initiation in a subset of glioblastomas harboring isocitrate dehydrogenase (IDH) mutations, but metabolic alterations in glioblastomas with wild-type IDH are poorly understood. MYC promotes metabolic reprogramming in cancer, but targeting MYC has proven notoriously challenging. Here, we link metabolic dysregulation in patient-derived brain tumor-initiating cells (BTIC) to a nexus between MYC and mevalonate signaling, which can be inhibited by statin or 6-fluoromevalonate treatment. BTICs preferentially express mevalonate pathway enzymes, which we find regulated by novel MYC-binding sites, validating an additional transcriptional activation role of MYC in cancer metabolism. Targeting mevalonate activity attenuated RAS-ERK-dependent BTIC growth and self-renewal. In turn, mevalonate created a positive feed-forward loop to activate MYC signaling via induction of miR-33b. Collectively, our results argue that MYC mediates its oncogenic effects in part by altering mevalonate metabolism in glioma cells, suggesting a therapeutic strategy in this setting. Cancer Res; 77(18); 4947-60. ©2017 AACR.

Spatiotemporal ablation of CXCR2 on oligodendrocyte lineage cells: Role in myelin repair

Background: Residual CXCR2 expression on CNS cells in Cxcr2+/−→Cxcr2−/− chimeric animals slowed remyelination after both experimental autoimmune encephalomyelitis and cuprizone-induced demyelination. Methods: We generated Cxcr2fl/−:PLPCre-ER(T) mice enabling an inducible, conditional deletion of Cxcr2 on oligodendrocyte lineage cells of the CNS. Cxcr2fl/−:PLPCre-ER(T) mice were evaluated in 2 demyelination/remyelination models: cuprizone-feeding and in vitro lysophosphatidylcholine (LPC) treatment of cerebellar slice cultures. Results: Cxcr2fl/−:PLPCre-ER(T)+ (termed Cxcr2-cKO) mice showed better myelin repair 4 days after LPC-induced demyelination of cerebellar slice cultures. Cxcr2-cKOs also displayed enhanced hippocampal remyelination after a 2-week recovery from 6-week cuprizone feeding. Conclusion: Using 2 independent demyelination/remyelination models, our data document enhanced myelin repair in Cxcr2-cKO mice, consistent with the data obtained from radiation chimerism studies of germline CXCR2. Further experiments are appropriate to explore how CXCR2 function in the oligodendrocyte lineage accelerates myelin repair.

Survey-based assessment of the relationship between cognitive impairment and mentally stimulating activity in multiple sclerosis

Abstract Objectives: Cognitive impairment (CI) is a common and potentially debilitating component of the disease course in multiple sclerosis (MS). However, therapeutic options remain limited. It is unknown whether cognitively enriching activities reduce the burden of CI in patients with MS, as is found in other neurologic diseases affecting cognition. The aim of this study was to determine whether participation in cognitively enriching activities decreased self-reported CI in MS patients. Methods: CI and activity levels were reported through electronic surveys completed by MS patients at the Cleveland Clinic. Responses were analyzed by univariable and multivariable regressions to identify factors associated with lower CI. Results: We received 316 survey responses. Use of an assistive device (β = 4.09; P = 0.033) and Internet use (β = 11.9; P = 0.017) were associated with higher reported CI, while employment correlated with reduced CI (β = −7.97; P < 0.0001). None of the cognitive activities surveyed were found to reduce CI. Discussion: This study did not identify a significant impact of cognitively enriching activities on reducing CI, suggesting that other prophylactic or therapeutic approaches should be investigated. A small portion of the population surveyed reported no or minimal CI, suggesting the existence of a resilient population.