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Caspase-8 is a cysteine protease that plays an essential role in apoptosis. Consistently with its canonical proapoptotic function, cancer cells may genetically or epigenetically downregulate its expression. Unexpectedly, Caspase-8 is often retained in cancer, suggesting the presence of alternative mechanisms that may be exploited by cancer cells to their own benefit. In this regard, we reported that Src tyrosine kinase, which is aberrantly activated in many tumors, promotes Caspase-8 phosphorylation on Tyrosine 380 (Y380) preventing its full activation. Here, we investigated the significance of Caspase-8 expression and of its phosphorylation on Y380 in glioblastoma, a brain tumor where both Caspase-8 expression and Src activity are often aberrantly upregulated. Transcriptomic analyses identified inflammatory response as a major target of Caspase-8, and in particular, NFκB signaling as one of the most affected pathways. More importantly, we could show that Src-dependent phosphorylation of Caspase-8 on Y380 drives the assembly of a multiprotein complex that triggers NFκB activation, thereby inducing the expression of inflammatory and pro-angiogenic factors. Remarkably, phosphorylation on Y380 sustains neoangiogenesis and resistance to radiotherapy. In summary, our work identifies a novel interplay between Src kinase and Caspase-8 that allows cancer cells to hijack Caspase-8 to sustain tumor growth.

Diffuse midline glioma (DMG) is a leading cause of brain tumor death in children. In addition to hallmark H3.3K27M mutations, significant subsets also harbor alterations of other genes, such asTP53andPDGFRA. Despite the prevalence of H3.3K27M, the results of clinical trials in DMG have been mixed, possibly due to the lack of models recapitulating its genetic heterogeneity. To address this gap, we developed human iPSC-derived tumor models harboring TP53R248Qwith or without heterozygous H3.3K27M and/or PDGFRAD842Voverexpression. The combination of H3.3K27M and PDGFRAD842Vresulted in more proliferative tumors when gene-edited neural progenitor (NP) cells were implanted into mouse brains compared to NP with either mutation alone. Transcriptomic comparison of tumors and their NP cells of origin identified conserved JAK/STAT pathway activation across genotypes as characteristic of malignant transformation. Conversely, integrated genome-wide epigenomic and transcriptomic analyses, as well as rational pharmacologic inhibition, revealed targetable vulnerabilities unique to the TP53R248Q; H3.3K27M; PDGFRAD842Vtumors and related to their aggressive growth phenotype. These includeAREG-mediated cell cycle control, altered metabolism, and vulnerability to combination ONC201/trametinib treatment. Taken together, these data suggest that cooperation between H3.3K27M and PDGFRA influences tumor biology, underscoring the need for better molecular stratification in DMG clinical trials.

Background Heterozygous TERT (telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis. However, no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. Method We established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis. Results Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs. Conclusion Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Heterozygous TERT (Telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis, however no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. To address this, we established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type (TPW) NPCs at initial in vivo passage which we attribute to relatively long telomeres. We further show that the TPM mutation recruited GA-Binding Protein (GABPA) and engendered low-level TERT expression, resulting in enhanced tumorigenesis upon secondary passage and maintenance of short telomere length as has been reported in human GBM. RNA sequencing of harvested tumors grown as secondary spheres demonstrated upregulated proliferation and mitosis pathway signatures in TPM cells, consistent with their increased in vitro proliferation relative to TPW counterparts. Finally, when secondary TPM and TPW sphere cultures were reinjected into mice, only the TPM led to tumor formation. In summary, our novel GBM model illustrates a growth advantage imparted by heterozygous TPMs in the context of GBM driver mutations relative to isogenic controls, and thereby allows for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

Telomerase reverse transcriptase promoter (TERTp) hotspot mutations are the most frequent mutations in primary glioblastomas (GBM). Previous studies have shown that the combination of TERTp and isocitrate dehydrogenase (IDH) status may serve as a useful diagnostic marker for oligodendroglioma and glioblastoma. In oligodendrogliomas, TERTp and IDH mutations, along with the 1p/19q codeletion, usually coexist and are likely to be founder mutations. However, in contrast to oligodendroglioma, the role of the TERTp status in GBM remains obscure. Here, we used Sanger sequencing, pyrosequencing, and digital PCR (dPCR) to examine the TERTp status in 15 pairs of frozen tissue samples from primary and recurrent IDH wild-type GBM, all of which were operated in a single institute. We showed that the TERTp status was stable between primary and recurrent GBM but this consistency was only detected by dPCR. The results suggest that dPCR is a powerful, highly sensitive tool to detect TERTp mutations, especially in a mixed cell population (e.g., a recurrent GBM tissue) where earlier treatment may have grossly altered the tumor microenvironment.

Histone H3 K27M mutation is a hallmark mutation for H3 K27M-mutant diffuse midline glioma (DMG), but targeting this mutation has yet to achieve a significant survival benefit in clinical trials. Recent analyses revealed alterations in several genes, such as NF1 and PDGFRA, are observed in substantial subpopulations of H3 K27M-mutant DMG patients in addition to H3 mutation and recurrent TP53 mutations, indicating patient-to-patient tumor heterogeneity and the potential necessity of tailored target therapy for the treatment of this disease. Here, using our human induced pluripotent stem cells (iPSC)-derived glioma avatar platform, we designed DMG models by introducing TP53R248Q with or without heterozygous H3 K27M mutation in combination with further genetic modifications of either NF1 knockout or PDGFRAD842V overexpression to recapitulate DMG subpopulations. Mice with TP53R248Q; H3F3AK27M (QM) tumors survived significantly longer than those harboring QM;NF1-/- (QMN) tumors and QM; PDGFRAD842Voe (QMP) tumors. RNA-sequencing of those induced DMG (iDMG) neurospheres revealed altered patterns of upregulation of MAPK pathway genes both in QMN and QMP-iDMG neurospheres compared to their H3 wildtype counterparts with the same combinations of genetic alterations, suggesting that those additional mutations modifies the oncogenic signaling associated with H3 K27M mutation. Further, differential expression analysis comparing QMN and QMP-iDMG neurospheres revealed 405 differentially expressed genes. Gene set enrichment analysis showed upregulation of transcriptional programs related to mesenchymal signature in QMN-iDMG neurospheres and proneural signature in QMP-iDMG neurosphere as expected. These data show that NF1 deletion and PDGFRAD842V overexpression significantly alter gene expression in H3 K27M-mutant iDMG tumors, potentially opening up a new therapeutic avenue in these devastating tumors with patient-to-patient heterogeneity. Further work using these models will shed light on the development of tailored therapy based on detailed genetic information on each patient sample, such as combining targeted kinase inhibition with HDAC inhibitors that have shown promise in the clinic.

Dysregulated RNA alternative splicing (AS) is a hallmark of cancer and has been studied in cancers including glioma. However, whether the dysregulated AS and the regulating RNA-binding proteins (RBPs) drive gliomagenesis and the role of glioma driver mutations in shaping the RBP/AS landscape have not been studied. Here we describe two AS-defined glioma subtypes that correlate with IDH1 mutation, tumor grade, and patient prognosis. Oncogenic AS isoforms are exclusively associated with AS subtype 2, IDH1 wild-type gliomas. Modulation of representative AS isoforms or subtype-associated RBPs regulate glioma stem-like cell (GSC) tumorgenicity. With a human induced pluripotent stem cell-derived glioma model system, we demonstrate that specific glioma driver mutations are involved in shaping the subtype-associated AS. Lastly, we target a sub2-associated RBP with anti-sense oligonucleotides to inhibit GSC tumorigenicity. Our data establish relationships between glioma driver mutations and AS programs and identify dysregulated RBP/AS network as a therapeutic vulnerability.

Glioblastoma is one of the most devastating human malignancies for which a novel efficient treatment is urgently required. This pre–clinical study shows that eribulin, a specific inhibitor of telomerase reverse transcriptase (TERT)‐RNA‐dependent RNA polymerase, is an effective anticancer agent against glioblastoma. Eribulin inhibited the growth of 4 TERT promoter mutation‐harboring glioblastoma cell lines in vitro at subnanomolar concentrations. In addition, it suppressed the growth of glioblastoma cells transplanted subcutaneously or intracerebrally into mice, and significantly prolonged the survival of mice harboring brain tumors at a clinically equivalent dose. A pharmacokinetics study showed that eribulin quickly penetrated brain tumors and remained at a high concentration even when it was washed away from plasma, kidney or liver 24 hours after intravenous injection. Moreover, a matrix‐assisted laser desorption/ionization mass spectrometry imaging analysis revealed that intraperitoneally injected eribulin penetrated the brain tumor and was distributed evenly within the tumor mass at 1 hour after the injection whereas only very low levels of eribulin were detected in surrounding normal brain. Eribulin is an FDA‐approved drug for refractory breast cancer and can be safely repositioned for treatment of glioblastoma patients. Thus, our results suggest that eribulin may serve as a novel therapeutic option for glioblastoma. Based on these data, an investigator‐initiated registration‐directed clinical trial to evaluate the safety and efficacy of eribulin in patients with recurrent GBM (UMIN000030359) has been initiated.

Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). However, H3.3K27M alone is insufficient for tumorigenesis in existing DIPG models, suggesting that it interacts with co-occurring oncogenic mutations. NF1 deletion co-occurs with TP53 mutation and H3.3K27M in DIPG, but the impact of these mutations individually and together are understudied. To address this gap, we designed a model of DIPG based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express either wild-type or deleted NF1 in conjunction with heterozygous H3.3K27M and a loss of function mutation in TP53. Edited iPSC were chemically differentiated into neural progenitor cells (iNPC), which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Mice with TP53 mut;H3.3K27M tumors survived significantly (p Citation Format: Kasey Skinner, Tomoyuki Koga, Shunichiro Miki, Robert F. Gruener, R. Stephanie Huang, Frank Furnari, Ryan Miller. NF1 deletion potentiates tumorigenesis and activates expression of cancer-related kinases in an iPSC-based model of H3.3K27M diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB067.

The authors describe a usual case of infiltrative low-grade glial neoplasm with a marked enhanced area which is difficult to diagnose. A 42-year-old man had the diffuse mass lesion partially with apparent enhancement in the right temporal lobe, the insular, and the basal ganglia regions. Final pathological diagnosis from the removed specimens was IDH-wildtype diffuse astrocytoma according to 2016 WHO classification, although it had unusual findings including gliomesenchymal reaction. The nodule-like enhancement was thought to be owing to the mesenchymal component. Several molecular analysis including pyrosequence analysis, Ion Torrent™ next-generation sequencing and multiplex ligation-dependent probe amplification analysis detected no genomic abnormality. IDH-wildtype diffuse astrocytoma is a heterogeneous category and has a minor subset with a silent genomic landscape like the present case.

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify SMPD1, an enzyme that regulates the conversion of sphingomyelin to ceramide and a critical regulator of plasma membrane structure and organization, as an actionable drug target in glioblastoma. We show that the safe and highly brain-penetrant antidepressant fluoxetine, potently inhibits SMPD1 activity, killing GBMs, in vitro and in patient-derived xenografts, through inhibition of EGFR signaling and via activation of lysosomal stress. Combining fluoxetine with the chemotherapeutic agent temozolomide, a standard of care for GBM patients, causes massive increases in GBM cell death, and complete and long-lived tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases, reveals significantly increased survival in glioblastoma patients treated with fluoxetine, which was not seen in patients treated with other SSRI anti-depressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for GBM patients and suggest prospective randomized clinical trials.

Summary The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). Existing DIPG models have provided insight into the role of H3.3K27M but have limitations: genetically engineered murine models often rely on overexpression of the mutant histone to form tumors; patient-derived xenografts (PDX) are more genetically faithful but preclude examination of the effect of individual mutations on pathogenesis. To address these shortcomings and better recapitulate the genetics of human tumors, we designed a novel DIPG model based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express heterozygous H3.3K27M. Edited iPSC were chemically differentiated into neural progenitor cells, which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Further, neurospheres cultured from primary tumors formed secondary tumors upon reimplantation with more diffuse invasion, suggesting in vivo evolution. To validate this model’s relevance to DIPG transcriptionally, we performed RNA-sequencing on a cohort of primary and secondary tumor neurospheres (termed primary and secondary iDIPG) and compared them to published RNA-seq data from pediatric PDX and patient tumor samples. Hierarchical clustering and principal component analysis on differentially expressed genes (P

Diffuse midline glioma is the leading cause of brain tumor death among the pediatric population. Drugs that show notable promise in preclinical models inevitably fail to demonstrate efficacy in clinical trials, likely due to the inadequacy of preclinical models. We have recently proposed glioblastoma models derived from human induced pluripotent stem cells (hiPSCs) genetically engineered with different combinations of glioblastoma-associated genetic alterations as a platform to search for therapeutic targets. These glioblastoma avatars authentically recapitulated the different pathobiology of glioblastoma subtypes, depending on what genetic alterations to be introduced. To investigate the biology and to develop novel therapeutics for diffuse midline glioma with H3K27M mutation, we have established a novel model by introducing H3.3 K27M mutation together with one of the most common concurrent genetic alterations, TP53 R248Q mutation, into hiPSCs through CRISPR/Cas9 genome engineering. Orthotopic engraftment of the neural progenitor cells derived from these edited hiPSCs formed diffusely invasive brainstem tumors with histological features of the diffuse midline glioma. These tumor avatars presented a global reduction in H3K27me3 accompanied by the expression of H3K27M. Transcriptome analyses of these models revealed that these avatars with H3K27M cluster apart from the pediatric glioma samples without this particular mutation, and that they present signatures of oligodendroglial progenitor differentiation as discovered in patient samples with this mutation. Using these models faithfully recapitulating histology and pathobiology of the patient tumors, we have performed drug screening and confirmed that their sensitivity to known drugs, including an EZH2 inhibitor and histone deacetylase inhibitors. On these faithful human avatars of diffuse midline glioma with H3K27M, we have applied bioinformatics algorithms of drug sensitivity prediction aiming at developing novel therapeutics for this devastating pediatric glioma.

Atypical teratoid rhabdoid tumors (ATRT) are challenging pediatric brain cancers which are predominantly associated with inactivation of the gene SMARCB1, a conserved subunit of the chromatin remodeling BAF complex, which has known contributions to developmental processes. To identify potential interactions between SMARCB1 loss and the process of neural development, we introduced an inducible SMARCB1 loss of function system into human induced pluripotent stem cells (iPSCs) which were subjected to either directed neuronal differentiation or differentiation into cerebral organoids. Using this system, we have identified substantial differences in the downstream effects of SMARCB1 loss depending on differentiation state and identified an interaction between SMARCB1 loss and neural differentiation pressure which causes a resistance to terminal differentiation and a defect in maintenance of a normal cell state. Our results provide insight into how SMARCB1 loss might interact with neural development in the process of ATRT tumorigenesis.

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation., The dearth of glioblastoma model systems that accurately recapitulate the disease remains a challenge. Here, the authors develop cancer avatars using genetically engineered human induced pluripotent cells.

Ionizing radiation (IR) and chemotherapy are standard of care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knock-in mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Glioblastomas (GBMs) are the most common malignant primary brain tumors, and the paucity of novel treatments warrants an investigation of its origins and development into aggressive, lethal tumors. Koga & Chaim et al. have recently shown that human pluripotent stem cells (hiPSCs) with different combinations of driver mutations can be differentiated into neural progenitor cells (NPCs) and engrafted into mice to form high grade gliomas (iHGGs). In this work, scRNA seq analysis was used to investigate the development of TP53−/−;PDGFRAΔ8–9 iHGGs, an avatar model that has been shown to recapitulate the proneural subtype of GBM. After re-engrafting the primary avatar cultures (secondary tumor stage), the TP53−/−;PDGFRAΔ8–9 iHGGs developed diverse transcriptional programming and acquired a subpopulation of cells with high expression of known GBM oncogenes, such as MYC, CDK4, and PDGFRA. Notably, when all datasets were aggregated, this oncogene amplifying transcriptional program became the largest source of variation between all stages and replicates of the TP53−/−;PDGFRAΔ8–9 iHGGs. Indicated by a larger total copy number variation (CNV), this oncogene-amplifying program was associated with a genomically unstable developmental state. Trajectory inference could track the development of this population from the initial primary culture of TP53−/−;PDGFRAΔ8–9 iHGG. Differential gene expression analysis identified distinct divergences in clonal evolution––e.g., high expression of the S100 protein family in one cluster––following the acquisition of this genomically unstable state. Lastly, genomic PCR was used to ascertain whether these changes in transcriptional programming were reflected in changes in DNA copy number and identified DNA amplifications of MYC and CDK4. Our scRNA seq analysis of the GBM avatar model platform provides novel insight into how oncogenic states in GBM develop from a small number of driver mutations.

Telomere reverse transcriptase (TERT) promotor mutations increase TERT expression and are known to be the most common single genetic abnormality in Glioblastoma (GBM). Recent studies have revealed this mutation to be an early event in gliomagenesis but it is detected subclonally in a considerable number of cases. Due to the long telomere length and TERT expression in somatic cells of mice, genetically engineered mouse models of GBM do not address the function of this mutation and thus an ideal heterozygous TERT promotor mutation model is yet to be established. Recently, our lab established strategies for generating GBM models using neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) that have been CRISPR/Cas9 engineered with different combinations of authentic GBM-related genetic drivers which can be used for introduction of mutant TERT promotor (MTP) sequence and examination of its function. We obtained multiple heterozygous MTP hiPS clones in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, using a two-step targeting approach. Two single-strand guide RNAs (sgRNAs) were used to delete the TERT promotor and a portion of exon 1 followed by repair of the locus with wild type TERT promoter (WTP) and MTP sequence. Following promoter reconstitution, TERT promotor function was tested in iPSCs, NPCs, and astrocytes. This analysis revealed successful TERT expression silencing in WTP astrocytes. Furthermore, orthotopic injection of WTP and MTP NPCs into immunodeficient mice formed pathologically similar GBM-like tumors in same period of time. TERT expression was only detected in cell lines derived from MTP tumors, indicating TERT promotor mutation has little impact in initiation of tumor formation from NPCs, explaining its subclonality in patient tumors. Our model enables further research on MTP function and dependency during gliomagenesis.

Many mouse and human glioma models have been utilized to study the genetic alterations involved in the genesis of these tumors, but they have not been fully evaluated for how closely they recapitulate pathobiology, including tumor heterogeneity, which is an inherent feature making patient treatment difficult. Here we present new glioma models using genetically engineered human pluripotent stem cells, in which authentic pathobiology is recapitulated through precision gene editing. Specifically, we show that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), with different combinations of genetic drivers introduced by CRISPR/Cas9-mediated editing give rise to distinct intracranial tumors recapitulating authentic pathobiology of the disease when engrafted in immunocompromised mice. NPCs deficient in PTEN and NF1, a genotype associated with the mesenchymal molecular subtype, and NPCs deficient in TP53 and expressing a PDGFRA activating mutation (PDGFRAΔ8–9), a genotype associated with the proneural glioblastoma molecular subtype, give rise to distinct tumors resembling glioblastoma. Both models presented inter and intra-tumor heterogeneity based on single-cell RNA sequencing, with the former model showing proneural signature and the latter a mesenchymal signature, and both having different degrees of cycling, and stem cell-enriched populations. The TP53/PDGFRA model had more clonal variability related with striking karyotype abnormalities including extrachromosomal DNA. Additionally, we expanded this approach to pediatric gliomas. Brainstem tumors derived from NPCs introduced with TP53 R248Q and H3F3A K27M mutations presented features of glial tumors with global expression of histone H3 K27M accompanied by suppression of histone H3K27 trimethylation, compatible with H3 K27M-mutant pediatric diffuse midline glioma. Using these isogenic human brain tumor models, we aim to advance our understanding of the pathobiology associated with different driver mutations and further, to provide a platform for development of targeted therapy.

Many current cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a new cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that capture authentic cancer pathobiology. Orthotopic engraftment of neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) result in formation of high-grade gliomas. As observed in GBM patient samples, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, and intra-tumor heterogeneity. Further, re-engraftment of primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. Thus, these cancer avatar models provide a platform for a comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.

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Background Gliomas that show extensive diffuse infiltration from the cerebellum to the brainstem without masslike expansion are extremely rare. The efficacy of bevacizumab treatment for diffusely infiltrating gliomas remains uncertain. Case Description A 75-year-old man presented with a cerebellar anaplastic astrocytoma showing diffuse infiltration to the brainstem without a definite mass. He had experienced rapidly progressive nausea and dysarthria, as well as vertigo and headache for 2 months. Magnetic resonance imaging (MRI) revealed a poorly demarcated T2 high-intensity area in the right cerebellum and brainstem. The tumor in the right cerebellum showed sparse enhancement with gadolinium (Gd). Suboccipital decompressive craniotomy and partial removal of the tumor was emergently performed because of the rapid progression of symptoms and severe tonsillar herniation demonstrated on MRI. The pathologic diagnosis was anaplastic astrocytoma, and genomic analyses revealed no mutation in IDH1, H3F3A, or BRAF. During concomitant chemoradiotherapy with temozolomide, rapid worsening of the neurologic symptoms developed and significant enlargement of the T2 high-intensity area extending to the cerebral peduncle was seen, as well as a new Gd-enhancing lesion in the midbrain. After administration of bevacizumab, the neurologic symptoms gradually improved, the T2 high-intensity area decreased, and the Gd-enhancing lesion disappeared. At follow-up 2 years after the operation, no worsening of neurologic symptoms was seen and the residual T2 high-intensity area remained unchanged on MRI. Conclusions Bevacizumab treatment may be a salvage treatment option for patients with diffusely infiltrating cerebellar gliomas that exhibits rapid progression during standard treatment.

We evaluated the feasibility of 5-aminolevulinic acid (5-ALA)-mediated photodynamic diagnosis (PDD) in the biopsy for primary central nervous system lymphoma (PCNSL). 5-ALA (20 mg kg(-1) ) was administered orally 4 hours preoperatively. Forty-one biopsies obtained under PDD in 47 consecutive biopsies (46 patients) that were finally pathologically diagnosed as PCNSL were evaluated. Positive fluorescence was observed in 34 of those 41 biopsies (82.9%). An intraoperative pathological diagnosis (IOD) of suspected PCNSL was made in 21 of the biopsies with positive fluorescence (61.8%). However, the eight IODs in the remaining 13 biopsies (23.5%) were not correct (atypical cell, 4; high-grade glioma, 1; gliosis, 1; unremarkable, 2). In those 8 biopsies, PCNSL was confirmed by the final pathological diagnosis. There was no difference in the mean Mib-1 labeling index between the biopsies with positive fluorescence (86.5%) and those without positive fluorescence (90.0%). IOD was not performed in 6 biopsies; however, 5 of those biopsies (83.3%) showed positive fluorescence and were finally pathologically diagnosed as PCNSL. Use of PDD in biopsies for patients with suspected PCNSL is a reliable way of obtaining specimens of adequate quality for the final pathological diagnosis and may lead to improved diagnostic yield in the biopsy of PCNSL.

A 48-year-old man with right hemi-facial palsy and cerebellar ataxia was referred to our hospital. Three years and 10 months earlier he had undergone gamma knife radiosurgery (GKRS) at the referring hospital for an 18 mm right vestibular schwannoma. Slight tumor enlargement had been observed on MRI performed at the referring hospital 3 years after the GKRS. On close follow-up after another 6 months an MRI showed an obvious enlargement of the tumor. An MRI on admission revealed an iso-intense mass lesion measuring 36 mm in maximum diameter at the right cerebellopontine angle. A two stage surgery was conducted using a retrosigmoid approach because bleeding from the tumor wall was difficult to control intraoperatively during the first operation. At the second operation, the majority of the tumor capsule had converted to necrotic tissue. A large hematoma cavity was present inside the tumor capsule which explained the rapid increase in size over a short period of time. Near total removal was achieved. Histopathological examination revealed massive intratumoral hemorrhage within a typical vestibular schwannoma with no malignancy. The complication of intratumoral hemorrhage is very rare and the utility of stereotactic radiation surgery/therapy, including GKRS, for vestibular schwannoma is well known. However, we must emphasize that careful follow-up is still required, even after several years.

Despite of recent intense investigations, no effective molecular targeting therapy has been successfully developed in patients with glioblastomas (GBMs). Approximately 60–80% of GBM harbor mutations in the promoter region of telomerase reverse transcriptase (TERT), which leads to its upregulation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity and TERT-RdRP activity controls cell cycle and maintains stem cell phenotype, suggesting that TERT can be directly targeted to suppress tumor cell proliferation. We have previously identified eribulin mesylate, which is an FDA-approved drug used to treat breast cancer and liposarcoma, as a specific inhibitor of RdRP activity of TERT and investigated the antitumor efficacy of eribulin using multiple human GBM cell lines in preclinical mouse brain tumor models. All GBM cell lines tested in vitro that harbored TERT promoter mutations were highly sensitive to eribulin with IC50 below 1nM. Intraperitoneal administration of eribulin (0.5 mg/kg), which was equivalent to the dose used in human (1.4 mg/m2), significantly prolonged the survival of multiple mouse brain tumor models established with U87MG or LN229 (p

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non-taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M-phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation-induced DNA damage. Several recent case reports have demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG and U118MG, and SJ28 cells, a patient-derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (P < .0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas.

Purpose To evaluate the clinical outcome and MRI findings after carotid artery stenting (CAS) without post-dilatation. Methods Between May 2005 and April 2012, a total of 169 consecutive patients (61.4% symptomatic) underwent 176 CAS procedures performed with an embolic protection device (GuardWire, n=116; FilterWire EZ, n=60). All stents were deployed without post-dilatation. Periprocedural complications and mid-term outcomes were analyzed. Results The stroke rate was 2.3% within 30 days post-CAS (asymptomatic patients 1.5%; symptomatic patients 2.8%). Cerebral infarction occurred in one asymptomatic patient (1.5%) and one symptomatic patient (0.9%). Intracranial hemorrhage occurred in two symptomatic patients (1.9%). Post-CAS diffusion-weighted imaging (DWI) revealed a high-intensity area in 26 of 176 procedures (14.8%). Ipsilateral stroke after 31 days occurred in two patients (1.1%) and restenosis occurred in six (3.4%). A post-CAS comparison of the embolic protection devices revealed no difference in stroke incidence within 30 days and in DWI high-intensity area. Conclusions Our CAS procedure without post-dilatation is feasible, safe and associated with a low incidence of stroke and restenosis.

Objective: Glioblastomas (GBMs) mainly occur locally, and develop distant or disseminated recurrence (non-local recurrence) in the time course of disease. Non-local recurrences indicate tumor cells invasion into normal brain or cerebrospinal fluid (CSF) space, and the treatment will be difficult. We studied the pattern of recurrence in GBMs treated with prolonged temozolomide (TMZ) maintenance and the risk factors to progress to non-local recurrence. Methods: We investigated 102 GBM patients with single lesion, who were treated with combined chemoradiotherapy using TMZ for a total of 24 cycles or until disease progression between May 2006 and May 2013. Pattern of recurrences were classified as local (at the regional tumor), distant (intraparenchymal, but distant from the original tumor site), and disseminated (distant from the original tumor and exposed to the CSF space, including subarachnoidal, subependymal, intraventricular, and spinal lesion). IDH1/2 mutation and O-6-methylguanine DNA ­methyltransferase (MGMT) promoter methylation were analyzed by direct sequencing and pyrosequencing, respectively. Results: Of 102 patients, 76 developed radiological recurrences including 48 (63.2%) local, 13 (17.1%) distant, and 15 (19.7%) disseminated. Of 48 the local recurrent tumors, 37 developed radiological recurrences including 24 (64.9%) local, 9 (24.3%) distant, and 5 (13.5%) disseminated. Finally, of the 76 recurrences, 50 (65.8%) developed non-local recurrences. The median time to local recurrence was 17.6 months, whereas that to disseminated was 42.4 months (p = 0.0023), and that to distant was not reached (p < 0.0001). Multivariate analyses of 76 patients who were available for IDH1/2 and MGMT promoter status analysis indicated that extent of resection, IDH1/2 mutations, and MGMT promoter methylation were correlated with non-local recurrence. In 45 GBMs with IDH1/2 wildtype and MGMT promoter unmethylation (IDH-WT-Unmet GBMs), the median time to local recurrence was 9.6 months, whereas that to disseminated was 24.5 months (p = 0.025), and that to distant was 17.4 months (p = 0.023). Conclusions: The predominant pattern of recurrence is local, and non-local recurrences increase cumulatively. The risk factors of developing non-local recurrence are partial/biopsy, IDH1/2 wildtype, and MGMT promoter unmethylation. Our results indicate that intensifying local tumor control before progression of non-local recurrence is the mainstay of treatment, especially in IDH-WT-Unmet GBMs.

BACKGROUND: Although it is recognized that methylation status of MGMT promoter is predictive for the response to temozolomide in newly diagnosed glioblastoma (GBM), at least in elderly patients, there is no study examining the ability of quantitative MGMT methylation status to predict volumetric changes of residual tumor in magnetic resonance images (MRIs) after surgery, radiation and temozolomide therapy. In this study, we performed retrospective volumetric analysis of the residual tumor in glioblastoma patients and investigated the relationship of MGMT status assessed by pyrosequencing. METHOD: Among 160 newly diagnosed GBM patients treated in our institute since 2006, volumetric analysis of the residual tumor was performed retrospectively in patients who were with residual unresected tumors after surgery, treated with standard radiation and temozolomide therapy. Patients who were without residual tumors or treated with bevacizumab were excluded. The MGMT methylation status was quantitatively assessed by averaging methylation at the 16 CpG sites measured by pyrosequencing. Enhanced tumor volume in MRI was measured at four time points of pre-operation, beginning of the treatments, three and six months after the initial treatment. RESULT: Forty four patients were enrolled (M:F = 25:19, median age: 61 y.o., median KPS: 70). The quantitative MGMT methylation data and tumor volume ratio, normalized to pre-treatment volume, were significantly correlated at 3 and 6 months after the initial treatment (p = 0.0002 and 0.0004, respectively). For the response rate, patients with lower MGMT methylation than 10% showed more progressive disease (defined as more than 50% gain in tumor volume) compared to patients with higher MGMT methylation than 10% at 3 and 6 months after initial treatment (3 months: 67.9% vs 18.8%, 6 months: 70.8% vs 15.4%, respectively). CONCLUSION: Quantitative MGMT methylation status analyzed by pyrosequencing may be useful to predict disease progression in glioblastomas treated with partial tumor removal followed by radiation and temozolomide therapy.

Background: The authors report a continuous case series of navigation-guided rigid endoscopic biopsy via the transcortical route for intraparenchymal brain lesions to assess the feasibility and efficacy of the method. Methods: Thirty-four patients with intraparenchymal brain lesions found on neurovisualization underwent navigation-guided rigid endoscopic biopsy. Most of the preoperative diagnoses were glioma WHO Grade II–IV (16 cases) or malignant lymphoma (15 cases). Intraoperative photodynamic diagnosis and intraoperative pathological diagnosis were used in 28 and 29 cases, respectively. In 2 cases with small and deep lesions, intraoperative magnetic resonance imaging was used for confirming the accuracy of the biopsy point. Results: The sampling accuracy determined by postoperative imaging and the definitive diagnosis ratio were 94% (32 out of 34 cases) and 97% (33 out of 34 cases), respectively. There was no postoperative mortality. In 2 patients, mild postoperative permanent morbidity (5.9%), presumably related to this technique, was observed in the early cases in the current group (34 case series). Conclusion: The method was estimated as safe and feasible for diagnostic tissue sampling of intraparenchymal brain lesions.

An 87-year-old woman presented with an atypical case of acute subdural hematoma (ASDH) manifesting as disturbance of consciousness and left hemiparesis. Computed tomography revealed a high density lentiform lesion in the right convexity, which was thought to be acute epidural hematoma preoperatively. Emergent decompressive craniotomy revealed an encapsulated solid fresh clot in the subdural space and a bleeding small cortical artery under the clot. The arachnoid membrane and the brain parenchyma were intact, and no other abnormal feature such as aneurysm or arteriovenous malformation was observed. The encapsulated ASDH was removed en bloc and the patient fully recovered. Histological examination confirmed that both the outer thicker and the inner membrane were fibrinous single structures without vasculature. The red blood cells constituting the clot in the capsule maintained their cell structure. The reported pathological mechanisms of lentiform ASDH are adhesion of the arachnoid membrane and the dura mater or intracapsular bleeding from sinusoidal vessels in the outer membrane of the chronic subdural hematoma. However, in our case, the arachnoid membrane had not adhered to the dura mater and the capsule was a fibrinous single structural membrane without vasculature, which probably resulted from a previous hematoma due to initial bleeding from the cortical artery. The possible mechanism in our case was that the re-bleeding dissected and flowed into the fibrinous single structural membrane, resulting in formation of the lentiform ASDH.

[Introduction] No effective molecular targeting therapy has been clinically developed for patients with glioblastoma (GBM) despite of intense genome analysis. Approximately 60-80% of GBM harbor mutations in the promoter region of TERT that leads to its upregulation, making it the most common single genetic abnormalities in GBM. TERT, a reverse-transcriptase subunit of telomerase, has been an attractive candidate for therapy target because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity, with which TERT is involved in maintenance of stem cell phenotype and mitotic progression. We have previously identified eribulin as a specific inhibitor of RdRP activity of TERT through drug screening and shown that eribulin suppressed growth of ovarian cancers with high TERT expression. In order to investigate an efficacy of eribulin as a novel TERT-targeted therapy against GBM, we investigated the antitumor effect of eribulin in cultured cell and brain tumor model using GBM cell lines. [Methods] The TERT promoter status was examined in 20 glioma cell lines. Seven cell lines were subjected to in vitro cytotoxicity assay. U87MG was either subcutaneously or intracranially transplanted in athymic mice. Tissue or plasma concentration of eribulin was measured by LC-MS/MS. [Results] All GBM cell lines tested that harbored TERT promoter mutations including U87MG, U251MG, U118MG as well as several patient-derived glioblastoma sphere culture cells were highly sensitive to eribulin, IC50 below 1nM. Intraperitoneal administration of eribulin completely suppressed the growth of U87MG subcutaneous tumor in nude mice, and the RdRP activities in treated tumors were significantly decreased in a dose-dependent manner. In the brain tumor model, a high concentration of eribulin was detected in the brain tumor tissues as early as 15 minutes after intravenous injection of eribulin, which remained stable even 24 hours later when the plasma concentration of eribulin became undetectable. Finally, intraperitoneal administration of eribulin significantly prolonged the survival of mice with intracerebrally transplanted U87MG xenograft (p [Conclusion] Our results showed that eribulin efficiently transfers into brain tumor tissues and has a strong antitumor effect against GBM cells through inhibition of RdRP activity. Eribulin thus appears to be a promising novel TERT-targeting therapeutic agent against GBM. A clinical trial is being scheduled. Citation Format: Masamichi Takahashi, Shunichiro Miki, Yuko Matsushita, Kohei Fukuoka, Yoshiko Maida, Mami Yasukawa, Mitsuhiro Hayashi, Akinobu Hamada, Akitake Mukasa, Ryo Nishikawa, Kenji Tamura, Yoshitaka Narita, Kenkichi Masutomi, Koichi Ichimura. Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2017-3117

Introduction: Despite of recent extensive genome analysis, no effective molecular targeting therapy has been successfully developed in patients with glioblastomas (GBMs). Approximately 60–80% of GBM harbour mutations in the promoter region of telomerase reverse transcriptase (TERT) that leads to upregulation of its protein production, making it the most common single genetic abnormalities in GBM. TERT has been an attractive candidate for molecular targeting therapy because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity, with which TERT is involved in maintenance of stem cell phenotype and mitotic progression. We have previously identified eribulin as a specific inhibitor of RdRP activity of TERT through drug screening and shown that eribulin suppressed growth of ovarian cancers with high TERT expression. In order to validate an efficacy of eribulin as a novel TERT-targeting therapy against GBM, we investigated the anti-tumor effect of eribulin in cultured cell and mouse brain tumor model using GBM cell lines. Methods: The TERT promoter status was examined in 20 glioma cell lines. Seven cell lines were subjected to in vitro cytotoxicity assay. U87MG cells were either subcutaneously or intracranially transplanted in athymic mice. Tissue or plasma concentration of eribulin was measured by LC-MS/MS. Results: All GBM cell lines tested that harboured TERT promoter mutations including U87MG, U251MG, U118MG as well as several patient-derived glioblastoma sphere culture cells were highly sensitive to eribulin, IC50 below 1nM. Intraperitoneal administration of eribulin completely suppressed the growth of U87MG subcutaneous tumors in athymic mice, and the RdRP activities in treated tumors were significantly decreased in a dose-dependent manner. In U87MG brain tumor model, a high concentration of eribulin was detected in the brain tumour tissues as early as 15 minutes after intravenous injection of eribulin, which remained stable even 24 hours later when the plasma concentration of eribulin became undetectable. Finally, intraperitoneal administration of eribulin significantly prolonged the survival of mice with intracranially transplanted U87MG xenograft (p

An 87-year-old woman presented with an atypical case of acute subdural hematoma (ASDH) manifesting as disturbance of consciousness and left hemiparesis. Computed tomography revealed a high density lentiform lesion in the right convexity, which was thought to be acute epidural hematoma preoperatively. Emergent decompressive craniotomy revealed an encapsulated solid fresh clot in the subdural space and a bleeding small cortical artery under the clot. The arachnoid membrane and the brain parenchyma were intact, and no other abnormal feature such as aneurysm or arteriovenous malformation was observed. The encapsulated ASDH was removed en bloc and the patient fully recovered. Histological examination confirmed that both the outer thicker and the inner membrane were fibrinous single structures without vasculature. The red blood cells constituting the clot in the capsule maintained their cell structure. The reported pathological mechanisms of lentiform ASDH are adhesion of the arachnoid membrane and the dura mater or intracapsular bleeding from sinusoidal vessels in the outer membrane of the chronic subdural hematoma. However, in our case, the arachnoid membrane had not adhered to the dura mater and the capsule was a fibrinous single structural membrane without vasculature, which probably resulted from a previous hematoma due to initial bleeding from the cortical artery. The possible mechanism in our case was that the re-bleeding dissected and flowed into the fibrinous single structural membrane, resulting in formation of the lentiform ASDH.

Recently, laparoscopy (also referred to as minimally invasive surgery) has been used during peritoneal catheter implantation in shunt placement for hydrocephalus; however, the procedure and devices for this technique have not yet been well established. We adopted umbilical and paraumbilical laparoscopy for peritoneal catheter insertion. In this paper, we describe the technique we used and its clinical results and benefits. Ten consecutive patients with hydrocephalus who underwent laparoscopic shunt surgery (6 cases of ventriculoperitoneal shunt and 4 of lumboperitoneal shunt) were enrolled for this study. The follow-up period ranged from 21 to 434 days (mean, 263 days). After a standard cranial/spinal procedure, an approximately 5-mm incision was made in the lateral side of the umbilicus, where the abdominal catheter was introduced subcutaneously. Thereafter, we inserted a laparoscope into the peritoneal cavity via a small incision beneath or just on the umbilicus. A shunt catheter was laparoscopically inserted through a peel-off cannula and placed after taking note of the outflow of cerebrospinal fluid (CSF) from the catheter tip. In all patients, the shunt was inserted with no complications, and good patency was achieved. Laparoscopy allows implantation of the catheter into the peritoneal cavity, and the outflow of CSF can be confirmed intraoperatively. Furthermore, the abdominal surgical wounds are minimal, even for obese patients, and fascia/muscle incisions are not needed. Laparoscopy-assisted shunt surgery for hydrocephalus is effective and safe and also has cosmetic advantages.