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    EGFR amplified and overexpressing glioblastomas and association with better response to adjuvant metronomic temozolomide. Cominelli Manuela,Grisanti Salvatore,Mazzoleni Stefania,Branca Caterina,Buttolo Luciano,Furlan Daniela,Liserre Barbara,Bonetti Maria Fausta,Medicina Daniela,Pellegrini Vilma,Buglione Michela,Liserre Roberto,Pellegatta Serena,Finocchiaro Gaetano,Dalerba Piero,Facchetti Fabio,Pizzi Marina,Galli Rossella,Poliani Pietro Luigi Journal of the National Cancer Institute BACKGROUND:Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment. METHODS:We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student's t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided. RESULTS:No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (P interaction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients. CONCLUSIONS:EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide-based therapies. 10.1093/jnci/djv041
    Redox-responsive magnetic nanoparticle for targeted convection-enhanced delivery of O6-benzylguanine to brain tumors. Stephen Zachary R,Kievit Forrest M,Veiseh Omid,Chiarelli Peter A,Fang Chen,Wang Kui,Hatzinger Shelby J,Ellenbogen Richard G,Silber John R,Zhang Miqin ACS nano Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes. 10.1021/nn503735w
    Understanding and targeting alkylator resistance in glioblastoma. Wick Wolfgang,Platten Michael Cancer discovery Alkylating chemotherapy is the mainstay in the treatment of pediatric and adult glioblastoma despite primary and acquired resistance and scientific efforts to precisely define therapies for individual patients. A focus on non-MGMT-mediated temozolomide resistance for pediatric glioblastoma suggests options for new drug combinations. 10.1158/2159-8290.CD-14-0918
    MGMT testing--the challenges for biomarker-based glioma treatment. Wick Wolfgang,Weller Michael,van den Bent Martin,Sanson Marc,Weiler Markus,von Deimling Andreas,Plass Christoph,Hegi Monika,Platten Michael,Reifenberger Guido Nature reviews. Neurology Many patients with malignant gliomas do not respond to alkylating agent chemotherapy. Alkylator resistance of glioma cells is mainly mediated by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in glioma cells compromises this DNA repair mechanism and increases chemosensitivity. MGMT promoter methylation is, therefore, a strong prognostic biomarker in paediatric and adult patients with glioblastoma treated with temozolomide. Notably, elderly patients (>65-70 years) with glioblastoma whose tumours lack MGMT promoter methylation derive minimal benefit from such chemotherapy. Thus, MGMT promoter methylation status has become a frequently requested laboratory test in neuro-oncology. This Review presents current data on the prognostic and predictive relevance of MGMT testing, discusses clinical trials that have used MGMT status to select participants, evaluates known issues concerning the molecular testing procedure, and addresses the necessity for molecular-context-dependent interpretation of MGMT test results. Whether MGMT promoter methylation testing should be offered to all individuals with glioblastoma, or only to elderly patients and those in clinical trials, is also discussed. Justifications for withholding alkylating agent chemotherapy in patients with MGMT-unmethylated glioblastomas outside clinical trials, and the potential role for MGMT testing in other gliomas, are also discussed. 10.1038/nrneurol.2014.100
    Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Wang Chao,Abegg Daniel,Hoch Dominic G,Adibekian Alexander Angewandte Chemie (International ed. in English) We present a novel chemical scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We demonstrate the tunability of the CMTs towards a specific biological target by synthesizing AA-CW236 as the first potent non-pseudosubstrate inhibitor of the O(6) -alkylguanine DNA methyltransferase (MGMT), a protein of major clinical significance for the treatment of several severe cancer forms. Using quantitative proteomics profiling techniques, we show that AA-CW236 exhibits a high degree of selectivity towards MGMT. Finally, we validate the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. Our results may open a new avenue towards the development of a clinically approved MGMT inhibitor. 10.1002/anie.201511301
    Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR. Jin Xing,Kang Soeun,Tanaka Shinya,Park Sunghyouk Angewandte Chemie (International ed. in English) The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real-time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U-(13) C]-labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS-producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti-glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up-regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox-dependent signaling pathways and drug resistance involving ROS. 10.1002/anie.201601026
    Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid. Pentsova Elena I,Shah Ronak H,Tang Jiabin,Boire Adrienne,You Daoqi,Briggs Samuel,Omuro Antonio,Lin Xuling,Fleisher Martin,Grommes Christian,Panageas Katherine S,Meng Fanli,Selcuklu S Duygu,Ogilvie Shahiba,Distefano Natalie,Shagabayeva Larisa,Rosenblum Marc,DeAngelis Lisa M,Viale Agnes,Mellinghoff Ingo K,Berger Michael F Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Cancer spread to the central nervous system (CNS) often is diagnosed late and is unresponsive to therapy. Mechanisms of tumor dissemination and evolution within the CNS are largely unknown because of limited access to tumor tissue. MATERIALS AND METHODS:We sequenced 341 cancer-associated genes in cell-free DNA from cerebrospinal fluid (CSF) obtained through routine lumbar puncture in 53 patients with suspected or known CNS involvement by cancer. RESULTS:We detected high-confidence somatic alterations in 63% (20 of 32) of patients with CNS metastases of solid tumors, 50% (six of 12) of patients with primary brain tumors, and 0% (zero of nine) of patients without CNS involvement by cancer. Several patients with tumor progression in the CNS during therapy with inhibitors of oncogenic kinases harbored mutations in the kinase target or kinase bypass pathways. In patients with glioma, the most common malignant primary brain tumor in adults, examination of cell-free DNA uncovered patterns of tumor evolution, including temozolomide-associated mutations. CONCLUSION:The study shows that CSF harbors clinically relevant genomic alterations in patients with CNS cancers and should be considered for liquid biopsies to monitor tumor evolution in the CNS. 10.1200/JCO.2016.66.6487
    Towards the overcoming of anticancer drug resistance mediated by p53 mutations. Cao Xin,Hou Jiayun,An Quanlin,Assaraf Yehuda G,Wang Xiangdong Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy Cancer continues to be a leading threat to human health and life. Resistance to anti-cancer drugs is a major impediment towards efficacious cancer treatment. p53 mutations play an important role in cancer cell resistance to chemotherapeutic drugs. The frequency of p53-based chemoresistance is highly associated with the chemical properties of the anticancer drug, the cellular drug target, the biological function being blocked by the chemotherapeutic agent, the genomic instability and alterations of the tumor, as well as its differentiation state. The p53-based molecular mechanisms of anticancer drug resistance are insufficiently understood. With a clear focus on the role of p53 mutations in anticancer drug resistance, the present article reviews the biological structure and function of p53, its regulatory mechanisms, as well as the molecular mechanisms underlying p53 mutation-dependent chemoresistance and possible modalities to surmount this drug resistance. We specifically discuss the roles of p53 in the development of chemoresistance to classical cytotoxic agents including for example cisplatin, doxorubicin, 5-fluorouracil, temozolomide, and paclitaxel. It is expected that the clinical manifestation of drug resistance can be integrated with data obtained from molecular multi-omics analyses addressing the alterations provoked by p53-driven resistance to discover the altered networks in these drug resistant tumors. Thus, novel drugs targeting mutant p53 or mutant p53-based dysregulated pathways, could be developed that may overcome well-defined mutant p53-mediated chemoresistance. Thus, an in-depth understanding of the p53-driven resistance modalities could facilitate the development of novel targeted antitumor drugs and strategies aimed at enhancing the efficacy of current cancer therapeutics. 10.1016/j.drup.2019.100671
    Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Johnson Brett E,Mazor Tali,Hong Chibo,Barnes Michael,Aihara Koki,McLean Cory Y,Fouse Shaun D,Yamamoto Shogo,Ueda Hiroki,Tatsuno Kenji,Asthana Saurabh,Jalbert Llewellyn E,Nelson Sarah J,Bollen Andrew W,Gustafson W Clay,Charron Elise,Weiss William A,Smirnov Ivan V,Song Jun S,Olshen Adam B,Cha Soonmee,Zhao Yongjun,Moore Richard A,Mungall Andrew J,Jones Steven J M,Hirst Martin,Marra Marco A,Saito Nobuhito,Aburatani Hiroyuki,Mukasa Akitake,Berger Mitchel S,Chang Susan M,Taylor Barry S,Costello Joseph F Science (New York, N.Y.) Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct from those in the initial tumor. To explore this hypothesis, we sequenced the exomes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF; this suggests that recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt-mTOR (mammalian target of rapamycin) pathways that bore the signature of TMZ-induced mutagenesis. 10.1126/science.1239947
    Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients. Adair Jennifer E,Johnston Sandra K,Mrugala Maciej M,Beard Brian C,Guyman Laura A,Baldock Anne L,Bridge Carly A,Hawkins-Daarud Andrea,Gori Jennifer L,Born Donald E,Gonzalez-Cuyar Luis F,Silbergeld Daniel L,Rockne Russell C,Storer Barry E,Rockhill Jason K,Swanson Kristin R,Kiem Hans-Peter The Journal of clinical investigation BACKGROUND:Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome. METHODS:We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated. RESULTS:Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG. CONCLUSION:These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT. TRIAL REGISTRATION:Clinicaltrials.gov NCT00669669. FUNDING:R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970. 10.1172/JCI76739
    ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents. Agnihotri Sameer,Burrell Kelly,Buczkowicz Pawel,Remke Marc,Golbourn Brian,Chornenkyy Yevgen,Gajadhar Aaron,Fernandez Nestor A,Clarke Ian D,Barszczyk Mark S,Pajovic Sanja,Ternamian Christian,Head Renee,Sabha Nesrin,Sobol Robert W,Taylor Michael D,Rutka James T,Jones Chris,Dirks Peter B,Zadeh Gelareh,Hawkins Cynthia Cancer discovery UNLABELLED:Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. SIGNIFICANCE:Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window. 10.1158/2159-8290.CD-14-0157
    A STAT3-based gene signature stratifies glioma patients for targeted therapy. Tan Melanie Si Yan,Sandanaraj Edwin,Chong Yuk Kien,Lim See Wee,Koh Lynnette Wei Hsien,Ng Wai Hoe,Tan Nguan Soon,Tan Patrick,Ang Beng Ti,Tang Carol Nature communications Intratumoral heterogeneity is a hallmark of glioblastoma (GBM) tumors, thought to negatively influence therapeutic outcome. Previous studies showed that mesenchymal tumors have a worse outcome than the proneural subtype. Here we focus on STAT3 as its activation precedes the proneural-mesenchymal transition. We first establish a STAT3 gene signature that stratifies GBM patients into STAT3-high and -low cohorts. STAT3 inhibitor treatment selectively mitigates STAT3-high cell viability and tumorigenicity in orthotopic mouse xenograft models. We show the mechanism underlying resistance in STAT3-low cells by combining STAT3 signature analysis with kinome screen data on STAT3 inhibitor-treated cells. This allows us to draw connections between kinases affected by STAT3 inhibitors, their associated transcription factors and target genes. We demonstrate that dual inhibition of IGF-1R and STAT3 sensitizes STAT3-low cells and improves survival in mice. Our study underscores the importance of serially profiling tumors so as to accurately target individuals who may demonstrate molecular subtype switching. 10.1038/s41467-019-11614-x
    Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment. Dirkse Anne,Golebiewska Anna,Buder Thomas,Nazarov Petr V,Muller Arnaud,Poovathingal Suresh,Brons Nicolaas H C,Leite Sonia,Sauvageot Nicolas,Sarkisjan Dzjemma,Seyfrid Mathieu,Fritah Sabrina,Stieber Daniel,Michelucci Alessandro,Hertel Frank,Herold-Mende Christel,Azuaje Francisco,Skupin Alexander,Bjerkvig Rolf,Deutsch Andreas,Voss-Böhme Anja,Niclou Simone P Nature communications The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment. 10.1038/s41467-019-09853-z
    Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance. Wickström Malin,Dyberg Cecilia,Milosevic Jelena,Einvik Christer,Calero Raul,Sveinbjörnsson Baldur,Sandén Emma,Darabi Anna,Siesjö Peter,Kool Marcel,Kogner Per,Baryawno Ninib,Johnsen John Inge Nature communications The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment. 10.1038/ncomms9904
    Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome. Favero F,McGranahan N,Salm M,Birkbak N J,Sanborn J Z,Benz S C,Becq J,Peden J F,Kingsbury Z,Grocok R J,Humphray S,Bentley D,Spencer-Dene B,Gutteridge A,Brada M,Roger S,Dietrich P-Y,Forshew T,Gerlinger M,Rowan A,Stamp G,Eklund A C,Szallasi Z,Swanton C Annals of oncology : official journal of the European Society for Medical Oncology BACKGROUND:Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. METHODS:We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. RESULTS:We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. CONCLUSION:This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. 10.1093/annonc/mdv127
    Spatiotemporal Evolution of the Primary Glioblastoma Genome. Kim Jinkuk,Lee In-Hee,Cho Hee Jin,Park Chul-Kee,Jung Yang-Soon,Kim Yanghee,Nam So Hee,Kim Byung Sup,Johnson Mark D,Kong Doo-Sik,Seol Ho Jun,Lee Jung-Il,Joo Kyeung Min,Yoon Yeup,Park Woong-Yang,Lee Jeongwu,Park Peter J,Nam Do-Hyun Cancer cell Tumor recurrence following treatment is the major cause of mortality for glioblastoma multiforme (GBM) patients. Thus, insights on the evolutionary process at recurrence are critical for improved patient care. Here, we describe our genomic analyses of the initial and recurrent tumor specimens from each of 38 GBM patients. A substantial divergence in the landscape of driver alterations was associated with distant appearance of a recurrent tumor from the initial tumor, suggesting that the genomic profile of the initial tumor can mislead targeted therapies for the distally recurred tumor. In addition, in contrast to IDH1-mutated gliomas, IDH1-wild-type primary GBMs rarely developed hypermutation following temozolomide (TMZ) treatment, indicating low risk for TMZ-induced hypermutation for these tumors under the standard regimen. 10.1016/j.ccell.2015.07.013
    New insights into acquired temozolomide resistance in glioblastoma? Happold Caroline,Weller Michael Brain : a journal of neurology 10.1093/brain/awv301
    AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Chow Ryan D,Guzman Christopher D,Wang Guangchuan,Schmidt Florian,Youngblood Mark W,Ye Lupeng,Errami Youssef,Dong Matthew B,Martinez Michael A,Zhang Sensen,Renauer Paul,Bilguvar Kaya,Gunel Murat,Sharp Phillip A,Zhang Feng,Platt Randall J,Chen Sidi Nature neuroscience A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo. 10.1038/nn.4620
    A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy. Yi Hee-Gyeong,Jeong Young Hun,Kim Yona,Choi Yeong-Jin,Moon Hyo Eun,Park Sung Hye,Kang Kyung Shin,Bae Mihyeon,Jang Jinah,Youn Hyewon,Paek Sun Ha,Cho Dong-Woo Nature biomedical engineering Patient-specific ex vivo models of human tumours that recapitulate the pathological characteristics and complex ecology of native tumours could help determine the most appropriate cancer treatment for individual patients. Here, we show that bioprinted reconstituted glioblastoma tumours consisting of patient-derived tumour cells, vascular endothelial cells and decellularized extracellular matrix from brain tissue in a compartmentalized cancer-stroma concentric-ring structure that sustains a radial oxygen gradient, recapitulate the structural, biochemical and biophysical properties of the native tumours. We also show that the glioblastoma-on-a-chip reproduces clinically observed patient-specific resistances to treatment with concurrent chemoradiation and temozolomide, and that the model can be used to determine drug combinations associated with superior tumour killing. The patient-specific tumour-on-a-chip model might be useful for the identification of effective treatments for glioblastoma patients resistant to the standard first-line treatment. 10.1038/s41551-019-0363-x
    Lnc-TALC promotes O-methylguanine-DNA methyltransferase expression via regulating the c-Met pathway by competitively binding with miR-20b-3p. Wu Pengfei,Cai Jinquan,Chen Qun,Han Bo,Meng Xiangqi,Li Yansheng,Li Ziwei,Wang Ruijia,Lin Lin,Duan Chunbin,Kang Chunsheng,Jiang Chuanlu Nature communications Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy. 10.1038/s41467-019-10025-2
    Multicenter Phase IB Trial of Carboxyamidotriazole Orotate and Temozolomide for Recurrent and Newly Diagnosed Glioblastoma and Other Anaplastic Gliomas. Omuro Antonio,Beal Kathryn,McNeill Katharine,Young Robert J,Thomas Alissa,Lin Xuling,Terziev Robert,Kaley Thomas J,DeAngelis Lisa M,Daras Mariza,Gavrilovic Igor T,Mellinghoff Ingo,Diamond Eli L,McKeown Andrew,Manne Malbora,Caterfino Andrew,Patel Krishna,Bavisotto Linda,Gorman Greg,Lamson Michael,Gutin Philip,Tabar Viviane,Chakravarty Debyani,Chan Timothy A,Brennan Cameron W,Garrett-Mayer Elizabeth,Karmali Rashida A,Pentsova Elena Journal of clinical oncology : official journal of the American Society of Clinical Oncology Purpose Carboxyamidotriazole orotate (CTO) is a novel oral inhibitor of non-voltage-dependent calcium channels with modulatory effects in multiple cell-signaling pathways and synergistic effects with temozolomide (TMZ) in glioblastoma (GBM) models. We conducted a phase IB study combining CTO with two standard TMZ schedules in GBM. Methods In cohort 1, patients with recurrent anaplastic gliomas or GBM received escalating doses of CTO (219 to 812.5 mg/m once daily or 600 mg fixed once-daily dose) combined with TMZ (150 mg/m 5 days during each 28-day cycle). In cohort 2, patients with newly diagnosed GBM received escalating doses of CTO (219 to 481 mg/m/d once daily) with radiotherapy and TMZ 75 mg/m/d, followed by TMZ 150 mg to 200 mg/m 5 days during each 28-day cycle. Results Forty-seven patients were enrolled. Treatment was well tolerated; toxicities included fatigue, constipation, nausea, and hypophosphatemia. Pharmacokinetics showed that CTO did not alter TMZ levels; therapeutic concentrations were achieved in tumor and brain. No dose-limiting toxicities were observed; the recommended phase II dose was 600 mg/d flat dose. Signals of activity in cohort 1 (n = 27) included partial (n = 6) and complete (n = 1) response, including in O-methylguanine-DNA methyltransferase unmethylated and bevacizumab-refractory tumors. In cohort 2 (n = 15), median progression-free survival was 15 months and median overall survival was not reached (median follow-up, 28 months; 2-year overall survival, 62%). Gene sequencing disclosed a high rate of responses among EGFR-amplified tumors ( P = .005), with mechanisms of acquired resistance possibly involving mutations in mismatch-repair genes and/or downstream components TSC2, NF1, NF2, PTEN, and PIK3CA. Conclusion CTO can be combined safely with TMZ or chemoradiation in GBM and anaplastic gliomas, displaying favorable brain penetration and promising signals of activity in this difficult-to-treat population. 10.1200/JCO.2017.76.9992
    expression promotes resistance to alkylating chemotherapy in gliomas. Wu Longtao,Bernal Giovanna M,Cahill Kirk E,Pytel Peter,Fitzpatrick Carrie A,Mashek Heather,Weichselbaum Ralph R,Yamini Bakhtiar Science translational medicine The response of patients with gliomas to alkylating chemotherapy is heterogeneous. However, there are currently no universally accepted predictors of patient response to these agents. We identify the nuclear factor κB (NF-κB) co-regulator B cell CLL/lymphoma 3 (BCL-3) as an independent predictor of response to temozolomide (TMZ) treatment. In glioma patients with tumors that have a methylated -methylguanine DNA methyltransferase () promoter, high BCL-3 expression was associated with a poor response to TMZ. Mechanistically, BCL-3 promoted a more malignant phenotype by inducing an epithelial-to-mesenchymal transition in glioblastomas through promoter-specific NF-κB dimer exchange. Carbonic anhydrase II (CAII) was identified as a downstream factor promoting BCL-3-mediated resistance to chemotherapy. Experiments in glioma xenograft mouse models demonstrated that the CAII inhibitor acetazolamide enhanced survival of TMZ-treated animals. Our data suggest that BCL-3 might be a useful indicator of glioma response to alkylating chemotherapy and that acetazolamide might be repurposed as a chemosensitizer for treating TMZ-resistant gliomas. 10.1126/scitranslmed.aar2238
    Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma. Yin Jianxing,Zeng Ailiang,Zhang Zhuoran,Shi Zhumei,Yan Wei,You Yongping EBioMedicine BACKGROUND:Although temozolomide (TMZ) resistance is a significant clinical problem in glioblastoma (GBM), its underlying molecular mechanisms are poorly understood. In this study, we identified the role of exosomal microRNAs (miRNAs) from TMZ-resistant cells as important mediators of chemoresistance in GBM cells. METHODS:Exosomes were isolated from TMZ-resistant GBM cells and characterized via scanning electron microscopy (SEM). Expression levels of miR-1238 in GBM cell lines and their exosomes, clinical tissues, and sera were evaluated by RT-qPCR. In vitro and in vivo experiments were performed to elucidate the function of exosomal miR-1238 in TMZ resistance in GBM cells. Co-immunoprecipitation assays and western blot analysis were used to investigate the potential mechanisms of miR-1238/CAV1 that contribute to TMZ resistance. FINDINGS:MiR-1238 levels were higher in TMZ-resistant GBM cells and their exosomes than in sensitive cells. Higher levels of miR-1238 were found in the sera of GBM patients than in healthy people. The loss of miR-1238 may sensitize resistant GBM cells by directly targeting the CAV1/EGFR pathway. Furthermore, bioactive miR-1238 may be incorporated into the exosomes shed by TMZ-resistant cells and taken up by TMZ-sensitive cells, thus disseminating TMZ resistance. INTERPRETATION:Our findings establish that miR-1238 plays an important role in mediating the acquired chemoresistance of GBM and that exosomal miR-1238 may confer chemoresistance in the tumour microenvironment. These results suggest that circulating miR-1238 serves as a clinical biomarker and a promising therapeutic target for TMZ resistance in GBM. FUND: This study was supported by the National Natural Science Foundation of China (No·81402056, 81472362, and 81772951) and the National High Technology Research and Development Program of China (863) (No·2012AA02A508). 10.1016/j.ebiom.2019.03.016
    Genome-Wide CRISPR-Cas9 Screening Identifies NF-κB/E2F6 Responsible for EGFRvIII-Associated Temozolomide Resistance in Glioblastoma. Huang Kai,Liu Xing,Li Yansheng,Wang Qixue,Zhou Junhu,Wang Yunfei,Dong Feng,Yang Chao,Sun Zhiyan,Fang Chuan,Liu Chaoyong,Tan Yanli,Wu Xudong,Jiang Tao,Kang Chunsheng Advanced science (Weinheim, Baden-Wurttemberg, Germany) Amplification of epidermal growth factor receptor (EGFR) and active mutant EGFRvIII occurs frequently in glioblastoma (GBM) and contributes to chemo/radio-resistance in various cancers, especially in GBM. Elucidating the underlying molecular mechanism of temozolomide (TMZ) resistance in GBM could benefit cancer patients. A genome-wide screening under a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 library is conducted to identify the genes that confer resistance to TMZ in EGFRvIII-expressing GBM cells. Deep sgRNA sequencing reveals 191 candidate genes that are responsible for TMZ resistance in EGFRvIII-expressing GBM cells. Notably, E2F6 is proven to drive a TMZ resistance, and E2F6 expression is controlled by the EGFRvIII/AKT/NF-κB pathway. Furthermore, E2F6 is shown as a promising therapeutic target for TMZ resistance in orthotopic GBM cell line xenografts and GBM patient-derived xenografts models. After integrating clinical data with paired primary-recurrent RNA sequencing data from 134 GBM patients who received TMZ treatment after surgery, it has been revealed that the E2F6 expression level is a predictive marker for TMZ response. Therefore, the inhibition of E2F6 is a promising strategy to conquer TMZ resistance in GBM. 10.1002/advs.201900782
    A novel enhancer regulates MGMT expression and promotes temozolomide resistance in glioblastoma. Chen Xiaoyue,Zhang Minjie,Gan Haiyun,Wang Heping,Lee Jeong-Heon,Fang Dong,Kitange Gaspar J,He Lihong,Hu Zeng,Parney Ian F,Meyer Fredric B,Giannini Caterina,Sarkaria Jann N,Zhang Zhiguo Nature communications Temozolomide (TMZ) was used for the treatment of glioblastoma (GBM) for over a decade, but its treatment benefits are limited by acquired resistance, a process that remains incompletely understood. Here we report that an enhancer, located between the promoters of marker of proliferation Ki67 (MKI67) and O6-methylguanine-DNA-methyltransferase (MGMT) genes, is activated in TMZ-resistant patient-derived xenograft (PDX) lines and recurrent tumor samples. Activation of the enhancer correlates with increased MGMT expression, a major known mechanism for TMZ resistance. We show that forced activation of the enhancer in cell lines with low MGMT expression results in elevated MGMT expression. Deletion of this enhancer in cell lines with high MGMT expression leads to a dramatic reduction of MGMT and a lesser extent of Ki67 expression, increased TMZ sensitivity, and impaired proliferation. Together, these studies uncover a mechanism that regulates MGMT expression, confers TMZ resistance, and potentially regulates tumor proliferation. 10.1038/s41467-018-05373-4
    c-Myc-miR-29c-REV3L signalling pathway drives the acquisition of temozolomide resistance in glioblastoma. Luo Hui,Chen Zhengxin,Wang Shuai,Zhang Rui,Qiu Wenjin,Zhao Lin,Peng Chenghao,Xu Ran,Chen Wanghao,Wang Hong-Wei,Chen Yuanyuan,Yang Jingmin,Zhang Xiaotian,Zhang Shuyu,Chen Dan,Wu Wenting,Zhao Chunsheng,Cheng Gang,Jiang Tao,Lu Daru,You Yongping,Liu Ning,Wang Huibo Brain : a journal of neurology Resistance to temozolomide poses a major clinical challenge in glioblastoma multiforme treatment, and the mechanisms underlying the development of temozolomide resistance remain poorly understood. Enhanced DNA repair and mutagenesis can allow tumour cells to survive, contributing to resistance and tumour recurrence. Here, using recurrent temozolomide-refractory glioblastoma specimens, temozolomide-resistant cells, and resistant-xenograft models, we report that loss of miR-29c via c-Myc drives the acquisition of temozolomide resistance through enhancement of REV3L-mediated DNA repair and mutagenesis in glioblastoma. Importantly, disruption of c-Myc/miR-29c/REV3L signalling may have dual anticancer effects, sensitizing the resistant tumours to therapy as well as preventing the emergence of acquired temozolomide resistance. Our findings suggest a rationale for targeting the c-Myc/miR-29c/REV3L signalling pathway as a promising therapeutic approach for glioblastoma, even in recurrent, treatment-refractory settings. 10.1093/brain/awv287
    Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2. Yi Guo-Zhong,Huang Guanglong,Guo Manlan,Zhang Xi'an,Wang Hai,Deng Shengze,Li Yaomin,Xiang Wei,Chen Ziyang,Pan Jun,Li Zhiyong,Yu Lei,Lei Bingxi,Liu Yawei,Qi Songtao Brain : a journal of neurology The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma. 10.1093/brain/awz202