bluuebunny/crossref_metadata_2025 huggingface dataset:
MGnify: the microbiome sequence data analysis resource in 2023
Authors: Richardson Lorna , Allen Ben , Baldi Germana , Beracochea Martin , Bileschi Maxwell L , Burdett Tony , Burgin Josephine , Caballero-Pérez Juan , Cochrane Guy , Colwell Lucy J , Curtis Tom , Escobar-Zepeda Alejandra , Gurbich Tatiana A , Kale Varsha , Korobeynikov Anton , Raj Shriya , Rogers Alexander B , Sakharova Ekaterina , Sanchez Santiago , Wilkinson Darren J , Finn Robert D
Published: December 2022

The MGnify platform (https://www.ebi.ac.uk/metagenomics) facilitates the assembly, analysis and archiving of microbiome-derived nucleic acid sequences. The platform provides access to taxonomic assignments and functional annotations for nearly half a million analyses covering metabarcoding, metatranscriptomic, and metagenomic datasets, which are derived from a wide range of different environments. Over the past 3 years, MGnify has not only grown in terms of the number of datasets contained but also increased the breadth of analyses provided, such as the analysis of long-read sequences. The MGnify protein database now exceeds 2.4 billion non-redundant sequences predicted from metagenomic assemblies. This collection is now organised into a relational database making it possible to understand the genomic context of the protein through navigation back to the source assembly and sample metadata, marking a major improvement. To extend beyond the functional annotations already provided in MGnify, we have applied deep learning-based annotation methods. The technology underlying MGnify's Application Programming Interface (API) and website has been upgraded, and we have enabled the ability to perform downstream analysis of the MGnify data through the introduction of a coupled Jupyter Lab environment.
The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation
Authors: Liu Wei , Shoji Keisuke , Naganuma Masahiro , Tomari Yukihide , Iwakawa Hiro-oki
Published: December 2022

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1–10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5′ nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5′ adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5′ A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.
TheEscherichia coliclamp loader rapidly remodels SSB on DNA to load clamps
Authors: Newcomb Elijah S P , Douma Lauren G , Morris Leslie A , Bloom Linda B
Published: December 2022

Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading.
Growth differentiation factor-15 predicts major bleeding, major adverse cardiac events and mortality in patients with end-stage kidney disease on haemodialysis: findings from the VIVALDI study
Authors: Nopp Stephan , Königsbrügge Oliver , Schmaldienst Sabine , Klauser-Braun Renate , Lorenz Matthias , Pabinger Ingrid , Säemann Marcus , Ay Cihan
Published: December 2022

Background

Patients with end-stage kidney disease (ESKD) are at high risk of cardiovascular events and bleeding. Optimizing risk assessment of ESKD patients regarding the risk of thromboembolism and bleeding complications in comorbid conditions, including atrial fibrillation and coronary heart disease, is challenging. To improve risk prediction we investigated growth differentiation factor-15 (GDF-15), a promising cardiovascular biomarker, and its relation to adverse outcomes.

Methods

In this prospective, multicentre, population-based cohort study, GDF-15 was measured in 594 ESKD patients on haemodialysis (median age 66 years, 38% female), who were followed up for a median of 3.5 years. The association of GDF-15 with major bleeding, arterial thromboembolism, major adverse cardiac events (MACE) and death was analysed within a competing risk framework. Further, we evaluated the additive predictive value of GDF-15 to cardiovascular and death risk assessment.

Results

GDF-15 levels were in median 5475 ng/l (25th–75th percentile 3964–7533) and independently associated with major bleeding {subdistribution hazard ratio [SHR] 1.31 per double increase [95% confidence interval (CI) 1.00–1.71]}, MACE [SHR 1.47 (95% CI 1.11–1.94)] and all-cause mortality [SHR 1.58 (95% CI 1.28–1.95)] but not arterial thromboembolism [SHR 0.91 (95% CI 0.61–1.36)]. The addition of GDF-15 to the HAS-BLED score significantly improved discrimination and calibration for predicting major bleeding [C-statistics increased from 0.61 (95% CI 0.52–0.70) to 0.68 (95% CI 0.61–0.78)]. Furthermore, we established an additive predictive value of GDF-15 beyond current risk models for predicting MACE and death.

Conclusion

GDF-15 predicts the risk of major bleeding, cardiovascular events and death in ESKD patients on haemodialysis and might be a valuable marker to guide treatment decisions in this challenging patient population.
Non-steroidal mineralocorticoid receptor antagonists and cardiorenal outcomes in chronic kidney disease
Authors: Lo Kevin Bryan , Rangaswami Janani , Vaduganathan Muthiah
Published: December 2022

The burden of adverse cardiorenal outcomes among patients with the trifecta of diabetes, heart failure (HF) and chronic kidney disease (CKD) remains high. Steroidal mineralocorticoid receptor antagonists (MRAs) have been shown to improve clinical outcomes in patients with HF, however, there is significant underutilization of these agents, especially in patients with advanced CKD. Non-steroidal MRAs are an emerging therapeutic option for patients with diabetic kidney disease and are now guideline-supported in this population. Non-steroidal MRAs have a unique pharmacological profile distinct from their steroidal counterparts that retains the class-specific cardiorenal benefits but may help mitigate adverse effects, especially hyperkalaemia, in patients with CKD. In this review we summarize the current evidence on the use of non-steroidal MRAs for improving cardiorenal outcomes in patients with CKD and diabetes, as well as for combination use alongside other foundational medical therapies used in HF and CKD.
ANGI-1 ASSESSING THE THERAPEUTIC EFFECT OF BEVACIZUMAB USING FMISO-PET IN GBM THERAPY
Authors: Suzuki Tomoya , Tanaka Toshihide , Takei Jun , Miyake Keisuke , Suzuki Kenta , Ogawa Daisuke , Akasaki Yasuharu , Yuichi Murayama
Published: December 2022

Background

Gd-enhanced MRI and T2WI/FLAIR images are used to determine the efficacy of bevacizumab(Bev) in glioblastoma(GBM). FMISO-PET reflects hypoxia in tumor tissue. In this study, we hypothesized that FMISO-PET could detect a series of changes in the hypoxic environment during the Bev effect and at the time of Bev recurrence, and compared images and pathological findings after initial Bev treatment and recurrence.

Subjects and Methods

Seven patients with first-episode GBM, IDH-wild type were included in the study. 3 patients underwent tumor resection after completion of Bev, radiation, and temozolomide treatment, and reoperation was performed at recurrence. FMISO-PET was performed at each time point. Four patients who underwent tumor resection after FMISO-PET in the absence of treatment were also included. The explanted specimens were analyzed for expression of hypoxia (CA9) and stem cell surface marker (FOXM1) by immunohistochemistry (IHC).

Results

All three patients treated with preoperative chemoradiotherapy showed reduced FMISO accumulation. Two of them showed recurrence and increased FMISO accumulation, and IHC showed increased CA9 and FOXM1 positive cells at the time of reoperation. There was a trend toward fewer CA9-positive cells in patients with low FMISO accumulation, including controls.

Conclusion

FMISO-PET was used to visualize the improvement of oxygenation in tumor tissues after preoperative chemoradiotherapy including Bev, and the increased FMISO accumulation at the time of Bev resistance suggested that FMISO-PET may be useful for monitoring the duration of Bev treatment effect by reflecting the oxygenation of tumor tissues. The results suggest that FMISO-PET may be useful for monitoring the duration of response to Bev therapy.
CBMS-1 TARGETING GLUTAMINE METABOLISM IN IDH-MUTANT GLIOMAS
Authors: Ohba Shigeo , Teranishi Takao , Hirayama Akiyoshi , Hitachi Keisuke , Yamaguchi Hisateru , Natsumeda Manabu , Tateishi Kensuke , Mukherjee Joydeep , Pieper Russell , Hirose Yuichi
Published: December 2022

IDH-wildtype and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. To reveal the difference in metabolites between IDH-wildtype and IDH-mutant glioma, two artificial cell lines made from normal human astrocyte, NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant), were investigated. Capillary electrophoresis- and ion chromatography-coupled mass spectrometry revealed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. Pharmacological or genetical inhibition of GLUD1 which converts glutamate to 2-oxoglutarate, suppressed proliferation of the cells by inducing ROS and apoptosis in NHAE6E7hTERTIDHmut cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells, revealing that cytotoxicity by GLUD1 inhibitor was at least partially due to the inhibitor-induced ROS. Exogeneous dimethyl 2-oxoglutarate rescued the cytotoxicity by GLUD1 inhibitor, suggesting decreased 2-oxoglutarate was associated with GLUD1 inhibitor-induced cytotoxicity. Mutant IDH1 overexpressed glioma cell line showed similar sensitivity to GLUD1 inhibitor to NHAE6E7hTERTIDHmut, which suggested that the difference of sensitivity to GLUD1 inhibitor was due to the status of mutant IDH. In conclusion, GLUD1 inhibitor will be new therapeutic options for IDH-mutant glioma.
CBMS-3 SEARCH FOR COMPOUNDS WITH ANTI-TUMOUR EFFECTS AGAINST GLIOBLASTOMA
Authors: Iwashimizu Sonoka , Ono Masaya , Sunagawa Yoichi , Hamabe Toshihide , Inai Kyoko , Katanasaka Yasufumi , Arakawa Yoshiki , Hasegawa Koji , Morimoto Tatsuya
Published: December 2022

Purpose

Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and the resistance to temozolomide, the standard treatment for GBM. Therefore, the development of new therapeutic agents for GBM is desired. We searched our compound library for compounds with anti-tumor activity against GBM and identified Curcumin (Cur) derivatives, Compound A and B. The purpose of this study was to investigate the antitumor activity of Compound A and B against GBM.

Methods and Results

To evaluate the antitumor activity of Compound A and B against GBM, we performed the MTT assay. Human glioblastoma cell lines, U87-MG and U251 cells, were treated with Compound A and B. After 96 hours, cell viability was evaluated using CCK-8, and the IC50 values of Compound A and B were calculated. In U87-MG, the IC50 of Cur was 9.78 μM, whereas the that of Compound A was 2.42 μM and that of Compound B was 1.28 μM. In U251 cells, the IC50 of Cur was 9.50 μM, whereas that of Compound A was 2.27 μM and that of Compound B was 0.64 μM. Next, to examine the effects of Compound A and B on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. At the concentrations at which antitumor effects were observed (Compound A; 3 μM, B; 1.5 μM), there was no reduction in cell viability in primary rat cultured astrocytes.

Discussion

The present study shows that Compound A and B exhibit antitumor activity against human glioblastoma cells at lower concentrations than Cur without affecting normal cells. We will examine the effects of Compound A and B in a mouse model of brain tumor transplanted with U87-RFP cells in the future, which may lead to the development of noble brain tumor therapeutics.
CBMS-4 CHROMOSOMAL INSTABILITY IN GLIOMA USING SPECTRAL KARYOTYPING METHOD
Authors: Negoto Tetsuya , Komaki Satoru , Hashimoto Aya , Yoshitake Hidenori , Moritsubo Mayuko , Furuta Takuya , Sakata Kiyohiko , Nakamura Hideo , Morioka Motohiro
Published: December 2022

Introduction

Chromosomal instability, the cell condition in which chromosome mis-segregation occur at a high frequency during cell division, has been considered to be involved in the molecular mechanisms that give rise to the complex genetic background of glioma. However, most of this phenomenon has been based on researches using cell line, and there have been few studies of chromosomal instability in clinical specimens of gliomas.

Methods

Primary cell culture was obtained from 11 glioma specimens (eight Glioblastoma (GBM), one Anaplastic PXA (aPXA), one Astrocytoma, and one Ependymoma), which was removed at our hospital, and chromosomes of up to five cells per case were analyzed by the SKY method. Chromosome instability was quantified by two parameters, one was AS (Aneuploidy score), which means the number of gain or loss of whole of the chromosome, and the other was SS: Structural abnormality score, which means the number of chromosome structural abnormality per cell. In addition, the phenotypes, which were characteristic of chromosomal instability were observed individually.

Results

Each quantitative value was as follows: GBM; AS:2.30±0.51 /SS:1.64±0.38, aPXA; AS :1.40±1.33 / SS:8.20±0.99, Astrocytoma and Ependymoma; AS:0.00 /SS:0.00, suggesting that chromosomal instability was associated with GBM and aPXA. Chromosome 7 amplification was most frequent in GBM (57%), and Mosaic loss of chromosome Y was also observed in 60% of males. Some characteristic karyotypes which suggest the phenomenon of Chromothripsis or Double minute were also observed. The karyotype concordance rate in the cases with p53 mutation was 60%, and that with p53 wild type was 100%, indicating that the p53 mutation increased the genotype heterogeneity in the same specimen.

Discussion

In clinical specimens of gliomas, aneuploidy and structural abnormalities were identified in GBM and aPXA, suggesting that chromosomal instability contributes to their cellular phenotype and malignancy.
CBMS-5 STEAROYL-COA DESATURASE INHIBITOR INDUCES APOPTOSIS VIA ENHANCING LIPOLYSIS IN IDH MUTANT GLIOMA
Authors: Zhang Lumin , Zhang Lumin , Dowdy Tyrone , Lita Adrian , Suzuki Ryoichi , Kida Satoru , Ooishi Tomoya , Koizumi Shinichiro , Kurozumi Kazuhiko , Larion Mioara
Published: December 2022

Background

Little is known about the antineoplastic effect and the mechanism of Stearoyl-CoA desaturase (SCD) inhibitor which catalyzes the biosynthesis of monounsaturated fatty acids (MUFA). Mutant isocitrate dehydrogenase (IDH) catalyzes the NADPH-mediated reduction of α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG) and causes metabolic reprograming of lipid. In this study, to develop a feasible drug for IDH mutant glioma, we have investigated the changes of the lipid distribution and the mechanism of antineoplastic effect of SCD inhibition in IDH mutant glioma.

Materials and Methods

We prepared genetically engineered glioma cell lines (U251 wild type: U251WT and U251 IDH mutant: U251mut) and patient derived cell lines (TS603 and GSC923). Lipid metabolic analysis was conducted by using Raman imaging spectroscopy and LC-MS, and functional analysis for the role of SCD expression in IDH mutant glioma was investigated by RNA sequence and Western-blotting. Results: In LC-MS analysis of the extracted Endoplasmic Reticulum, MUFAs were distributed significantly higher in IDH mutant than wild type. SCD expression was increased in IDH mutant compared to wild type due to 2HG-mediated upregulation of SCD. Therefore, IDH mutant in which SCD expression level was high indicated high sensitivity to SCD inhibitor, and apoptosis was highly induced in IDH mutant compared to wild type. RNA sequencing was performed in U251mut treated with SCD inhibitor compared to U251mut treated with DMSO, and lipid droplet metabolism-associated RNA expression was significantly changed in U251mut treated with SCD inhibitor. We checked lipid droplet in U251mut with presence or absence of SCD inhibitor, and lipolysis was induced by SCD inhibitor treatment, suggesting that SCD inhibition is associated with the apoptosis in IDH mutant via enhanced lipolysis mechanism.

Conclusions

2HG produced in IDH mutant glioma directly induces SCD expression and enhances sensitivity to SCD inhibitor, which suggests that SCD inhibitor is an IDH mutant glioma-specific treatment strategy.
CBMS-9 INFLUENCE OF HYPOXIA ON PHOTODYNAMIC THERAPY WITH 5-AMINOLEVULINIC ACID FOR MALIGNANT GLIOMA STEM CELLS
Authors: Nonoguchi Naosuke , Ihata Tomohiro , Omura Naoki , Wanibuchi Masahiko
Published: December 2022

Background

Glioblastoma is a highly malignant brain tumor refractory to standard treatment and its refractoriness is attributed to the presence of a small number of glioma stem cells (GSCs) that survive in a harsh microenvironment. The aim of this study was to investigate the effect of hypoxic conditions on the sensitivity of GSCs to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT).

Materials and Methods

Six human GSClines: three GSCs classified as Mesenchymal subtype and three GSCs classified as Proneural subtype, were divided into normoxia-GSCs (O2: 21%) and hypoxia-GSCs (O2: 5%) groups. To compare the effects of different oxygen partial pressures on protoporphyrin-IX (PpIX) biosynthetic activity, the expression levels of PpIX biosynthetic enzymes and transporters were examined by qRT-PCR and intracellular PpIX concentration was measured by flow cytometry. In addition, the sensitivity of these two cell groups to ALA-PDT was assessed in vitro.

Results

Hypoxia-GSCs showed higher mRNA levels of FECH (ferrochelatase), which is required for iron synthesis to convert PpIX to haem, compared to Normoxia-GSCs. Flow cytometry revealed that the accumulation of PpIX from exogenous ALA in Hypoxia-GSCs was reduced compared to in Normoxia-GSCs. Despite this, no Hypoxia-GSC lines showed significantly reduced sensitivity to ALA-PDT compared to Normoxia-GSCs.

Conclusion

Hypoxia-GSCs had lower intracellular PpIX accumulation than Normoxia-GSCs due to increased gene expression of FECH, and that their sensitivity to ALA- PDT was reduced less, despite accumulating lower concentrations of PpIX. ALA-PDT is at least a potentially effective therapy for hypoxia-tolerant GSCs that exist in hypoxia at 5% oxygen concentration.
CBMS-10 INFLUENCE OF HYPOXIA ON PHOTODYNAMIC THERAPY WITH 5-AMINOLEVULINIC ACID FOR MALIGNANT GLIOMA STEM CELLS
Authors: Nonoguchi Naosuke , Ihata Tomohiro , Omura Naoki , Wanibuchi Masahiko
Published: December 2022

Background

Glioblastoma is a highly malignant brain tumor refractory to standard treatment and its refractoriness is attributed to the presence of a small number of glioma stem cells (GSCs) that survive in a harsh microenvironment. The aim of this study was to investigate the effect of hypoxic conditions on the sensitivity of GSCs to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT).

Materials and Methods

Six human GSClines: three GSCs classified as Mesenchymal subtype and three GSCs classified as Proneural subtype, were divided into normoxia-GSCs (O2: 21%) and hypoxia-GSCs (O2: 5%) groups. To compare the effects of different oxygen partial pressures on protoporphyrin-IX (PpIX) biosynthetic activity, the expression levels of PpIX biosynthetic enzymes and transporters were examined by qRT-PCR and intracellular PpIX concentration was measured by flow cytometry. In addition, the sensitivity of these two cell groups to ALA-PDT was assessed in vitro.Results:Hypoxia-GSCs showed higher mRNA levels of FECH (ferrochelatase), which is required for iron synthesis to convert PpIX to haem, compared to Normoxia-GSCs. Flow cytometry revealed that the accumulation of PpIX from exogenous ALA in Hypoxia-GSCs was reduced compared to in Normoxia-GSCs. Despite this, no Hypoxia-GSC lines showed significantly reduced sensitivity to ALA-PDT compared to Normoxia-GSCs.Conclusion:Hypoxia-GSCs had lower intracellular PpIX accumulation than Normoxia-GSCs due to increased gene expression of FECH, and that their sensitivity to ALA- PDT was reduced less, despite accumulating lower concentrations of PpIX. ALA-PDT is at least a potentially effective therapy for hypoxia-tolerant GSCs that exist in hypoxia at 5% oxygen concentration.
SPDR-2 DEVELOPMENT OF A NOVEL THERAPEUTIC STRATEGY AGAINST MEDULLOBLASTOMA TARGETING MYC
Authors: Ito Hiroshi , Nakahara Yukiko , Namikawa Hiroki , Masuoka Jun , Abe Tatsuya
Published: December 2022

Medulloblastoma (MB) is the most common pediatric malignant brain tumor, with aggressive behavior and central nervous system dissemination. Standard of care for medulloblastoma include surgery, chemotherapy with platinum agent, and craniospinal radiation. If patients survived with these intensive treatments, frequently faced treatment-related adverse events. Therefore, development of novel therapeutic strategy is an important issue. Driver mutation underlying MB pathogenesis have been discovered, and molecular profiling led to the identification of subgroups; Wingless (WNT), Sonic Hedgehog(SHH), Group3, and Group4. Group 3, especially the group with MYC/MYCN amplification/overexpression confer a poor prognosis. Myc is attracting as a therapeutic target, however, to date, no drugs have been developed that target myc. We had found that N-myc downstream regulated gene-1 (NDRG1) protein expression is a good prognostic factor in glioblastoma, a representative of adult malignant brain tumors, and that Differentiation inducing factor-1 (DIF-1) increased NDRG1 expression. We reported that DIF-1 crossed the Blood-Brain Barrier and could be an effective treatment for GBM in vivo. In this research process, we found that DIF-1 markedly reduced Myc expression in patient-derived glioblastoma stem cells with MYC/MYCN amplification. Based on this result, we found that DIF-1 decreased Myc expression and inhibited cell survival/proliferation even in medulloblastoma cell lines with MYC/MYCN amplification.

In this presentation, we will present the results obtained in the research of a novel Myc-targeted therapy using DIF-1 for MB with MYC/MYCN amplification.
SPDR-4 SLFN11 RENDERS MEDULLOBLASTOMA SENSITIVE TO DNA DAMAGE CHEMOTHERAPIES
Authors: Nakata Satoshi , Natsumeda Manabu , Murai Junko , Okada Masayasu , Fujii Yukihiko
Published: December 2022

Background

Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma.

Methods

SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo.

Results

High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells.

Conclusions

High SLFN11 expression renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma through enhancing response to cisplatin.
GEN-1 ORIGINALLY DEVELOPED TUMOR SAMPLER TO REVEAL INTRATUMORAL HETEROGENEITY IN BRAIN TUMORS
Authors: Otani Ryohei , Yajima Hirohisa , Yamada Ryoji , Shinoura Nobusada
Published: December 2022

Intratumor heterogeneity has been analyzed in brain tumors, and it has revealed that clonal evolution results in the formation of genetically diverse cell populations, which is a major factor in treatment resistance and recurrence. However, intratumoral heterogeneity has not been analyzed in routine clinical practice and remains at the research level. Here, we developed a novel tumor sampler to establish the analysis of intratumoral heterogeneity as a routine clinical procedure and to clarify its clinical significance in brain tumors. Our novel tumor sampler consists of a hollow cylinder with a lid, which enabled us to obtain a cylindrical tumor sample cross-sectionally by inserting from the tumor surface to the depth, like the geological borehole test. The obtained cylindrical sample was divided vertically into two, one for pathological analysis and one for genetic analysis, which allowed us to compare genetic findings and pathological findings in arbitrary site of the sample. The sampling procedure was simple and completed in a short time, allowing comparison of heterogeneity among a large number of cases. We collected samples from four cases of Glioblastoma and three cases of brain metastasis using the sampler, and analyzed intratumoral heterogeneity by identifying somatic mutations at multiple sites by whole exome sequencing. We evaluated genetic and morphological heterogeneity among each site, generated a phylogenetic tree of clonal evolution within the tumor, and confirmed that these data could be compared among the cases. Using our novel tumor sampler could establish the analysis of intratumoral heterogeneity as a routine clinical procedure, and it expected to have a significant impact on treatment strategies for brain tumors.
GEN-8 COMPREHENSIVE WHOLE GENOME SEQUENCING ANALYSIS ELUCIDATES STRUCTURAL VARIANTS IN MEDULLOBLASTOMA
Authors: Funakoshi Yusuke , Nakashima Takuma , Uneda Atsuhito , Yoshimoto Koji , Narita Yoshitaka , Suzuki Hiromichi
Published: December 2022

Introduction

Medulloblastoma is the most common malignant pediatric brain tumor with a poor prognosis. International collaborative efforts for sequencing the genomes of medulloblastomas have delineated the landscape of coding mutations. However, little is yet known about structural variants (SVs). Here, we analyzed whole-genome sequencing (WGS) data of medulloblastoma to reveal the comprehensive genetic aberrations, including SVs.

Methods

We collected and analyzed publically available WGS data of 432 cases with medulloblastoma from MAGIC consortium, ICGC, and St Jude cloud.

Results

The median coverage is 36.4x with no difference among cohorts. The median number of SV per case is 1.0 (WNT), 5.0 (SHH), 6.0 (Group 3), and 5.0 (Group 4), respectively. SHH medulloblastoma with TP53 mutation has significantly a greater number of SVs (77.0/sample), suggesting that genome instability by TP53 mutations gives rise to SVs. At least one SV involved in already known driver genes in 10.9% of the SHH cases, supporting a model where SV could lead to tumor initiation. Since some of those SVs are caused by copy-neutral translocation, detecting those alterations is challenging by other than WGS. Complex SVs involving TERT focal amplification are identified in 11.5% of the SHHα subtype which usually does not have TERT promoter mutation. Although TERT promoter mutation is enriched predominantly in the SHHδ subtype, overexpression of TERT may be necessary for tumor maintenance in some of the SHHα cases. Unbalanced amplifications are commonly observed in known drive genes such as PPM1D, CCND2, and PVT1 where a part of exons are exclusively amplified, implying that functionally important regions are selectively altered by SVs which could promote tumor development.

Conclusions

SV affects not just copy number changes but also the structure of the genome, where breakpoints exist based on gene function and regulations. SVs along with genetic mutations contribute to medulloblastoma pathogenesis.
GEN-9 WHOLE GENOME SEQUENCING ANALYSIS REVEALS STRUCTURAL VARIANTS CONTRIBUTE TO TUMOR EVOLUTION IN IDH-MUTANT GLIOMA
Authors: Funakoshi Yusuke , Nakashima Takuma , Uneda Atsuhito , Nambu Shohei , Tanaka Shota , Ishida Joji , Saito Ryuta , Hanaya Ryosuke , Yoshimoto Koji , Narita Yoshitaka , Suzuki Hiromichi
Published: December 2022

Introduction

Recent large-scale sequencing projects for IDH-mutant adult-type diffuse glioma (Astrocytoma, IDH-mutant and Oligodendroglioma, IDH-mutant and 1p/19q-codeleted) have revealed the genetic landscape of coding mutations. However, little is yet known about the non-coding regions and structural variants (SVs). We analyzed deep (> x120) whole-genome sequencing (WGS) to delineate the comprehensive genetic lesions of IDH-mutant gliomas.

Methods

We investigated WGS of 228 cases with IDH-mutant glioma (204 cases in our cohort along with 24 publically available cases).

Results

The median tumor mutational burden in astrocytoma and oligodendroglioma was 2.0/Mb and 1.7/Mb, respectively. The median number of SV per case was 15.0 and 4.5, respectively. SV was involved in known driver genes in 7.7% of cases, supporting a model in which accumulation of SV as well as mutation drives tumor initiation and/or progression. The distribution of SV is biased on each chromosome, suggesting that each chromosome has a distinct susceptibility for SV. In IDH-mutant astrocytoma, complex SVs are significantly enriched on chromosome 12 which frequently involves CDK4, suggesting that SVs could lead to tumor evolution. The numbers of SVs and single nucleotide variants (SNVs) per case are correlated, presuming that IDH-mutant glioma can progress by acquiring both SNV and SV in a time-dependent manner.

Conclusion

SV could contribute to the development of IDH-mutant gliomas as well as mutations. Since WGS has a great resolution for genetic alterations, further analysis would enable us to uncover glioma pathogenesis.
GEN-10 WHOLE GENOME LANDSCAPE OF GLIOBLASTOMA, IDH-WILD TYPE
Authors: Nakashima Takuma , Funakoshi Yusuke , Uneda Atsuhito , Nambu Shohei , Tanaka Shota , Ishida Joji , Saito Ryuta , Hanaya Ryosuke , Yoshimoto Koji , Narita Yoshitaka , Suzuki Hiromichi
Published: December 2022

Introduction: Glioblastoma, IDH-wild type (GBM) is the most common malignant brain tumor with a dismal prognosis. Although its coding region is well-analyzed, little is yet known about the landscape of whole-genome in GBM. Here, we analyzed whole-genome sequencing data from large cohorts to completely uncover the genetic aberrations in GBM. Methods: We analyzed 281 whole-genome sequencing data of patients with GBM, of which 152 cases are from our cohort with deep coverage (×120) and 129 cases are from a public database. Results: The median numbers of somatic mutations and structural variants (SVs) per case are 3.0/Mb and 62.5, respectively. While a complex SV is rare in other malignant brain tumors such as IDH-mutant glioma (35% of samples), a large proportion of GBM cases (85%) have complex SV with ≥10 breakpoints. CDKN2A/B homozygous deletions (HDs) are usually comprised of a simple deletion in IDH-mutant glioma whereas about a quarter of CDKN2A/B HDs in GBM are induced by complex SVs. In addition, 30.5% of extrachromosomal DNA (ecDNA) involves multiple chromosomes. Taken together, complex SVs could play a key role in the initiation and progression of GBM. Our deep WGS enables us to delineate a fine view of clonal architecture, where mutational signature varies between clonal and subclonal mutations. The majority of clonal mutations consist of the clock-like signature, whereas subclonal mutations have a relatively low proportion of the clock-like signature. Instead, several other signatures including the APOBEC signature significantly increase in subclones, presuming that various mutational processes along with the clock-like signature contribute to the GBM pathogenesis in its progression phase. Conclusions: GBM evolves through exploiting complex structural variants involving multiple driver genes and the accumulation of genetic mutations caused by distinct mechanisms depending on its developmental stage.
ET-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS
Authors: Tamura Ryota , Yo Masahiro , Miyoshi Hiroyuki , Sampetrean Oltea , Saya Hideyuki , Okano Hideyuki , Toda Masahiro
Published: December 2022

Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.
ET-4 BASIC RESEARCH OF BORON NEUTRON CAPTURE THERAPY USING A NOVEL BORON COMPOUND TARGETED TO INTEGRIN
Authors: Tsujino Kohei , Kawabata Shinji , Kashiwagi Hideki , Yoshimura Kohei , Kayama Ryo , Fukuo Yusuke , Kanemitsu Takuya , Hiramatsu Ryo , Hu Naonori , Miyatake Shin-Ichi , Nishimura Kai , Takata Takushi , Tanaka Hiroki , Suzuki Minoru , Nakamura Hiroyuki , Wanibuchi Masahiko
Published: December 2022

Background

Boron neutron capture therapy (BNCT) is a particle radiation modality capable of selectively destroying tumor cells. The most commonly used boron compound for BNCT is boronphenylalanine (BPA). BPA is taken up into the tumor cell via the L-type aminoacid transporter (LAT-1). However, there are some BPA-refractory situations. Therefore, a novel boron compound is expected to improve the therapeutic performance of BNCT. We focused on integrinαvβ3, which is overexpressed in malignant gliomas as in many cancer cells, and have developed cRGD-MID-AC, a conjugate of cyclic RGD (cRGD), which selectively inhibited integrinαvβ3, and MID-AC, which we have already reported as effective on BNCT as BPA as a boron compound in F98 rat glioma models. We evaluated the efficacy of BNCT using this novel compound.Methods: F98 glioma cells were exposed to BPA, cRGD-MID-AC, and cRGD-MID for cellular uptake and neutron irradiation experiment. Intracellular boron concentrations and compound biological effectiveness (CBE) for each boron compound was calculated. After intravenous administration (i.v.) of cRGD-MID-AC or BPA, the biodistribution of boron compounds was measured and neutron irradiation experiment were performed in F98 rat glioma models.

Results

Intracellular boron concentrations of BPA and cRGD-MID-AC were increased gradually at all exposed time, and CBE for cRGD-MID-AC was comparable to that for BPA. In cRGD-MID-AC, the boron concentration in the tumor was the highest at 8 h after i.v. and tended to be retained longer at 24h. In vivo neutron irradiation experiment, long-term survival was observed only in the group irradiated 8 h after cRGD-MID-AC i.v.. These experiments suggest that cRGD-MID-AC has sufficient cell-killing effect and may be more effective in vivo.

Conclusion

cRGD-MID-AC has a tumor accumulation mechanism different from that of BPA, and could be an effective boron carrier in BNCT for malignant gliomas.
ET-5 POTENT BYSTANDER EFFECT IN SUICIDE GENE THERAPY USING TK-EXPRESSING STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH
Authors: Koizumi Shinichiro , Horikawa Makoto , Yamamoto Taisuke , Oishi Tomoya , Yamasaki Tomohiro , Kida Satoru , Namba Hiroki , Kurozumi Kazuhiko
Published: December 2022

Introduction

We investigated HSVTK/GCV suicide gene therapy for malignant glioma, and demonstrated the migration ability and antitumor effect of various tissue-derived pluripotent stem cells. In recent years, stem cells from human exfoliated deciduous teeth (SHED), which have excellent ethical and self-renewal ability, have attracted attention, especially in regenerative medicine. In this study, using SHEDTK transfected with TK, we examined the migration ability and antitumor effect against malignant glioma and metastasis models.

Methods

In vitro assay: Using Matrigel chamber, the migration ability of SHEDTK to conditioned medium (CM) of glioma cells, lung carcinoma cells, and various tumor growth factors (TGF) was examined. The antitumor effect was examined for cell viability by co-culturing SHEDTK and each tumor cell under the addition of GCV. in vivo assay: Using glioma model mice, SHEDTK migrating around the tumor was confirmed in brain sections. In addition, we co-implanted SHEDTK and administered GCV to glioma and metastasis model as therapeutic models, evaluated tumor reduction effect by bioluminescence, and confirmed survival curves.

Results

In vitro assay: SHEDTK significantly migrated to CM and TGF compared to control. The antitumor effect was observed even when the ratio of each tumor cell to SHEDTK was 128-256 times. in vivo assay: In glioma model mice, SHEDTK migrated around contralateral tumor. In the treatment experiment, the control group died of tumors, whereas the tumor in the treatment group disappeared within the period of GCV administration, and they survived without neurological deficits until 100 days later, and the overall survival time was improved with a statistically significant difference.

Conclusions

We confirmed the migration ability and antitumor effect of SHEDTK on malignant glioma and metastasis models. Suicide gene therapy using SHEDTK was suggested as novel gene therapy. In the future, we plan to conduct translational research such as preclinical studies and physician -initiated clinical trials.
ET-6 EFFICACY OF NEAR-INFRARED PHOTOIMMUNOTHERAPY TARGETING PODOPLANIN IN MALIGNANT GLIOMAS
Authors: Natsumeda Manabu , On Jotaro , Watanabe Jun , Ando Kazuhiro , Tsukamoto Yoshihiro , Okada Masayasu , Ogura Ryosuke , Oishi Makoto , Fujii Yukihiko
Published: December 2022

Background

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment based on a conjugate of photosensitizer IR700 and an antibody which selectively binds to the surface antigen of cancer cells. Upon exposure to near infrared light, which does not harm normal tissues, cancer cells are selectively killed. Clinical application for EGFR amplified head and neck cancer has already been realized but is still in the preclinical stages of research for brain tumors. Finding a surface antigen which is selective to cancer cells is key to the effective application of NIR-PIT. In IDH-wildtype gliomas, we found that podoplanin (PDPN) is a promising candidate. PDPN is a transmembrane sialoglycoprotein highly expressed in IDH-wildtype gliomas and other cancers.

Methods

Three PDPN-positive (NGT-11, NGT-41, LN319) and three PDPN-negative (T98G, U87MG, U251MG) glioblastoma cell lines were treated with PDPN-IR700 conjugate and exposed to near infrared light. Cytotoxicity was assessed by cell viability assays. Next, we treated subcutaneous mouse xenografts by NIR-PIT.

Results

Cytotoxicity was only observed after NIR-PIT using PDPN-conjugate in cell lines expressing PDPN. This cytotoxicity was dose-dependent to near infrared light exposure, and most pronounced in LN319 cell line, which expressed the highest levels of PDPN. Time lapse videos revealed instantaneous expanding, followed by rounding of dead cells after treatment. NIR-PIT treated subcutaneous xenografts showed significantly smaller tumor sizes compared to control, and pathological investigation revealed selective and extensive necrosis of tumor tissue.

Discussion/conclusion

PDPN is highly expressed in IDH-wildtype gliomas, and we found that NIR-PIT was highly effective in treating PDPN expressing glioblastoma cell lines. NIR-PIT is anticipated to be more selective and cytotoxic than photodynamic therapy and is a promising new treatment strategy for IDH-wildtype gliomas.
TB-1 ADDITIONAL GENETIC ALTERATIONS DIFFERENTIALLY ALTER THE TRANSCRIPTOMIC LANDSCAPE OF H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA
Authors: Miki Shunichiro , Koga Tomoyuki , Skinner Kasey R , Gruener Robert F , Kawauchi Daisuke , Huang R Stephanie , Miller C Ryan , Furnari Frank
Published: December 2022

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.
TB-3 A GINGER EXTRACT COMPOUND A REVEALED ANTITUMOR ACTIVITY AGAINST GLIOMA
Authors: Inai Kyoko , Ono Masaya , Iwashimizu Sonoka , Hamabe-Horiike Toshihide , Sunagawa Yoichi , Katanasaka Yasufumi , Arakawa Yoshiki , Hasegawa Koji , Morimoto Tatsuta
Published: December 2022

Purpose

Malignant neoplasms arising in the brain and central nervous system have a poor prognosis and present with symptoms such as headache, epileptic seizures, and paralysis of the arms and legs. Treatment of glioma, the most frequent primary brain tumor, is based on surgical removal of the tumor, radiation therapy, and chemotherapy. However, since gliomas grow invasively, surgical removal of the entire tumor is considered difficult. In addition, the standard treatment, temozolomide, possesses the problem of resistance. Therefore, the risk of recurrence is high, and the development of noble therapeutic agents for Glioma is required. We have screened for compounds with anti-tumor activity against Glioma from our natural compound library and focused on ginger extract, Compound A. In this study, we investigated the effect of Compound A on Glioma cell lines.

Methods and Results

To evaluate the antitumor activity of Compound A on Glioma in vitro , MTT assay was performed. Human glioma cell lines U87-MG and U251 cells were treated with Compound A. After 96 hours, cell viability was evaluated using CCK-8. The IC50 value of Compound A in U87-MG cells was calculated to be 16.3 μM. The IC50 of Compound A in U251 cells was 10.8 μM. Next, to examine the effect of Compound A on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. The MTT assay showed no decrease in cell viability in primary cultured rat astrocytes at 15 μM, whereas an antitumor effect was observed in a human glioma cell line.

Discussion

In this study, Compound A showed antitumor activity against Glioma without affecting normal cells. In the future, we will examine the effects of Compound A in an immunocompromised mouse brain in which U87-MG-RFP cells are implanted, which may lead to the development of therapeutic agents against Glioma.