bims-malgli Biomed News
on Biology of malignant gliomas
Issue of 2022‒05‒08
fourteen papers selected by
Oltea Sampetrean
Keio University

  1. Brain Tumor Pathol. 2022 May 05.
      The characteristic features of plasticity and heterogeneity in glioblastoma (GB) cells cause therapeutic difficulties. GB cells are exposed to various stimuli from the tumor microenvironment and acquire the potential to resist chemoradiotherapy. To investigate how GB cells acquire stem cell-like phenotypes, we focused on ribosomal proteins, because ribosome incorporation has been reported to induce stem cell-like phenotypes in somatic cells. Furthermore, dysregulation of ribosome biogenesis has been reported in several types of cancer. We focused on ribosomal protein S6, which promotes sphere-forming ability and stem cell marker expression in GB cells. We expect that investigation of dysregulation of ribosome biogenesis and extra-ribosomal function in GB will provide new insights about the plasticity, heterogeneity, and therapeutic resistance of GB cells, which can potentially lead to revolutionary therapeutic strategies.
    Keywords:  Glioblastoma; Glioma stem cells; Plasticity; Ribosomal protein S6; Ribosome
  2. STAR Protoc. 2022 Jun 17. 3(2): 101331
      Glioblastoma (GBM) cells invade the brain by following linear structures like blood vessel walls and white matter tracts by using specific motility modes. In this protocol, we describe two micropatterning techniques allowing recapitulation of these linear tracks in vitro: micro-contact printing and deep UV photolithography. We also detail how to maintain, transfect, and prepare human glioma propagating cells (hGPCs) for migration assays on linear tracks, followed by image acquisition and analysis, to measure key parameters of their motility. For complete details on the use and execution of this protocol, please refer to Monzo et al. (2016) and Monzo et al. (2021a).
    Keywords:  Biophysics; Biotechnology and bioengineering; Cancer; Cell Biology; Cell culture; Cell-based Assays; Microscopy
  3. Neuro Oncol. 2022 May 02. pii: noac108. [Epub ahead of print]
      BACKGROUND: Seizures can present at any time before or after the diagnosis of a glioma. Roughly, 25-30 % of glioblastoma (GBM) patients initially present with seizures, and an additional 30 % develop seizures during the course of the disease. Early studies failed to show an effect of general administration of anti-epileptic drugs for glioblastoma patients, since they were unable to stratify patients into high- or low-risk seizure groups.METHODS: 111 patients, who underwent surgery for a GBM, were included. Genome-wide DNA methylation profiling was performed, before methylation subclasses and copy number changes inferred from methylation data were correlated with clinical characteristics. Independently, global gene expression was analyzed in GBM methylation subclasses from TCGA datasets (n=68).
    RESULTS: Receptor tyrosine Kinase (RTK) II GBM showed a significantly higher incidence of seizures than RTK I and mesenchymal (MES) GBM (p<0.01). Accordingly, RNA expression datasets revealed an upregulation of genes involved in neurotransmitter synapses and vesicle transport in RTK II glioblastomas. In a multivariate analysis, temporal location (p=0.02, OR 5.69) and RTK II (p=0.03, OR 5.01) were most predictive for preoperative seizures. During postoperative follow-up, only RTK II remained significantly associated with the development of seizures (p<0.01, OR 8.23). Consequently, the need for antiepileptic medication and its increase due to treatment failure was highly associated with the RTK II methylation subclass (p<0.01).
    CONCLUSION: Our study shows a strong correlation of RTK II glioblastomas with preoperative and long-term seizures. These results underline the benefit of molecular glioblastoma profiling with important implications for postoperative seizure control.
    Keywords:  Glioma; Glioma-related seizures; Methylation; RTKII
  4. Glia. 2022 May 07.
      Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.
    Keywords:  H3K27M; diffuse intrinsic pontine glioma; diffuse midline glioma; oligodendrocyte progenitor cells
  5. Cancer Res. 2022 May 02. pii: canres.3868.2021. [Epub ahead of print]
      Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by re-expression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKAs), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, co-treatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable.
  6. Neuro Oncol. 2022 May 02. pii: noac116. [Epub ahead of print]
      BACKGROUND: Nearly all patients with newly diagnosed glioblastoma experience recurrence following standard-of-care radiotherapy (RT) + temozolomide (TMZ). The purpose of the phase 3 randomized CheckMate 548 study was to evaluate RT+TMZ combined with the immune checkpoint inhibitor nivolumab (NIVO) or placebo (PBO) in patients with newly diagnosed glioblastoma with methylated MGMT promoter (NCT02667587).METHODS: Patients (N=716) were randomized 1:1 to NIVO [(240 mg every 2 weeks ×8, then 480 mg every 4 weeks) + RT (60 Gy over 6 weeks) + TMZ (75 mg/m 2 once daily during RT, then 150-200 mg/m 2 once daily days 1-5 of every 28-day cycle ×6)] or PBO+RT+TMZ following the same regimen. The primary endpoints were progression-free survival (PFS) and overall survival (OS) in patients without baseline corticosteroids and in all randomized patients.
    RESULTS: As of December 22, 2020, median (m)PFS (blinded independent central review) was 10.6 months (95% CI, 8.9-11.8) with NIVO+RT+TMZ vs 10.3 months (95% CI, 9.7-12.5) with PBO+RT+TMZ (HR, 1.1; 95% CI, 0.9-1.3) and mOS was 28.9 months (95% CI, 24.4-31.6) vs 32.1 months (95% CI, 29.4-33.8), respectively (HR, 1.1; 95% CI, 0.9-1.3). In patients without baseline corticosteroids, mOS was 31.3 months (95% CI, 28.6-34.8) with NIVO+RT+TMZ vs 33.0 months (95% CI, 31.0-35.1) with PBO+RT+TMZ (HR, 1.1; 95% CI, 0.9-1.4). Grade 3/4 treatment-related adverse event rates were 52.4% vs 33.6%, respectively.
    CONCLUSIONS: NIVO added to RT+TMZ did not improve survival in patients with newly diagnosed glioblastoma with methylated or indeterminate MGMT promoter. No new safety signals were observed.
    Keywords:   MGMT promoter; PD-L1; glioblastoma; nivolumab; temozolomide
  7. Cell Discov. 2022 Apr 30. 8(1): 39
      The entity of DNA N6-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N6-methyladenine (m6A) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of m6A nucleoside. The released m6A nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.
  8. Genomics. 2022 May 02. pii: S0888-7543(22)00123-9. [Epub ahead of print] 110378
      Glioblastoma (GBM) is one of the most malignant and intractable central nervous system tumors with high recurrence, low survival rate, and poor prognosis. Despite the advances of aggressive, multimodal treatment, a successful treatment strategy is still elusive, often leading to therapeutic resistance and fatality. Thus, it is imperative to search for and identify novel markers critically associated with GBM pathogenesis to improve the existing trend of diagnosis, prognosis, and treatment. Seven publicly available GEO microarray datasets containing 409 GBM samples were integrated and further data mining was conducted using several bioinformatics tools. A total of 209 differentially expressed genes (DEGs) were identified in the GBM tissue samples compared to the normal brains. Gene Ontology (GO) enrichment analysis of the DEGs revealed association of the upregulates genes with extracellular matrix (ECM), conceivably contributing to the invasive nature of GBM while downregulated DEGs were found to be predominantly related to neuronal processes and structures. Alongside, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway analyses described the involvement of the DEGs with various crucial contributing pathways (PI3K-Akt signaling pathway, p53 signaling pathway, insulin secretion, etc.) in GBM progression and pathogenesis. Protein-protein interaction (PPI) network containing 879 nodes and 1237 edges revealed 3 significant modules and consecutive KEGG pathway analysis of these modules showed a significant connection to gliomagenesis. Later, 10 hub genes were screened out based on degree and their expressions were externally validated. Surprisingly, only fibronectin 1 (FN1) high expression appeared to be related to poor prognosis. Subsequently, 109 transcription factors and 211 miRNAs were detected to be involved with the hub genes where FN1 demonstrated the highest number of interactions. Considering its high connectivity and potential prognostic value FN1 could be a novel biomarker providing new insights into the prognosis and treatment for GBM, although experimental validation is required.
    Keywords:  Bioinformatics; Differentially expressed genes; Fibronectin 1; Glioblastoma multiforme; Prognosis
  9. Clin Cancer Res. 2022 May 05. pii: clincanres.2830.2021. [Epub ahead of print]
      PURPOSE: The immunosuppressive tumor microenvironment present in the majority of diffuse glioma limits therapeutic response to immunotherapy. As the determinants of the glioma-associated immune response are relatively poorly understood, the study of glioma with more robust tumor-associated immune responses may be particularly useful to identify novel immunomodulatory factors that can promote T cell effector function in glioma.EXPERIMENTAL DESIGN: We used multiplex immune-profiling, proteomic profiling, and gene expression analysis to define the tumor-associated immune response in two molecular subtypes of glioma and identify factors that may modulate this response. We then used patient-derived glioma cultures and an immunocompetent murine model for malignant glioma to analyze the ability of tumor-intrinsic factors to promote a CD8+ T cell response.
    RESULTS: As compared with IDH-mutant astrocytoma, MAPK-activated pleomorphic xanthoastrocytoma (PXA) harbored increased numbers of activated cytotoxic CD8+ T cells and Iba1+ microglia/macrophages, increased MHC class I expression, enrichment of genes associated with antigen presentation and processing, and increased tumor cell secretion of the chemokine CXCL14. CXCL14 promoted activated CD8+ T cell chemotaxis in vitro, recruited tumor-infiltrating CD8+ T cells in vivo, and prolonged overall survival in a cytotoxic T cell-dependent manner. The immunomodulatory molecule B7-H3 was also highly expressed in PXA.
    CONCLUSIONS: We identify the MAPK-activated lower grade astrocytoma PXA, as having an immune-rich tumor microenvironment and suggest this tumor may be particularly vulnerable to immunotherapeutic modulation. We also identify CXCL14 as an important determinant of the glioma-associated immune microenvironment, sufficient to promote an anti-tumor CD8+ T cell response.
  10. Cancer Immunol Res. 2022 May 04. pii: canimm.CIR-21-1050-E.2021. [Epub ahead of print]
      Glioblastoma (GBM) is an immunologically "cold" tumor characterized by poor responsiveness to immunotherapy. Standard-of-care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g. mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex immunohistochemistry. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo and rGBM and identify perivascular T cells as potential therapeutic targets.
  11. Stem Cell Reports. 2022 Apr 25. pii: S2213-6711(22)00196-5. [Epub ahead of print]
      The insulin receptor (INSR) is an evolutionarily conserved signaling protein that regulates development and cellular metabolism. INSR signaling promotes neurogenesis in Drosophila; however, a specific role for the INSR in maintaining adult neural stem cells (NSCs) in mammals has not been investigated. We show that conditionally deleting the Insr gene in adult mouse NSCs reduces subventricular zone NSCs by ∼70% accompanied by a corresponding increase in progenitors. Insr deletion also produced hyposmia caused by aberrant olfactory bulb neurogenesis. Interestingly, hippocampal neurogenesis and hippocampal-dependent behaviors were unperturbed. Highly aggressive proneural and mesenchymal glioblastomas had high INSR/insulin-like growth factor (IGF) pathway gene expression, and isolated glioma stem cells had an aberrantly high ratio of INSR:IGF type 1 receptor. Moreover, INSR knockdown inhibited GBM tumorsphere growth. Altogether, these data demonstrate that the INSR is essential for a subset of normal NSCs, as well as for brain tumor stem cell self-renewal.
    Keywords:  IGF-II; cancer stem cell; glioblastomas; insulin receptor; insulin-like growth factors; mouse; stem cell niche; subgranular zone; subventricular zone
  12. Neuro Oncol. 2022 May 03. pii: noac111. [Epub ahead of print]
      BACKGROUND: IDH-mutant gliomas are separate based on the codeletion of the chromosomal arms 1p and 19q into oligodendrogliomas IDH-mutant 1p/19q-codeleted and astrocytomas IDH-mutant. While nuclear loss of ATRX expression excludes 1p/19q codeletion, its limited sensitivity prohibits to conclude on 1p/19q status in tumors with retained nuclear ATRX expression.METHODS: Employing mass spectrometry based proteomic analysis in a discovery series containing 35 fresh frozen and 72 formalin fixed and paraffin embedded tumors with established IDH and 1p/19q status, potential biomarkers were discovered. Subsequent validation immunohistochemistry was conducted on two independent series (together 77 oligodendrogliomas IDH-mutant 1p/19q-codeleted and 92 astrocytomas IDH-mutant.
    RESULTS: We detected highly specific protein patterns distinguishing oligodendroglioma and astrocytoma. In these patterns high HIP1R and low vimentin levels were observed in oligodendroglioma while low HIP1R and high vimentin levels occurred in astrocytoma. Immunohistochemistry for HIP1R and vimentin expression in 35 cases from the FFPE discovery series confirmed these findings. Blinded evaluation of the validation cohorts predicted the 1p/19q status with a positive and negative predictive value as well as an accuracy of 100% in the first cohort and with a positive predictive value of 83%; negative predictive value of 100% and an accuracy of 92% in the second cohort. Nuclear ATRX loss as marker for astrocytoma increased the sensitivity to 96% and the specificity to 100%.
    CONCLUSIONS: We demonstrate that immunohistochemistry for HIP1R, vimentin and ATRX predict 1p/19q status with 100% specificity and 95% sensitivity and therefore, constitutes a simple and inexpensive approach to the classification of IDH-mutant glioma.
    Keywords:  1p/19q; ATRX; HIP1R; IDH; Vimentin; proteomics
  13. Clin Cancer Res. 2022 05 02. pii: clincanres.4053.2021. [Epub ahead of print]
      PURPOSE: Despite dramatic growth in the number of small molecule drugs developed to treat solid tumors, durable therapeutic options to control primary central nervous system malignancies are relatively scarce. Chemotherapeutic agents which appear biologically potent in model systems have often been found to be marginally effective at best when given systemically in clinical trials. This work presents for the first time an ultrasmall (&lt; 8 nm) multimodal core-shell silica nanoparticle, Cornell prime dots (or C' dots), for the efficacious treatment of high-grade gliomas.EXPERIMENTAL DESIGN: This work presents first-in-kind renally-clearable ultrasmall (&lt; 8 nm) multimodal Cornell prime dots (or C' dots) with surface-conjugated doxorubicin via pH-sensitive linkers for the efficacious treatment in two different clinically relevant high-grade glioma models.
    RESULTS: Optimal drug-per-particle ratios of as-developed nanoparticle-drug conjugates were established and used to obtain favorable pharmacokinetic profiles. The in vivo efficacy results showed significantly improved biological, therapeutic, and toxicological properties over the native drug after intravenous administration in platelet-derived growth factor-driven genetically engineered mouse model, and an epidermal growth factor expressing patient-derived xenograft (EGFR PDX) model.
    CONCLUSIONS: Ultrasmall C' dot-drug conjugates showed great translational potential over doxorubicin for improving the therapeutic outcome of patients with high-grade gliomas, even without a cancer-targeting moiety.