Anaplerotic nutrient stress drives synergy of angiogenesis inhibitors with therapeutics targeting tumor metabolism.

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as an induced vulnerability to therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells.

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.

Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5-15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer.

Spatial concordance of DNA methylation classification in diffuse glioma.

BACKGROUND: Intratumoral heterogeneity is a hallmark of diffuse gliomas. DNA methylation profiling is an emerging approach in the clinical classification of brain tumors. The goal of this study is to investigate the effects of intratumoral heterogeneity on classification confidence. METHODS: We used neuronavigation to acquire 133 image-guided and spatially separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma (7 IDH-wildtype and 2 IDH-mutant glioblastoma, 6 diffuse astrocytoma, IDH-mutant and 1 oligodendroglioma, IDH-mutant and 1p19q codeleted), which we characterized using DNA methylation arrays. Samples were obtained from regions with and without abnormalities on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery MRI. Methylation profiles were analyzed to devise a 3-dimensional reconstruction of (epi)genetic heterogeneity. Tumor purity was assessed from clonal methylation sites. RESULTS: Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and explains a substantial part of apparent epigenetic spatial heterogeneity. We observed that DNA methylation subtypes are often, but not always, conserved in space taking tumor purity and prediction accuracy into account. CONCLUSION: Our results underscore the infiltrative nature of diffuse gliomas and suggest that DNA methylation subtypes are relatively concordant in this tumor type, although some heterogeneity exists.

Glioblastoma in adults: a Society for Neuro-Oncology (SNO) and European Society of Neuro-Oncology (EANO) consensus review on current management and future directions.

Glioblastomas are the most common form of malignant primary brain tumor and an important cause of morbidity and mortality. In recent years there have been important advances in understanding the molecular pathogenesis and biology of these tumors, but this has not translated into significantly improved outcomes for patients. In this consensus review from the Society for Neuro-Oncology (SNO) and the European Association of Neuro-Oncology (EANO), the current management of isocitrate dehydrogenase wildtype (IDHwt) glioblastomas will be discussed. In addition, novel therapies such as targeted molecular therapies, agents targeting DNA damage response and metabolism, immunotherapies, and viral therapies will be reviewed, as well as the current challenges and future directions for research.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Molecular and clonal evolution in recurrent metastatic gliosarcoma.

We discuss the molecular evolution of gliosarcoma, a mesenchymal type of glioblastoma (GBM), using the case of a 37-yr-old woman who developed two recurrences and an extracranial metastasis. She was initially diagnosed with isocitrate dehydrogenase (IDH) wild-type gliosarcoma in the frontal lobe and treated with surgery followed by concurrent radiotherapy with temozolomide. Five months later the tumor recurred in the left frontal lobe, outside the initially resected area, and was treated with further surgery and radiotherapy. Six months later the patient developed a second left frontal recurrence and was again treated with surgery and radiotherapy. Six weeks later, further recurrence was observed in the brain and bone, and biopsy confirmed metastases in the pelvic bones. To understand the clonal relationships between the four tumor instances and the origin of metastasis, we performed whole-genome sequencing of the intracranial tumors and the tumor located in the right iliac bone. We compared their mutational and copy-number profiles and inferred the clonal phylogeny. The tumors harbored shared alterations in GBM driver genes, including mutations in TP53, NF1, and RB1, and CDKN2A deletion. Whole-genome doubling was identified in the first recurrence and the extracranial metastasis. Comparisons of the metastatic to intracranial tumors highlighted a high similarity in molecular profile but contrasting evidence regarding the origin of the metastasis. Subclonal reconstruction suggested a parallel evolution of the recurrent tumors, and that the metastatic tumor was largely derived from the first recurrence. We conclude that metastasis in glioma can be a late event in tumorigenesis.

Longitudinal molecular trajectories of diffuse glioma in adults.

The evolutionary processes that drive universal therapeutic resistance in adult patients with diffuse glioma remain unclear1,2. Here we analysed temporally separated DNA-sequencing data and matched clinical annotation from 222 adult patients with glioma. By analysing mutations and copy numbers across the three major subtypes of diffuse glioma, we found that driver genes detected at the initial stage of disease were retained at recurrence, whereas there was little evidence of recurrence-specific gene alterations. Treatment with alkylating agents resulted in a hypermutator phenotype at different rates across the glioma subtypes, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent gliomas and was characterized by IDH mutation but without co-deletion of chromosome arms 1p/19q, and further converged with acquired alterations in the cell cycle and poor outcomes. The clonal architecture of each tumour remained similar over time, but the presence of subclonal selection was associated with decreased survival. Finally, there were no differences in the levels of immunoediting between initial and recurrent gliomas. Collectively, our results suggest that the strongest selective pressures occur during early glioma development and that current therapies shape this evolution in a largely stochastic manner.

Evolving Insights into the Molecular Neuropathology of Diffuse Gliomas in Adults.

Recent advances in molecular analysis and genome sequencing have prompted a paradigm shift in neuropathology. This article discusses the discovery and clinical relevance of molecular biomarkers in diffuse gliomas in adults and how these biomarkers led to revision of the World Health Organization classification of these tumors. We relate progress in clinical classification to an overview of studies using molecular profiling to study gene expression and DNA methylation to categorize diffuse gliomas in adults and issues dealing with intratumoral heterogeneity. These efforts will refine the taxonomy of diffuse gliomas, facilitate selection of appropriate treatment regimens, and ultimately improve patient's lives.

The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations.

The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, we stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ∼11 kb TDs feature loss of TP53 and BRCA1. TDPs with ∼231 kb and ∼1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. We demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, we show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.

Reconstructing the molecular life history of gliomas.

At the time of their clinical manifestation, the heterogeneous group of adult and pediatric gliomas carries a wide range of diverse somatic genomic alterations, ranging from somatic single-nucleotide variants to structural chromosomal rearrangements. Somatic abnormalities may have functional consequences, such as a decrease, increase or change in mRNA transcripts, and cells pay a penalty for maintaining them. These abnormalities, therefore, must provide cells with a competitive advantage to become engrained into the glioma genome. Here, we propose a model of gliomagenesis consisting of the following five consecutive phases that glioma cells have traversed prior to clinical manifestation: (I) initial growth; (II) oncogene-induced senescence; (III) stressed growth; (IV) replicative senescence/crisis; (V) immortal growth. We have integrated the findings from a large number of studies in biology and (neuro)oncology and relate somatic alterations and other results discussed in these papers to each of these five phases. Understanding the story that each glioma tells at presentation may ultimately facilitate the design of novel, more effective therapeutic approaches.

Systematic analysis of telomere length and somatic alterations in 31 cancer types.

Cancer cells survive cellular crisis through telomere maintenance mechanisms. We report telomere lengths in 18,430 samples, including tumors and non-neoplastic samples, across 31 cancer types. Telomeres were shorter in tumors than in normal tissues and longer in sarcomas and gliomas than in other cancers. Among 6,835 cancers, 73% expressed telomerase reverse transcriptase (TERT), which was associated with TERT point mutations, rearrangements, DNA amplifications and transcript fusions and predictive of telomerase activity. TERT promoter methylation provided an additional deregulatory TERT expression mechanism. Five percent of cases, characterized by undetectable TERT expression and alterations in ATRX or DAXX, demonstrated elongated telomeres and increased telomeric repeat-containing RNA (TERRA). The remaining 22% of tumors neither expressed TERT nor harbored alterations in ATRX or DAXX. In this group, telomere length positively correlated with TP53 and RB1 mutations. Our analysis integrates TERT abnormalities, telomerase activity and genomic alterations with telomere length in cancer.

Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes.