Deep learning-based feature discovery for decoding phenotypic plasticity in pediatric high-grade gliomas single-cell transcriptomics.

303 ISSN 2045-0907 10.2217/CNS.13.21 © 2013 Future Medicine Ltd CNS Oncol. (2013) 2(4), 303–306 High-grade gliomas (HGGs) are categorized by WHO as grade III–IV glial malignancies and account for roughly 15–20% of all pediatric CNS tumors [1]. For pediatric patients diagnosed with supratentorial HGGs, the 2-year survival rates range from 10 to 30%. The prognosis for patients with diffuse brainstem gliomas is even more dismal, as less than 10% will survive more than 2 years after diagnosis [1]. The current treatment for newly diagnosed pediatric HGGs is safe maximal surgical resection and radiotherapy. The addition of chemotherapy to this regimen is debatable, reflecting the lack of consensus among pediatric neuro-oncologists regarding the optimal protocol/treatment plan for newly diagnosed patients [2]. Despite these efforts, marginal impact has been made on the overall survival rates of these patients. This begs the question: ‘how do we change the way in which we view and treat pediatric HGGs?’ To fully understand the complexities of pediatric HGGs and subsequently identify therapeutic vulnerabilities, we must first understand the full molecular, cellular and physiological context of these malignancies. While it has long been thought that both pediatric and adult gliomas are the same disease affecting different patient populations, recently, multiple groups including the Canadian Paediatric Cancer Genome Consortium and the St Jude Children’s Research Hospital–Washington University in St Louis Pediatric Cancer Genome Project have challenged this notion by revealing striking differences in the mutation spectrum and frequencies between pediatric and adult HGGs [3–11]. The use of high-resolution genomic approaches has uncovered potential therapeutic targets for pediatric gliomas such as PDGFRA, MET and IGF1R (recurrent focal amplification in HGGs and pontine gliomas) [7,10,12], BRAF and CDK2NA (BRAF-V600E mutations with concomitant CDKN2A loss in a subset of malignant astrocytomas) [11] and FGFR1 (intragenic duplications of the tyrosine kinase domain in low-grade gliomas) [3]. Alone, the identification and characterization of the somatic DNA alterations is insufficient. Pediatric HGGs harbor

Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq, and multiplexed immunofluorescence staining. The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Tumor-associated macrophages (TAMs) residing in the tumor microenvironment (TME) are characterized by their pivotal roles in tumor progression, antitumor immunity, and TME remodeling. However, a thorough comparative characterization of tumor-TAM crosstalk across IDH-defined categories of glioma remains elusive, likely contributing to mixed outcomes in clinical trials. We delineated the phenotypic heterogeneity of TAMs across IDH-stratified gliomas. Notably, two TAM subsets with a mesenchymal phenotype were enriched in IDH-WT glioblastoma (GBM) and correlated with poorer patient survival and reduced response to anti-PD-1 immune checkpoint inhibitor (ICI). We proposed SLAMF9 receptor as a potential therapeutic target. Inference of gene regulatory networks identified PPARG, ELK1, and MXI1 as master transcription factors of mesenchymal BMD-TAMs. Our analyses of reciprocal tumor-TAM interactions revealed distinct crosstalk in IDH-WT tumors, including ANXA1-FPR1/3, FN1-ITGAVB1, VEGFA-NRP1, and TNFSF12-TNFRSF12A with known contribution to immunosuppression, tumor proliferation, invasion and TAM recruitment. Spatially resolved transcriptomics further elucidated the architectural organization of highlighted communications. Furthermore, we demonstrated significant upregulation of ANXA1, FN1, NRP1, and TNFRSF12A genes in IDH-WT tumors using bulk RNA-seq and RT-qPCR. Longitudinal expression analysis of candidate genes revealed no difference between primary and recurrent tumors indicating that the interactive network of malignant states with TAMs does not drastically change upon recurrence. Collectively, our study offers insights into the unique cellular composition and communication of TAMs in glioma TME, revealing novel vulnerabilities for therapeutic interventions in IDH-WT GBM.

Twist2-driven chromatin remodeling governs the postnatal maturation of dermal fibroblasts.

BACKGROUND Recent advances in genetics and epigenetics research have underscored the heterogeneity of pediatric brain tumors, characterized by overlapping morphological and molecular features across various tumor types. This complexity presents substantial challenges for precision diagnosis in routine diagnostics. In this study, we collected a cohort of pediatric high-grade brain tumors that remain unresolved after next-generation sequencing (NGS). Our goal is try to refine diagnosis by DNA methylation profiling, thereby evaluating the utility of this new diagnostic tool against unresolved and challenging cases in standard clinical practice. METHODS In this retrospective analysis, we selected 10 cases from local pathology archives based on the following criteria: (1) age at diagnosis< 18 years; (2) brain tumor with high-grade evidence; (3) NGS was employed but failed to provide a definitive diagnosis or not aligned to WHO CNS tumor classification. All cases were subjected to the Illumina Infinium MethylationEPIC v2.0 BeadChip, DNA methylation profiling was conducted by using an customized pipeline alongside the DKFZ brain tumor classifier. NGS was previously done by utilizing a DNA-panel targeting over 500 genes associated with tumor-related genetic variations. The collection of tumor samples and clinical data was processed in accordance with standards approved by the local ethical committees. RESULT All cases were supratentorial high-grade tumors, aged 1 to 16 years (n=10). Utilizing DNA methylation profiling, definitive diagnoses were established in 60% of cases: two cases of embryonal tumor with multilayered rosettes (ETMR), one pediatric diffuse high-grade glioma MYCN subtype, one pleomorphic xanthoastrocytoma (PXA), one medulloblastoma non-WNT/non-SHH subgroup 7, and one alveolar rhabdomyosarcoma. The remaining cases (n=4) neither yielded convincing calibrated scores from the DKFZ brain tumor classifier (v12.8) nor provided valuable insights from methylation based t-SNE clustering or copy-number profiling analysis. These 4 cases includes: a pediatric high grade glioma (pHGG) harboring CDK12, DICER1, JAK3, NF1, NTRK2, RB1 and SPEN mutation; a pHGG harboring PIK3CA, NF1 and TP53 mutation; a malignant embryonal tumor in the pineal gland harboring ATRX, BCL2, BLM, CYSLTER2, GPR101, HIF1A and SHOC2 mutation; and a malignant embryonal tumor with TERT, SMARCA4 mutation and EML4-ALK fusion. CONCLUSION In confronting the diagnostic complexities of pediatric high-grade brain tumors, DNA methylation profiling may provide more critical evidence that can address and unravel intricate clinical diagnostic challenges. SUPPORT/DISCLOSURE This research was supported by Natural Science Foundation of Chongqing (No.CSTB2023NSCQ-BHX0105).

Pediatric high-grade gliomas (World Health Organization grades III and IV astrocytomas) remain tumors with a very poor prognosis for which novel therapeutic strategies are needed. Poly(ADP-ribose) polymerase (PARP) is known to have multiple functions in tumors, including single-strand DNA repair and induction of caspase-independent apoptosis. PARP has been suggested as a therapeutic target in adult malignancies, and this study examines whether it could also be a potential target in pediatric high-grade glioma. Tissue microarrays containing 150 formalin-fixed pediatric high-grade gliomas were examined by immunohistochemistry for levels of PARP and expression of apoptosis inducing factor (AIF). Full retrospective clinical and survival data were available for this cohort. Stratification and statistical analysis was performed to assess the effect of PARP status on prognosis. The level of PARP immunopositivity had a statistically significant inverse correlation (P = .019) with survival in supratentorial pediatric high-grade glioma. AIF staining was notable for its absence in the majority of tumors but with moderate levels of expression in surrounding normal brain. PARP is expressed at high levels in many pediatric high-grade gliomas, and in these tumors, the ability of PARP to activate AIF appears to have been lost. PARP may therefore represent a promising therapeutic target for these lesions and warrants evaluation in clinical trials.

Simple SummaryPediatric high-grade gliomas are incurable brain tumors for which there is a critical need for new therapeutic strategies as well as treatment-predictive biomarkers. This study examined the expression of DNA repair and cell cycle genes in pediatric high-grade gliomas with distinct driving mutations. The aim is to propose a novel classification of these tumors based on sub-groups exposing therapeutic vulnerabilities. Several DNA repair factors were identified that might become new diagnostic markers.Background: Pediatric high-grade gliomas (pHGGs) are the leading cause of mortality in pediatric neuro-oncology, displaying frequent resistance to standard therapies. Profiling DNA repair and cell cycle gene expression has recently been proposed as a strategy to classify adult glioblastomas. To improve our understanding of the DNA damage response pathways that operate in pHGGs and the vulnerabilities that these pathways might expose, we sought to identify and characterize a specific DNA repair and cell-cycle gene expression signature of pHGGs. Methods: Transcriptomic analyses were performed to identify a DNA repair and cell-cycle gene expression signature able to discriminate pHGGs (n = 6) from low-grade gliomas (n = 10). This signature was compared to related signatures already established. We used the pHGG signature to explore already transcriptomic datasets of DIPGs and sus-tentorial pHGGs. Finally, we examined the expression of key proteins of the pHGG signature in 21 pHGG diagnostic samples and nine paired relapses. Functional inhibition of one DNA repair factor was carried out in four patients who derived H3.3 K27M mutant cell lines. Results: We identified a 28-gene expression signature of DNA repair and cell cycle that clustered pHGGs cohorts, in particular sus-tentorial locations, in two groups. Differential protein expression levels of PARP1 and XRCC1 were associated to TP53 mutations and TOP2A amplification and linked significantly to the more radioresistant pHGGs displaying the worst outcome. Using patient-derived cell lines, we showed that the PARP-1/XRCC1 expression balance might be correlated with resistance to PARP1 inhibition. Conclusion: We provide evidence that PARP1 overexpression, associated to XRCC1 expression, TP53 mutations, and TOP2A amplification, is a new theranostic and potential therapeutic target.

Editor's evaluation: Regionally distinct trophoblast regulate barrier function and invasion in the human placenta

Decision letter: Regionally distinct trophoblast regulate barrier function and invasion in the human placenta

Diffuse hemispheric H3 G34R/V mutant gliomas (G34R/V DHG) are lethal brain malignancies occurring in the cerebral hemisphere of adolescent patients with no effective treatment options. To date, standard care is adapted from adult treatment protocols which include maximal tumor resection, radiotherapy, and chemotherapy with temozolomide (TMZ). In a recent study led by the Children’s Oncology Group (COG, ACNS0423), the combination of the alkylators TMZ and lomustine (CCNU) showed improved event-free survival for pediatric high-grade glioma patients. Still, patient benefits remain limited and thus demand further improvement of treatment protocols. Therefore, the aim of this study is to identify FDA-approved blood-brain-barrier crossing compounds that specifically targeting known vulnerabilities of pediatric high-grade glioma that achieve synergistic treatment results when combined with the double alkylator approach. We conducted a compound screen in combination with TMZ and CCNU on G34R/V-tumor derived gliomaspheres to identify synergies based on cell viability assays in vitro. We identified three candidate agents - Fimepinostat, trametinib, and avapritinib, which inhibit different key oncogenic targets in pediatric high-grade glioma - Pi3K and histone-deacetylases, MAPKs, and PDGFRA, respectively. Our ongoing in vivo study elaborates the translatability of the identified combinatorial therapies in patient derived G34R/V DHG orthotopic xenograft models. We are comparing different study arms of single agent treatment and double as well as triple combinations of alkylators and the candidate agents. This study will serve to determine (i) treatment efficacy, as measured by tumor growth and survival, (ii) occurring side effects, especially alkylator-induced suppression of platelet counts, and (iii) molecular changes upon different treatments using single cell transcriptomics and immunohistochemistry. In summary, we present valuable pre-clinical data supporting the combination of alkylators with targeted agents as a novel treatment strategy in G34R/V DHG, which might be translated into a promising strategy of a new comparative study based on COG trial ACNS0423.

Pediatric high-grade gliomas represent 8–12% of all primary tumors of the nervous system in children. Five-year survival for these pediatric aggressive tumors is poor (15–35%) indicating the need to develop better treatments for pediatric high-grade gliomas. In this work we used SF188 and SJ-GBM2 cell lines to study the function of the ubiquitin carboxyl-terminal esterase L1 (UCHL1), a deubiquitinase de-regulated in several cancers, in pediatric high-grade gliomas. UCHL1 depletion in SF188 and SJ-GBM2 glioma cells was associated with decreased cell proliferation and invasion, along with a reduced ability to grow in soft agar and to form spheres (i.e. self-renewal measure). A 70% reduction in Wnt signaling was also observed in the SF188 and SJ-GBM2 UCHL1 knockdowns (KDs) using a TCF-dependent TOPflash reporter assay. Transcriptome comparisons of UCHL1 KDs versus vector control identified a list of 306 differentially expressed genes (at least 2-fold change; p <0.05) which included genes known to be involved in cancer like ACTA2, POSTN, LIF, FBXL7, FBXW11, GDF15, HEY2, but also potential novel genes such us IGLL5, ABCA4, AQP3, AQP4, CALB1, and ALK. Bioinformatics gene ontology (GO) analysis of these 306 genes revealed significant enrichment in “signal peptides”, “extracellular matrix”and “secreted proteins” GO Terms. “Angiogenesis and blood vessel development”, “neuron differentiation/development”, cell adhesion”, and “cell migration” also showed significant enrichment in our GO analysis. Top canonical pathways identified by Ingenuity Pathway Analysis (IPA) included “Clathrin-mediated Endocytosis Signaling” (p = 5.14x10-4), “Virus Entry via Endocytic Pathways” (p = 6.15x 10−4), and “High Mobility Group-Box 1 (HMGB1) Signaling” (p = 6.15x10-4). While FGF2, IL1B, TNF and PDGFB were predicted as top upstream regulators (p < 2x10-16) of the UCHL1 KD-associated transcriptome. Aberrant expression of UCHL1 in pediatric high-grade gliomas may promote cell invasion, transformation, and self-renewal properties, at least in part, by modulating Wnt/Beta catenin activity. UCHL1 might act as an oncogene in glioma within the gene network that imparts stem-like characteristics to these cancer cells.

DDIT4 (DNA Damage Inducible Transcript 4), a well-established inhibitor of the PI3K-Akt/mTOR pathway, is upregulated under cellular stress conditions. Extensive research has demonstrated that DDIT4 expression is aberrantly elevated in various malignancies, where it exhibits context-dependent roles in either tumor promotion or suppression. However, the mechanisms underlying how DDIT4 is involved in tumor immune regulation remain to be fully elucidated. This review systematically summarizes the multifaceted mechanisms by which DDIT4 participates in tumor immunomodulation, primarily through its inhibition of the PI3K-Akt/mTOR pathway to induce autophagy activation and metabolic reprogramming; furthermore, it comprehensively examines DDIT4’s regulatory effects on various components within the tumor immune microenvironment, including tumor cells, both innate and adaptive immune cells, and immunomodulatory cytokines. This comprehensive analysis aims to establish a theoretical foundation for considering DDIT4 as a potential therapeutic target in tumor immunotherapy.

Epigenetics provides the critical connection between environmental influence and gene expression, where environmental stressors could modulate expression of specific genes in particular scenarios using molecular markers etched at the genome level. Hence, epigenetics likely play important roles in potentiating the development of specific lineages, cell fate or cellular differentiation. For example, when specific environmental stressor is present, epigenetic markers in the genome receive a signal for either activating or deactivating expression of particular sets of genes, which may be linked to the developmental trajectory of the organism. Using Escherichia coli as model organism, a possible study may investigate the role of epigenetics in influencing cellular differentiation of the bacterium. Specifically, a single E. coli cell would be propagated into a consortium of 12 or more bacterial cells in a microfluidics growth chamber. Genetic material extracted would be sent for single cell genomics, transcriptomics, and chromatin immunoprecipitation sequencing (ChIP-seq). After profiling, the residual population would be diverted by microchannels to 6 different cell growth chambers, where they would be cultivated under identical conditions for understanding possible triggers to cell differentiation. At suitable time points of 2, 4, 6, 8, 10, 12 hours, single cell would be extracted from each growth chamber for profiling single cell genomics, transcriptomics, and epigenetics markers. Optical and confocal laser scanning microscopy would provide readout of cell morphologies. Comparison of the readout between the original clonal population and those of the different growth chambers may provide important points for correlating epigenetic markers with gene expression and phenotypic readout in cell lineage, fate and differentiation. In subsequent experiments, different environmental stressors such as pH, imbalance nutrient composition between carbon and nitrogen, nanoparticles or heavy metals, could be used as triggers for specific cell growth response guided by epigenetic programmes embedded within the epigenome of the bacterium. Collectively, epigenetics hold influence for cellular differentiation in view of specific environmental stressors, where epigenetic markers on the genome communicate specific environmental factor's effect on the organism through altering expression of particular sets of genes, that result in different cell fate, lineage and differentiation. Using modern single cell techniques at the genomics, transcriptomics and epigenomics level, the study hopes to elucidate epigenetic potentiators of cellular differentiation in E. coli with and without environmental stressors such as nutrient deprivation, pH and toxic metals.

SummaryDickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on β-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of β-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic β-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits β-catenin-dependent transcriptional output.

Wnt signaling regulates cell fate decisions in diverse contexts during development and disease. In the mouse cranial mesenchyme (CM) Wnt signaling pathway components and reporters are spatially distributed during calvarial osteoblast fate selection. Within 24 hours, loss of Wnt signaling in the mouse embryonic CM results in a robust and binary cell fate switch, from calvarial bone to ectopic cartilage. The mechanism by which Wnt signaling regulates this cell fate switch is not clear. Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) activation is required for bone-cartilage cell fate decisions in long bone perichondrium, and we have demonstrated that ERK1/2 activation is present in calvarial osteoblasts. Here, we test the hypothesis that ERK signaling is a mediator of binary Wnt-dependent bone-cartilage cell fate decisions. First, we used three distinct Wnt signaling loss-of-function mouse models to demonstrate that Wnt signaling is required for activation of ERK signaling in calvarial bone progenitors. Second, we showed that loss of ERK signaling precedes formation of ectopic cartilage in CM-Wnt signaling mutants and ERK activation is highly sensitive to levels of Wnt signaling within the cranial mesenchyme. Third, loss of Erk1/2 in the CM results in elevated levels of the master cartilage determinant, SOX9, by E13.5 and loss of calvarial bone by E16.5. These results demonstrate a link between the Wnt and ERK signaling pathways in regulating calvarial bone cell fate decisions in vivo. We propose a new model whereby reciprocal regulation of both canonical and non-canonical Wnt pathways in the CM generates a gradient of Wnt signaling and utilizes ERKs to reinforce binary cell fate decisions in vivo. This offers a new opportunity for therapeutic targeting of Wnt signaling in craniofacial skeletal defects and disease.