The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action

Heavy ions and X-rays in brain tumor treatment : a comparison of their biological effects on tissue slice cultures

Background: In this interdisciplinary project, the biological effects of heavy ions are compared to those of X-rays using tissue slice culture preparations from rodents and humans. Advantages of this biological model are the conservation of an organotypic environment and the independency from genetic immortalization strategies used to generate cell lines. Its open access allows easy treatment and observation via live-imaging microscopy. Materials and methods: Rat brains and human brain tumor tissue are cut into 300 micro m thick tissue slices. These slices are cultivated using a membrane-based culture system and kept in an incubator at 37°C until treatment. The slices are treated with X-rays at the radiation facility of the University Hospital in Frankfurt at doses of up to 40 Gy. The heavy ion irradiations were performed at the UNILAC facility at GSI with different ions of 11.4 A MeV and fluences ranging from 0.5–10 x 106 particles/cm². Using 3D-confocal microscopy, cell-death and immune cell activation of the irradiated slices are analyzed. Planning of the irradiation experiments is done with simulation programs developed at GSI and FIAS. Results: After receiving a single application of either X-rays or heavy ions, slices were kept in culture for up to 9d post irradiation. DNA damage was visualized using gamma H2AXstaining. Here, a dose-dependent increase and time-dependent decrease could clearly be observed for the X-ray irradiation. Slices irradiated with heavy ions showed less gamma H2AX-positive cells distributed evenly throughout the slice, even though particles were calculated to penetrate only 90–100 micro m into the slice. Conclusions: Single irradiations of brain tissue, even at high doses of 40 Gy, will result neither in tissue damage visible on a macroscopic level nor necrosis. This is in line with the view that the brain is highly radio-resistant. However, DNA damage can be detected very well in tissue slices using gamma H2AX-immuno staining. Thus, slice cultures are an excellent tool to study radiation-induced damage and repair mechanisms in living tissues

Neurovascular EGFL7 regulates adult neurogenesis in the subventricular zone and thereby affects olfactory perception

Adult neural stem cells reside in a specialized niche in the subventricular zone (SVZ). Throughout life they give rise to adult-born neurons in the olfactory bulb (OB), thus contributing to neural plasticity and pattern discrimination. Here, we show that the neurovascular protein EGFL7 is secreted by endothelial cells and neural stem cells (NSCs) of the SVZ to shape the vascular stem-cell niche. Loss of EGFL7 causes an accumulation of activated NSCs, which display enhanced activity and re-entry into the cell cycle. EGFL7 pushes activated NSCs towards quiescence and neuronal progeny towards differentiation. This is achieved by promoting Dll4-induced Notch signalling at the blood vessel-stem cell interface. Fewer inhibitory neurons form in the OB of EGFL7-knockout mice, which increases the absolute signal conducted from the mitral cell layer of the OB but decreases neuronal network synchronicity. Consequently, EGFL7-knockout mice display severe physiological defects in olfactory behaviour and perception

ATP synthase deficiency due to TMEM70 mutation leads to ultrastructural mitochondrial degeneration and is amenable to treatment.

TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy) ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G) in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment

Molecular matched targeted therapies for primary brain tumors—a single center retrospective analysis

PURPOSE: Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. METHODS: Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. RESULTS: 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. CONCLUSION: A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11060-022-04049-w

TERT promoter mutation and chromosome 6 loss define a high-risk subtype of ependymoma evolving from posterior fossa subependymoma

Subependymomas are benign tumors characteristically encountered in the posterior fossa of adults that show distinct epigenetic profiles assigned to the molecular group "subependymoma, posterior fossa" (PFSE) of the recently established DNA methylation-based classification of central nervous system tumors. In contrast, most posterior fossa ependymomas exhibit a more aggressive biological behavior and are allocated to the molecular subgroups PFA or PFB. A subset of ependymomas shows epigenetic similarities with subependymomas, but the precise biology of these tumors and their potential relationships remain unknown. We therefore set out to characterize epigenetic traits, mutational profiles, and clinical outcomes of 50 posterior fossa ependymal tumors of the PFSE group. On histo-morphology, these tumors comprised 12 ependymomas, 14 subependymomas and 24 tumors with mixed ependymoma-subependymoma morphology. Mixed ependymoma-subependymoma tumors varied in their extent of ependymoma differentiation (2-95%) but consistently exhibited global epigenetic profiles of the PFSE group. Selective methylome analysis of microdissected tumor components revealed CpG signatures in mixed tumors that coalesce with their pure counterparts. Loss of chr6 (20/50 cases), as well as TERT mutations (21/50 cases), were frequent events enriched in tumors with pure ependymoma morphology (p < 0.001) and confined to areas with ependymoma differentiation in mixed tumors. Clinically, pure ependymoma phenotype, chr6 loss, and TERT mutations were associated with shorter progression-free survival (each p < 0.001). In conclusion, our results suggest that subependymomas may acquire genetic and epigenetic changes throughout tumor evolution giving rise to subclones with ependymoma morphology (resulting in mixed tumors) that eventually overpopulate the subependymoma component (pure PFSE ependymomas)

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.publishedVersio

DNA methylation-based prediction of response to immune checkpoint inhibition in metastatic melanoma

Background Therapies based on targeting immune checkpoints have revolutionized the treatment of metastatic melanoma in recent years. Still, biomarkers predicting long-term therapy responses are lacking. Methods A novel approach of reference-free deconvolution of large-scale DNA methylation data enabled us to develop a machine learning classifier based on CpG sites, specific for latent methylation components (LMC), that allowed for patient allocation to prognostic clusters. DNA methylation data were processed using reference-free analyses (MeDeCom) and reference-based computational tumor deconvolution (MethylCIBERSORT, LUMP). Results We provide evidence that DNA methylation signatures of tumor tissue from cutaneous metastases are predictive for therapy response to immune checkpoint inhibition in patients with stage IV metastatic melanoma. Conclusions These results demonstrate that LMC-based segregation of large-scale DNA methylation data is a promising tool for classifier development and treatment response estimation in cancer patients under targeted immunotherapy

Genetic and epigenetic characterization of posterior pituitary tumors

Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors (14 PITUI; 12 GCT; 21 SCO) to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (n = 23) contained most PITUIs and a subset of SCOs and was enriched for pathogenic mutations within genes in the MAPK/PI3K pathways (12/17 [71%] of sequenced tumors: FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1)) and two with accompanying TERT promoter mutation. The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group (copy number imbalances present). Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets