Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma.

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.

Consequences of IDH1/2 Mutations in Gliomas and an Assessment of Inhibitors Targeting Mutated IDH Proteins.

Isocitrate dehydrogenases (IDH) 1 and 2 are key metabolic enzymes that generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) to maintain a pool of reduced glutathione and peroxiredoxin, and produce α-ketoglutarate, a co-factor of numerous enzymes. IDH1/2 is mutated in ~70⁻80% of lower-grade gliomas and the majority of secondary glioblastomas. The mutant IDH1 (R132H), in addition to losing its normal catalytic activity, gains the function of producing the d-(R)-2-hydroxyglutarate (2-HG). Overproduction of 2-HG in cancer cells interferes with cellular metabolism and inhibits histone and DNA demethylases, which results in histone and DNA hypermethylation and the blockade of cellular differentiation. We summarize recent findings characterizing molecular mechanisms underlying oncogenic alterations associated with mutated IDH1/2, and their impact on tumor microenvironment and antitumor immunity. Isoform-selective IDH inhibitors which suppress 2-HG production and induce antitumor responses in cells with IDH1 and IDH2 mutations were developed and validated in preclinical settings. Inhibitors of mutated IDH1/2 enzymes entered clinical trials and represent a novel drug class for targeted therapy of gliomas. We describe the development of small-molecule compounds and peptide vaccines targeting IDH-mutant gliomas and the results of their testing in preclinical and clinical studies. All those results support the translational potential of strategies targeting gliomas carrying IDH1 mutations.

Mapping chromatin accessibility and active regulatory elements reveals pathological mechanisms in human gliomas.

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.

Injections of vehicle, but not cyclosporin A or tacrolimus (FK506), afford neuroprotection following injury in the developing rat brain.

Susceptibility of the injured rat brain to seizures depends on the developmental stage at which the injury had been inflicted. Our previous study shows that tacrolimus (FK506) and cyclosporin A (CsA) applied following the injury can also decrease or increase the seizure susceptibility in an age-dependent way. To find possible neuronal substrates of the effects, we examined influences of the agents on the injured brain and on its neuronal population. Rat brains were mechanically injured on postnatal days 6 (P6) or 30 (P30). Twenty minutes and 24 hours following the injury, FK506 or CsA were injected in clinically used pharmaceutical formulations (Prograf or Sandimmun, respectively). The brains were fixed on postnatal day 60 and processed for histological examinations. To detect if negative effects of the injury could be abolished by the treatments, we examined the brain weight, the size of the injured region, and the nerve cell density, including the density of calretinin- and parvalbumin-immunopositive cells. We have found that long-term effects of treatments with the FK506- and CsA-containing pharmaceutical formulations were never better than those of the vehicle alone (Cremophor and ethanol mixture). Moreover, the treatments could even amplify negative consequences of the injury alone. It could, therefore, be concluded that all the neuroprotective effects observed in the present study resulted exclusively from the influence of the vehicle alone. These effects of the brain injury and of subsequent treatments performed at different developmental stages were considered as possible determinants of further increase or decrease in susceptibility to seizures observed in adulthood.

Different effects of neuroprotectants FK-506 and cyclosporin A on susceptibility to pilocarpine-induced seizures in rats with brain injured at different developmental stages.

Susceptibility of the injured brain to epileptic seizures depends on the developmental stage at which the injury had been inflicted (our previous paper published in Epilepsy Res. 53 (2003) 216-224). The present study was designed to examine whether neuroprotective agents applied following the injury can decrease the seizure susceptibility. In order to solve this problem, the left cerebral hemisphere was mechanically injured in 6- and 30-day-old Wistar rats. Neuroprotectants FK506 or Cyclosporin A (CsA) were injected 20 min and 24h following the injury. On postnatal day 60, all the animals received single i.p. pilocarpine injections to evoke epileptic seizures. During a 6h period following the injection, the animals were observed continuously and pilocarpine-induced symptoms were recorded and rated. The animals were sacrificed 7 days after pilocarpine injection. In rats injured on postnatal days 6 or 30 (P6 or P30, respectively) and injected with FK-506 after the injury, signs of amelioration in the course of epilepsy were observed. Generally, proportions of rats suffering from heavy seizures were lower and/or their survival periods were longer. Following treatment with CsA, proportions of rats displaying heavy seizures were greater. It was accompanied by extremely high mortality (in rats injured on P6) or a longer duration of seizures (in rats injured on P30). The results appear to point to age-dependent differences between the mechanisms of action of the two neuroprotectants.

MK-801, a NMDA receptor antagonist, increases phosphorylation of histone H3 in the rat medial prefrontal cortex.

BACKGROUND: The present study investigated whether MK-801, when given in doses that cause psychomimetic effects in rats, could alter the phosphorylation of histone 3 (H3) at serine 10 (H3S10p) and the acetylation of H3 at lysine 14 (H3K14ac) in the medial prefrontal cortex (mPFC). These posttranslational modifications of H3 promote chromatin relaxation and increase the probability of gene expression. METHODS: Stereological counting, immunoblot analysis and confocal laser scanning microscopy. RESULTS: Treatment with MK-801 (0.4 mg/kg) evoked a time-dependent increase in the number of H3S10p positive nuclei in both the II/III and V/VI layers of the mPFC, reaching the peak of activation 30 min after injection. MK-801 treatment (0.4 mg/kg) failed to alter H3K14ac. These effects were confirmed by immunoblot analysis on tissue samples from the mPFC. Analysis of cortical cells expressing H3S10p positive nuclei revealed that constitutive and MK-801-induced expression of H3S10p was observed only in neurons and not in glia cells (H3S10p colocalized with NeuN but not with S-100ß). Moreover, it has been found that H3S10p is exclusively present in pyramidal (glutamate-positive) but not in cortical GABA-ergic interneurons (GABA-positive). The effects of MK-801 can be attenuated or blocked by the neuroleptic drug risperidone. In the cortical layer II/III, risperidone was effective at doses of 0.2 and 1 mg/kg, while it was only active at a dose of 1 mg/kg in the V/VI layer. Again, these stereological data were confirmed by immunoblot analysis. CONCLUSIONS: Our results indicate that MK-801 may increase the transcriptional activity of mPFC via the activation of the epigenetic program associated with H3S10p phosphorylation during the course of experimental psychosis.