ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response.

AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

Selective inhibitors of bromodomain BD1 and BD2 of BET proteins modulate radiation-induced profibrotic fibroblast responses.

Radiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation-induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan-BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I-BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan-inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET-BD1 [GSK778] and iBET-BD2 [GSK046]). Here, their potential to attenuate radiation-induced fibroblast activation with low-toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I-BET151 and iBET-BD1, but not with iBET-BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I-BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET-BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor-treatment. In conclusion, iBET-BD2 efficiently suppressed radiation-induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2-selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy-induced fibrosis.

ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

Identification of therapeutic targets of the hijacked super-enhancer complex in EVI1-rearranged leukemia.

Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1-G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Diacylglycerol Kinase Alpha in Radiation-Induced Fibrosis: Potential as a Predictive Marker or Therapeutic Target.

Radiotherapy is an efficient tool in cancer treatment, but it brings along the risk of side effects such as fibrosis in the irradiated healthy tissue thus limiting tumor control and impairing quality of life of cancer survivors. Knowledge on radiation-related fibrosis risk and therapeutic options is still limited and requires further research. Recent studies demonstrated that epigenetic regulation of diacylglycerol kinase alpha (DGKA) is associated with radiation-induced fibrosis. However, the specific mechanisms are still unknown. In this review, we scrutinized the role of DGKA in the radiation response and in further cellular functions to show the potential of DGKA as a predictive marker or a novel target in fibrosis treatment. DGKA was reported to participate in immune response, lipid signaling, exosome production, and migration as well as cell proliferation, all processes which are suggested to be critical steps in fibrogenesis. Most of these functions are based on the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) at plasma membranes, but DGKA might have also other, yet not well-known functions in the nucleus. Current evidence summarized here underlines that DGKA activation may play a central role in fibrosis formation post-irradiation and shows a potential of direct DGKA inhibitors or epigenetic modulators to attenuate pro-fibrotic reactions, thus providing novel therapeutic choices.

DNA methylation profiling to explore colorectal tumor differences according to menopausal hormone therapy use in women.

Aim: Use of menopausal hormone therapy (MHT) has been associated with a reduced risk for colorectal cancer, but mechanisms underlying this relationship are not well understood. In the colon, MHT appears to act through estrogen receptor ß (ERß) which may influence DNA methylation by binding to DNA. Using genome-wide methylation profiling data, we aimed to identify genes that may be differentially methylated according to MHT use. Materials & methods: DNA methylation was measured using Illumina HumanMethylation450k arrays in two independent tumor sample sets of colorectal cancer patients. Differential methylation was determined using R/limma. Results: In the discovery analysis, two CpG sites showed differential DNA methylation according to MHT use, both were not replicated. In stratified analyses, 342 CpG sites were associated with current MHT use only in ERß-positive tumors. Conclusion: The suggestive findings of differential methylation according to current MHT use in ERß-positive tumors warrant further investigation.

DNA methylation at an enhancer of the three prime repair exonuclease 2 gene (TREX2) is linked to gene expression and survival in laryngeal cancer.

BACKGROUND: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). RESULTS: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. CONCLUSIONS: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies.

DNA damage in human whole blood caused by radiopharmaceuticals evaluated by the comet assay.

Radiopharmaceuticals used for diagnosis or therapy induce DNA strand breaks, which may be detectable by single-cell gel electrophoresis (called comet assay). Blood was taken from patients before and at different time points after treatment with radiopharmaceuticals; blood cells were investigated by the comet assay using the percentage of DNA in the tail as the critical parameter. Whereas [225Ac]Ac-prostate-specific membrane antigen (PSMA)-617 alpha therapy showed no difference relative to the blood sample taken before treatment, beta therapy with [177Lu]Lu-PSMA-617 3 h post-injection revealed a small but significant increase in DNA strand breaks. In blood of patients who underwent positron emission tomography (PET) with either [18F]2-fluor-2-deoxy-D-glucose (FDG) or [68Ga]Ga-PSMA-11, an increase of DNA migration determined by the comet assay was not found when analysed at different time points (2-70 min) after intravenous tracer injection. Human whole blood was incubated with the targeted clinically relevant therapeutic radiopharmaceuticals [225Ac]Ac-PSMA-617, [177Lu]Lu-PSMA-617 and [90Y]Y-DOTA(0)-Phe(1)-Tyr(3)-octreotide (DOTA-TOC) at different activity concentrations (kBq/ml) for 5 days and then analysed by the comet assay. DNA damage increased with higher concentrations of all radiolabeled compounds tested. [177Lu]Lu-PSMA-617 caused higher blood cell radiotoxicity than equal activity concentrations of [90Y]Y-DOTA-TOC. Likewise, whole human blood was exposed to the positron emitters [18F]FDG and [68Ga]Ga-PSMA-11 in vitro for 24 h with activity concentrations ranging between 5 and 40 MBq/ml. The same activity concentration dependent elevated DNA migration was observed for both compounds although decay energies are different. This study demonstrated that the amount of DNA damage detected by the comet assay in whole human blood is similar among different positron emitters and divergent by a factor of 200 between alpha particles and beta radiation.

DNA damage response of clinical carbon ion versus photon radiation in human glioblastoma cells.

BACKGROUND AND PURPOSE: Carbon ion radiotherapy is a promising therapeutic option for glioblastoma patients due to its high physical dose conformity and greater biological effectiveness than photons. However, the biological effects of carbon ion radiation are still incompletely understood. Here, we systematically compared the biological effects of clinically used carbon ion radiation to photon radiation with emphasis on DNA repair. MATERIALS AND METHODS: Two human glioblastoma cell lines (U87 and LN229) were irradiated with carbon ions or photons and DNA damage response was systematically analyzed, including clonogenic survival, induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and apoptosis or autophagy. γH2AX foci were analyzed by flow cytometry, conventional light microscopy and 3D superresolution microscopy. RESULTS: DSBs were repaired delayed and with slower kinetics after carbon ions versus photons. Carbon ions caused stronger and longer-lasting cell cycle delays, predominantly in G2 phase, and a higher rate of apoptosis. Compared to photons, the effectiveness of carbon ions was less cell cycle-dependent. Homologous recombination (HR) appeared to be more important for DSB repair after carbon ions versus photons in phosphatase and tensin homolog (PTEN)-deficient U87 cells, as opposed to PTEN-proficient LN229 cells. CONCLUSION: Carbon ions induced more severe DSB damage than photons, which was repaired less efficiently in both cell lines. Thus, carbon ion radiotherapy may help to overcome resistance mechanisms of glioblastoma associated with DNA repair for example in combination with repair pathway-specific drugs in the context of personalized radiotherapy.

Genome-wide DNA methylation differences according to oestrogen receptor beta status in colorectal cancer.

Involvement of sex hormones in colorectal cancer (CRC) development has been linked to oestrogen receptor ß (ERß). Expression of ERß is found reduced in tumour tissue and inversely related to mortality. However, mechanisms are not well understood. Our study aimed to detect differentially methylated genes associated with ERß expression, which could point to mechanisms by which ERß could influence risk and prognosis of CRC. Epigenome-wide DNA methylation profiling was performed using Illumina HumanMethylation450k BeadChip arrays in two independent tumour sample sets of CRC patients recruited in 2003-2010 by the German DACHS study (discovery cohort n = 917, replication cohort n = 907). ERß expression was measured using immunohistochemistry and scored as negative, moderate and high. Differentially methylated CpG sites and genomic regions were determined using limma in the R-package RnBeads. For the comparison of tumours with moderate/high ERß versus negative expression, differentially methylated CpG sites were identified but not confirmed by replication. Comparing tumours of high with tumours of negative ERß expression revealed 2,904 differentially methylated CpG sites of which 403 were replicated (FDR adjusted p-value<0.05). Replicated CpGs were annotated to genes such as CD36, HK1 or LRP5. A survival analysis indicates that 30 of the replicated CpGs are also associated with overall survival (FDR-adjusted p-value<0.05). The regional analysis identified 60 differentially methylated promotor regions. The epigenome-wide analysis identified both novel genes as well as genes already implicated in CRC. Follow-up mechanistic studies to better understand the regulatory role of ERß could inform potential targets for improving treatment or prevention of CRC.

Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain.

The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.

Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance.

Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.

Genome-wide genetic and epigenetic analyses of pancreatic acinar cell carcinomas reveal aberrations in genome stability.

Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, to identify potential targets for personalized treatment, we perform integrative genome-wide and epigenome-wide analyses. The results show frequently aberrant DNA methylation, abundant chromosomal amplifications and deletions, and mutational signatures suggesting defective DNA repair. In contrast to pancreatic ductal adenocarcinoma, no recurrent point mutations are detected. The tumor suppressors ID3, ARID1A, APC, and CDKN2A are frequently impaired also on the protein level and thus potentially affect ACC tumorigenesis. Consequently, this work identifies promising therapeutic targets in ACC for drugs recently approved for precision cancer therapy.

BET-bromodomain inhibitors modulate epigenetic patterns at the diacylglycerol kinase alpha enhancer associated with radiation-induced fibrosis.

BACKGROUND AND PURPOSE: Fibrosis is a frequent adverse effect of radiotherapy and no effective treatments are currently available to prevent or reverse fibrotic disease. We have previously identified altered epigenetic patterns at a gene enhancer of the diacylglycerol kinase alpha (DGKA) locus in normal skin fibroblasts derived from fibrosis patients. An open chromatin pattern related to radiation-inducibility of DGKA is associated with onset of radiation-induced fibrosis. Here, we explore epigenetic modulation of DGKA as a way to mitigate predisposition to fibrosis. MATERIAL AND METHODS: We studied the effect of the BET-bromodomain inhibitors (JQ1, PFI-1) on DGKA inducibility in primary fibroblasts. Hence, DGKA transcription was additionally induced by the radiomimetic drug bleomycin, and DGKA mRNA expression, histone H3K27 acetylation and downstream markers of profibrotic fibroblast activation after BET-bromodomain inhibition were determined. RESULTS: BET-bromodomain inhibition suppressed induction of DGKA in bleomycin-treated fibroblasts, reduced H3K27ac at the DGKA enhancer and repressed collagen marker gene expression. Alterations in fibroblast morphology and reduction of collagen deposition were observed. CONCLUSION: For the DGKA enhancer, we show that BET-bromodomain inhibitors can alter the epigenetic landscape of fibroblasts, thus counteracting profibrotic transcriptional events. Interference with epigenetic patterns of fibrosis predisposition may provide novel preventive therapies that improve radiotherapy.

Integrative Genome-Scale Analysis Identifies Epigenetic Mechanisms of Transcriptional Deregulation in Unfavorable Neuroblastomas.

The broad clinical spectrum of neuroblastoma ranges from spontaneous regression to rapid progression despite intensive multimodal therapy. This diversity is not fully explained by known genetic aberrations, suggesting the possibility of epigenetic involvement in pathogenesis. In pursuit of this hypothesis, we took an integrative approach to analyze the methylomes, transcriptomes, and copy number variations in 105 cases of neuroblastoma, complemented by primary tumor- and cell line-derived global histone modification analyses and epigenetic drug treatment in vitro We found that DNA methylation patterns identify divergent patient subgroups with respect to survival and clinicobiologic variables, including amplified MYCN Transcriptome integration and histone modification-based definition of enhancer elements revealed intragenic enhancer methylation as a mechanism for high-risk-associated transcriptional deregulation. Furthermore, in high-risk neuroblastomas, we obtained evidence for cooperation between PRC2 activity and DNA methylation in blocking tumor-suppressive differentiation programs. Notably, these programs could be re-activated by combination treatments, which targeted both PRC2 and DNA methylation. Overall, our results illuminate how epigenetic deregulation contributes to neuroblastoma pathogenesis, with novel implications for its diagnosis and therapy. Cancer Res; 76(18); 5523-37. ©2016 AACR.

Combined Treatment of ATRA with Epigenetic Drugs Increases Aggressiveness of Glioma Xenografts.

BACKGROUND/AIM: Recently, anti-tumourigenic effects of all-trans-retinoic-acid (ATRA) on glioblastoma stem cells were demonstrated. Therefore we investigated if these beneficial effects could be enhanced by co-medication with epigenetic drugs such as the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) or the DNA-methyltransferase inhibitor 5-aza-2'deoxycytidine (5-AZA). MATERIALS AND METHODS: Glioma stem cell xenografts were treated for 42 days with ATRA plus SAHA or ATRA plus 5-AZA or the correspondent monotherapies. Tumour sizes, histological features, proliferation and apoptosis rates were assessed. RESULTS: Neither SAHA nor 5-AZA were able to enhance the anti-tumourigenic effect of ATRA. Instead, tumours became more aggressive. Combination of ATRA plus 5-AZA increased tumour size (p<0.05) and induced more frequent and larger necroses (p<0.05) and tumours were more invasive (p<0.05) in comparison to controls. A similar trend was observed for the combination of ATRA plus SAHA. CONCLUSION: Combining ATRA with epigenetic drug therapies led to the unwanted opposite effect and increased aggressiveness of glioma xenografts, arguing against future clinical applications of such combinations.

LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients.

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.