Alpha-thalassemia/mental Retardation X-linked Research Articles

Investigating the structural and functional consequences of germline single nucleotide polymorphisms located in the genes of the alternative lengthening of telomere (ALT) pathway

BackgroundThe Alternative Lengthening of Telomeres (ALT) pathway represents a non-canonical mechanism of telomere maintenance that operates independently of the conventional telomerase activity. The three biologically significant proteins, designated as SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1), DAXX (Death domain-associated protein 6) and ATRX (alpha-thalassemia/mental retardation, X-linked) are associated with ALT in certain cancer types. The purpose of this study was to identify the most high-risk nsSNPs (non-synonymous Single Nucleotide Polymorphisms) within these three genes and assess their impacts on the structure and function of the proteins they encode. MethodsThe reported genetic polymorphisms of SMARCAL1, DAXX and ATRX genes were retrieved from the Ensembl database. Later, various computational tools like PROVEAN, PolyPhen2, SNPs and GO, SNAP2, Predict-SNP, Panther and PMut were used to predict the most deleterious nsSNPs. MutPred was used to understand the underlying molecular reasons of those nsSNPs being deleterious, followed by prediction of Post Translational Modification Sites (PTMs) using ModPred. I-Mutant and MUpro were used to predict the effect of SNP on energy stability. Later, 3D clustering analysis was done using Mutation 3D server. Moreover, ConSurf was utilized to identify the conservation scores of wild-type amino acids. Additionally, the NCBI conserved domain search tool was employed to pinpoint conserved domains within these three proteins. Project-Hope helped for biophysical validation, followed by prediction of these genes’ interaction and function by using GeneMANIA. ResultAnalysis on SMARCAL1 protein revealed that among 665 nsSNPs, four were identified as the most deleterious: L578S, T581S, P582A, and P582S. Similarly, within the DAXX protein, among a pool of 480 nsSNPs, P284S, R230C, and R230S were found out to be the most deleterious variants. In case of ATRX protein, V178D, R246C, and V277G, from the total of 1009 nsSNPs, were predicted to be the most deleterious. All these nsSNPs were found to occur at residue positions that are 100 % conserved within protein domains and were predicted to be most damaging from both structural and functional perspectives and highly destabilizing to their corresponding proteins. ConclusionComputational investigation on the 3 proteins-SMARCAL1, DAXX and ATRX through different bioinformatics analysis tools concludes that the identified high risk nsSNPs of these proteins are pathogenic SNPs. These variants potentially exert functional and structural influences, thus making them valuable candidates for future genetic studies.

Mutation analysis of the TERT gene in ovarian cancer patients of the Turkish population by next generation sequencing method.

Ovarian cancer (OC) ranks seventh among malignant tumors worldwide. As one of the most common gynecological malignancies, ovarian cancer has the second-highest mortality rate, after cervical and uterine cancer. Next-Generation Sequencing (NGS) technology has enhanced multi-gene panel analysis and its clinical utility for identifying cancer-causing gene mutations. This study aimed to determine the presence of significant and nonsense mutations in telomerase reverse transcriptase (TERT), alpha-thalassemia/mental retardation, X-linked (ATRX), O-6-methylguanine-DNA methyltransferase (MGMT), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes using the Next-Generation Sequencing (NGS) method. A cohort of 33 patients diagnosed with ovarian cancer was included in this investigation, and peripheral blood samples were collected from all participants. Significant and nonsense mutations in TERT, ATRX, MGMT, IDH1, and IDH2 genes were detected using the Next-Generation Sequencing method. Bioinformatics analysis was conducted using the QIAGEN Clinical Insight system. Twenty-four patients exhibited seven different TERT mutations, occurring in both exonic and intronic regions. One patient displayed a c.699-3delC deletion in the intronic region of the IDH1 gene, and the c.532G > A (p.V178I) mutation observed in three patients was assessed as potentially harmful. Additionally, novel mutations c.881A > G and c.995A > G were observed in the ATRX gene. The heterozygous novel mutation identified in the ATRX gene was confirmed through Sanger sequencing. These mutations were not previously associated with ovarian cancer and are considered novel candidate markers for ovarian cancer susceptibility. Confirmation of these results through larger cohort studies or functional investigations will contribute to a better understanding of the molecular mechanisms underlying ovarian cancer.

The Chromatin Remodeler ATRX: Role and Mechanism in Biology and Cancer

The alpha-thalassemia mental retardation X-linked (ATRX) syndrome protein is a chromatin remodeling protein that primarily promotes the deposit of H3.3 histone variants in the telomere area. ATRX mutations not only cause ATRX syndrome but also influence development and promote cancer. The primary molecular characteristics of ATRX, including its molecular structures and normal and malignant biological roles, are reviewed in this article. We discuss the role of ATRX in its interactions with the histone variant H3.3, chromatin remodeling, DNA damage response, replication stress, and cancers, particularly gliomas, neuroblastomas, and pancreatic neuroendocrine tumors. ATRX is implicated in several important cellular processes and serves a crucial function in regulating gene expression and genomic integrity throughout embryogenesis. However, the nature of its involvement in the growth and development of cancer remains unknown. As mechanistic and molecular investigations on ATRX disclose its essential functions in cancer, customized therapies targeting ATRX will become accessible.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT)

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Immunohistochemical Expression of IDH1, ATRX, Ki67, GFAP, and Prognosis in Indonesian Glioma Patients.

The current World Health Organization (WHO) 2021 classification of human glioma is based on key molecular biomarkers to define neoplastic entities. This review further delineates mutant IDH (isocitrate dehydrogenase) from wild-type IDH disease, a necessity given the large survival gap between mutant IDH and wild-type IDH tumors. In Indonesia, there are currently few reports on the distribution and significance of these mutations. Therefore, this research aims to determine the relationship between IDH mutations, as well as clinicopathological and prognostic factors in patients with gliomas. Other immunohistochemical markers including ATRX (alpha-thalassemia/mental retardation, X-linked), Ki67 and GFAP (glial fibrillary acidic protein) expression were also evaluated. Forty-two glioma samples were collected from patients who underwent surgery at Dr. Kariadi General Hospital in Semarang, Central Java, Indonesia. Fresh and paraffin-embedded, formalin-fixed tissue samples were removed and sectioned for hematoxylin and eosin staining, immunohistochemistry, and IDH analysis of mutation. Medical records were used to collect clinicopathological and survival data. IDH1 mutations were discovered in 32 (76,1%) patients, and those with IDH1 mutation had longer overall survival when corresponded to patients with IDH1-wild-type. Lower expression of Ki67 was discovered to be very associated with a better prognosis. IDH1 mutations status showed a significant relationship with prognosis in patients with glioma. Meanwhile, other markers (ATRX, Ki67, and GFAP) did not correlate with the prognosis.

ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN.

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

Expression of neuroendocrine markers predicts increased survival in triple-negative breast cancer patients.

The significance of neuroendocrine (NE) markers in triple-negative breast cancer (TNBC) patients has not been investigated. This study aims to clarify the incidence and prognostic significance of NE marker expression in TNBC, determine its association with other clinicopathological parameters, and further explore the pathological features and potential treatment options for TNBC patients expressing NE markers. Clinicopathological data were collected from 396 TNBC patients undergoing radical breast cancer surgery at Peking Union Medical College Hospital from January 2002 to December 2014, with a final follow-up in July 2019. Immunohistochemistry (IHC) staining was performed for NE markers including chromogranin A (CgA) and synaptophysin (Syn). For TNBC patients with positive NE marker expression, IHC staining was then performed for alpha-thalassemia/mental retardation X-linked (ATRX), O(6)-methylguanine-methyltransferase (MGMT), somatostatin receptor 2 (SSTR2), and programmed death receptor-ligand 1 (PD-L1). The chi-square or Fisher exact test was used to evaluate the correlations between NE marker expression and other parameters. Survival curves were plotted using the Kaplan-Meier (K-M) method to assess the prognostic significance of NE markers in TNBC. NE marker-positive staining was observed in 7.6% (30/396) of all TNBC cases. Only 0.5% (2/396) cases had ≥ 90% neoplastic cells expressing NE markers. Positive NE marker expression was associated with negative basal-like marker expression. K-M survival analysis showed that the NE marker-positive TNBC patients had higher disease-free survival (DFS) rates than the NE marker-negative patients at the same stage. Among the 30 NE marker-positive TNBC cases, 13.3% and 26.7% showed negative IHC staining for ATRX and MGMT, respectively, while 13.3% had a 3+ score for SSTR2 IHC staining. For PD-L1 IHC staining, 13.3% of the 30 TNBC cases were higher than 10 scores in Combined Positive Score (CPS), and 10.0% were higher than 10% in Tumor Cell Proportion Score (TPS). There was a small proportion of TNBC patients expressing NE markers. TNBC patients with positive NE marker expression had a better prognosis than the negative group at the same stage. TNBC cases with positive NE marker expression may potentially benefit from immunotherapy or somatostatin analogue treatment.

Multicenter clinical radiomics-integrated model based on [18F]FDG PET and multi-modal MRI predict ATRX mutation status in IDH-mutant lower-grade gliomas.

To develop a clinical radiomics-integrated model based on 18 F-fluorodeoxyglucose positron emission tomography ([18F]FDG PET) and multi-modal MRI for predicting alpha thalassemia/mental retardation X-linked (ATRX) mutation status of IDH-mutant lower-grade gliomas (LGGs). One hundred and two patients (47 ATRX mutant-type, 55 ATRX wild-type) diagnosed with IDH-mutant LGGs (CNS WHO grades 1 and 2) were retrospectively enrolled. A total of 5540 radiomics features were extracted from structural MR (sMR) images (contrast-enhanced T1-weighted imaging, CE-T1WI; T2-weighted imaging, and T2WI), functional MR (fMR) images (apparent diffusion coefficient, ADC; cerebral blood volume, CBV), and metabolic PET images ([18F]FDG PET). The random forest algorithm was used to establish a clinical radiomics-integrated model, integrating the optimal multi-modal radiomics model with three clinical parameters. The predictive effectiveness of the models was evaluated by receiver operating characteristic (ROC) and decision curve analysis (DCA). The optimal multi-modal model incorporated sMR (CE-T1WI), fMR (ADC), and metabolic ([18F]FDG) images ([18F]FDG PET+ADC+ CE-T1WI) with the area under curves (AUCs) in the training and test groups of 0.971 and 0.962, respectively. The clinical radiomics-integrated model, incorporating [18F]FDG PET+ADC+CE-T1WI, three clinical parameters (KPS, SFSD, and ATGR), showed the best predictive effectiveness in the training and test groups (0.987 and 0.975, respectively). The clinical radiomics-integrated model with metabolic, structural, and functional information based on [18F]FDG PET and multi-modal MRI achieved promising performance for predicting the ATRX mutation status of IDH-mutant LGGs. • The clinical radiomics-integrated model based on [18F]FDG PET and multi-modal MRI achieved promising performance for predicting ATRX mutation status in LGGs. • The study investigated the value of multicenter clinical radiomics-integrated model based on [18F]FDG PET and multi-modal MRI in LGGs regarding ATRX mutation status prediction. • The integrated model provided structural, functional, and metabolic information simultaneously and demonstrated with satisfactory calibration and discrimination in the training and test groups (0.987 and 0.975, respectively).

ATRX protein is a potential prognostic marker in clear cell renal cell carcinoma.

Cancer cells activate either telomerase or alternative lengthening of telomeres (ALT) to maintain telomere length and achieve immortalization. Alpha thalassemia/mental retardation X-linked (ATRX) is involved in chromatin remodeling. Mutations in ATRX genes are associated with the loss of nuclear expression and correlated with the ALT phenotype. ATRX expression has been evaluated in various cancers, especially sarcoma and neuroendocrine tumors, and its clinical significance has been shown to be diverse, depending on the tumor types. The role and prognostic value of ATRX expression in clear cell renal cell carcinoma (CCRCC) have not been elucidated. We investigated the messenger RNA (mRNA) expression levels of ATRX using the gene expression profiling interactive analysis (GEPIA) database and evaluated the expression of ATRX using immunohistochemical (IHC) staining in 302 CCRCC cases. Loss of ATRX expression was significantly associated with larger tumor size, higher nuclear grade (NG), lymphovascular invasion (LVI), pathologic T (pT) stage, recurrence/metastasis, and stage. Although ATRX was not an independent prognostic factor, patients with loss of ATRX expression showed poor survival. Our findings suggest that loss of ATRX expression could be a potential biomarker for predicting aggressive tumor behavior and poor clinical outcomes in CCRCC.

Determination of IDH1, IDH2, MGMT, TERT and ATRX Gene Mutations in Glial Tumors.

The most frequent and dangerous kind of primary brain tumor is glioblastoma multiforme (GBM). The survival rates associated with GBM are very short and molecular markers for predicting survival are needed. The aim of our study was to evaluate isocitrate dehydrogenase 1 and 2 (IDH1, IDH2), telomerase reverse transcriptase (TERT), O-6- methylguanine-DNA methyltransferase (MGMT) and alpha-thalassemia/mental retardation, X-linked (ATRX) genes with next-generation sequencing (NGS) to find potential pathological mutations and their effect on survival. Thirty patients who had undergone craniotomy and were diagnosed with high-grade glioma were evaluated for this study. Peripheral blood samples were obtained from all participants. IDH1, IDH2, TERT, MGMT and ATRX genes were evaluated with next-generation sequencing from the samples. Survival analysis evaluated the effects of all these mutations on survival. The median age of the patients was 58.5 (range=11- 74) years, and 56.7% (n=17) were under 60 years of age. According to sex, male patients comprised 66.7%. Targeted NGS detected 21 chromosomal aberrations. When more than three chromosomal anomalies were accepted as a reference, anomaly in three or fewer chromosomes negatively affected overall survival (hazard ratio=2.83). Targeted NGS generates therapeutically meaningful information, providing better prognostic information than conventional histology. Our study shows that NGS provides important information on survival by helping to detect chromosomal changes that can be detected in routine blood samples. It is clear that incorporating molecular diagnostics into our standard-of-care routine will help us better understand our patients' outcomes.

Clinical implications of cell-of-origin epigenetic characteristics in non-functional pancreatic neuroendocrine tumors.

Primary non-functional pancreatic neuroendocrine tumors (NF-PanNETs) are a heterogeneous group of neuroendocrine neoplasms that display highly variable clinical behavior. Therefore, NF-PanNETs often present clinical teams with a dilemma: the uncertain metastatic potential of the tumor has to be weighed against the morbidity associated with surgical resection. Thus, rather than utilizing current radiologic thresholds, there is an urgent need for improved prognostic biomarkers. Recent studies aimed at understanding the epigenetic underpinnings of NF-PanNETs have led to the identification of tumor subgroups based on histone modification and DNA methylation patterns. These molecular profiles tend to resemble the cellular origins of PanNETs. Subsequent retrospective analyses have demonstrated that these molecular signatures are of prognostic value and, importantly, may be useful in the preoperative setting. These studies have highlighted that sporadic NF-PanNETs displaying biomarkers associated with disease progression and poor prognosis, such as alternative lengthening of telomeres, inactivating alpha thalassemia/mental retardation X-linked (ATRX) or death domain-associated protein (DAXX) gene mutations, or copy number variations, more often display alpha cell characteristics. Conversely, NF-PanNETs with beta cell characteristics often lack these unfavorable biomarkers. Alternative lengthening of telomeres, transcription factor protein expression, and possibly DNA methylation can be assessed in endoscopic ultrasound-guided tumor biopsies. Prospective studies focusing on cell-of-origin and epigenetic profile-driven decision making prior to surgery are likely to be routinely implemented into clinical practice in the near future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Circular RNA mitochondrial translation optimization 1 homologue (CircMTO1) induced by zinc finger protein 460 (ZNF460) promotes oral squamous cell carcinoma progression through the microRNA miR-320a / alpha thalassemia/mental retardation, X-linked (ATRX) axis

ABSTRACT Oral squamous cell carcinoma (OSCC) is one of the most common cancer types of head and neck cancer, accounting for 95% of all cases. However, the mechanisms underlying the pathogenesis of OSCC remain unclear. Circular RNA (CircRNA) has been extensively studied in the past decades and is a promising direction for the development of OSCC therapeutic targets. In this study, we aimed to investigate the role of circMTO1 in OSCC progression. First, we validated the characterization and expression of circMTO1 in OSCC. It was found that circMTO1 was upregulated in OSCC tumor tissues and cells. Subsequently, we conducted biological experiments. It was found that circMTO1 knockdown inhibited OSCC cell proliferation, migration, and invasion. Furthermore, we conducted a series of experiments to elucidate the underlying mechanisms. A novel circMTO1/miR-320a/ATRX axis was identified. Our results suggest that circMTO1 modulates ATRX expression to accelerate OSCC progression by sponging miR-320a. Moreover, we found that circMTO1 expression in OSCC was transcriptionally regulated by Zinc Finger Protein 460 (ZNF460). Our study showed a novel ZNF460/circMTO1/miR-320a/ATRX signaling in OSCC development.

Prognostic Significance of Altered ATRX/DAXX Gene in Pancreatic Neuroendocrine Tumors: A Meta-Analysis.

BackgroundPancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of neoplasms with increasing incidence and unpredictable behavior. Whole-exome sequencing recently has shown very frequent somatic mutations in the alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes in PanNETs. And the prognostic significance of altered ATRX/DAXX genes in PanNETs patients have been revealed in several reports. However, many of these include small sample size and hold controversial opinions. To increase statistical power, we performed a systematic review and meta-analysis to determine a pooled conclusion. We examined the impact of altered ATRX/DAXX genes mainly on overall survival (OS), disease-free survival (DFS) and relapse-free survival (RFS) in PanNETs.MethodsEligible studies were identified and quality was assessed using multiple search strategies (last search May 2021). Data were collected from studies about prognostic significance of altered ATRX/DAXX in PanNETs. Studies were pooled, and combined hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate strength of the associations.ResultsFourteen studies involving 2313 patients treated for PanNETs were included. After evaluating for publication bias, disease-free survival and relapse-free survival was significantly shortened in patients with altered ATRX/DAXX gene, with combined HR 5.05 (95% confidence interval (CI): 1.58-16.20, P = 0.01) and 3.21 (95% confidence interval (CI): 1.44-7.16, P < 0.01) respectively. However, the combined data showed there were no difference between patients with altered ATRX/DAXX gene or not in overall survival, with a combined HR 0.71 (95% confidence interval (CI): 0.44-1.15, P = 0.23). We also performed a subgroup analysis with metastatic patients in overall survival, showing a combined HR 0.22 (95% confidence interval (CI): 0.11-0.48, P = 0.96). The small number of studies and paucity of multivariate analyses are the limitations of our study.ConclusionsThis is the first rigorous pooled analysis assessing ATRX/DAXX mutation as prognostic biomarkers in PanNETs. Patients with altered ATRX/DAXX gene would have poor DFS according to the combined data. And altered ATRX/DAXX genes in metastatic patients showed a trend towards improved overall survival, although the difference did not reach statistical significance.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic...

Plasma amino acids indicate glioblastoma with ATRX loss.

Glioblastoma (GB) is the most common primary brain tumour in adults. The lack of molecular biomarker, non-specific symptoms and fast growth rate often result in asignificant delay in diagnosis. Despite multimodal treatment, the prognosis remains poor. Here, we verified the hypothesis that amino acids (AA) regulating the critical metabolic pathways necessary for maintenance, growth, reproduction, and immunity of an organism, may constitute afavourable target in GB biomarker research. We measured the plasma amino acids levels in 18GB patients and 15 controls and performed the quantitative and qualitative metabolomic analysis of free AA applying high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). We present both the raw data and the results of our statistical analysis. The majority of AA were lowered in the study group in comparison to the control group. Five of these (arginine, glutamic acid, glutamine, glycine, and histidine) differed significantly (all p < 10-5 and AUC > 0.9). Plasma levels of leucine and phenylalanine decreased in the case of GB with lost alpha-thalassemia/mental retardation X-linked (ATRX) expression on immunohistochemistry (p = 0.003 and 0.045, respectively). We demonstrated for the first time that certain plasma-free AA levels of GB patients were significantly different from those in healthy volunteers. Target profiling of plasma-free AA, identified utilizing LC-QTOF-MS, may present prognostic value by indicating GB patients with lost ATRX expression. Theon-going quest for glioma biomarkers still aims to determine the detailed metabolic profile and evaluate its impact on therapy and prognosis.

ATRX In-Frame Fusion Neuroblastoma Is Sensitive to EZH2 Inhibition via Modulation of Neuronal Gene Signatures

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

Expression and prognostic impact of alpha thalassemia/mental retardation X-linked and death domain-associated protein in human lung cancer

Molecular characterization of lung cancer specimens after radical surgery offers additional prognostic information and may help to guide adjuvant therapeutic procedures. The transcriptional regulators alpha thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) have recently been described in different cancer entities as a useful prognostic biomarker. This study was initiated to explore their protein expression patterns and prognostic value in patients with operable lung cancer disease.The protein abundance (in the following text also named protein expression) of ATRX and DAXX were analyzed by immunohistochemistry in 194 samples of squamous cell lung carcinoma (SQCLC), 111 samples of pulmonary adenocarcinoma (AC) and 40 samples of small cell lung cancer (SCLC). The protein levels of ATRX and DAXX were correlated with clinicopathological characteristics and patient outcome.ATRX showed strong protein expression in 16.2% of AC, 11.9% of SQCLC, and 42.5% of SCLC. DAXX was highly expressed in 54.9% of AC, 76.2% of SQCLC, and 82.5% of SCLC. Immunostaining of both ATRX and DAXX were seen in 14.4% of AC, 11.3% of SQCLC, and 42.5% of SCLC. High protein expression of ATRX was a favorable prognostic marker for patients with AC (hazard ratio 0.38, P = .02). Sub-group analyses showed a significant correlation between ATRX and the clinical stage of SQCLC and SCLC. Histological grading and ATRX were also significantly associated in cases of SQCLC.The presence of ATRX and DAXX are correlated with lung cancer histology. Strong ATRX protein expression is associated with a significantly longer overall survival in patients with AC.

Abstract 3414: Identifying dysregulated lncRNAs and miRNAs in low grade glioma: Uncovering signaling pathways and novel therapeutic targets

Abstract A molecular subtype of low grade gliomas (LGG) is characterized by mutations in Alpha-Thalassemia Mental Retardation X-linked (ATRX), TP53 and isocitrate dehydrogenase 1 (IDH1R132H, mIDH1). Our lab has generated a genetically engineered mouse model that harbors the characteristic genetic lesions found in this molecular subtype of LGG. We determined that mouse neurospheres (NS) bearing these mutations showed downregulation of signaling pathways involved in oligodendrocyte differentiation. The production of 2-hydroxyglutarate (2HG) by mIDH1 induces histone and DNA hypermethylation, which has been shown to impair neural precursor cells’ differentiation. We aim to investigate how the presence of mIDH1 impacts the epigenetic regulation of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs in human mutant IDH1 glioma cell cultures obtained from patients at the time of surgery. We hypothesize that the dysregulation/remodeling of lncRNAs, miRNAs and mRNAs contributes to the invasiveness, differentiation status and stemness of glioma cells. Only a handful of lncRNAs have been studied in relation to their role in neural cells’ lineage commitment and glioma stem cells’ biology. We identified 127 differentially expressed (DE) lncRNA (FDR&amp;lt;0.01) in a human primary glioma cell culture SJGBM2, which has a ATRX mutation and was stably-transfected to express mIDH1. Of the 14 DE miRNAs identified, hsa-miR-210-3p and hsa-miR-4461, which have been linked to tumor invasiveness. The results from this work will reveal novel mechanistic pathways by which lncRNA/miRNAs and differentially expressed mRNAs impact downstream signaling pathways in LGG and uncover new therapeutic targets for this molecular glioma subtype. SC is supported by the T32 Cancer Biology Training Grant, the work is supported by grants from NIH/NINDS to MGC and PRL. Citation Format: Stephen Carney, Felipe Nuñez, Pedro R. Lowenstein, Maria G. Castro. Identifying dysregulated lncRNAs and miRNAs in low grade glioma: Uncovering signaling pathways and novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3414.

Abstract 482: Recurrent glioma with lineage conversion from oligodendroglioma to astrocytoma: Another facet of glioma evolution during recurrence

Abstract Background: Following the introduction of the molecular classification of gliomas by the WHO in 2016, molecularly-proven lineage conversion during glioma recurrence has never been reported. Methods: We analyzed two cases of recurrent glioma that began as oligodendroglioma, both histologically and molecularly, but converted to astrocytoma over episodes of recurrence. Results: The cases were initially diagnosed as oligodendroglioma with 1p/19q-codeletion and mutation of isocitrate dehydrogenase 1 (IDH1)-R132H. The recurrent tumors showed loss of alpha-thalassemia/mental retardation X-linked (ATRX) expression, strong P53 positivity, and 1p/19q-nondeletion. Next generation sequencing (NGS) analysis performed on the first case confirmed the transition of molecular traits from oligodendroglioma to astrocytoma. An IDH mutation of R132H was preserved in the episodes of recurrence, but ATRX and TP53 mutations were newly acquired and TERT promoter mutation C228T was lost at the most recent recurrence. Conclusions: The issue in question for the presented cases is whether the original tumors were pure oligodendrogliomas that then transdifferentiated into astrocytomas, or whether the original tumor was an oligoastrocytoma having oligodendroglioma cells that outnumbered the astrocytoma cells and where the astrocytoma cells becoming more dominant over the episodes of recurrence. With the recognition of the possibility of lineage conversion, our study suggests that molecular examination should be performed to adjust therapeutic strategies in recurrent gliomas. Indeed, our observation of lineage conversion in glioma recurrence calls into question the current distinction drawn between oligodendroglioma, astrocytoma and oligoastrocytoma, rather than simply bidding “farewell to oligoastrocytoma”. Citation Format: Kyung-Hwa Lee, Tae-Young Jung, Shin Jung, Jae-Hyuk Lee, Kyung-Keun Kim, Hangun Kim, Kyung-Sub Moon. Recurrent glioma with lineage conversion from oligodendroglioma to astrocytoma: Another facet of glioma evolution during recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 482.

High frequency of H3K27M immunopositivity in adult thalamic glioblastoma.

Adult thalamic glioblastomas (GBM) are uncommon tumors with limited available molecular data. One of the reported molecular alterations in these tumors is the H3K27M mutation. It has been documented that H3K27M mutation is found in a high proportion of pediatric thalamic gliomas. In this study, we have analyzed the molecular alterations exclusive to adult thalamic GBM. This is a 6 years retrospective study of adult thalamic GBM patients who underwent surgical decompression of the tumor. Clinical data were obtained from the case records. Immunohistochemistry (IHC) was performed on the tumors using antibodies directed against the gene products of R132H mutant isocitrate dehydrogenase 1 (IDH1), alpha-thalassemia/mental retardation X-linked (ATRX), p53, H3K27M, H3K27me3, and V600E mutant BRAF. Molecular analyses were carried out to detect other IDH1 and IDH2 mutations, O6 -methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation, and epidermal growth factor gene (EGFR) and telomerase reverse transcriptase gene (TERT) promoter mutations. A total of 42 cases of adult thalamic GBM were studied. The mean age of presentation was 42 years with age range of 19-58 years. Male predominance was noted. All the tumors were IDH wild-type, BRAF (V600E)-immunonegative and unmethylated for MGMT promoter. H3K27M immunopositivity was noted in 60% of tumors. Of these 33.3% were from older adults above the age of 50 years. Of the H3K27M-immunopositive cases, ATRX loss of expression was seen in 32%, p53 immunopositivity in 24% and EGFR amplification in 12%. Higher frequency of TERT promoter mutations was noted in H3K27M-immunonegative cases (58.8%) compared to immunopositive cases (20%). Ours is one of the few studies elucidating the molecular alterations exclusive to adult thalamic GBM. We show a high frequency of H3K27M immunopositivity, suggestive of its mutational status in these tumors, including in older adults.