ATRX restricts Human Cytomegalovirus (HCMV) viral DNA replication through heterochromatinization and minimizes unpackaged viral genomes.

ATRX limits the accumulation of human cytomegalovirus (HCMV) Immediate Early (IE) proteins at the start of productive, lytic infections, and thus is a part of the cell-intrinsic defenses against infecting viruses. ATRX is a chromatin remodeler and a component of a histone chaperone complex. Therefore, we hypothesized ATRX would inhibit the transcription of HCMV IE genes by increasing viral genome heterochromatinization and decreasing its accessibility. To test this hypothesis, we quantitated viral transcription and genome structure in cells replete with or depleted of ATRX. We found ATRX did indeed limit viral IE transcription, increase viral genome chromatinization, and decrease viral genome accessibility. The inhibitory effects of ATRX extended to Early (E) and Late (L) viral protein accumulation, viral DNA replication, and progeny virion output. However, we found the negative effects of ATRX on HCMV viral DNA replication were independent of its effects on viral IE and E protein accumulation but correlated with viral genome heterochromatinization. Interestingly, the increased number of viral genomes synthesized in ATRX-depleted cells were not efficiently packaged, indicating the ATRX-mediated restriction to HCMV viral DNA replication may benefit productive infection by increasing viral fitness. Our work mechanistically describes the antiviral function of ATRX and introduces a novel, pro-viral role for this protein, perhaps explaining why, unlike during infections with other herpesviruses, it is not directly targeted by a viral countermeasure in HCMV infected cells.

The DNA methylome of pediatric brain tumors appears shaped by structural variation and predicts survival.

Structural variation heavily influences the molecular landscape of cancer, in part by impacting DNA methylation-mediated transcriptional regulation. Here, using multi-omic datasets involving >2400 pediatric brain and central nervous system tumors of diverse histologies from the Children's Brain Tumor Network, we report hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of somatic structural variant (SV) breakpoints is recurrently associated with altered expression or DNA methylation, respectively, including tumor suppressor genes ATRX and CDKN2A. Altered DNA methylation near enhancers associates with nearby somatic SV breakpoints, including MYC and MYCN. A subset of genes with SV-CGI methylation associations also have expression associations with patient survival, including BCOR, TERT, RCOR2, and PDLIM4. DNA methylation changes in recurrent or progressive tumors compared to the initial tumor within the same patient can predict survival in pediatric and adult cancers. Our comprehensive and pan-histology genomic analyses reveal mechanisms of noncoding alterations impacting cancer genes.

Multi-pool chemical exchange saturation transfer MRI in glioma grading, molecular subtyping and evaluating tumor proliferation.

PURPOSE: To evaluate the performance of multi-pool Chemical exchange saturation transfer (CEST) MRI in prediction of glioma grade, isocitrate dehydrogenase (IDH) mutation, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss and Ki-67 labeling index (LI), based on the fifth edition of the World Health Organization classification of central nervous system tumors (WHO CNS5). METHODS: 95 patients with adult-type diffuse gliomas were analyzed. The amide, direct water saturation (DS), nuclear Overhauser enhancement (NOE), semi-solid magnetization transfer (MT) and amine signals were derived using Lorentzian fitting, and asymmetry-based amide proton transfer-weighted (APTwasym) signal was calculated. The mean value of tumor region was measured and intergroup differences were estimated using student-t test. The receiver operating curve (ROC) and area under the curve (AUC) analysis were used to evaluate the diagnostic performance of signals and their combinations. Spearman correlation analysis was performed to evaluate tumor proliferation. RESULTS: The amide and DS signals were significantly higher in high-grade gliomas compared to low-grade gliomas, as well as in IDH-wildtype gliomas compared to IDH-mutant gliomas (all p < 0.001). The DS, MT and amine signals showed significantly differences between ATRX loss and retention in grade 2/3 IDH-mutant gliomas (all p < 0.05). The combination of signals showed the highest AUC in prediction of grade (0.857), IDH mutation (0.814) and ATRX loss (0.769). Additionally, the amide and DS signals were positively correlated with Ki-67 LI (both p < 0.001). CONCLUSION: Multi-pool CEST MRI demonstrated good potential to predict glioma grade, IDH mutation, ATRX loss and Ki-67 LI.

Evaluation of Microvascular Density in Glioblastomas in Relation to p53 and Ki67 Immunoexpression.

Glioblastoma is the most aggressive tumor in the central nervous system, with a survival rate of less than 15 months despite multimodal therapy. Tumor recurrence frequently occurs after removal. Tumoral angiogenesis, the formation of neovessels, has a positive impact on tumor progression and invasion, although there are controversial results in the specialized literature regarding its impact on survival. This study aims to correlate the immunoexpression of angiogenesis markers (CD34, CD105) with the proliferation index Ki67 and p53 in primary and secondary glioblastomas. This retrospective study included 54 patients diagnosed with glioblastoma at the Pathology Department of County Emergency Clinical Hospital Târgu Mureș. Microvascular density was determined using CD34 and CD105 antibodies, and the results were correlated with the immunoexpression of p53, IDH1, ATRX and Ki67. The number of neoformed blood vessels varied among cases, characterized by different shapes and calibers, with endothelial cells showing modified morphology and moderate to marked pleomorphism. Neovessels with a glomeruloid aspect, associated with intense positivity for CD34 or CD105 in endothelial cells, were observed, characteristic of glioblastomas. Mean microvascular density values were higher for the CD34 marker in all cases, though there were no statistically significant differences compared to CD105. Mutant IDH1 and ATRX glioblastomas, wild-type p53 glioblastomas, and those with a Ki67 index above 20% showed a more abundant microvascular density, with statistical correlations not reaching significance. This study highlighted a variety of percentage intervals of microvascular density in primary and secondary glioblastomas using immunohistochemical markers CD34 and CD105, respectively, with no statistically significant correlation between evaluated microvascular density and p53 or Ki67.

Orphan nuclear receptors-induced ALT-associated PML bodies are targets for ALT inhibition.

Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2 and TR4, are implicated in telomerase-negative cancers that maintain their telomeres through the alternative lengthening of telomeres (ALT) mechanism. However, how telomere association of orphan NRs is involved in ALT activation remains unclear. Here, we demonstrate that telomeric tethering of orphan NRs in human fibroblasts initiates formation of ALT-associated PML bodies (APBs) and features of ALT activity, including ALT telomere DNA synthesis, telomere sister chromatid exchange, and telomeric C-circle generation, suggesting de novo ALT induction. Overexpression of orphan NRs exacerbates ALT phenotypes in ALT cells, while their depletion limits ALT. Orphan NRs initiate ALT via the zinc finger protein 827, suggesting the involvement of chromatin structure alterations for ALT activation. Furthermore, we found that orphan NRs and deficiency of the ALT suppressor ATRX-DAXX complex operate in concert to promote ALT activation. Moreover, PML depletion by gene knockout or arsenic trioxide treatment inhibited ALT induction in fibroblasts and ALT cancer cells, suggesting that APB formation underlies the orphan NR-induced ALT activation. Importantly, arsenic trioxide administration abolished APB formation and features of ALT activity in ALT cancer cell line-derived mouse xenografts, suggesting its potential for further therapeutic development to treat ALT cancers.

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response.

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

ATRX guards against aberrant differentiation in mesenchymal progenitor cells.

Alterations in the tumor suppressor ATRX are recurrently observed in mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks. We additionally observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Finally, we demonstrated that loss of ATRX in a mesenchymal malignancy, undifferentiated pleomorphic sarcoma, results in similar epigenetic disruption and de-repression of transposable elements. Together, our results reveal a role for ATRX in maintaining epigenetic states and transcriptional repression in mesenchymal progenitors and tumor cells and in preventing aberrant differentiation in the progenitor context.

Image Omics Nomogram Based on Incoherent Motion Diffusion-Weighted Imaging in Voxels Predicts ATRX Gene Mutation Status of Brain Glioma Patients.

This study aimed to construct an imaging genomics nomogram based on intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) to predict the status of the alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene in patients with brain gliomas. We retrospectively analyzed routine MR and IVIM-DWI data from 85 patients with pathologically confirmed brain gliomas from January 2017 to May 2023. The data were divided into a training set (N=61) and a test set (N=24) in a 7:3 ratio. Regions of interest (ROIs) of brain gliomas, including the solid tumor region (rCET), edema region (rE), and necrotic region (rNec), were delineated using 3D-Slicer software and projected onto the D, D*, and f sequences. A total of 1037 features were extracted from each ROI, resulting in 3111 features per patient. Age was incorporated in the calculation of the Radscore, and a clinical-imaging genomics combined model was constructed, from which a nomogram graph was generated. Separate models were built for the D, D*, and f parameters. The AUC value of the D parameter model was 0.97 (95% CI: 0.93-1.00) in the training set and 0.91 (95% CI: 0.79-1.00) in the validation set, which was significantly higher than that of the D* parameter model (0.90, 0.82) and the f parameter model (0.89, 0.91). The imaging genomics nomogram based on IVIM-DWI can effectively predict the ATRX gene status of patients with brain gliomas, with the D parameter showing the highest efficacy.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice.

Myelin, an extension of the oligodendrocyte plasma membrane, wraps around axons to facilitate nerve conduction. Myelination is compromised in ATR-X intellectual disability syndrome patients, but the causes are unknown. We show that loss of ATRX leads to myelination deficits in male mice that are partially rectified upon systemic thyroxine administration. Targeted ATRX inactivation in either neurons or oligodendrocyte progenitor cells (OPCs) reveals OPC-intrinsic effects on myelination. OPCs lacking ATRX fail to differentiate along the oligodendrocyte lineage and acquire a more plastic state that favors astrocytic differentiation in vitro and in vivo. ATRX chromatin occupancy in OPCs greatly overlaps with that of the chromatin remodelers CHD7 and CHD8 as well as H3K27Ac, a mark of active enhancers. Overall, our data indicate that ATRX regulates the onset of myelination systemically via thyroxine, and by promoting OPC differentiation and suppressing astrogliogenesis. These functions of ATRX identified in mice could explain white matter pathogenesis observed in ATR-X syndrome patients.

Multiple Roles of dXNP and dADD1-Drosophila Orthologs of ATRX Chromatin Remodeler.

The Drosophila melanogaster dADD1 and dXNP proteins are orthologues of the ADD and SNF2 domains of the vertebrate ATRX (Alpha-Thalassemia with mental Retardation X-related) protein. ATRX plays a role in general molecular processes, such as regulating chromatin status and gene expression, while dADD1 and dXNP have similar functions in the Drosophila genome. Both ATRX and dADD1/dXNP interact with various protein partners and participate in various regulatory complexes. Disruption of ATRX expression in humans leads to the development of α-thalassemia and cancer, especially glioma. However, the mechanisms that allow ATRX to regulate various cellular processes are poorly understood. Studying the functioning of dADD1/dXNP in the Drosophila model may contribute to understanding the mechanisms underlying the multifunctional action of ATRX and its connection with various cellular processes. This review provides a brief overview of the currently available information in mammals and Drosophila regarding the roles of ATRX, dXNP, and dADD1. It discusses possible mechanisms of action of complexes involving these proteins.

General Clinico-Pathological Characteristics in Glioblastomas in Correlation with p53 and Ki67.

Introduction: A glioblastoma is an intra-axial brain tumour of glial origin that belongs to the category of diffuse gliomas and is the most common malignant neoplasia of the central nervous system. The rate of survival at 5 years, from the moment of diagnosis, is not higher than 10%. Materials and methods: In this retrospective study, fifty-four patients diagnosed with glioblastoma, from the Pathology Department of the County Emergency Clinical Hospital of Târgu Mureș, between 2014 and 2017 were included. We studied the clinico-pathological data (age, gender, location, and laterality) and, respectively, the immunoexpression of p53, Ki67, ATRX, and IDH-1 proteins. Results: We observed a statistically significant association between the laterality of the tumour according to the age groups, with the localization on the right side being more frequent in the age group below 65 years of age, while the involvement of the left hemisphere was more prevalent in those over 65 years. Out of the total 54 cases, 87.04% were found to be primary glioblastomas; more than 70% of the cases were ATRX immunopositive; almost 80% of the glioblastomas studied had wild-type p53 profile; and 35% of the cases were found to have a Ki67 index greater than 20%. A statistically significant association between gender and ATRX mutation was found; female cases were ATRX immunopositive in 92% of the cases. Almost 70% of the cases were both IDH-1 and p53 wild-type, and we observed the presence of both mutations in only 3.7% of the cases. Approximately 83% of primary glioblastomas were ATRX positive, respectively, and all IDH-1 mutant cases were ATRX negative. Conclusions: Glioblastomas still represent a multidisciplinary challenge considering their reserved prognosis. In this study, we described the most common clinico-pathological characteristics and IHC marker expression profiles, highlighting a variety of percentage ranges in primary and secondary glioblastomas. Given the small number of studied cases, further prospective studies on larger cohorts are needed in the future to evaluate the role of these immunohistochemical markers as prognostic factors for survival or recurrence.

An immune signature to predict the prognosis of ATRX-wildtype glioma patients and guide immune checkpoint blockade therapy.

Immune and stromal cells contribute to glioma progression by infiltrating the tumor microenvironment. We used clinical characteristics, RNA sequencing data and the ESTIMATE algorithm to obtain stromal and immune scores for alpha thalassemia retardation syndrome X-linked (ATRX)-mutation-type (ATRX-mt) and ATRX-wildtype (ATRX-wt) glioma tissues from The Cancer Genome Atlas. To identify specific immune biomarkers of glioma, we compared the gene expression profiles of ATRX-wt glioma tissues with high vs. low immune/stromal scores, and discovered 162 differentially expressed genes. The protein-protein interaction network based on these results contained 80 interacting genes, of which seven (HOXA5, PTPN2, WT1, HOXD10, POSTN, ADAMDEC1 and MYBPH) were identified as key prognostic genes via LASSO and Cox regression analyses. A risk model constructed using the expression of these seven genes could predict survival for ATRX-wt glioma patients, but was ineffective for ATRX-mt patients. T cells and macrophages were more prevalent in low-risk than in high-risk glioma tissues. Immune checkpoint blockade treatment was highly beneficial for patients with low risk scores. High-risk gliomas were predicted to be more sensitive to rapamycin, dasatinib, 5-fluorouracil and gemcitabine. Thus, our model can be used for the diagnosis, prognostic prediction and treatment planning of ATRX-wt glioma patients.

Inhibition of p53 and ATRX increases telomeric recombination in primary fibroblasts.

Telomere length can be maintained either by the telomerase enzyme or by alternative lengthening of telomeres (ALT), which is based on telomeric recombination. However, both mechanisms are inactive in most human somatic cells. ATRX has been previously identified as an ALT repressor gene. Nonetheless, TP53 is also deficient in most ALT cell lines, and previous works showed that it is an inhibitor of homologous recombination (HR). Despite this, the role of p53 as an ALT repressor has not been previously examined. Therefore, we investigated the effects of p53 and ATRX inhibition on normal human fibroblasts (devoid of any mutation), in the presence or absence of X-ray-induced telomeric damage. Performing immunofluorescence with antibodies for RAD51, H2AX, and TRF1 (for studying HR-mediated DNA damage repair) and CO-FISH (for telomeric sister chromatid exchanges), we observed that HR is a normal mechanism for the repair of telomeric damage, present also in noncancer cells. Moreover, we discovered that telomeric HR, as for HR in general, is significantly inhibited by p53. Indeed, we observed that inhibition of p53 drastically increases telomeric sister chromatid exchanges. We also confirmed that ATRX inhibition increases telomeric recombination. In particular, we observed an increase in crossover products, but a much higher increase in noncrossover products.

LncRNA PWRN2 promotes polycystic ovary syndrome progression via epigenetically reducing ATRX by recruiting LSD1.

Long non-coding RNA has been shown to mediate the progression of polycystic ovary syndrome (PCOS). However, the role and mechanism of Prader-Willi region nonprotein coding RNA 2 (PWRN2) in PCOS progression remain unclear. In our study, Sprague-Dawley rat was injected with dehydroepiandrosterone to mimic PCOS rat models. HE staining was used to assess the number of benign granular cells, and serum insulin and hormone levels were detected by ELISA kit. The expression of PWRN2 was examined by qRT-PCR. Ovarian granulosa cells (GCs) proliferation and apoptosis were examined by CCK-8 assay and flow cytometry. The protein levels of apoptosis markers and Alpha thalassemia retardation syndrome X-linked (ATRX) were determined by western blot. The interaction between lysine-specific demethylase 1 (LSD1) and PWRN2 or ATRX was confirmed by RIP and ChIP assay. Our data showed that PWRN2 was upregulated and ATRX was downregulated in the ovarium tissues and serum of PCOS rat. PWRN2 knockdown promoted GCs proliferation and inhibited apoptosis. In the mechanism, PWRN2 inhibited ATRX transcription by binding with LSD1. In addition, downregulation of ATRX also eliminated the effect of sh-PWRN2 on GCs growth. In conclusion, our data suggested that PWRN2 might restrain GCs growth to promote PCOS progression, which was achieved by binding with LSD1 to inhibit ATRX transcription.

Endoscopic ultrasound fine-needle biopsy to assess DAXX/ATRX expression and alternative lengthening of telomeres status in non-functional pancreatic neuroendocrine tumors.

BACKGROUND/OBJECTIVES: Death domain-associated protein (DAXX) and/or α-thalassemia/mental retardation X-linked (ATRX) chromatin remodeling genes mutations and alternative lengthening of telomeres (ALT) activation are associated with more aggressive behavior of non-functional pancreatic neuroendocrine tumors (NF-PanNETs). We aimed to evaluate the reliability of such markers on endoscopic-ultrasound fine-needle biopsy (EUS-FNB) specimens. METHODS: Patients who underwent EUS-FNB and subsequent surgical resection for PanNETs between January 2017 and December 2019 were retrospectively identified. Immunohistochemistry (IHC) to evaluate DAXX/ATRX expression and fluorescence in situ hybridization (FISH) for ALT status were performed. Primary outcome was the concordance rate of markers expression between EUS-FNB and surgical specimens. Secondary aims were association between markers and lesion aggressiveness, their diagnostic performance in predicting aggressiveness, and agreement of preoperative and post-surgical Ki67-based grading. RESULTS: Forty-one NF-PanNETs (mean diameter 36.1 ± 26.5 mm) were included. Twenty-four showed features of lesion aggressiveness. Concordance of expressions of DAXX, ATRX, and ALT status between EUS-FNB and surgical specimens were 95.1% (κ = 0.828; p < 0.001), 92.7% (κ = 0.626; p < 0.001), and 100% (κ = 1; p < 0.001), respectively. DAXX/ATRX loss and ALT-positivity were significantly (p < 0.05) associated with metastatic lymphnodes and lymphovascular invasion. The combination of all tumor markers (DAXX/ATRX loss + ALT-positivity + grade 2) reached an accuracy of 73.2% (95%CI 57.1-85.8) in identifying aggressive lesions. Pre- and post-operative ki-67-based grading was concordant in 80.5% of cases (k = 0.573; p < 0.001). CONCLUSION: DAXX/ATRX expression and ALT status can be accurately evaluated in a preoperative setting on EUS-FNB samples, potentially improving the identification of patients with increased risk and poorer prognosis.

Somatostatin receptor activity assessed by 68Ga-DOTATOC PET can preoperatively predict DAXX/ATRX loss of expression in well-differentiated pancreatic neuroendocrine tumors.

PURPOSE: To evaluate the role of 68Ga-DOTATOC PET parameters in predicting DAXX/ATRX loss of expression in patients with Pancreatic neuroendocrine tumors (PanNET) candidate to surgery. METHODS: This retrospective study included 72 consecutive patients with PanNET (January 2018-March 2022) who underwent to 68Ga-DOTATOC PET for preoperative staging. Image analysis: qualitative assessment and extraction of SUVmax, SUV mean, somatostatin receptor density (SRD), and total lesion somatostatin receptor density (TLSRD) from primary PanNET. Radiological diameter and biopsy information (grade, Ki67) were collected. Loss of expression (LoE) of DAXX/ATRX was assessed by immunohistochemistry on surgical specimen. Student t-test, univariate and multivariate logistic regression and ROC curves have been used to investigate the predictive value of PET parameters on DAXX/ATRX LoE. RESULTS: Forty-two/72 patients had a G1, 28/72 a G2, and 2/72 a G3 PanNET. Seven/72 patients had DAXX LoE, 10/72 ATRX LoE, and 2/72 DAXX/ATRX LoE. SRD and TLSRD could predict DAXX LoE (p = 0.002, p = 0.018, respectively). By evaluating SRD in combination with radiological diameter, only SRD maintained statistical significance (multivariate logistic regression: p = 0.020, OR = 1.05), providing the best prediction (AUC-ROC = 79.01%; cut-off = 46.96; sensitivity = 77.78%; specificity = 88.89%). In the sub-analysis performed on 55 patients with biopsy availability, SRD demonstrated its role in providing useful and additional information (multivariate logistic regression: SRD p = 0.007; grade p = 0.040). CONCLUSION: SRD has a predictive role on DAXX LoE in PanNETs, with higher probability of LoE at increasing SRD values. SRD provides complementary/additional information to grade assessed on biopsy material, and the combined use of these approaches might support patients' management by preoperatively identifying subjects with more aggressive diseases.

Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.

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.

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.