Death Domain-associated Protein Research Articles

CALML3-AS1 enhances malignancies and stemness of small cell lung cancer cells through interacting with DAXX protein and promoting GLUT4-mediated aerobic glycolysis

The lncRNA CALML3 antisense RNA 1 (CALML3-AS1) is a biomarker for various cancers, including non-small cell lung cancer (NSCLC). However, the role of CALM3-AS1 in small cell lung cancer (SCLC) is still unclear. Here, we found that the CALML3-AS1 was upregulated in SCLC tissues and cells. SCLC cells (NCI-H69 and NCI-H466 cells) were transfected with small interfering RNA of CALML-AS1 (si-CALML3-AS1) and Death domain-associated protein (DAXX) (si-DAXX) or an overexpression vector of CALML-AS1 (dCas9-CALML3-AS1) and DAXX (dCas9-DAXX). The results showed that silencing CALML3-AS1 inhibited SCLC cell proliferation, colony formation, migration, invasion, and spheroid formation, and reduced the expression of stemness marker proteins (Nanog. Oct4, and Lin28). Moreover, silencing CALML3-AS1 reduced glycolysis rate, glucose utilization, and lactate production, and decreased the levels of key glycolytic regulatory proteins (GLUT1, GLUT4, HK2, and PKM2) in SCLC cells, while overexpression of CALML3-AS1 promoted malignant growth and stemness and enhanced glucose transporters type 4 (GLUT4)-mediated aerobic glycolysis by interacting with DAXX in NCI-H69 and NCI-H466 cells. Silencing DAXX or GLUT4, or treatment with 2-Deoxy-d-glucose (2-DG, a glycolysis inhibitor) reversed the effects of CALML3-AS1 overexpression on aerobic glycolysis, malignant growth, and stemness of SCLC cells. Finally, NCI-H69 cells transfected with CALML3-AS1, sh-CALML3-AS1, and sh-DAXX lentiviral vectors were subcutaneously injected into nude mice to construct xenograft models. Knockdown of CALML3-AS1 or DAXX inhibited tumor growth in SCLC in vivo. In conclusion, CALML3-AS1, an oncogene, promotes the malignancy and stemness of SCLC cells by interacting with DAXX to enhance GLUT4-mediated aerobic glycolysis, thereby promoting SCLC progression.

Prognostic and predictive significance of p53 and ATRX in neuroendocrine neoplasms of GIT and pancreas and their utility as an adjunct to accurate diagnosis-An eight-year retrospective study.

Neuroendocrine neoplasms of gastrointestinal tract (GIT)and pancreas are heterogenous tumors. World Health Organization (WHO)2019 classification introduced Grade (G)3 neuroendocrine tumor (NET) distinct from neuroendocrine carcinoma (NEC), based on molecular differences and to triage the patients for appropriate therapy. This distinction largely relies on morphology, which can be challenging at times. Genomic profiling has revealed TP53andRB1mutations in NECs, while death domain-associated protein 6 (DAXX) and alpha-thalassemia/mental retardation X-linked (ATRX),in G3NET. Their role as biological markers in differentiating these entities and their significance as prognostic markers are not yet established. This study aims at analyzing the diagnostic and prognostic role of p53 and ATRX in neuroendocrine neoplasms of GIT and pancreas. A single-centre, eight-year retrospective study of neuroendocrine neoplasm of GIT and pancreas comprised G2NET, G3NET and NEC. Tumor slides were stained by immunohistochemistry for p53 and ATRX. Strong nuclear staining of > 50% of tumor cells for p53 was considered mutated. Nuclear staining of ATRX in < 5% of tumor cells was considered ATRX loss. Expression of p53 and ATRX was analyzed and correlated with tumor grades and patient survival. Fifty-five patients with gastro-entero-pancreatic neuroendocrine neoplasm were studied, comprising G2NET (58%), G3NET (16%) and NEC (26%). Median age of diagnosis was 59years with male predominance. The pancreas was the most common site followed by the small bowel. NEC showed lower survival compared to G3 and G2NET. Mutated p53 immunohistochemical expression was more frequent among NEC than G3NET. Patients with mutated p53 had significantly lower survival irrespective of the grade (p = 0.001). There was no association of ATRX loss with grade or survival. G3NETs are genetically different from NECs. Use of immunohistochemistry for p53 in addition to histomorphology may facilitate accurate categorization of NEC and G3NET. Mutated p53 may also be used as an independent prognostic marker in neuroendocrine tumors of GIT and pancreas.

The Impact of DAXX, HJURP and CENPA Expression in Uveal Melanoma Carcinogenesis and Associations with Clinicopathological Parameters.

Uveal melanomas (UMs) represent rare malignant tumors associated with grim prognosis for the majority of patients. DAXX (Death Domain-Associated Protein), HJURP (Holliday Junction Recognition Protein) and CENPA (Centromere Protein A) proteins are implicated in epigenetic mechanisms, now in the spotlight of cancer research to better understand the molecular background of tumorigenesis. Herein, we investigated their expression in UM tissues using immunohistochemistry and explored possible correlations with a multitude of clinicopathological and survival parameters. The Cancer Genome Atlas Program (TCGA) was used for the investigation of their mRNA levels in UM cases. Nuclear DAXX expression correlated with an advanced T-stage (p = 0.004), while cytoplasmic expression marginally with decreased disease-free survival (DFS) (p = 0.084). HJURP nuclear positivity also correlated with advanced T-status (p = 0.054), chromosome 3 loss (p = 0.042) and increased tumor size (p = 0.03). More importantly, both nuclear and cytoplasmic HJURP immunopositivity correlated with decreased overall survival (OS) (p = 0.011 and 0.072, respectively) and worse DFS (p = 0.071 and 0.019, respectively). Lastly, nuclear CENPA overexpression was correlated with presence of irido-corneal angle involvement (p = 0.015) and loss of chromosome 3 (p = 0.041). Nuclear and cytoplasmic CENPA immunopositivity associated with decreased OS (p = 0.028) and DFS (p = 0.018), respectively. HJURP and CENPA mRNA overexpression exhibited strong association with tumor epithelioid histology and was linked to worse prognosis. Our results show the compounding role of DAXX, HJURP and CENPA in UM carcinogenesis, designating them as potential biomarkers for assessing prognosis and possible targets for novel therapeutic interventions.

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.

Estrogen signalling and Alzheimer's disease: Decoding molecular mechanisms for therapeutic breakthrough.

In females, Alzheimer's disease (AD) incidences increases as compared to males due to estrogen deficiency after menopause. Estrogen therapy is the mainstay therapy for menopause and associated complications. Estrogen, a hormone with multifaceted physiological functions, has been implicated in AD pathophysiology. Estrogen plays a crucial role in amyloid precursor protein (APP) processing and overall neuronal health by regulating various factors such as brain-derived neurotrophic factor (BDNF), intracellular calcium signalling, death domain-associated protein (Daxx) translocation, glutamatergic excitotoxicity, Voltage-Dependent Anion Channel, Insulin-Like Growth Factor 1 Receptor, estrogen-metabolising enzymes and apolipoprotein E (ApoE) protein polymorphisms. All these factors impact the physiology of postmenopausal women. Estrogen replacement therapies play an important treatment strategy to prevent AD after menopause. However, use of these therapies may lead to increased risks of breast cancer, venous thromboembolism and cardiovascular disease. Various therapeutic approaches have been used to mitigate the effects of estrogen on AD. These include hormone replacement therapy, Selective Estrogen Receptor Modulators (SERMs), Estrogen Receptor Beta (ERβ)-Selective Agonists, Transdermal Estrogen Delivery, Localised Estrogen Delivery, Combination Therapies, Estrogen Metabolism Modulation and Alternative Estrogenic Compounds like genistein from soy, a notable phytoestrogen from plant sources. However, mechanism via which these approaches modulate AD in postmenopausal women has not been explained earlier thoroughly. Present review will enlighten all the molecular mechanisms of estrogen and estrogen replacement therapies in AD. Along-with this, the association between estrogen, estrogen-metabolising enzymes and ApoE protein polymorphisms will also be discussed in postmenopausal AD.

The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Abstract 4451: Regulation of lipid metabolism and ferroptosis by DAXX

Abstract Intracellular lipid production in cancer cells supplies lipids to synthesize cell membranes and signaling molecules during rapid cell proliferation and tumor growth. Cancer cells also utilize fatty acid oxidation (FAO) to generate ATP to meet their energy demand. Notably, lipid metabolites can inhibit and trigger ferroptosis due to iron-dependent oxidation of polyunsaturated fatty acids (PUFAs). Therefore, identifying regulators that maintain the intricate balance of lipid biosynthesis required for cell proliferation and survival is critical in cancer biology and therapy. Lipid metabolism is regulated by two oncogenic signaling pathways: the RAS-RAF-MEK-MAPK and the mammalian target of rapamycin (mTOR) pathways. About 30% of all cancers harbor constitutively active mutations in KRAS, HRAS, or NRAS, resulting in hyperactive RAS-RAF-MEK-MAPK signaling to drive tumorigenesis, metastatic progression, immune evasion, and resistance to therapy. KRAS regulates lipid uptake, lipid synthesis, and FAO. mTOR is a serine/threonine kinase acting as a key intracellular signaling hub to regulate nutrient homeostasis, metabolism, protein synthesis, and autophagy. The mTORC1 complex promotes lipogenesis. The RAS and mTOR signaling pathways exhibit both positive and negative cross-regulation. The coordinated activity of both pathways is critical to sustained tumor growth. Notably, mTORC1 signaling inhibition enhances RAS-RAF-MEK-MAPK signaling to promote cancer cell survival and proliferation. Furthermore, constitutive mTORC1 signaling induces cell death when the supply of unsaturated FAs is limited. However, the molecular link for coordinating the activity of the RAS and mTOR signaling pathways remains poorly defined. Death domain-associated protein (DAXX) is essential for mouse embryonic development and has ill-defined pro-cell survival functions. Our lab has a long-standing interest in deciphering DAXX’s complex biological functions. Our recent study shows that DAXX drives tumorigenesis by promoting lipogenic gene expression and lipid synthesis through interacting with sterol regulatory element-binding proteins SREBP1 and SREBP2 (SREBP1/2) (Mahmud et al., 2023, PMID 37045819). Unexpectedly, our new data reveal that DAXX inhibits ferroptosis. Significantly, phosphorylation via RAS signaling appears to regulate DAXX’s activity in lipid synthesis and resistance to ferroptosis. Remarkably, disruption of DAXX-mediated lipid synthesis potentiates ferroptosis due to mTORC1 activity. Based on these observations, we propose that stimulated by the RAS-RAF-MEK-MAPK oncogenic signaling, DAXX promotes tumorigenesis through upregulating genes for lipid synthesis and resistance to ferroptosis, and that the lack of DAXX-mediated lipid synthesis leads to sensitization of cells to ferroptosis due to mTORC1’s proliferative effects. (Supported by FDOH grant 23K03). Citation Format: Wenlin Yang, Nikee Awasthee, Qi Chen, Seth Hale, Daiqing Liao. Regulation of lipid metabolism and ferroptosis by DAXX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4451.

X Ray-Induced Insulinoma Cell Line Rin-5F Has a Novel Mutation Site, C.A1459G (P.T487A), in Death Domain Associated Protein (DAXX) Gene

A popular toxicological and pharmacological research cell line is the insulin-secreting pancreatic cell line Rin-5F. The cell line originates from insulinomas induced by X-ray exposure. The author of this report looked at the mutation status of the DAXX gene in the Rin-5F cell line clone.&lt;em&gt; &lt;/em&gt;The complete DNA and RNA were extracted from the cultivated cells as well. Double-stranded cDNA was then synthesized using the RNA template. Sequencing was done using a 3730xl DNA Analyzer.&lt;em&gt; &lt;/em&gt;In the present study, c.A1459G (p.T487A) in Exon 5 in the DAXX gene was detected in Rin-5F cell lines, one of the X-ray-induced insulinomas. An NCBI homology search reveals that the 487 amino acid site in rats is the 497 amino acid of humans, based on the genomic cDNA homology between rats and humans. In humans, the COSMIC database suggests that mutations involving 497 amino acids have not been detected in all human cancers. However, the mutation of 496 amino acids was detected in human stomach and colon cancers. This is the first account of the DAXX gene's state in a cell line created by exposure to X-rays. This may point to the need for additional data and research on unique gene alterations involved in the development of X-ray-induced insulinoma tumors.

Death domain-associated protein (Daxx) impairs colon cancer chemotherapy by inhibiting the cGAS-STING pathway

Death domain-associated protein (Daxx) impairs colon cancer chemotherapy by inhibiting the cGAS-STING pathway

Supplementation of dietary areca nut extract modulates the growth performance, cecal microbiota composition, and immune function in Wenchang chickens.

The study was aimed at evaluating the effects of areca nut extract (ANE) on the growth performance, cecal microbiota, and immunity of Wenchang chickens. For this study, 42-day-old healthy Wenchang chickens (n = 450) with similar body weight were chosen. The animals were randomly divided into five groups, with six replicates per group and 15 chickens per replicate. One group was fed a basal diet (control; CCK). The remaining four groups were fed a basal diet supplemented with varying ANE concentrations: 0.038, 0.063, 0.100, and 0.151 g/kg, with the groups denoted as CNT1, CNT2, CNT3, and CNT4, respectively. The feeding experiment lasted 35 days. The ligated cecum segments of the control and experimental groups were collected for metabolomic and metagenomic analysis, while the bone marrow samples were extracted for tandem mass tag (TMT)-based proteomic analysis. All the experimental groups exhibited significantly higher average daily gain (ADG) and significantly lower feed-to-weight (F/G) ratios than CCK. Metabolomic screening of the cecum contents revealed the presence of 544 differential metabolites, including several gut health-related metabolites, such as xanthine, hydroxy hypoxanthine, 2,5-dimethylhydrazine, ganoderic acid, and 2-aminohexanoic acid. Metagenomic analysis of the cecum contents showed an upregulation in the abundance of Prevotella spp. in the experimental groups. However, we observed no significant differences in the abundances of other cecal microbes at phylum and genus levels. Furthermore, we observed significant associations between Prevotella spp. and the differentially abundant metabolites, such as cherubins, thiaburimamide, and 3,4-dihydroxy-L-phenylalanine, (r)-mevalonate, 5-O-methylalloptaeroxylin, nalidixic acid, and deoxyloganin (p < 0.05). Proteomic analysis revealed that the differentially expressed proteins (such as interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), MHC-BF1, and death domain-associated protein (Daxx)) in the bone marrow of the chickens were primarily enriched in the immune network for IgA production and B cell receptor signaling pathway. In conclusion, dietary ANE supplementation was found to enhance metabolic activity and energy utilization, improve growth performance, modulate cecal microbiota, and strengthen the immunity of Wenchang chickens.

The Clinical Impact of Death Domain-Associated Protein and Holliday Junction Recognition Protein Expression in Cancer: Unmasking the Driving Forces of Neoplasia.

Death domain-associated protein (DAXX) and Holliday junction recognition protein (HJURP) act as chaperones of H3 histone variants H3.3 and centromere protein A (CENPA), respectively, and are implicated in many physiological processes, including aging and epigenetic regulation, by controlling various genes' transcription and subsequently protein expression. Research has highlighted both these biomolecules as participants in key procedures of tumorigenesis, including cell proliferation, chromosome instability, and oncogene expression. As cancer continues to exert a heavy impact on patients' well-being and bears substantial socioeconomic ramifications, the discovery of novel biomarkers for timely disease detection, estimation of prognosis, and therapy monitoring remains of utmost importance. In the present review, we present data reported from studies investigating DAXX and HJURP expression, either on mRNA or protein level, in human tissue samples from various types of neoplasia. Of note, the expression of DAXX and HJURP has been associated with a multitude of clinicopathological parameters, including disease stage, tumor grade, patients' overall and disease-free survival, as well as lymphovascular invasion. The data reveal the tumor-promoting properties of DAXX and HJURP in a number of organs as well as their potential use as diagnostic biomarkers and underline the important association between aberrations in their expression and patients' prognosis, rendering them as possible targets of future, personalized and precise therapeutic interventions.

HPV16 E7 protein antagonizes TNF-α-induced apoptosis of cervical cancer cells via Daxx/JNK pathway

Human papillomavirus (HPV) E7 protein as an important viral factor was involved in the progression of cervical cancer by mediating the cellular signaling pathways. Daxx (Death domain-associated protein) can interact with a variety of proteins to affect the viral infection process. However, the interaction and its related function between HPV16 E7 and Daxx have not been adequately investigated. Here, it was found that HPV16 E7 can interact with Daxx in HeLa or C33A cells by co-immunoprecipitation. HPV16 E7 protein treatment can up-regulate Daxx protein expression, while the interference in Daxx expression and the agonists for JNK can both reduce the antagonistic effects of HPV16 E7 on TNF-α-induced apoptosis, suggesting that Daxx/JNK pathway may be involved in the anti-apoptotic activity of HPV16 E7.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.

RIPK3 activation promotes DAXX-dependent neuronal necroptosis after intracerebral hemorrhage in mice.

Necroptosis induced by receptor-interacting protein kinase 3 (RIPK3) is engaged in intracerebral hemorrhage (ICH) pathology. In this study, we explored the impact of RIPK3 activation on neuronal necroptosis and the mechanism of the death domain-associated protein (DAXX)-mediated nuclear necroptosis pathway after ICH. Potential molecules linked to the progression of ICH were discovered using RNA sequencing. The level of DAXX was assessed by quantitative real-time PCR, ELISA, and western blotting. DAXX localization was determined by immunofluorescence and immunoprecipitation assays. The RIPK3 inhibitor GSK872 and DAXX knockdown with shRNA-DAXX were used to examine the nuclear necroptosis pathway associated with ICH. Neurobehavioral deficit assessments were performed. DAXX was increased in patients and mice after ICH. In an ICH mouse model, shRNA-DAXX reduced brain water content and alleviated neurologic impairments. GSK872 administration reduced the expression of DAXX. shRNA-DAXX inhibited the expression of p-MLKL. Immunofluorescence and immunoprecipitation assays showed that RIPK3 and AIF translocated into the nucleus and then bound with nuclear DAXX. RIPK3 revitalization promoted neuronal necroptosis in ICH mice, partially through the DAXX signaling pathway. RIPK3 and AIF interacted with nuclear DAXX to aggravate ICH injury.

ATR protects centromere identity by promoting DAXX association with PML nuclear bodies

Centromere protein A (CENP-A) defines centromere identity and nucleates kinetochore formation for mitotic chromosome segregation. Here, we show that ataxia telangiectasia and Rad3-related (ATR) kinase, a master regulator of the DNA damage response, protects CENP-A occupancy at interphase centromeres in a DNA damage-independent manner. In unperturbed cells, ATR localizes to promyelocytic leukemia nuclear bodies (PML NBs), which house the histone H3.3 chaperone DAXX (death domain-associated protein 6). We find that ATR inhibition reduces DAXX association with PML NBs, resulting in the DAXX-dependent loss of CENP-A and an aberrant increase in H3.3 at interphase centromeres. Additionally, we show that ATR-dependent phosphorylation within the C terminus of DAXX regulates CENP-A occupancy at centromeres and DAXX localization. Lastly, we demonstrate that acute ATR inhibition during interphase leads to kinetochore formation defects and an increased rate of lagging chromosomes. These findings highlight a mechanism by which ATR protects centromere identity and genome stability.

The Impact of Human Mammary Tumor Virus (HMTV) on the Expression of Epidermal Growth Factor Receptor (EGFR) and Death-Domain Associated Protein (DAXX) in Breast Carcinoma Tissues.

The presence and characteristics of HMTV in Iraqi breast cancer women are still unknown. Furthermore, the identification of HMTV in human breast carcinoma tissue of patients differs by country, and the factors that influence it are still unknown. In many epithelial tumor types, the EGFR and its signaling pathways outcomes are necessary for the behavior of cells and regulating their proliferation, and it has been discovered that DAXX has strong carcinogenic characteristics and could be a new treatment target. This case-control retrospective study investigated the presence of HMTV in paraffin-embedded blocks (FFPT) of tumor samples from 60 Iraqi women patients diagnosed with primary breast cancer and 20 cases of benign tumors as a control group. HMTV env sequences were identified by Real-time PCR. EGFR and DAXX expression were immun0-detected by the immuno-histochemistry technique. HMTV sequences were detected in 15 (25%) samples of malignant breast tumors and 8 (40%) samples of benign breast tumors. There was no statistically significant association between the detection of env sequences of HMTV and age, grade, hormone receptors, EGFR, or DAXX expression compared to clinicopathological characteristics. However, statistically, the data showed a highly significant difference in the Expression of EGFR between study groups, age, and histological types (P=0.0001), and a significant negative association was observed between EGFR and both Her2 and TNBC. There was a statistically significant difference between DAXX (+) and DAXX (-) in study groups (P=0.002), and it was significantly associated with age and histological types of breast cancer (P=0.031 and 0.007, respectively). No significant association was found between DAXX and EGFR, grade, Her2. TNBC of breast cancer. The current study found HMTV env sequences in breast tumors of Iraqi women, suggesting that a larger sample size is needed to illustrate the potential causative role of HMTV in the development of human breast malignancy. Moreover, a negative association was found between HMTV and DAXX and EGFR Expression.

The role of DNA methylation in human pancreatic neuroendocrine tumours.

Pancreatic neuroendocrine tumours (PNETs) are the second most common pancreatic tumour. However, relatively little is known about their tumourigenic drivers, other than mutations involving the multiple endocrine neoplasia 1 (MEN1), ATRX chromatin remodeler, and death domain-associated protein genes, which are found in ~40% of sporadic PNETs. PNETs have a low mutational burden, thereby suggesting that other factors likely contribute to their development, including epigenetic regulators. One such epigenetic process, DNA methylation, silences gene transcription via 5'methylcytosine (5mC), and this is usually facilitated by DNA methyltransferase enzymes at CpG-rich areas around gene promoters. However, 5'hydroxymethylcytosine, which is the first epigenetic mark during cytosine demethylation, and opposes the function of 5mC, is associated with gene transcription, although the significance of this remains unknown, as it is indistinguishable from 5mC when conventional bisulfite conversion techniques are solely used. Advances in array-based technologies have facilitated the investigation of PNET methylomes and enabled PNETs to be clustered by methylome signatures, which has assisted in prognosis and discovery of new aberrantly regulated genes contributing to tumourigenesis. This review will discuss the biology of DNA methylation, its role in PNET development, and impact on prognostication and discovery of epigenome-targeted therapies.

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.

Stitched peptides as potential cell permeable inhibitors of oncogenic DAXX protein.

DAXX (Death Domain Associated Protein 6) is frequently upregulated in various common cancers, and its suppression has been linked to reduced tumor progression. Consequently, DAXX has gained significant interest as a therapeutic target in such cancers. DAXX is known to function in several critical biological pathways including chromatin remodelling, transcription regulation, and DNA repair. Leveraging structural information, we have designed and developed a novel set of stapled/stitched peptides that specifically target a surface on the N-terminal helical bundle domain of DAXX. This surface serves as the anchor point for binding to multiple interaction partners, such as Rassf1C, p53, Mdm2, and ATRX, as well as for the auto-regulation of the DAXX N-terminal SUMO interaction motif (SIM). Our experiments demonstrate that these peptides effectively bind to and inhibit DAXX with a higher affinity than the known interaction partners. Furthermore, these peptides release the auto-inhibited SIM, enabling it to interact with SUMO-1. Importantly, we have developed stitched peptides that can enter cells, maintaining their intracellular concentrations at nanomolar levels even after 24 hours, without causing any membrane perturbation. Collectively, our findings suggest that these stitched peptides not only serve as valuable tools for probing the molecular interactions of DAXX but also hold potential as precursors to the development of therapeutic interventions.

Energy landscape quantifications of histone H3.3 recognition by chaperone DAXX reveal an uncoupled binding specificity and affinity.

Histone variant H3.3 differs from the canonical histone H3.1 by only five amino acids, yet its chaperone death domain-associated protein (DAXX) can specifically recognize H3.3 over H3.1, despite having a large DAXX-interacting surface on the H3.3-H4 heterodimer common to that on the H3.1-H4 complex. This observation gives rise to the question of, from the binding energy point view, how high binding specificity may be achieved with small differences of the overall binding energy for protein-protein interactions in general. Here we investigate the mechanism of coupling of binding specificity and affinity in protein-protein interactions using the DAXX-H3.3-H4 complex as a model. Using a multi-scale method, we found that the hydrophobic interactions between DAXX and the H3.3-specific region contributed to their initial binding process. And the structural flexibility of the interacting partners contributed to the binding affinity after their encounter. By quantifying the free energy landscape, we revealed that the interaction between the specific residues of H3.3 and DAXX decreased the encounter barrier height while the folding of H3.3-H4 and DAXX increased the depth of the free energy basin of the final binding state. The encounter barrier height, which is not coupled to the thermodynamic stability of the final binding state, had a marked effect on the initial binding rate of flexible histones and chaperones. Based on the energy landscape theory, we found that the intrinsic binding energy funnel of this uncoupled recognition process was affected by the structural flexibility and the flexibility modulated the degree of coupling between binding specificity and affinity. Our work offers a biophysical explanation of the specific recognition between the histones and their chaperones, and also extends the use of energy landscape theory for understanding molecular recognitions in general.