Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

[Characterization of sequences, expression profiling, and natural allelic variation analysis of the MYC gene family in sorghum (Sorghum bicolor)].

Sorghum aphid (Melanaphis sacchari) and head smut fungi (Sporisorium reilianum) infesting sorghum cause delayed growth and development, and reduce yield and quality. This study use bioinformatics and molecular biological approaches to profile the gene expression pattern during sorghum development and under pest infestation, and analyzed the natural allelic DNA variation of sorghum MYC gene family. The findings provide insights for potential application in breeding the stress resistant and high productivity sorghum varieties. The results indicated that there are 28 MYC genes identified in sorghum genome, distributed on 10 chromosomes. The bHLH_MYC_N and HLH domains are the conserved domains of the MYC gene in sorghum. Gene expression analysis showed that SbbHLH35.7g exhibited high expression levels in leaves, SbAbaIn showed strong expression in early grains, and SbMYC2.1g showed high expression levels in mature pollen. In anti-aphid strains at the 5-leaf stage, SbAbaIn, SbLHW.4g and SbLHW.2g were significantly induced in leaves, while SbbHLH35.7g displayed the highest expression level in panicle tissue, which was significantly induced by the infection of head smut. Promoter cis-element analysis identified methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA) and MYB-binding sites related to drought-stress inducibility. Furthermore, genomic resequencing data analysis revealed natural allelic DNA variations such as single nucleotide polymorphism (SNP) and insertion-deletion (INDEL) for the key SbMYCs. Protein interaction network analysis using STRING indicated that SbAbaIn interacts with TIFYdomain protein, and SbbHLH35.7g interacts with MDR and imporin. SbMYCs exhibited temporal and spatial expression patterns and played vital roles during the sorghum development. Infestation by sugarcane aphids and head smut fungi induced the expression of SbAbaIn and SbbHLH35.7g, respectively. SbAbaIn modulated the jasmonic acid (JA) pathway to regulate the expression of defensive genes, conferring resistance to insects. On the other hand, SbbHLH35.7g participated in detoxification reactions to defend against pathogens.

EIF2Ss, a Novel c-Myc-Correlated Gene Family, is Associated with Poor Prognosis and Immune Infiltration in Pancreatic Adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor in urgent need of novel diagnostics, prognostic markers, and treatments. Eukaryotic translation initiation factor 2 subunits (EIF2Ss), comprising Eukaryotic translation initiation factor 2 subunit alpha (EIF2S1), Eukaryotic translation initiation factor 2 subunit beta (EIF2S2), and Eukaryotic translation initiation factor 2 subunit gamma (EIF2S3), is a family of eukaryotic initiation factors that participate in early protein synthesis and are crucial for tumor initiation and progression. However, the role of EIF2Ss in PAAD has yet to be reported. The aim of this study was therefore to analyze EIF2Ss in relation to the diagnosis, prognosis, and treatment of PAAD. METHODS: The cancer genome atlas (TCGA) database was used to investigate gene expression and patient survival. Gene alterations, immune cell infiltration, and immune checkpoints in PAAD were also evaluated. Univariate and multivariate analysis, nomograms, calibration curves, and Decision Curve Analysis (DCA) diagrams were used to develop and evaluate a prediction model for patient outcome. Single-cell RNA-seq (scRNA) analysis, functional enrichment, co-IP assay, mass spectrometry, and western blot were used to study the relationship between EIF2Ss and c-myc in PAAD. RESULTS: EIF2Ss are over-expressed in PAAD tissue and are associated with poor prognosis. The frequency of EIF2S1, EIF2S2, and EIF2S3 gene alteration in PAAD was 0.2%, 0.4%, and 0.2%, respectively. High EIF2Ss expression was associated with Th2 cell infiltration, whereas low expression was associated with pDC infiltration. Moreover, EIF2Ss expression was positively correlated with the expression of the NT5E, ULBP1, PVR, CD44, IL10RB, and CD276 checkpoints. A prediction model developed using EIF2Ss and important clinicopathologic features showed good predictive value for the overall survival of PAAD patients. ScRNA-Seq data showed that EIF2Ss was associated with enrichment for endothelial cells, fibroblasts, malignant cells, and ductal cells. EIF2Ss expression was also correlated with adipogenesis, interferon-alpha response, epithelial-mesenchymal transition, myc targets, G2M checkpoint, oxidative phosphorylation, and hypoxia. Functional enrichment analysis of EIF2Ss showed a close correlation with the myc pathway, and interactions between EIF2Ss and c-myc were confirmed by co-IP assay and mass spectrometry. Importantly, knockdown of c-myc decreased the expression of EIF2S1, EIF2S2, and EIF2S3 in PAAD cells. CONCLUSIONS: EIF2Ss were found to have significant clinical implications for the prognosis and treatment of PAAD. Inhibition of c-myc caused the downregulation of EIF2S1, EIF2S2, and EIF2S3 expression.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer.

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3'-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Stratification of Colorectal Patients Based on Survival Analysis Shows the Value of Consensus Molecular Subtypes and Reveals the CBLL1 Gene as a Biomarker of CMS2 Tumours.

The consensus molecular subtypes (CMSs) classification of colorectal cancer (CRC) is a system for patient stratification that can be potentially applied to therapeutic decisions. Hakai (CBLL1) is an E3 ubiquitin-ligase that induces the ubiquitination and degradation of E-cadherin, inducing epithelial-to-mesenchymal transition (EMT), tumour progression and metastasis. Using bioinformatic methods, we have analysed CBLL1 expression on a large integrated cohort of primary tumour samples from CRC patients. The cohort included survival data and was divided into consensus molecular subtypes. Colon cancer tumourspheres were used to analyse the expression of stem cancer cells markers via RT-PCR and Western blotting. We show that CBLL1 gene expression is specifically associated with canonical subtype CMS2. WNT target genes LGR5 and c-MYC show a similar association with CMS2 as CBLL1. These mRNA levels are highly upregulated in cancer tumourspheres, while CBLL1 silencing shows a clear reduction in tumoursphere size and in stem cell biomarkers. Importantly, CMS2 patients with high CBLL1 expression displayed worse overall survival (OS), which is similar to that associated with CMS4 tumours. Our findings reveal CBLL1 as a specific biomarker for CMS2 and the potential of using CMS2 with high CBLL1 expression to stratify patients with poor OS.

Nuclear to cytoplasmic transport is a druggable dependency in MYC-driven hepatocellular carcinoma.

The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). MYC is considered undruggable to date. Here, we comprehensively identify genes essential for survival of MYChigh but not MYClow cells by a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen uncovers novel MYC synthetic lethal (MYC-SL) interactions and identifies most MYC-SL genes described previously. In particular, the screen reveals nucleocytoplasmic transport to be a MYC-SL interaction. We show that the majority of MYC-SL nucleocytoplasmic transport genes are upregulated in MYChigh murine HCC and are associated with poor survival in HCC patients. Inhibiting Exportin-1 (XPO1) in vivo induces marked tumor regression in an autochthonous MYC-transgenic HCC model and inhibits tumor growth in HCC patient-derived xenografts. XPO1 expression is associated with poor prognosis only in HCC patients with high MYC activity. We infer that MYC may generally regulate and require altered expression of nucleocytoplasmic transport genes for tumorigenesis.

Synthesis of DNA-Boron Cluster Composites and Assembly into Functional Nanoparticles with Dual, Anti-EGFR, and Anti-c-MYC Oncogene Silencing Activity.

A versatile method for the automated synthesis of composites containing DNA-oligonucleotides and boron cluster scaffolds and their assembly into functional nanoparticles is described. The obtained, torus-like nanoparticles carry antisense oligonucleotides that target two different oncogenes simultaneously. The nanoparticles exhibited notable silencing efficiency in vitro in a pancreatic carcinoma cell line PANC-1 toward EGFR and c-Myc genes at the mRNA level, and a significant efficiency at the protein level. The proposed approach may be an attractive alternative to methods currently used, including one therapeutic nucleic acid, one genetic target, or the use of cocktails of therapeutic nucleic acids.

Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

Molecular Insights into the Specific Targeting of c-MYC G-Quadruplex by Thiazole Peptides.

Stabilization of a G-quadruplex (G4) in the promotor of the c-MYC proto-oncogene leads to inhibition of gene expression, and it thus represents a potentially attractive new strategy for cancer treatment. However, most G4 stabilizers show little selectivity among the many G4s present in the cellular complement of DNA and RNA. Intriguingly, a crescent-shaped cell-penetrating thiazole peptide, TH3, preferentially stabilizes the c-MYC G4 over other promotor G4s, but the mechanisms leading to this selective binding remain obscure. To investigate these mechanisms at the atomic level, we performed an in silico comparative investigation of the binding of TH3 and its analogue TH1 to the G4s from the promotors of c-MYC, c-KIT1, c-KIT2, and BCL2. Molecular docking and molecular dynamics simulations, combined with in-depth analyses of non-covalent interactions and bulk and per-nucleotide binding free energies, revealed that both TH3 and TH1 can induce the formation of a sandwich-like framework through stacking with both the top and bottom G-tetrads of the c-MYC G4 and the adjacent terminal capping nucleotides. This framework produces enhanced binding affinities for c-MYC G4 relative to other promotor G4s, with TH3 exhibiting an outstanding binding priority. Van der Waals interactions were identified to be the key factor in complex formation in all cases. Collectively, our findings fully agree with available experimental data. Therefore, the identified mechanisms leading to specific binding of TH3 towards c-MYC G4 provide valuable information to guide the development of new selective G4 stabilizers.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

Background: As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. Methods: We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. Results: The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3 °C, and that of D. japonicus was 77.7-78.0 °C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Conclusion: Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

MYC Oncogene: A Druggable Target for Treating Cancers with Natural Products.

Various diseases, including cancers, age-associated disorders, and acute liver failure, have been linked to the oncogene, MYC. Animal testing and clinical trials have shown that sustained tumor volume reduction can be achieved when MYC is inactivated, and different combinations of therapeutic agents including MYC inhibitors are currently being developed. In this review, we first provide a summary of the multiple biological functions of the MYC oncoprotein in cancer treatment, highlighting that the equilibrium points of the MYC/MAX, MIZ1/MYC/MAX, and MAD (MNT)/MAX complexes have further potential in cancer treatment that could be used to restrain MYC oncogene expression and its functions in tumorigenesis. We also discuss the multifunctional capacity of MYC in various cellular cancer processes, including its influences on immune response, metabolism, cell cycle, apoptosis, autophagy, pyroptosis, metastasis, angiogenesis, multidrug resistance, and intestinal flora. Moreover, we summarize the MYC therapy patent landscape and emphasize the potential of MYC as a druggable target, using herbal medicine modulators. Finally, we describe pending challenges and future perspectives in biomedical research, involving the development of therapeutic approaches to modulate MYC or its targeted genes. Patients with cancers driven by MYC signaling may benefit from therapies targeting these pathways, which could delay cancerous growth and recover antitumor immune responses.

Transcriptional activation of the Myc gene by glucose in ß-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes.

OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.

Epigenetic Dysregulation in MYCN-Amplified Neuroblastoma.

Neuroblastoma (NB), a childhood cancer arising from the neural crest, poses significant clinical challenges, particularly in cases featuring amplification of the MYCN oncogene. Epigenetic factors play a pivotal role in normal neural crest and NB development, influencing gene expression patterns critical for tumorigenesis. This review delves into the multifaceted interplay between MYCN and known epigenetic modifications during NB genesis, shedding light on the intricate regulatory networks underlying the disease. We provide an extensive survey of known epigenetic mechanisms, encompassing DNA methylation, histone modifications, non-coding RNAs, super-enhancers (SEs), bromodomains (BET), and chromatin modifiers in MYCN-amplified (MNA) NB. These epigenetic changes collectively contribute to the dysregulated gene expression landscape observed in MNA NB. Furthermore, we review emerging therapeutic strategies targeting epigenetic regulators, including histone deacetylase inhibitors (HDACi), histone methyltransferase inhibitors (HMTi), and DNA methyltransferase inhibitors (DNMTi). We also discuss and summarize current drugs in preclinical and clinical trials, offering insights into their potential for improving outcomes for MNA NB patients.

Optimal combination of MYCN differential gene and cellular senescence gene predicts adverse outcomes in patients with neuroblastoma.

Introduction: Neuroblastoma (NB) is a common extracranial tumor in children and is highly heterogeneous. The factors influencing the prognosis of NB are not simple. Methods: To investigate the effect of cell senescence on the prognosis of NB and tumor immune microenvironment, 498 samples of NB patients and 307 cellular senescence-related genes were used to construct a prediction signature. Results: A signature based on six optimal candidate genes (TP53, IL-7, PDGFRA, S100B, DLL3, and TP63) was successfully constructed and proved to have good prognostic ability. Through verification, the signature had more advantages than the gene expression level alone in evaluating prognosis was found. Further T cell phenotype analysis displayed that exhausted phenotype PD-1 and senescence-related phenotype CD244 were highly expressed in CD8+ T cell in MYCN-amplified group with higher risk-score. Conclusion: A signature constructed the six MYCN-amplified differential genes and aging-related genes can be used to predict the prognosis of NB better than using each high-risk gene individually and to evaluate immunosuppressed and aging tumor microenvironment.

Analysis of the Polymorphisms and Expression Levels of the BCL2, BAX and c-MYC Genes in Patients with Ovarian Cancer.

Ovarian cancer (OC) is one of the biggest problems in gynecological oncology and is one of the most lethal cancers in women worldwide. Most patients with OC are diagnosed at an advanced stage; therefore, there is an urgent need to find new biomarkers for this disease. Gene expression profiling is proving to be a very effective tool for exploring new molecular markers for OC patients, although the relationship between such markers and patient survival and clinical outcomes is still elusive. Moreover, polymorphisms in genes encoding both apoptosis-associated proteins and oncoproteins may serve as key markers of cancer susceptibility. The aim of our study was to analyze the polymorphisms and expressions of the BCL2, BAX and c-MYC genes in a group of 198 women, including 98 with OC. The polymorphisms and mRNA expressions of the BCL2, BAX and c-MYC genes were analyzed using real-time PCR. The analysis of the BAX (rs4645878; G>A) and c-MYC (rs4645943; C>T) polymorphisms showed no association with ovarian cancer risk. The BCL2 polymorphism (rs2279115; C>A) showed a significant difference in the frequency of genotypes between the studied groups (CC: 23.47% vs. 16.00%, AA: 25.51% vs. 37.00%; p = 0.046; OR = 1.61). Furthermore, the expression levels of the BCL2 and c-MYC genes showed a decrease at the transcript level for OC patients compared to the control group (BCL2: 17.46% ± 3.26 vs. 100% ± 8.32; p < 0.05; c-MYC: 37.56% ± 8.16 vs. 100% ± 9.12; p < 0.05). No significant changes in the mRNA level were observed for the BAX gene (104.36% ± 9.26 vs. 100% ± 9.44; p > 0.05). A similar relationship was demonstrated in the case of the protein expressions of the studied genes. These findings suggest that the CC genotype and C allele of the BCL2 polymorphism could be genetic risk factors for OC development. A gene expression analysis indicated that BCL2 and c-MYC are associated with OC risk.

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt's lymphoma.

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.

[Genetic analysis of two cases with MYC "negative" Burkitt lymphoma].

OBJECTIVE: To carry out combined genetic analysis on two patients suspected for Burkitt lymphoma to facilitate their diagnosis and treatment. METHODS: G banded karyotyping and interphase and metaphase fluorescence in situ hybridization (FISH) were used to detect the specific sites of chromosomes by using separate and fusion probes. RESULTS: The separate probe showed no presence of MYC gene abnormality, while fusion probe confirmed the IGH::MYC translocation in the samples. Combined with the clinical features and pathological characteristics, the two patients were finally diagnosed with Burkitt lymphoma, which was confirmed by targeted capture next generation sequencing. CONCLUSION: The separate probe for the MYC gene has some shortcomings and should be used together with dual fusion probe to improve the accuracy of diagnosis.