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Significant racial disparities exist between Black and White patients with uterine serous carcinoma (USC). While the reasons for these disparities are unclear, several studies have demonstrated significantly different rates of driver mutations between racial groups, including TP53. However, limited research has investigated the transcriptional differences of tumors or the composition of the tumor microenvironment (TME) between these groups. Here, we report the single-nuclei RNA-sequencing profiles of primary USC tumors from diverse racial backgrounds. We find that there are significant differences between the tumors of Black and White patients. Tumors from Black patients exhibited higher expression of specific genes associated with aggressiveness, such as PAX8, and axon guidance and synaptic signaling pathways. We also demonstrated that T cell populations are reduced in the tumor tissue compared to matched benign, while anti-inflammatory macrophage populations are retained within the TME. Furthermore, we investigated the connection between PAX8 overexpression and immunosuppression in USC through regulation of several cytokines and chemokines. Notably, we show that PAX8 activity can influence macrophage gene expression and protein secretion. These studies provide a detailed understanding of the USC transcriptome and TME, and identify differences in tumor biology from patients of different racial backgrounds.
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Factor de Transcripción PAX8 , Transducción de Señal , Microambiente Tumoral , Neoplasias Uterinas , Humanos , Femenino , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Transducción de Señal/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/inmunología , Regulación Neoplásica de la Expresión Génica , Población Blanca/genética , Análisis de la Célula Individual , Persona de Mediana EdadRESUMEN
BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.
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Cromatina , Lamina Tipo B , Animales , Lamina Tipo B/genética , Heterocromatina , Hibridación Fluorescente in Situ , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminas , Expresión Génica , Mamíferos/genéticaRESUMEN
Uterine fibroids (UF), that can disrupt normal uterine function and cause significant physical and psychological health problems, are observed in nearly 70% of women of reproductive age. Although heritable genetics is a significant risk factor, specific genetic variations and gene targets causally associated with UF are poorly understood. Here, we performed a meta-analysis on existing fibroid genome-wide association studies (GWAS) and integrated the identified risk loci and potentially causal single nucleotide polymorphisms (SNPs) with epigenomics, transcriptomics, 3D chromatin organization from diverse cell types as well as primary UF patient's samples. This integrative analysis identifies 24 UF-associated risk loci that potentially target 394 genes, of which 168 are differentially expressed in UF tumors. Critically, integrating this data with single-cell gene expression data from UF patients reveales the causal cell types with aberrant expression of these target genes. Lastly, CRISPR-based epigenetic repression (dCas9-KRAB) or activation (dCas9-p300) in a UF disease-relevant cell type further refines and narrows down the potential gene targets. Our findings and the methodological approach indicate the effectiveness of integrating multi-omics data with locus-specific epigenetic editing approaches for identifying gene- and celt type-targets of disease-relevant risk loci.
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Estudio de Asociación del Genoma Completo , Leiomioma , Humanos , Femenino , Epigenómica , Leiomioma/patología , Factores de Riesgo , Perfilación de la Expresión Génica , Polimorfismo de Nucleótido SimpleRESUMEN
Following a period of slow progress, the completion of genome sequencing and the paradigm shift relative to the cell of origin for high grade serous ovarian cancer (HGSOC) led to a new perspective on the biology and therapeutic solutions for this deadly cancer. Experimental models were revisited to address old questions, and improved tools were generated. Additional pathways emerging as drivers of ovarian tumorigenesis and key dependencies for therapeutic targeting, in particular, VEGF-driven angiogenesis and homologous recombination deficiency, were discovered. Molecular profiling of histological subtypes of ovarian cancer defined distinct genetic events for each entity, enabling the first attempts toward personalized treatment. Armed with this knowledge, HGSOC treatment was revised to include new agents. Among them, PARP inhibitors (PARPis) were shown to induce unprecedented improvement in clinical benefit for selected subsets of patients. Research on mechanisms of resistance to PARPis is beginning to discover vulnerabilities and point to new treatment possibilities. This Review highlights these advances, the remaining challenges, and unsolved problems in the field.
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Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , BiologíaRESUMEN
BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTS: Paired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONS: Our findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.
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Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.
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Leiomioma , Humanos , Leiomioma/genética , Miocitos del Músculo Liso , Mutación , Genómica , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Factores de Transcripción , Complejo Mediador/genéticaRESUMEN
Endometriosis and adenomyosis are closely related disorders. Their pathophysiologies are extremely similar. Both tissues originate from the eutopically located intracavitary endometrium. Oligoclones of endometrial glandular epithelial cells with somatic mutations and attached stromal cells may give rise to endometriosis if they travel to peritoneal surfaces or the ovary via retrograde menstruation and/or may be entrapped in the myometrium to give rise to adenomyosis. In both instances, the endometrial cell populations possess survival and growth capabilities conferred by somatic epithelial mutations and epigenetic abnormalities in stromal cells. Activating mutations of KRAS are the most commonly found genetic variant in endometriotic epithelial cells, whereas the adenomyotic epithelial cells almost exclusively bear KRAS mutations. Epigenetic abnormalities in the stromal cells of endometriosis and adenomyosis are very similar and involve an abnormal expression pattern of nuclear receptors, including the steroid receptors. These epigenetic defects give rise to excessive local estrogen biosynthesis by aromatase and abnormal estrogen action via estrogen receptor-ß. Deficient progesterone receptor expression results in progesterone resistance in both endometriosis and adenomyosis.
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Adenomiosis , Endometriosis , Enfermedades Uterinas , Femenino , Humanos , Endometriosis/metabolismo , Adenomiosis/genética , Adenomiosis/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Enfermedades Uterinas/metabolismo , Endometrio/metabolismo , EstrógenosRESUMEN
Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the MED12 gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.
RESUMEN
OBJECTIVE: To assess the cellular and molecular landscape of adenomyosis. DESIGN: Single-cell analysis of genome-wide messenger RNA (mRNA) expression (single-cell RNA sequencing) of matched tissues of endometrium, adenomyosis, and myometrium using relatively large numbers of viable cells. SETTING: Not applicable. PATIENT(S): Patients (n = 3, age range 40-44 years) undergoing hysterectomy for diffuse adenomyosis. MAIN OUTCOME MEASURE(S): Definition of the molecular landscape of matched adenomyotic, endometrial and myometrial tissues from the same uterus using single-cell RNA sequencing and comparison of distinct cell types in these tissues to identify disease-specific cell populations, abnormal gene expression and pathway activation, and mesenchymal-epithelial interactions. RESULT(S): The largest cell population in the endometrium was composed of closely clustered fibroblast groups, which comprise 36% of all cells and seem to originate from pericyte progenitors differentiating to estrogen/progesterone receptor-expressing endometrial stromal- cells. In contrast, the entire fibroblast population in adenomyosis comprised a larger (50%) portion of all cells and was not linked to any pericyte progenitors. Adenomyotic fibroblasts eventually differentiate into extracellular matrix protein-expressing fibroblasts and smooth muscle cells. Hierarchical clustering of mRNA expression revealed a unique adenomyotic fibroblast population that clustered transcriptomically with endometrial fibroblasts, suggestive of an endometrial stromal cell population serving as progenitors of adenomyosis. Four other adenomyotic fibroblast clusters with disease-specific transcriptomes were distinct from those of endometrial or myometrial fibroblasts. The mRNA levels of the natural WNT inhibitors, named, secreted frizzled-related proteins 1, 2, and 4, were higher in these 4 adenomyotic fibroblast clusters than in endometrial fibroblast clusters. Moreover, we found that multiple WNTs, which originate from fibroblasts and target ciliated and unciliated epithelial cells and endothelial cells, constitute a critical paracrine signaling network in adenomyotic tissue. Compared with endometrial tissue, unciliated and ciliated epithelial cells in adenomyosis comprised a significantly smaller portion of this tissue and exhibited molecular evidence of progesterone resistance and diminished regulation of estrogen signaling. CONCLUSION(S): We found a high degree of heterogeneity in fibroblast-like cells in the adenomyotic uterus. The WNT signaling involving differential expression of secreted frizzled-related proteins, which act as decoy receptors for WNTs, in adenomyotic fibroblasts may have a key role in the pathophysiology of this disease.
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Adenomiosis , Endometriosis , Femenino , Humanos , Adulto , Adenomiosis/genética , Adenomiosis/metabolismo , Vía de Señalización Wnt/genética , Células Endoteliales , Transcriptoma , Endometrio/metabolismo , Estrógenos , ARN Mensajero/genética , Endometriosis/metabolismoRESUMEN
The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and the reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.
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Histonas , Proteínas Serina-Treonina Quinasas , Acetilación , Centrómero/metabolismo , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis , Fosforilación , Treonina/genética , Treonina/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.
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Carcinoma Ductal Pancreático , Proteínas con Homeodominio LIM , Proteínas del Tejido Nervioso , Neoplasias Pancreáticas , Factores de Transcripción , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Genes Supresores de Tumor , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin-state and gene expression analyses in ovarian CSCs versus non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (encoded by ERN1) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation and sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish what we believe is a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.
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Factores de Transcripción Forkhead , Neoplasias Ováricas , Respuesta de Proteína Desplegada , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Humanos , Neoplasias Ováricas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.
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Alérgenos , Hipersensibilidad , Alérgenos/química , Alérgenos/genética , Animales , Biología , Sistemas CRISPR-Cas/genética , Gatos , Edición Génica , Glicoproteínas/química , Glicoproteínas/genética , Hipersensibilidad/genética , Hipersensibilidad/terapiaRESUMEN
Genome-wide association studies have identified SNPs associated with glioma risk on 9p21.3, but biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 9p21.3 affects activity of an enhancer, causing altered expression of nearby genes. We considered all SNPs in linkage disequilibrium with the 9p21.3 sentinel SNP rs634537 that mapped to putative enhancers. An enhancer containing rs1537372 exhibited allele-specific effects on luciferase activity. Deletion of this enhancer in GBM cell lines correlated with decreased expression of CDKN2B-AS1. Expression quantitative trait loci analysis using non-diseased brain samples showed rs1537372 to be a consistently significant eQTL for CDKN2B-AS1. Additionally, our analysis of Hi-C data generated in neural progenitor cells showed that the bait region containing rs1537372 interacted with the CDKN2B-AS1 promoter. These data suggest rs1537372, a SNP at the 9p21.3 risk locus, is a functional variant that modulates expression of CDKN2B-AS1.
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Glioma , ARN Largo no Codificante , Elementos de Facilitación Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Glioma/genética , Humanos , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante/genéticaRESUMEN
BACKGROUND: Adenomyosis, characterized by the presence of islands of endometrial tissue surrounded by hypertrophic smooth muscle cells within the myometrium, is one of the most challenging uterine disorders in terms of diagnosis and management. Adenomyosis presents with pelvic pain, excessive uterine bleeding, anemia and infertility. The relative contributions of abnormal endometrial tissue and myometrial smooth muscle cells to the development and growth of adenomyosis are not well understood. Moreover, there is continuing debate on the origins of adenomyosis; two competing theories describe the invagination of basal endometrium into the myometrium or the metaplastic differentiation of remnant endometrial stem/progenitor cells within the myometrium. OBJECTIVE AND RATIONALE: A recent series of next-generation sequencing (NGS) studies have provided the best scientific evidence thus far regarding the cellular origins of adenomyosis and the contributions of new signaling pathways to its pathogenesis, survival, and growth. These seminal studies on endometrium, adenomyosis and endometriosis demonstrate or support the following key points. (i) Mutations of KRAS map to both intracavitary endometrial tissue and proximally located adenomyotic samples, supporting the invagination theory of pathogenesis. Driver mutations found in smooth muscle cells of uterine fibroids are absent in adenomyosis. (ii) KRAS and other less frequent mutations are limited to endometrial-type epithelial cells. They are also observed in endometriosis, indicating that the disease process in adenomyosis is similar to that in endometriosis and distinct from that of uterine fibroids. (iii) Activating mutations of KRAS stimulate specific pathways to increase cell survival and proliferation and are associated with progesterone resistance in adenomyosis. Together, these findings suggest that distinct cell populations in eutopic endometrial tissue play key roles in the etiology of adenomyosis. Dependence on ovarian steroids and ovulatory cycles for disease severity is a unique feature of adenomyosis. In this context, common patterns of aberrant gene expression have been reported both in adenomyosis and endometriosis. These include pathways that favor increased estrogen biosynthesis, decreased estradiol metabolism, a unique estrogen receptor beta (ESR2)-driven inflammatory process, and progesterone resistance due to decreased progesterone receptor expression. Since adenomyosis exhibits a uniquely estrogen-driven inflammatory process and progesterone resistance, we discuss the interactions between these molecular characteristics and signaling pathways induced by the newly discovered KRAS mutations. SEARCH METHODS: We conducted a comprehensive search using PubMed for human and animal studies published until 2020 in the following areas: adenomyosis, endometriosis, endometrium, NGS, whole-exome sequencing, whole-genome sequencing, RNA sequencing, targeted deep sequencing, epigenetics, driver mutation, KRAS, progesterone resistance, estrogen action and steroid production. OUTCOMES: Targeted deep sequencing analyses of epithelial cells in adenomyosis and adjacent basalis endometrial glands demonstrated recurring KRAS mutations in both cell types. This finding suggests that adenomyosis originates from basalis endometrium. Epithelial cells of the endometrium, adjacent adenomyosis and co-occurring endometriosis also share identical KRAS mutations. These findings suggest both adenomyosis and endometriosis are oligoclonal tissues that arise from endometrial cell populations carrying a specific driver mutation that most commonly affects the KRAS gene. WIDER IMPLICATIONS: Adenomyosis usually follows an event such as pregnancy that has disrupted the integrity of the endometrial-myometrial junction followed by repetitious menstrual episodes that increase the likelihood of the entrapment of the basalis endometrium within the myometrium. Glandular epithelial cells carrying KRAS mutations and located within the deep crypts of basalis endometrium may become entrapped and invade myometrial tissue to give rise to adenomyosis. Evidence suggests that KRAS mutations may be responsible, in part, for previously observed phenomena such as prolonged cell survival and progesterone resistance in adenomyosis.
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Adenomiosis , Endometriosis , Enfermedades Uterinas , Adenomiosis/genética , Adenomiosis/patología , Animales , Endometriosis/patología , Endometrio/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Uterinas/patologíaRESUMEN
Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk-associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele-specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele-specific effects on activity. Deletion of this enhancer by CRISPR-Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1-TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild-type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild-type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.
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Estudio de Asociación del Genoma Completo , Glioma , Alelos , Predisposición Genética a la Enfermedad , Glioma/genética , Glioma/metabolismo , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.
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Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferasas , Animales , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desarrollo de Medicamentos , Técnicas de Inactivación de Genes , Humanos , Ratones Desnudos , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Much of the international community opposes editing the human germline. Yet, given enough experience to become better acquainted with strengths and limitations, prominent international figures are cautiously optimistic about using CRISPR-like novel technologies for clinical applications. Not only might such applications be morally (ethically) permissible, but clinical trials for therapeutic aims could be necessary. Here, we assess critical dimensions of early-phase trials deploying germline-editing technologies for "bench-to-bedside" translation. While assuming no overarching position favoring or opposing such research, our discussion primarily focuses on normative considerations. First, we evaluate the imperative of conducting trials to produce reliable, reproducible knowledge and advancement, if possible, for human diseases that are incurable and/or whose treatments are deficient. Second, we address complexities in assessing risk and potential-benefit profiles. Third, we review the moral foundations of trial participation through well-established and accepted bioethical principles: autonomy, nonmaleficence, beneficence, and distributive justice. Finally, we raise critical questions about the scope of regulatory authority and investigator and funder accountability for these applications that could have everlasting impacts.
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Ensayos Clínicos como Asunto/ética , Edición Génica/ética , Mutación de Línea Germinal/genética , Beneficencia , Sistemas CRISPR-Cas/genética , Ensayos Clínicos como Asunto/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Células Germinativas/metabolismo , HumanosRESUMEN
Whole-genome sequencing efforts of tumors and normal tissues have identified numerous genetic mutations, both somatic and germline, that do not overlap with coding genomic sequences. Attributing a functional role to these noncoding mutations and characterizing them using experimental methods has been more challenging compared with coding mutations. In this review, we provide a brief introduction to the world of noncoding mutations. We discuss recent progress in identifying noncoding mutations and the analytic and experimental approaches utilized to interpret their functional roles. We also highlight the potential mechanisms by which a noncoding mutation may exert its effect and discuss future challenges and opportunities.
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Genoma Humano , Mutación , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias/genética , Sitios de Carácter Cuantitativo , Telomerasa/genéticaRESUMEN
Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. SIGNIFICANCE: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.