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1.
ACS Cent Sci ; 10(8): 1640-1656, 2024 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-39220711

RESUMEN

Glioblastoma (GBM) is the most aggressive and frequently occurring type of malignant brain tumor in adults. The initiation, progression, and recurrence of malignant tumors are known to be driven by a small subpopulation of cells known as tumor-initiating cells or cancer stem cells (CSCs). GBM CSCs play a pivotal role in orchestrating drug resistance and tumor relapse. As a prospective avenue for GBM intervention, the targeted suppression of GBM CSCs holds considerable promise. In this study, we found that rocaglates, compounds which are known to inhibit translation via targeting of the DEAD-box helicase eIF4A, exert a robust, dose-dependent cytotoxic impact on GBM CSCs with minimal killing of nonstem GBM cells. Subsequent optimization identified novel rocaglate derivatives (rocaglate acyl sulfamides or Roc ASFs) that selectively inhibit GBM CSCs with nanomolar EC50 values. Furthermore, comparative evaluation of a lead CSC-optimized Roc ASF across diverse mechanistic and target profiling assays revealed suppressed translation inhibition relative to that of other CSC-selective rocaglates, with enhanced targeting of the DEAD-box helicase DDX3X, a recently identified secondary target of rocaglates. Overall, these findings suggest a promising therapeutic strategy for targeting GBM CSCs.

2.
bioRxiv ; 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39345542

RESUMEN

Cell survival depends upon the ability to adapt to changing environments. Environmental stressors trigger an acute stress response program that rewires cell physiology, downregulates proliferation genes and pauses the cell cycle until the cell adapts. Here, we show that dynamic phosphorylation of the yeast cell cycle-regulatory transcription factor Hcm1 is required to maintain fitness in chronic stress. Hcm1 is activated by cyclin dependent kinase (CDK) and inactivated by the phosphatase calcineurin (CN) in response to stressors that signal through increases in cytosolic Ca 2+ . Expression of a constitutively active, phosphomimetic Hcm1 mutant reduces fitness in stress, suggesting Hcm1 inactivation is required. However, a comprehensive analysis of Hcm1 phosphomutants revealed that Hcm1 activity is also important to survive stress, demonstrating that Hcm1 activity must be toggled on and off to promote gene expression and fitness. These results suggest that dynamic control of cell cycle regulators is critical for survival in stressful environments.

3.
Sci Transl Med ; 16(762): eadj9366, 2024 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-39196958

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) has quickly risen to become the third leading cause of cancer-related death in the United States. This is in part because of its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here, we investigated an immunotherapy approach combining delivery of stimulator of interferon genes (STING) and Toll-like receptor 4 (TLR4) innate immune agonists by lipid-based nanoparticle (NP) coencapsulation with senescence-inducing RAS-targeted therapies, which can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other proinflammatory signaling pathways, increased antigen presentation by tumor cells and antigen-presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and type I interferon-mediated tumor regression and long-term survival in preclinical PDAC models dependent on both tumor and host STING activation. STING and TLR4-mediated type I interferon signaling was also associated with enhanced natural killer and CD8+ T cell immunity in human PDAC samples. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can orchestrate a coordinated type I interferon-driven innate and adaptive immune response with durable antitumor efficacy against PDAC.


Asunto(s)
Carcinoma Ductal Pancreático , Inmunidad Innata , Nanopartículas , Neoplasias Pancreáticas , Receptor Toll-Like 4 , Microambiente Tumoral , Animales , Inmunidad Innata/efectos de los fármacos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Humanos , Nanopartículas/química , Microambiente Tumoral/efectos de los fármacos , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/terapia , Ratones , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/agonistas , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Interferón Tipo I/metabolismo , Línea Celular Tumoral , Inmunoterapia/métodos , Senescencia Celular/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos
4.
BioTech (Basel) ; 13(3)2024 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-39189209

RESUMEN

RNA sequencing (RNA-seq) has become a standard method for profiling gene expression, yet genomic DNA (gDNA) contamination carried over to the sequencing library poses a significant challenge to data integrity. Detecting and correcting this contamination is vital for accurate downstream analyses. Particularly, when RNA samples are scarce and invaluable, it becomes essential not only to identify but also to correct gDNA contamination to maximize the data's utility. However, existing tools capable of correcting gDNA contamination are limited and lack thorough evaluation. To fill the gap, we developed CleanUpRNAseq, which offers a comprehensive set of functionalities for identifying and correcting gDNA-contaminated RNA-seq data. Our package offers three correction methods for unstranded RNA-seq data and a dedicated approach for stranded data. Through rigorous validation on published RNA-seq datasets with known levels of gDNA contamination and real-world RNA-seq data, we demonstrate CleanUpRNAseq's efficacy in detecting and correcting detrimental levels of gDNA contamination across diverse library protocols. CleanUpRNAseq thus serves as a valuable tool for post-alignment quality assessment of RNA-seq data and should be integrated into routine workflows for RNA-seq data analysis. Its incorporation into OneStopRNAseq should significantly bolster the accuracy of gene expression quantification and differential expression analysis of RNA-seq data.

5.
bioRxiv ; 2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39091883

RESUMEN

Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Significance: Though immune checkpoint blockade (ICB) therapies can achieve curative responses in many treatment-refractory cancers, they have limited efficacy in CRPC. Here we identify a genetic mechanism by which VEGF contributes to T cell suppression, and demonstrate that VEGFR2 blockade can potentiate the effects of PD-L1 ICB to immunologically treat CRPC.

6.
Mol Biol Cell ; 35(10): ar123, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39083354

RESUMEN

Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl2 stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl2 impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms-one that requires the downstream transcription factor Crz1 and another that is Crz1 independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl2-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.


Asunto(s)
Adaptación Fisiológica , Calcineurina , Mitocondrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estrés Fisiológico , Factores de Transcripción , Calcineurina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Mitocondrias/metabolismo , Adaptación Fisiológica/fisiología , Factores de Transcripción/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cloruro de Calcio/farmacología , Ciclo Celular , Regulación Fúngica de la Expresión Génica , Proteínas de Unión al ADN
7.
Cell Rep ; 43(5): 114211, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38722741

RESUMEN

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.


Asunto(s)
Mieloma Múltiple , Proteínas Proto-Oncogénicas c-myc , Proteína Quinasa Deficiente en Lisina WNK 1 , Mieloma Múltiple/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Humanos , Animales , Ratones , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Cadenas Pesadas de Inmunoglobulina/genética , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Nat Commun ; 15(1): 4410, 2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38782979

RESUMEN

Pancreatic ß cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. Here, we show that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis.


Asunto(s)
Encéfalo , Proteínas de Drosophila , Drosophila melanogaster , Secreción de Insulina , Insulina , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Encéfalo/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Eje Cerebro-Intestino/fisiología , Lipasa/metabolismo , Lipasa/genética , Grasas de la Dieta/metabolismo , Glucosa/metabolismo , Cuerpo Adiposo/metabolismo , Lipoproteína Lipasa/metabolismo , Lipoproteína Lipasa/genética , Masculino
9.
bioRxiv ; 2024 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-38562881

RESUMEN

Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long-term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl2 stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl2 impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms - one that requires the downstream transcription factor Crz1 and another that is Crz1-independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl2-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione (GSH) biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.

10.
Cell Rep ; 43(4): 114041, 2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38573857

RESUMEN

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.


Asunto(s)
Aciltransferasas , Antígeno CD24 , Neoplasias Ováricas , Fagocitosis , Animales , Femenino , Humanos , Ratones , Aciltransferasas/metabolismo , Amidohidrolasas/metabolismo , Amidohidrolasas/genética , Antígeno CD24/metabolismo , Línea Celular Tumoral , Glicosilfosfatidilinositoles/metabolismo , Macrófagos/metabolismo , Macrófagos/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia
11.
Leukemia ; 38(5): 951-962, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38553571

RESUMEN

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas con Dominio LIM , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteína 1 de la Leucemia Linfocítica T Aguda , Animales , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas con Dominio LIM/metabolismo , Proteínas con Dominio LIM/genética , Timo/metabolismo , Timo/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología
12.
Cell Rep ; 42(11): 113347, 2023 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-37910503

RESUMEN

Understanding the cell biological mechanisms that enable tumor cells to persist after therapy is necessary to improve the treatment of recurrent disease. Here, we demonstrate that transient receptor potential channel 6 (TRPC6), a channel that mediates calcium entry, contributes to the properties of breast cancer stem cells, including resistance to chemotherapy, and that tumor cells that persist after therapy are dependent on TRPC6. The mechanism involves the ability of TRPC6 to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated calcium entry represses the epithelial splicing factor ESRP1 (epithelial splicing regulatory protein 1), which enables expression of the integrin α6B splice variant. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic inhibition of TRPC6 sensitizes triple-negative breast cancer (TNBC) cells and tumors to chemotherapy by targeting the splicing of α6 integrin mRNA and inducing Myc. These data reveal a Ca2+-dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing.


Asunto(s)
Antineoplásicos , Canales de Potencial de Receptor Transitorio , Canales de Calcio/metabolismo , Integrina alfa6 , Canal Catiónico TRPC6 , Calcio/metabolismo , Canales Catiónicos TRPC/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
13.
Cell Rep Med ; 4(11): 101285, 2023 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-37951219

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective therapies. Here, we demonstrate that the transcription factor, homeobox C6 (HOXC6), is overexpressed in most PDACs, and its inhibition blocks PDAC tumor growth and metastasis. HOXC6 transcriptionally activates tumor-promoting kinase MSK1 and suppresses tumor-inhibitory protein PPP2R2B in PDAC. HOXC6-induced PPP2R2B suppression causes mammalian target of rapamycin (mTOR) pathway activation, which facilitates PDAC growth. Also, MSK1 upregulation by HOXC6 is necessary for PDAC growth because of its ability to suppress apoptosis via its substrate DDX17. Combinatorial pharmacological inhibition of MSK1 and mTOR potently suppressed PDAC tumor growth and metastasis in PDAC mouse models. PDAC cells with acquired resistance to MSK1/mTOR-inhibitors displayed activated insulin-like growth factor 1 receptor (IGF1R) signaling and were successfully eradicated by IGF1R inhibitor. Furthermore, MEK inhibitor trametinib enhanced the efficacy of dual MSK1 and mTOR inhibition. Collectively, these results identify therapeutic vulnerabilities of PDAC and an approach to overcome acquired drug resistance to prolong therapeutic benefit.


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Proliferación Celular , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Serina-Treonina Quinasas TOR/farmacología , Serina-Treonina Quinasas TOR/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Neoplasias , Mamíferos
14.
bioRxiv ; 2023 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-37790484

RESUMEN

Pancreatic ductal adenocarcinoma has quickly risen to become the 3rd leading cause of cancer-related death. This is in part due to its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here we investigated an innovative immunotherapy approach combining local delivery of STING and TLR4 innate immune agonists via lipid-based nanoparticles (NPs) co-encapsulation with senescence-inducing RAS-targeted therapies that can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other pro-inflammatory signaling, increased antigen presentation by tumor cells and antigen presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and Type I interferon-dependent tumor regressions and long-term survival in preclinical PDAC models. STING and TLR4-mediated Type I interferon signaling were also associated with enhanced NK and CD8+ T cell immunity in human PDAC. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can synergize to orchestrate a coordinated innate and adaptive immune assault to overcome immune suppression and activate durable anti-tumor T cell responses against PDAC.

15.
bioRxiv ; 2023 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-37905100

RESUMEN

Inflammatory stresses underlie endothelial dysfunction and contribute to the development of chronic cardiovascular disorders such as atherosclerosis and vascular fibrosis. The initial transcriptional response of endothelial cells to pro-inflammatory cytokines such as TNF-alpha is well established. However, very few studies uncover the effects of inflammatory stresses on chromatin architecture. We used integrative analysis of ATAC-seq and RNA-seq data to investigate chromatin alterations in human endothelial cells in response to TNF-alpha and febrile-range heat stress exposure. Multi-omics data analysis suggests a correlation between the transcription of stress-related genes and endothelial dysfunction drivers with chromatin regions exhibiting differential accessibility. Moreover, microscopy identified the dynamics in the nuclear organization, specifically, the changes in a subset of heterochromatic nucleoli-associated chromatin domains, the centromeres. Upon inflammatory stress exposure, the centromeres decreased association with nucleoli in a p38-dependent manner and increased the number of transcripts from pericentromeric regions. Overall, we provide two lines of evidence that suggest chromatin alterations in vascular endothelial cells during inflammatory stresses.

16.
Nature ; 623(7987): 625-632, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37880368

RESUMEN

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.


Asunto(s)
Neoplasias , Uridina Difosfato Ácido Glucurónico , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Uridina Difosfato Ácido Glucurónico/biosíntesis , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato Xilosa/biosíntesis , Uridina Difosfato Xilosa/metabolismo , Adenocarcinoma del Pulmón , Neoplasias Pulmonares
17.
bioRxiv ; 2023 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-37292647

RESUMEN

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

18.
Nat Cancer ; 4(6): 872-892, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37142692

RESUMEN

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Ratones , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Fenotipo Secretor Asociado a la Senescencia , Microambiente Tumoral/genética
19.
Nat Methods ; 20(6): 898-907, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37156841

RESUMEN

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.


Asunto(s)
Edición Génica , Genoma , Ratones , Animales , Edición Génica/métodos , ADN/genética , Roturas del ADN de Doble Cadena , Línea Celular , Sistemas CRISPR-Cas , Mamíferos/genética
20.
Sci Transl Med ; 15(694): eade5855, 2023 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-37134151

RESUMEN

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.


Asunto(s)
Neoplasias de la Próstata , Factor A de Crecimiento Endotelial Vascular , Masculino , Animales , Humanos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Transducción de Señal , Antígeno B7-H1/genética , Neoplasias de la Próstata/metabolismo
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