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BACKGROUND: Cyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates. METHODS: The activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches. RESULTS: CDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo. CONCLUSION: Our work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.
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There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Proteína p300 Asociada a E1A , Receptores Androgénicos , Transducción de Señal , Masculino , Humanos , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Animales , Proteína p300 Asociada a E1A/metabolismo , Proteína p300 Asociada a E1A/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Regulación Neoplásica de la Expresión Génica , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas que Contienen Bromodominio , Proteína de Unión a CREBRESUMEN
Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.
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Leishmania donovani , Leishmania infantum , Leishmaniasis Visceral , Humanos , Leishmaniasis Visceral/tratamiento farmacológico , Antiparasitarios/farmacología , Quinazolinas/farmacología , Quinazolinas/uso terapéuticoRESUMEN
Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled "Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?" at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.
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Descubrimiento de Drogas , Opinión Pública , Descubrimiento de Drogas/métodos , FenotipoRESUMEN
The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.
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Biogénesis de Organelos , Orgánulos , Orgánulos/metabolismo , Peroxisomas/metabolismo , Aparato de Golgi/metabolismo , Mitocondrias/metabolismo , LípidosRESUMEN
Peptide receptor radionuclide therapy (PRRT) using 177Lutetium-DOTA-octreotate (LuTate) for neuroendocrine tumours (NET) is now an approved treatment available in many countries, though primary or secondary resistance continue to limit its effectiveness or durability. We hypothesised that a genome-wide CRISPR/Cas9 screen would identify key mediators of response to LuTate and gene targets that might offer opportunities for novel combination therapies for NET patients. Methods: We utilised a genome-wide CRISPR-Cas9 screen in LuTate-treated cells to identify genes that impact on the sensitivity or resistance of cells to LuTate. Hits were validated through single-gene knockout. LuTate-resistant cells were assessed to confirm LuTate uptake and retention, and persistence of somatostatin receptor 2 (SSTR2) expression. Gene knockouts conferring LuTate sensitivity were further characterised by pharmacological sensitisation using specific inhibitors and in vivo analysis of the efficacy of these inhibitors in combination with LuTate. Results: The CRISPR-Cas9 screen identified several potential targets for both resistance and sensitivity to PRRT. Two gene knockouts which conferred LuTate resistance in vitro, ARRB2 and MVP, have potential mechanisms related to LuTate binding and retention, and modulation of DNA-damage repair (DDR) pathways, respectively. The screen showed that sensitivity to LuTate treatment in vitro can be conferred by the loss of a variety of genes involved in DDR pathways, with loss of genes involved in Non-Homologous End-Joining (NHEJ) being the most lethal. Loss of the key NHEJ gene, PRKDC (DNA-PK), either by gene loss or inhibition by two different inhibitors, resulted in significantly reduced cell survival upon exposure of cells to LuTate. In SSTR2-positive xenograft-bearing mice, the combination of nedisertib (a DNA-PK specific inhibitor) and LuTate produced a more robust control of tumour growth and increased survival compared to LuTate alone. Conclusions: DDR pathways are critical for sensing and repairing radiation-induced DNA damage, and our study shows that regulation of DDR pathways may be involved in both resistance and sensitivity to PRRT. Additionally, the use of a DNA-PK inhibitor in combination with LuTate PRRT significantly improves the efficacy of the treatment in pre-clinical models, providing further evidence for the clinical efficacy of this combination.
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Lutecio , Tumores Neuroendocrinos , Animales , Humanos , Ratones , Sistemas CRISPR-Cas/genética , ADN , Lutecio/metabolismo , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/radioterapia , Octreótido/uso terapéutico , Radioisótopos/uso terapéuticoRESUMEN
Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.
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Endometriosis, defined as the growth of hormonally responsive endometrial-like tissue outside of the uterine cavity, is an estrogen-dependent, chronic, pro-inflammatory disease that affects up to 11.4% of women of reproductive age and gender-diverse people with a uterus. At present, there is no long-term cure, and the identification of new therapies that provide a high level of efficacy and favourable long-term safety profiles with rapid clinical access are a priority. In this study, quantitative high-throughput compound screens of 3517 clinically approved compounds were performed on patient-derived immortalized human endometrial stromal cell lines. Following assay optimization and compound criteria selection, a high-throughput screening protocol was developed to enable the identification of compounds that interfered with estrogen-stimulated cell growth. From these screens, 23 novel compounds were identified, in addition to their molecular targets and in silico cell-signalling pathways, which included the neuroactive ligand-receptor interaction pathway, metabolic pathways, and cancer-associated pathways. This study demonstrates for the first time the feasibility of performing large compound screens for the identification of new translatable therapeutics and the improved characterization of endometriosis molecular pathophysiology. Further investigation of the molecular targets identified herein will help uncover new mechanisms involved in the establishment, symptomology, and progression of endometriosis.
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Endometriosis , Humanos , Femenino , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Ensayos Analíticos de Alto Rendimiento , Estrógenos/metabolismo , Endometrio/metabolismo , ÚteroRESUMEN
Transcriptomic profiling has fundamentally influenced our understanding of cancer pathophysiology and response to therapeutic intervention and has become a relatively routine approach. However, standard protocols are usually low-throughput, single-plex assays and costs are still quite prohibitive. With the evolving complexity of in vitro cell model systems, there is a need for resource-efficient high-throughput approaches that can support detailed time-course analytics, accommodate limited sample availability, and provide the capacity to correlate phenotype to genotype at scale. MAC-seq (multiplexed analysis of cells) is a low-cost, ultrahigh-throughput RNA-seq workflow in plate format to measure cell perturbations and is compatible with high-throughput imaging. Here we describe the steps to perform MAC-seq in 384-well format and apply it to 2D and 3D cell cultures. On average, our experimental conditions identified over ten thousand expressed genes per well when sequenced to a depth of one million reads. We discuss technical aspects, make suggestions on experimental design, and document critical operational procedures. Our protocol highlights the potential to couple MAC-seq with high-throughput screening applications including cell phenotyping using high-content cell imaging.
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Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , RNA-Seq/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Perfilación de la Expresión Génica/métodos , Fenotipo , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodosRESUMEN
Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood. Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema. Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury. Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema.
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Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.
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Leucemia Mieloide Aguda , Receptores de Citocinas , Humanos , Receptores de Citocinas/uso terapéutico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Fosforilación , Transducción de Señal , Proliferación Celular , Células Madre NeoplásicasRESUMEN
BACKGROUND: Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. METHODS: As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms. RESULTS: A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.
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Cobre , Neoplasias , Animales , Ratones , Diferenciación Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Transducción de SeñalRESUMEN
We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.
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Neoplasias de la Mama Triple Negativas , Humanos , Proteína de Unión al GTP rac1/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido/metabolismo , Movimiento Celular , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.
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Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal/genética , Nucléolo Celular/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
The gut fermentation product butyrate displays anti-cancer properties in the human proximal colon, including the ability to inhibit proliferation and induce apoptosis in colorectal cancer (CRC) cells. A natural histone deacetylase inhibitor (HDACi), butyrate can alter histone acetylation patterns in CRC cells, and thereby regulate global gene expression, including the non-coding transcriptome and microRNAs (miRNAs). Dysregulated miRNA expression affects CRC development and progression; however, the interplay between miRNA activity and butyrate response remains to be elucidated. A high-throughput functional screen was employed to identify miRNAs that can act as enhancers of the anti-cancer properties of butyrate. Validation studies confirmed that several miRNAs, including miR-125b, miR-181a, miR-593, and miR-1227, enhanced apoptosis, decreased proliferation, and promoted cell-cycle arrest in the presence of butyrate. Pathway analyses of predicted miRNA target genes highlighted their likely involvement in critical cancer-related growth pathways, including WNT and PI3K signaling. Several cancer-associated miRNA targets, including TRIM29, COX2, PIK3R3, CCND1, MET, EEF2K, DVL3, and NUP62 were synergistically regulated by the combination of cognate miRNAs and butyrate. Overall, this study has exposed the potential of miRNAs to act as enhancers of the anti-cancer effects of HDAC inhibition and identifies specific miRNAs that might be exploited for therapeutic benefit.
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The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.
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High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.
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Linfocitos B , Epigénesis Genética , Animales , Humanos , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Fenotipo , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Visceral leishmaniasis is a potentially fatal disease caused by the parasitic protists, Leishmania donovani and L. infantum. Current treatments remain unsuitable due to cost, the need for hospitalization, variable efficacy against different species, toxicity and emerging resistance. Herein, we report the SAR exploration of the novel hit 4-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-1H-imidazole-2-yl)benzamide [1] previously identified from a high throughput screen against Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani. An extensive and informative set of analogues were synthesized incorporating key modifications around the scaffold resulting in improved potency, whilst the majority of compounds maintained low cytotoxicity against human THP-1 macrophages that are target cells for these pathogens. New lead compounds identified within this study also maintained desirable physicochemical properties, improved metabolic stability in vitro and displayed no significant mitotoxicity against HepG2 cell lines. This compound class warrants continued investigation towards development as a novel treatment for Visceral Leishmaniasis.
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Antiprotozoarios , Leishmania donovani , Leishmaniasis Visceral , Trypanosoma cruzi , Antiprotozoarios/química , Humanos , Imidazoles/uso terapéutico , Leishmaniasis Visceral/tratamiento farmacológicoRESUMEN
Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by residual disease that ultimately results in relapse. This residual disease is often characterized by non-genetic adaptive resistance, that in melanoma is characterised by altered metabolism. Here, we examine how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNA interference (RNAi) screen and global gene expression profiling. Using this systematic approach we demonstrate post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA processing kinase U2AF homology motif kinase 1 (UHMK1) associates with mRNAs encoding metabolism proteins and selectively controls their transport and translation during adaptation to BRAF-targeted therapy. UHMK1 inactivation induces cell death by disrupting therapy induced metabolic reprogramming, and importantly, delays resistance to BRAF and MEK combination therapy in multiple in vivo models. We propose selective mRNA processing and translation by UHMK1 constitutes a mechanism of non-genetic resistance to targeted therapy in melanoma by controlling metabolic plasticity induced by therapy.