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1.
Cell ; 182(2): 297-316.e27, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32619424

RESUMEN

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.


Asunto(s)
Memoria Inmunológica/fisiología , Linfoma de Células B Grandes Difuso/patología , Proteínas Nucleares/genética , Células Precursoras de Linfocitos B/inmunología , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cromatina/química , Cromatina/metabolismo , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear/química , Co-Represor 2 de Receptor Nuclear/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transcripción Genética
2.
Immunity ; 54(8): 1807-1824.e14, 2021 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-34380064

RESUMEN

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.


Asunto(s)
Linfocitos B/citología , Proteína Forkhead Box O1/genética , Centro Germinal/inmunología , Linfoma de Células B/patología , Animales , Linfocitos B/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Linfoma de Células B/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología
3.
Cell ; 160(6): 1246-60, 2015 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-25748654

RESUMEN

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.


Asunto(s)
Sistemas CRISPR-Cas , Carcinoma de Pulmón de Células no Pequeñas/genética , Técnicas de Inactivación de Genes , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias Pulmonares/patología , Ratones , ARN Guía de Kinetoplastida
4.
Mol Cell ; 82(10): 1865-1877.e4, 2022 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-35366394

RESUMEN

RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.


Asunto(s)
Proteínas Asociadas a CRISPR , ARN Guía de Kinetoplastida , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , ADN/genética , ARN Guía de Kinetoplastida/metabolismo , Ribonucleasas/metabolismo
5.
Mol Cell ; 82(23): 4487-4502.e7, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36427491

RESUMEN

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.


Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/genética , Plásmidos , ARN , ARN Guía de Kinetoplastida/metabolismo
6.
Cell ; 154(6): 1380-9, 2013 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-23992846

RESUMEN

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.


Asunto(s)
Roturas del ADN de Doble Cadena , Marcación de Gen/métodos , Genoma , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Cigoto/metabolismo , ARN Pequeño no Traducido
7.
Nature ; 609(7929): 1021-1028, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36131014

RESUMEN

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.


Asunto(s)
Diferenciación Celular , Neoplasias Cerebelosas , Meduloblastoma , Metencéfalo , Diferenciación Celular/genética , Linaje de la Célula , Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Cerebelo/embriología , Cerebelo/patología , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Proteínas Hedgehog/metabolismo , Histona Demetilasas , Humanos , Antígeno Ki-67/metabolismo , Meduloblastoma/clasificación , Meduloblastoma/genética , Meduloblastoma/patología , Metencéfalo/embriología , Metencéfalo/patología , Proteínas Musculares , Mutación , Factores de Transcripción Otx/deficiencia , Factores de Transcripción Otx/genética , Proteínas Represoras , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción
8.
Mol Cell ; 77(1): 120-137.e9, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31733993

RESUMEN

Phenotypic and metabolic heterogeneity within tumors is a major barrier to effective cancer therapy. How metabolism is implicated in specific phenotypes and whether lineage-restricted mechanisms control key metabolic vulnerabilities remain poorly understood. In melanoma, downregulation of the lineage addiction oncogene microphthalmia-associated transcription factor (MITF) is a hallmark of the proliferative-to-invasive phenotype switch, although how MITF promotes proliferation and suppresses invasion is poorly defined. Here, we show that MITF is a lineage-restricted activator of the key lipogenic enzyme stearoyl-CoA desaturase (SCD) and that SCD is required for MITFHigh melanoma cell proliferation. By contrast MITFLow cells are insensitive to SCD inhibition. Significantly, the MITF-SCD axis suppresses metastasis, inflammatory signaling, and an ATF4-mediated feedback loop that maintains de-differentiation. Our results reveal that MITF is a lineage-specific regulator of metabolic reprogramming, whereby fatty acid composition is a driver of melanoma phenotype switching, and highlight that cell phenotype dictates the response to drugs targeting lipid metabolism.


Asunto(s)
Adaptación Fisiológica/fisiología , Ácidos Grasos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo/fisiología , Humanos , Ratones , Invasividad Neoplásica/patología , Fenotipo , Transducción de Señal/fisiología
9.
Nature ; 589(7841): 299-305, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33299181

RESUMEN

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.


Asunto(s)
Transformación Celular Neoplásica/genética , Cromatina/química , Cromatina/genética , Histonas/deficiencia , Histonas/genética , Linfoma/genética , Linfoma/patología , Alelos , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Autorrenovación de las Células , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Centro Germinal/patología , Histonas/metabolismo , Humanos , Linfoma/metabolismo , Ratones , Mutación , Células Madre/metabolismo , Células Madre/patología
10.
Proc Natl Acad Sci U S A ; 121(18): e2320421121, 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38662551

RESUMEN

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.


Asunto(s)
Linfoma de Células B Grandes Difuso , FN-kappa B , Transducción de Señal , Factor 3 Asociado a Receptor de TNF , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Humanos , FN-kappa B/metabolismo , Quinasa de Factor Nuclear kappa B , Línea Celular Tumoral , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proliferación Celular
11.
Blood ; 2024 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-38820500

RESUMEN

While initial therapy of mantle cell lymphoma (MCL) is not standardized, bendamustine-rituximab (BR) is commonly used in older patients. Rituximab (R) maintenance following induction is often utilized. Thus, the open-label, randomized phase II ECOG-ACRIN Cancer Research Group E1411 trial was designed to test two questions: 1) Does addition of bortezomib to BR induction (BVR) and/or 2) addition of lenalidomide to rituximab (LR) maintenance improve progression-free survival (PFS) in patients with treatment-naïve MCL? From 2012-2016, 373 previously untreated patients, 87% ≥ 60 years old, were enrolled in this trial. At a median follow up of 7.5 years, there is no difference in the median PFS of BR compared to BVR (5.5 yrs vs. 6.4 yrs, HR 0.90, 90% CI 0.70, 1.16). There were no unexpected additional toxicities with BVR treatment compared to BR, with no impact on total dose/duration of treatment received. Independent of the induction treatment, addition of lenalidomide to rituximab did not significantly improve PFS, with median PFS in R vs LR (5.9 yrs vs 7.2 yrs, HR 0.84 90% CI 0.62, 1.15). The majority of patients completed the planned 24 cycles of LR at the scheduled dose. In summary, adding bortezomib to BR induction does not prolong PFS in treatment-naïve MCL, and LR maintenance was not associated with longer PFS compared with rituximab alone following BR. Nonetheless, the > 5 year median PFS outcomes in this prospective cooperative group trial indicate the efficacy of BR followed by rituximab maintenance as highly effective initial therapy for older MCL patients. (NCT01415752).

12.
Blood ; 2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38701426

RESUMEN

Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit": HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of, and mechanisms driving, IG vs non-IG MYC rearrangements have not been elucidated. Here we used custom targeted capture and/or whole genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, while BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because one IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

13.
Nature ; 580(7805): 663-668, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32152607

RESUMEN

On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.


Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Simulación del Acoplamiento Molecular/métodos , Programas Informáticos , Interfaz Usuario-Computador , Acceso a la Información , Automatización/métodos , Automatización/normas , Nube Computacional , Simulación por Computador , Bases de Datos de Compuestos Químicos , Descubrimiento de Drogas/normas , Evaluación Preclínica de Medicamentos/normas , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ligandos , Simulación del Acoplamiento Molecular/normas , Terapia Molecular Dirigida , Factor 2 Relacionado con NF-E2/metabolismo , Reproducibilidad de los Resultados , Programas Informáticos/normas , Termodinámica
14.
Mol Cell ; 70(2): 327-339.e5, 2018 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-29551514

RESUMEN

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , ARN Bacteriano/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Bases de Datos Genéticas , Escherichia coli/enzimología , Escherichia coli/genética , Eubacterium/enzimología , Eubacterium/genética , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Dominios Proteicos , Estructura Secundaria de Proteína , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Ruminococcus/enzimología , Ruminococcus/genética , Relación Estructura-Actividad
15.
Proc Natl Acad Sci U S A ; 120(3): e2218332120, 2023 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-36626549

RESUMEN

O-GlcNAc transferase (OGT) modifies serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We performed a genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferation induced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cells stems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.


Asunto(s)
Linfocitos T CD8-positivos , Complejo de la Endopetidasa Proteasomal , Animales , Ratones , Acetilglucosamina/metabolismo , Linfocitos T CD8-positivos/metabolismo , Supervivencia Celular , Mitocondrias/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo
16.
J Biol Chem ; 300(6): 107300, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38641066

RESUMEN

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.


Asunto(s)
Fibroblastos , Galectina 3 , Fibrosis Pulmonar Idiopática , Factor de Crecimiento Transformador beta1 , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Galectina 3/metabolismo , Galectina 3/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Pulmón/patología , Transducción de Señal , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Galectinas/metabolismo , Colágeno Tipo I/metabolismo , Células Cultivadas , Proteínas Sanguíneas
17.
Bioinformatics ; 40(1)2024 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-38152895

RESUMEN

MOTIVATION: Single cell segmentation is critical in the processing of spatial omics data to accurately perform cell type identification and analyze spatial expression patterns. Segmentation methods often rely on semi-supervised annotation or labeled training data which are highly dependent on user expertise. To ensure the quality of segmentation, current evaluation strategies quantify accuracy by assessing cellular masks or through iterative inspection by pathologists. While these strategies each address either the statistical or biological aspects of segmentation, there lacks a unified approach to evaluating segmentation accuracy. RESULTS: In this article, we present ESQmodel, a Bayesian probabilistic method to evaluate single cell segmentation using expression data. By using the extracted cellular data from segmentation and a prior belief of cellular composition as input, ESQmodel computes per cell entropy to assess segmentation quality by how consistent cellular expression profiles match with cell type expectations. AVAILABILITY AND IMPLEMENTATION: Source code is available on Github at: https://github.com/Roth-Lab/ESQmodel.


Asunto(s)
Programas Informáticos , Células Secretoras de Somatostatina , Teorema de Bayes , Entropía , Procesamiento de Imagen Asistido por Computador
18.
PLoS Pathog ; 19(11): e1011781, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37976321

RESUMEN

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.


Asunto(s)
Citomegalovirus , Herpesviridae , Humanos , Núcleo Celular/metabolismo , Herpesviridae/metabolismo , Replicación Viral , Simplexvirus , Acrilamidas/metabolismo , Antivirales/farmacología , Antivirales/metabolismo
19.
Blood ; 142(6): 561-573, 2023 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-37084389

RESUMEN

Follicular lymphoma (FL) accounts for ∼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.


Asunto(s)
Linfoma Folicular , Linfoma de Células B Grandes Difuso , Humanos , Linfoma Folicular/patología , Mutación , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología
20.
Blood ; 141(20): 2493-2507, 2023 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-36302166

RESUMEN

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.


Asunto(s)
Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Humanos , Hibridación Fluorescente in Situ , Estudios Prospectivos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/epidemiología , Linfoma de Células B Grandes Difuso/genética , Pronóstico
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