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1.
Nat Commun ; 13(1): 4053, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35831288

RESUMO

The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética
3.
Genome Biol ; 23(1): 113, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35538548

RESUMO

BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.


Assuntos
Neoplasias Colorretais , Complemento C1q , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Complemento C1q/genética , Complemento C1q/uso terapêutico , Humanos , Instabilidade de Microssatélites , Transcriptoma , Microambiente Tumoral/genética
4.
Cell Rep ; 36(4): 109429, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34320344

RESUMO

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.


Assuntos
Neoplasias/patologia , Organoides/patologia , Medicina de Precisão , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fluorescência , Genômica , Antígenos HLA/genética , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Neoplasias/genética , Redes Neurais de Computação , Transcriptoma/genética
5.
Cell Rep ; 23(2): 361-375, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29641997

RESUMO

Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.


Assuntos
Reprogramação Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Antígenos de Superfície/metabolismo , Linhagem Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fosforilação , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
6.
Proc Natl Acad Sci U S A ; 114(2): 340-345, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28003464

RESUMO

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Fator de Iniciação Eucariótico 4G/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Ligação Proteica/fisiologia , Ribossomos/metabolismo , Proteína SOS1/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia
7.
Curr Protoc Hum Genet ; 88: 21.4.1-21.4.23, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26724721

RESUMO

Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Genoma Humano/genética , Células-Tronco Pluripotentes/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Mutação INDEL , Reprodutibilidade dos Testes
8.
Curr Protoc Hum Genet ; 87: 21.2.1-21.2.21, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26439714

RESUMO

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genética , Técnicas de Cultura de Células , Separação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Transfecção
9.
Cell Stem Cell ; 14(1): 40-52, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24239284

RESUMO

Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) promotes a broad array of cellular changes. Here we show that the let-7 family of microRNAs acts as an inhibitory influence on the reprogramming process through a regulatory pathway involving prodifferentiation factors, including EGR1. Inhibiting let-7 in human cells promotes reprogramming to a comparable extent to c-MYC when combined with OCT4, SOX2, and KLF4, and persistence of let-7 inhibits reprogramming. Inhibiting let-7 during reprogramming leads to an increase in the level of the let-7 target LIN-41/TRIM71, which in turn promotes reprogramming and is important for overcoming the let-7 barrier to reprogramming. Mechanistic studies revealed that LIN-41 regulates a broad array of differentiation genes, and more specifically, inhibits translation of EGR1 through binding its cognate mRNA. Together our findings outline a let-7-based pathway that counteracts the activity of reprogramming factors through promoting the expression of prodifferentiation genes.


Assuntos
Diferenciação Celular , Reprogramação Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Ubiquitina-Proteína Ligases/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética
10.
Cell ; 123(4): 621-9, 2005 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-16271385

RESUMO

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Here, we show that both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC.


Assuntos
Proteínas de Drosophila/metabolismo , Interferência de RNA , Complexo de Inativação Induzido por RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Linhagem Celular , Sistema Livre de Células , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo
11.
Proc Natl Acad Sci U S A ; 101(40): 14385-9, 2004 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-15452342

RESUMO

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.


Assuntos
Proteínas de Drosophila/isolamento & purificação , Interferência de RNA , Complexo de Inativação Induzido por RNA/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas Argonautas , Linhagem Celular , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/isolamento & purificação , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/genética , Homologia de Sequência de Aminoácidos , Cloreto de Sódio
12.
Science ; 301(5641): 1921-5, 2003 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-14512631

RESUMO

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.


Assuntos
Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endorribonucleases/metabolismo , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Argonautas , Biotinilação , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Linhagem Celular , Precipitação Química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Cinética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , RNA Helicases/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Complexo de Inativação Induzido por RNA/isolamento & purificação , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Recombinantes/metabolismo , Ribonuclease III
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