RESUMO
Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.
Assuntos
Interferons , Metiltransferases , Animais , Camundongos , Interferons/genética , Metiltransferases/genética , Metiltransferases/metabolismo , RNA de Cadeia DuplaRESUMO
Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.
Assuntos
Células-Tronco Pluripotentes , Fatores de Transcrição , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Genoma , Regiões Promotoras GenéticasRESUMO
There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
Assuntos
Proteínas F-Box , Leucemia Mieloide Aguda , Oxirredutases do Álcool , Proteínas de Ligação a DNA , Proteínas F-Box/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária , Proteína-Arginina N-Metiltransferases/metabolismo , Recidiva , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.
Assuntos
Ferro , Proteína Killer-Antagonista Homóloga a bcl-2 , Apoptose , Morte Celular , Humanos , Ferro/metabolismo , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoAssuntos
Núcleo Celular , Neoplasias , Instabilidade Cromossômica , Instabilidade Genômica , HumanosRESUMO
The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.
Assuntos
Células Sanguíneas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endotélio/metabolismo , Hematopoese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição/metabolismo , Saco Vitelino/metabolismo , Células Sanguíneas/citologia , Diferenciação Celular , Linhagem da Célula , Células Eritroides/citologia , Células Eritroides/metabolismo , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Humanos , Modelos Biológicos , Transcrição GênicaRESUMO
Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.
Assuntos
Endotélio/citologia , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Desenvolvimento Embrionário/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Organoides/citologiaRESUMO
AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disorder characterised by loss of insulin-producing beta cells of the pancreas. Progress in understanding the cellular and molecular mechanisms underlying the human disease has been hampered by a dearth of appropriate human experimental models. We previously reported the characterisation of islet-infiltrating CD4+ T cells from a deceased organ donor who had type 1 diabetes. METHODS: Induced pluripotent stem cell (iPSC) lines derived from the above donor were differentiated into CD14+ macrophages and tested for their capacity to present antigen to T cell receptors (TCRs) derived from islet-infiltrating CD4+ T cells from the same donor. RESULTS: The iPSC macrophages displayed typical macrophage morphology, surface markers (CD14, CD86, CD16 and CD11b) and were phagocytic. In response to IFNγ treatment, iPSC macrophages upregulated expression of HLA class II, a characteristic that correlated with their capacity to present epitopes derived from proinsulin C-peptide to a T cell line expressing TCRs derived from islet-infiltrating CD4+ T cells of the original donor. T cell activation was specifically blocked by anti-HLA-DQ antibodies but not by antibodies directed against HLA-DR. CONCLUSIONS/INTERPRETATION: This study provides a proof of principle for the use of iPSC-derived immune cells for modelling key cellular interactions in human type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Ilhotas Pancreáticas/metabolismo , Macrófagos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Ilhotas Pancreáticas/imunologia , Macrófagos/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we used a simultaneous reprogramming and CRISPR-Cas9 genome editing method to produce an iPSC line with the heterozygous patient mutation (COL1A1 c. 3936 G>T) along with an isogenic gene-corrected control iPSC line. Both IPSC lines had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This osteogenesis imperfecta mutant and isogenic iPSC control line will be of use in exploring disease mechanisms and therapeutic approaches in vitro.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Reprogramação Celular , Colágeno Tipo I , Células-Tronco Pluripotentes Induzidas , Mutação de Sentido Incorreto , Osteogênese Imperfeita , Substituição de Aminoácidos , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologiaRESUMO
To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we have used gene editing to produce a facsimile of the patient heterozygous COL1A1 mutation in an established control iPSC line. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line and the isogenic parental iPSC line will be of use in exploring osteogenesis imperfecta disease mechanisms and therapeutic approaches in vitro.
Assuntos
Sistemas CRISPR-Cas , Colágeno Tipo I/genética , Edição de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Leucócitos Mononucleares/patologia , Mutação , Osteogênese Imperfeita/genética , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Cadeia alfa 1 do Colágeno Tipo I , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Osteogênese Imperfeita/patologia , FenótipoRESUMO
Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.
Assuntos
Técnicas de Cultura de Células , Regulação da Expressão Gênica no Desenvolvimento , Rim/citologia , Células-Tronco Pluripotentes/citologia , Albuminas/metabolismo , Diferenciação Celular , Células Cultivadas , Doxorrubicina/farmacologia , Humanos , Néfrons/metabolismo , Organoides , Transdução de Sinais , Transcrição Gênica , Proteínas Wnt/metabolismoRESUMO
The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
Assuntos
Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Células-Tronco Pluripotentes/metabolismo , Transgenes , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Células-Tronco Pluripotentes/citologia , Nucleases dos Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ß-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.
Assuntos
Caderinas/genética , Diferenciação Celular/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Western Blotting , Caderinas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sobrevivência Celular/genética , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Microscopia Confocal , Neuritos/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de Tempo , beta Catenina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Chemotherapy fails to provide durable cure for the majority of cancer patients. To identify mechanisms associated with chemotherapy resistance, we identified genes differentially expressed before and after chemotherapeutic treatment of breast cancer patients. Treatment response resulted in either increased or decreased cell cycle gene expression. Tumors in which cell cycle gene expression was increased by chemotherapy were likely to be chemotherapy sensitive, whereas tumors in which cell cycle gene transcripts were decreased by chemotherapy were resistant to these agents. A gene expression signature that predicted these changes proved to be a robust and novel index that predicted the response of patients with breast, ovarian, and colon tumors to chemotherapy. Investigations in tumor cell lines supported these findings, and linked treatment induced cell cycle changes with p53 signaling and G1/G0 arrest. Hence, chemotherapy resistance, which can be predicted based on dynamics in cell cycle gene expression, is associated with TP53 integrity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antineoplásicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Imuno-Histoquímica , Células MCF-7 , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
A suitable culture condition using advanced biomaterials has the potential to improve stem cell differentiation into selective lineages. In this study, we evaluated the effects of recombinant extracellular matrix (ECM) components on the mouse embryonic stem (mES) and induced pluripotent stem (miPS) cells' self-renewal and differentiation into neural progenitors, comparing conventional culture substrata. The recombinant ECMs were established by immobilizing two chimera proteins of cadherin molecules, E-cadherin-Fc and N-cadherin-Fc, either alone or in combination. We report that the completely homogeneous population of mES and miPS cells could be maintained on E-cadherin-based substrata under feeder- and serum-free culture conditions to initiate neural differentiation. Using defined monolayer differentiation conditions on E-cadherin and N-cadherin (E-/N-cad-Fc) hybrid substratum, we routinely obtained highly homogeneous population of primitive ectoderm and neural progenitor cells. Moreover, the differentiated cells with higher expression of ßIII-tubulin, Pax6, and tyrosine hydroxylase (TH) in absence of GFAP (a glial cell marker) expression suggesting the presence of a lineage restricted to neural cells. Our improved culture method should provide a homogeneous microenvironment for differentiation and obviate the need for protocols based on stromal feeders or embryoid bodies.