RESUMO
To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.
Assuntos
Melanoma , Humanos , Redes Reguladoras de Genes , Imunoterapia , Melanócitos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição 4/genética , Microambiente TumoralRESUMO
Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasia Residual/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor X Retinoide gama/antagonistas & inibidores , Animais , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos SCID , Mutação , Neoplasia Residual/metabolismo , Neoplasia Residual/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.
Assuntos
Linfócitos T CD4-Positivos , Morte Celular , Imunoterapia , Inflamação , Neoplasias , Microambiente Tumoral , Humanos , Células Apresentadoras de Antígenos/imunologia , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata , Inflamação/imunologia , Interferons/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Células Th1/citologia , Células Th1/imunologiaRESUMO
Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.
Assuntos
Proliferação de Células , Melanoma , Metástase Neoplásica , Animais , Comunicação Celular , Diferenciação Celular , Linhagem da Célula , Rastreamento de Células , Reprogramação Celular , Células Endoteliais , Melanoma/genética , Melanoma/patologia , Mesoderma/patologia , Camundongos , Metástase Neoplásica/patologia , Crista Neural/embriologia , Fenótipo , Análise de Célula Única , Transcriptoma , Microambiente TumoralRESUMO
An incomplete view of the mechanisms that drive metastasis, the primary cause of cancer-related death, has been a major barrier to development of effective therapeutics and prognostic diagnostics. Increasing evidence indicates that the interplay between microenvironment, genetic lesions, and cellular plasticity drives the metastatic cascade and resistance to therapies. Here, using melanoma as a model, we outline the diversity and trajectories of cell states during metastatic dissemination and therapy exposure, and highlight how understanding the magnitude and dynamics of nongenetic reprogramming in space and time at single-cell resolution can be exploited to develop therapeutic strategies that capitalize on nongenetic tumor evolution.
Assuntos
Plasticidade Celular , Melanoma/fisiopatologia , Metástase Neoplásica/fisiopatologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/terapia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Células-Tronco Neoplásicas/citologia , Fenótipo , Microambiente TumoralRESUMO
DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
Assuntos
Metilação de DNA , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Timina DNA Glicosilase/metabolismo , 5-Metilcitosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citidina Desaminase/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Timina DNA Glicosilase/genética , Transcrição GênicaRESUMO
Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing, which is fundamentally driven by aberrant cell motility behaviors. Discovering "migrastatics" targets, specifically controlling invasion and dissemination of melanoma cells during metastasis, is therefore of primary importance. Here, we uncover the prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic dissemination. In vitro as well as in vivo, TRPV2 activity is sufficient to confer both migratory and invasive potentials, while conversely TRPV2 silencing in highly metastatic melanoma cells prevents aggressive behavior. In invasive melanoma cells, TRPV2 channel localizes at the leading edge, in dynamic nascent adhesions, and regulates calcium-mediated activation of calpain and the ensuing cleavage of the adhesive protein talin, along with F-actin organization. In human melanoma tissues, TRPV2 overexpression correlates with advanced malignancy and poor prognosis, evoking a biomarker potential. Hence, by regulating adhesion and motility, the mechanosensitive TRPV2 channel controls melanoma cell invasiveness, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Melanoma/genética , Membrana Celular/metabolismo , Neoplasias Cutâneas/genética , Canais de Cátion TRPV/genética , Movimento Celular/genética , Invasividade Neoplásica/patologia , Cálcio/metabolismoRESUMO
Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.
Assuntos
Códon/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Biossíntese de Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Códon/efeitos dos fármacos , Feminino , Humanos , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transdução de Sinais , Fatores de Elongação da Transcrição , Uridina/química , Uridina/genética , Uridina/metabolismo , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Peixe-Zebra/genéticaRESUMO
The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD(+)/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatin-bound MDM2 in cancer cell metabolism.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Cromatina/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Glicina/metabolismo , Células HCT116 , Homeostase , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Nus , Mutação , Oxirredução , Estresse Oxidativo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Animais , Cromatina/genética , Feminino , Inativação Gênica , Genes Ligados ao Cromossomo X/genética , Histona Desacetilases/genética , Camundongos , Ativação Transcricional/genética , Cromossomo X/genéticaRESUMO
NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5' part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA Longo não Codificante/genética , Processamento Alternativo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Exossomos/metabolismo , Técnicas de Inativação de Genes , Homeostase , Humanos , Hibridização in Situ Fluorescente , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Estabilidade de RNA , Estresse Fisiológico/genética , TranscriptomaRESUMO
We present SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data (http://scenic.aertslab.org). On a compendium of single-cell data from tumors and brain, we demonstrate that cis-regulatory analysis can be exploited to guide the identification of transcription factors and cell states. SCENIC provides critical biological insights into the mechanisms driving cellular heterogeneity.
Assuntos
Redes Reguladoras de Genes , Análise de Célula Única , Algoritmos , Animais , Encéfalo/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , CamundongosRESUMO
Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.
Assuntos
Acetaldeído , Melanoma , Peixe-Zebra , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Melanoma/tratamento farmacológico , Acetaldeído/metabolismo , Acetaldeído/farmacologia , Animais , Humanos , Linhagem Celular Tumoral , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , Histonas/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Transcrição Gênica/efeitos dos fármacos , Crista Neural/metabolismo , Crista Neural/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
The ecosystem of brain tumors is considered immunosuppressed, but our current knowledge may be incomplete. Here we analyzed clinical cell and tissue specimens derived from patients presenting with glioblastoma or nonmalignant intracranial disease to report that the cranial bone (CB) marrow, in juxtaposition to treatment-naive glioblastoma tumors, harbors active lymphoid populations at the time of initial diagnosis. Clinical and anatomical imaging, single-cell molecular and immune cell profiling and quantification of tumor reactivity identified CD8+ T cell clonotypes in the CB that were also found in the tumor. These were characterized by acute and durable antitumor response rooted in the entire T cell developmental spectrum. In contrast to distal bone marrow, the CB niche proximal to the tumor showed increased frequencies of tumor-reactive CD8+ effector types expressing the lymphoid egress marker S1PR1. In line with this, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker indicating focal association with improved progression-free survival. The data of this study advocate preservation and further exploitation of these cranioencephalic units for the clinical care of glioblastoma.
RESUMO
BACKGROUND: One of the key limitations of targeted cancer therapies is the rapid onset of therapy resistance. Taking BRAF-mutant melanoma as paradigm, we previously identified the lipogenic regulator SREBP-1 as a central mediator of resistance to MAPK-targeted therapy. Reasoning that lipogenesis-mediated alterations in membrane lipid poly-unsaturation lie at the basis of therapy resistance, we targeted fatty acid synthase (FASN) as key player in this pathway to evoke an exquisite vulnerability to clinical inducers of reactive oxygen species (ROS), thereby rationalizing a novel clinically actionable combination therapy to overcome therapy resistance. METHODS: Using gene expression analysis and mass spectrometry-based lipidomics of BRAF-mutant melanoma cell lines, melanoma PDX and clinical data sets, we explored the association of FASN expression with membrane lipid poly-unsaturation and therapy-resistance. Next, we treated therapy-resistant models with a preclinical FASN inhibitor TVB-3664 and a panel of ROS inducers and performed ROS analysis, lipid peroxidation tests and real-time cell proliferation assays. Finally, we explored the combination of MAPK inhibitors, TVB-3664 and arsenic trioxide (ATO, as a clinically used ROS-inducer) in Mel006 BRAF mutant PDX as a gold model of therapy resistance and assessed the effect on tumor growth, survival and systemic toxicity. RESULTS: We found that FASN expression is consistently increased upon the onset of therapy resistance in clinical melanoma samples, in cell lines and in Mel006 PDX and is associated with decreased lipid poly-unsaturation. Forcing lipid poly-unsaturation in therapy-resistant models by combining MAPK inhibition with FASN inhibition attenuated cell proliferation and rendered cells exquisitely sensitive to a host of ROS inducers. In particular, the triple combination of MAPK inhibition, FASN inhibition, and the clinical ROS-inducing compound ATO dramatically increased survival of Mel006 PDX models from 15 to 72% with no associated signs of toxicity. CONCLUSIONS: We conclude that under MAPK inhibition the direct pharmacological inhibition of FASN evokes an exquisite vulnerability to inducers of ROS by increasing membrane lipid poly-unsaturation. The exploitation of this vulnerability by combining MAPK and/or FASN inhibitors with inducers of ROS greatly delays the onset of therapy resistance and increases survival. Our work identifies a clinically actionable combinatorial treatment for therapy-resistant cancer.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Lipídeos de Membrana/farmacologia , Lipídeos de Membrana/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos AntineoplásicosRESUMO
BACKGROUND: Immune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy. METHODS: We used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs. RESULTS: Here, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry). CONCLUSION: As HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Glucocorticoides , Imunoterapia , Melanoma , Animais , Camundongos , Linfócitos T CD8-Positivos , Glucocorticoides/uso terapêutico , Interferon gama/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Reposicionamento de MedicamentosRESUMO
Recent studies suggest that BRAFV600-mutated melanomas in particular respond to dual anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibition (ICI). Here we identified an over-representation of interleukin (IL)-17-type 17 helper T (TH17) gene expression signatures (GES) in BRAFV600-mutated tumors. Moreover, high baseline IL-17 GES consistently predicted clinical responses in dual-ICI-treated patient cohorts but not in mono anti-CTLA-4 or anti-PD-1 ICI cohorts. High IL-17 GES corresponded to tumor infiltration with T cells and neutrophils. Accordingly, high neutrophil infiltration correlated with clinical response specifically to dual ICI, and tumor-associated neutrophils also showed strong IL-17-TH17 pathway activity and T cell activation capacity. Both the blockade of IL-17A and the depletion of neutrophils impaired dual-ICI response and decreased T cell activation. Finally, high IL-17A levels in the blood of patients with melanoma indicated a higher global TH17 cytokine profile preceding clinical response to dual ICI but not to anti-PD-1 monotherapy, suggesting a future role as a biomarker for patient stratification.
Assuntos
Interleucina-17 , Melanoma , Humanos , Interleucina-17/genética , Interleucina-17/uso terapêutico , Antígeno CTLA-4/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas B-raf/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genéticaRESUMO
Epithelial-to-mesenchymal transition (EMT), a process through which epithelial tumor cells acquire mesenchymal phenotypic properties, contributes to both metastatic dissemination and therapy resistance in cancer. Accumulating evidence indicates that nonepithelial tumors, including melanoma, can also gain mesenchymal-like properties that increase their metastatic propensity and decrease their sensitivity to therapy. In this review, we discuss recent findings, illustrating the striking similarities-but also knowledge gaps-between the biology of mesenchymal-like state(s) in melanoma and mesenchymal state(s) from epithelial cancers. Based on this comparative analysis, we suggest hypothesis-driven experimental approaches to further deepen our understanding of the EMT-like process in melanoma and how such investigations may pave the way towards the identification of clinically relevant biomarkers for prognosis and new therapeutic strategies.