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1.
Cell ; 185(14): 2591-2608.e30, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35803246

RESUMO

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.


Assuntos
Neoplasias Encefálicas , Melanoma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Linfócitos T CD8-Positivos/patologia , Ecossistema , Humanos , RNA-Seq
2.
Nature ; 595(7865): 114-119, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33915568

RESUMO

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.


Assuntos
COVID-19/patologia , COVID-19/virologia , Pulmão/patologia , SARS-CoV-2/patogenicidade , Análise de Célula Única , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Atlas como Assunto , Autopsia , COVID-19/imunologia , Estudos de Casos e Controles , Feminino , Fibroblastos/patologia , Fibrose/patologia , Fibrose/virologia , Humanos , Inflamação/patologia , Inflamação/virologia , Macrófagos/patologia , Macrófagos/virologia , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Linfócitos T/imunologia
3.
Immunity ; 47(4): 789-802.e9, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29045907

RESUMO

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.


Assuntos
Imunoterapia/métodos , Neoplasias Experimentais/terapia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
4.
Genes Immun ; 25(1): 82-84, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38082156

RESUMO

Immune evasion is a hallmark of cancer, yet the underlying mechanisms are often unknown in many patients. Using single-cell transcriptomics analysis, we previously identified the co-stimulator CD58 as part of a cancer cell-intrinsic immune checkpoint resistance signature in patient melanoma tissue. We subsequently validated CD58 loss as a driver of immune evasion using a patient-derived co-culture model of cancer and cytotoxic tumor-infiltrating lymphocytes in a pooled single-cell perturbation experiment, where we additionally observed concurrent upregulation of PD-L1 protein expression in melanoma cells with CD58 loss. In our most recent study, we uncovered the mechanisms of immune evasion mediated by CD58 loss, including impaired T cell activation and infiltration within tumors, as well as inhibitory signaling by PD-L1 via a shared regulator, CMTM6. Thus, cancer cell-intrinsic reduction of CD58 represents a multi-faceted determinant of immune evasion. Furthermore, its reciprocal interaction with PD-L1 via CMTM6 provides critical insights into how co-inhibitory and co-stimulatory immune cues are regulated.


Assuntos
Antígeno B7-H1 , Melanoma , Humanos , Antígeno B7-H1/genética , Melanoma/genética , Evasão da Resposta Imune , Linhagem Celular Tumoral , Transdução de Sinais
5.
Int J Cancer ; 150(1): 142-151, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34528710

RESUMO

Most melanoma-associated deaths result from the early development of metastasis. Toll-like receptor 4 (TLR4) expression on nontumor cells is well known to contribute to tumor development and metastatic progression. The role of TLR4 expression on tumor cells however is less well understood. Here we describe TLR4 as a driver of tumor progression and metastatic spread of melanoma cells by employing a transplantable mouse melanoma model. HCmel12 melanoma cells lacking functional TLR4 showed increased sensitivity to tumor necrosis factor α induced cell killing in vitro compared to cells with intact TLR4. Interestingly, TLR4 knockout melanoma cells also showed impaired migratory capacity in vitro and a significantly reduced ability to metastasize to the lungs after subcutaneous transplantation in vivo. Finally, we demonstrate that activation of TLR4 also promotes migration in a subset of human melanoma cell lines. Our work describes TLR4 as an important mediator of melanoma migration and metastasis and provides a rationale for therapeutic inhibition of TLR4 in melanoma.


Assuntos
Movimento Celular , Neoplasias Pulmonares/secundário , Melanoma/patologia , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose , Sistemas CRISPR-Cas , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Células Tumorais Cultivadas
7.
Nature ; 507(7490): 109-13, 2014 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-24572365

RESUMO

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.


Assuntos
Inflamação/etiologia , Neoplasias Pulmonares/secundário , Melanoma/irrigação sanguínea , Melanoma/patologia , Neoplasias Cutâneas/patologia , Queimadura Solar/etiologia , Raios Ultravioleta , Animais , Movimento Celular/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Modelos Animais de Doenças , Progressão da Doença , Feminino , Proteína HMGB1/metabolismo , Imunidade Inata/efeitos da radiação , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/etiologia , Masculino , Melanócitos/patologia , Melanócitos/efeitos da radiação , Melanoma/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/etiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/etiologia , Queimadura Solar/complicações , Receptor 4 Toll-Like/metabolismo
8.
Nature ; 490(7420): 412-6, 2012 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-23051752

RESUMO

Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.


Assuntos
Desdiferenciação Celular , Imunoterapia , Inflamação/patologia , Melanoma/patologia , Melanoma/terapia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Transferência Adotiva , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Antígeno gp100 de Melanoma/metabolismo
9.
Nat Biotechnol ; 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38783148

RESUMO

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

10.
Nat Cancer ; 5(3): 433-447, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38286827

RESUMO

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.


Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Insulina , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
11.
bioRxiv ; 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36711647

RESUMO

Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios - KLRG1 + subset of tumor-infiltrating regulatory T cells (Tregs) was associated with tumor progression from immune equilibrium to escape, and were also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumorinfiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1 + CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker and/or target discovery.

12.
J Immunother Cancer ; 11(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37657842

RESUMO

Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios-KLRG1+ subset of tumor-infiltrating regulatory T cells was associated with tumor progression from immune equilibrium to escape and was also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumor-infiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1+ CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker discovery.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Linfócitos T CD4-Positivos , Subpopulações de Linfócitos T , Imunoterapia , Biomarcadores , Receptores Imunológicos , Lectinas Tipo C
13.
Cancer Cell ; 41(5): 933-949.e11, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-37116491

RESUMO

Due to their immunosuppressive role, tumor-infiltrating regulatory T cells (TI-Tregs) represent attractive immuno-oncology targets. Analysis of TI vs. peripheral Tregs (P-Tregs) from 36 patients, across four malignancies, identified 17 candidate master regulators (MRs) as mechanistic determinants of TI-Treg transcriptional state. Pooled CRISPR-Cas9 screening in vivo, using a chimeric hematopoietic stem cell transplant model, confirmed the essentiality of eight MRs in TI-Treg recruitment and/or retention without affecting other T cell subtypes, and targeting one of the most significant MRs (Trps1) by CRISPR KO significantly reduced ectopic tumor growth. Analysis of drugs capable of inverting TI-Treg MR activity identified low-dose gemcitabine as the top prediction. Indeed, gemcitabine treatment inhibited tumor growth in immunocompetent but not immunocompromised allografts, increased anti-PD-1 efficacy, and depleted MR-expressing TI-Tregs in vivo. This study provides key insight into Treg signaling, specifically in the context of cancer, and a generalizable strategy to systematically elucidate and target MR proteins in immunosuppressive subpopulations.


Assuntos
Neoplasias , Linfócitos T Reguladores , Humanos , Linfócitos T Reguladores/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Proteínas Repressoras/metabolismo
14.
Nat Commun ; 14(1): 8435, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38114518

RESUMO

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Terapia Neoadjuvante , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Ensaios Clínicos Fase II como Assunto
15.
Cancer Cell ; 41(7): 1207-1221.e12, 2023 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-37327789

RESUMO

The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.


Assuntos
Antígeno B7-H1 , Melanoma , Camundongos , Animais , Antígeno B7-H1/genética , Linfócitos T , Antígenos CD58/química , Antígenos CD58/metabolismo , Melanoma/genética , Melanoma/metabolismo , Ativação Linfocitária
16.
Clin Transl Immunology ; 10(4): e1276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968406

RESUMO

OBJECTIVES: Type I interferons are evolutionally conserved cytokines, with broad antimicrobial and immunoregulatory functions. Despite well-characterised role in spontaneous cancer immunosurveillance, the function of type I IFNs in cancer immunotherapy remains incompletely understood. METHODS: We utilised genetic mouse models to explore the role of the type I IFN system in CD8+ T-cell immunotherapy targeting the melanocytic lineage antigen gp100. RESULTS: The therapeutic efficacy of adoptively transferred T cells was found to depend on a functional type I IFN system in myeloid immune cells. Compromised type I IFN signalling in myeloid immune cells did not prevent expansion, tumor infiltration or effector function of melanoma-specific Pmel-1 CD8+ T cells. However, melanomas growing in globally (Ifnar1-/-) or conditionally (Ifnar1ΔLysM) type I IFN system-deficient mice displayed increased myeloid infiltration, hypoxia and melanoma cell dedifferentiation. Mechanistically, hypoxia was found to induce dedifferentiation and loss of the gp100 target antigen in melanoma cells and type I IFN could directly inhibit the inflammatory activation of myeloid cells. Unexpectedly, the immunotherapy induced significant reduction in tumor blood vessel density and whereas host type I IFN system was not required for the vasculosculpting, it promoted vessel permeability. CONCLUSION: Our results substantiate a complex and plastic phenotypic interconnection between melanoma and myeloid cells in the context of T-cell immunotherapy. Type I IFN signalling in myeloid cells was identified as a key regulator of the balance between antitumor immunity and disease-promoting inflammation, thus supporting the development of novel combinatorial immunotherapies targeting this immune cell compartment.

17.
Nat Genet ; 53(3): 332-341, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33649592

RESUMO

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.


Assuntos
Antígenos CD58/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Melanoma/patologia , Análise de Célula Única/métodos , Evasão Tumoral , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultura , Biologia Computacional/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Epitopos/genética , Técnicas de Inativação de Genes , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/patologia , Melanoma/tratamento farmacológico , Melanoma/imunologia , Análise de Sequência de RNA , Evasão Tumoral/genética
18.
Cancer Res ; 80(4): 798-810, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31882401

RESUMO

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo-expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. SIGNIFICANCE: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor-resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.


Assuntos
Azepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Azepinas/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Indóis/uso terapêutico , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/antagonistas & inibidores
19.
Nat Med ; 26(8): 1271-1279, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32572264

RESUMO

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.


Assuntos
Ascite/genética , Cistadenoma Seroso/genética , Neoplasias Ovarianas/genética , Análise de Célula Única , Ascite/patologia , Linhagem Celular Tumoral , Cistadenoma Seroso/patologia , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Janus Quinase 1/genética , Gradação de Tumores , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Prognóstico , Fatores de Transcrição STAT/genética , Análise de Sequência de RNA , Transdução de Sinais/genética
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