RESUMO
Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.
Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Resistencia a Medicamentos Antineoplásicos/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/transplante , Animais , Apoptose , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Proliferação de Células , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Prognóstico , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.
RESUMO
Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.
RESUMO
BACKGROUND: Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer. METHODS: To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing. RESULTS: Our studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration. CONCLUSIONS: Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations.
Assuntos
Sistemas CRISPR-Cas , Genômica , Neoplasias/genética , Evasão Tumoral/genética , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/genética , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/terapia , Proteína-Arginina N-Metiltransferases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Repressoras/genética , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
PURPOSE: OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy.Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kß inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. RESULTS: We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kß-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells. CONCLUSIONS: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Melanoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Receptores OX40/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Receptores OX40/antagonistas & inibidoresRESUMO
Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti-PD-1-treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3' untranslated region (UTR) of HLA-A to destabilize the mRNA.Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3' UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366-76. ©2018 AACRSee related commentary by Kalbasi and Ribas, p. 3239.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos HLA-A/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Biomarcadores Tumorais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/genética , Genes Reporter , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Humanos , Interferon gama/biossíntese , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Ligação Proteica , Proteínas de Ligação a RNA/metabolismoRESUMO
Background: Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods: Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results: We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett's test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor-treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey's test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions: We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.
Assuntos
Imunoterapia Adotiva/métodos , Melanoma/terapia , Linfócitos T Citotóxicos/transplante , Inibidores da Topoisomerase I/uso terapêutico , Animais , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Terapia Combinada , Feminino , Humanos , Irinotecano/uso terapêutico , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Topotecan/uso terapêutico , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.
Assuntos
Glicólise , Imunoterapia Adotiva , Neoplasias Pulmonares/terapia , Melanoma/terapia , Linfócitos T/transplante , Animais , Linhagem Celular Tumoral , Quimiocina CXCL10/metabolismo , Feminino , Humanos , Fator Regulador 1 de Interferon/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ipilimumab/farmacologia , Melanoma/terapia , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Imunoterapia , Interferons/farmacologia , Estimativa de Kaplan-Meier , Melanoma/genética , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Regulação para CimaRESUMO
UNLABELLED: T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kß inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.
Assuntos
Anticorpos/administração & dosagem , Melanoma/tratamento farmacológico , Melanoma/genética , PTEN Fosfo-Hidrolase/deficiência , Linfócitos T/imunologia , Aminopiridinas/administração & dosagem , Aminopiridinas/uso terapêutico , Animais , Anticorpos/uso terapêutico , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Melanoma/imunologia , Camundongos , Morfolinas/administração & dosagem , Morfolinas/uso terapêutico , Receptor de Morte Celular Programada 1/imunologiaRESUMO
PURPOSE: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. EXPERIMENTAL DESIGN: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2D(b) to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. RESULTS: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. CONCLUSION: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell-based immunotherapy for the treatment of patients with melanoma.