RESUMO
Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Doxorrubicina/farmacologia , Feminino , Células HCT116 , Células HT29 , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos Nus , Microscopia Confocal , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Despite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge in the field of immunotherapy; thus, more optimized translational models could strongly influence clinical trial development. To address this unmet need, we designed a preclinical model reflecting the heterogeneity in melanoma patients' clinical responses that can be used to evaluate novel immunotherapies and synergistic combinatorial treatment strategies. Using our all-autologous humanized melanoma mouse model, we examined the efficacy of a novel engineered interleukin 2 (IL-2)-based cytokine variant immunotherapy. METHODS: To study immune responses and antitumor efficacy for human melanoma tumors, we developed an all-autologous humanized melanoma mouse model using clinically annotated, matched patient tumor cells and peripheral blood mononuclear cells (PBMCs). After inoculating immunodeficient NSG mice with patient tumors and an adoptive cell transfer of autologous PBMCs, mice were treated with anti-PD-1, a novel investigational engineered IL-2-based cytokine (nemvaleukin), or recombinant human IL-2 (rhIL-2). The pharmacodynamic effects and antitumor efficacy of these treatments were then evaluated. We used tumor cells and autologous PBMCs from patients with varying immunotherapy responses to both model the diversity of immunotherapy efficacy observed in the clinical setting and to recapitulate the heterogeneous nature of melanoma. RESULTS: Our model exhibited long-term survival of engrafted human PBMCs without developing graft-versus-host disease. Administration of an anti-PD-1 or nemvaleukin elicited antitumor responses in our model that were patient-specific and were found to parallel clinical responsiveness to checkpoint inhibitors. An evaluation of nemvaleukin-treated mice demonstrated increased tumor-infiltrating CD4+ and CD8+ T cells, preferential expansion of non-regulatory T cell subsets in the spleen, and significant delays in tumor growth compared with vehicle-treated controls or mice treated with rhIL-2. CONCLUSIONS: Our model reproduces differential effects of immunotherapy in melanoma patients, capturing the inherent heterogeneity in clinical responses. Taken together, these data demonstrate our model's translatability for novel immunotherapies in melanoma patients. The data are also supportive for the continued clinical investigation of nemvaleukin as a novel immunotherapeutic for the treatment of melanoma.
Assuntos
Inibidores de Checkpoint Imunológico , Imunoterapia , Interleucina-2 , Melanoma , Neoplasias Cutâneas , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Melanoma/terapia , Animais , Camundongos , Inibidores de Checkpoint Imunológico/administração & dosagem , Inibidores de Checkpoint Imunológico/uso terapêutico , Interleucina-2/administração & dosagem , Interleucina-2/uso terapêutico , Neoplasias Cutâneas/terapia , Imunoterapia/métodos , Autoenxertos , Camundongos Endogâmicos NODRESUMO
A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8(+) T cell responses to a dominant peptide (RNEU(420-429)) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N-derived CD4(+)CD25(+) T cells. RNEU(420-429)-specific CD8(+) T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU(420-429)-specific CD8(+) T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4(+)CD25(+) T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8(+) T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Epitopos de Linfócito T/imunologia , Ativação Linfocitária/imunologia , Neoplasias Mamárias Experimentais/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/genética , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Ciclofosfamida/administração & dosagem , Citotoxicidade Imunológica , Epitopos de Linfócito T/genética , Feminino , Genes erbB-2/imunologia , Imunossupressores/administração & dosagem , Imunoterapia Adotiva , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Transgênicos , Receptores de Interleucina-2/imunologia , VacinaçãoRESUMO
Subclinical doses of Paclitaxel (PTX) given 1day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu(+) tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8(+) T cells with enhanced lytic activity against neu(+) tumors. PTX treatment also enhances maturation marker expression on CD11c(+) DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8(+) T cells with greater lytic capability than DC's from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-gamma-secreting CD8(+) T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer , Células Dendríticas/efeitos dos fármacos , Paclitaxel/farmacologia , Animais , Antígenos CD/biossíntese , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Citocinas/genética , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Quimioterapia Combinada , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/terapia , Receptor ErbB-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
Immune-checkpoint blockade enhances antitumor responses against cancers. One cancer type that is sensitive to checkpoint blockade is squamous cell carcinoma of the head and neck (SCCHN), which we use here to study limitations of this treatment modality. We observed that CD8+ tumor-infiltrating lymphocytes (TILs) in SCCHN and melanoma express excess immune checkpoints components PD-1 and Tim-3 and are also CD27-/CD28-, a phenotype we previously associated with immune dysfunction and suppression. In ex vivo experiments, patients' CD8+ TILs with this phenotype suppressed proliferation of autologous peripheral blood T cells. Similar phenotype and function of TILs was observed in the TC-1 mouse tumor model. Treatment of TC-1 tumors with anti-PD-1 or anti-Tim-3 slowed tumor growth in vivo and reversed the suppressive function of multi-checkpoint+ CD8+ TIL. Similarly, treatment of both human and mouse PD-1+ Tim-3+ CD8+ TILs with anticheckpoint antibodies ex vivo reversed their suppressive function. These suppressive CD8+ TILs from mice and humans expressed ligands for PD-1 and Tim-3 and exerted their suppressive function via IL10 and close contact. To model therapeutic strategies, we combined anti-PD-1 blockade with IL7 cytokine therapy or with transfer of antigen-specific T cells. Both strategies resulted in synergistic antitumor effects and reduced suppressor cell function. These findings enhance our understanding of checkpoint blockade in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptor de Morte Celular Programada 1/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunoterapia , Interleucina-10/imunologia , Interleucina-7/farmacologia , Interleucina-7/uso terapêutico , Ligantes , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Microambiente Tumoral/imunologiaRESUMO
Nearly half of melanoma patients develop brain metastases during the course of their disease. Despite advances in both localized radiation and systemic immunotherapy, brain metastases remain difficult to treat, with most patients surviving less than 5 months from the time of diagnosis. While both treatment regimens have individually shown considerable promise in treating metastatic melanoma, there is interest in combining these strategies to take advantage of potential synergy. In order to study the ability of local radiation and anti-PD-1 immunotherapy to induce beneficial anti-tumor immune responses against distant, unirradiated tumors, we used two mouse models of metastatic melanoma in the brain, representing BRAF mutant and non-mutant tumors. Combination treatments produced a stronger systemic anti-tumor immune response than either treatment alone. This resulted in reduced tumor growth and larger numbers of activated, cytotoxic CD8+ T cells, even in the unirradiated tumor, indicative of an abscopal effect. The immune-mediated effects were present regardless of BRAF status. These data suggest that irradiation of brain metastases and anti-PD-1 immunotherapy together can induce abscopal anti-tumor responses that control both local and distant disease.
RESUMO
BACKGROUND: The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8+ T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8+ T cells are tumor-derived exosomes (TDEs). METHODS: Membrane vesicles and TDEs from multiple head and neck cancer cell line's conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3+CD8+ T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8+ T cells followed by suppression analysis. RESULTS: Using fractionated conditioned growth media, factors >200 kDa induced CD8+ T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8+ T cells. CONCLUSIONS: For the first-time, TDEs have been identified to induce a SP in CD8+ T cells and their mode of action may be synergistic effects from exosomal proteins and RNA. One protein in particular, galectin-1, appears to play a significant role in inducing T cell SP. Therefore, tumor-derived immunosuppressive exosomes are a potential therapeutic target to prevent T cell dysfunction and enhance anti-tumor immune responses.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Exossomos/genética , Galectina 1/genética , Neoplasias de Cabeça e Pescoço/imunologia , Antígenos CD28/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Exossomos/metabolismo , Galectina 1/metabolismo , Humanos , Espectrometria de Massas , Microambiente Tumoral , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Mcl-1, a Bcl-2 family member, is highly expressed in a variety of human cancers and is believed to enhance tumorigenic potential and chemotherapy resistance through the inhibition of apoptosis and senescence. We previously reported that Mcl-1's regulation of chemotherapy-induced senescence (CIS) is dependent on its ability to prevent reactive oxygen species (ROS) generation. In this report, we demonstrate that Mcl-1-regulated CIS requires not only ROS, but specifically mitochondrial ROS, and that these events are upstream of activation of the DNA damage response, another necessary step toward senescence. Mcl-1's anti-senescence activity also involves the unique ability to inhibit ROS formation by preventing the upregulation of pro-oxidants. Specifically, we found that NADPH oxidases (NOXs) are regulated by Mcl-1 and that NOX4 expression in particular is a required step for CIS induction that is blocked by Mcl-1. Lastly, we illustrate that by preventing expression of NOX4, Mcl-1 limits its availability in the mitochondria, thereby lowering the production of mitochondrial ROS during CIS. Our studies not only define the essential role of Mcl-1 in chemoresistance, but also for the first time link a key pro-survival Bcl-2 family member with the NOX protein family, both of which have significant ramifications in cancer progression.
Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , NADPH Oxidase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , NADPH Oxidase 4/genética , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Despite multiple studies demonstrating the importance of the anti-apoptotic protein Mcl-1 in tumor cell survival and treatment resistance, a clinically important inhibitor has yet to be developed. A recent study by Guo Wei and colleagues published in Cancer Cell has utilized a novel high-throughput approach to identify compounds that act as transcriptional repressors of MCL1 expression. Their findings identified a number of candidate drugs to be tested for clinical relevance in human cancers dependent on MCL1 expression.
RESUMO
Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53⺠cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53⻠cells restored CIS to similar levels as p53⺠cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53⺠cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.
Assuntos
Senescência Celular/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Genes p53 , Humanos , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologiaRESUMO
PURPOSE: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways. EXPERIMENTAL DESIGN: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection. RESULTS: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3ß, which, in turn, prevented the phosphorylation and loss of Mcl-1. We further showed a key role for Mcl-1 in that its knockdown or inhibition abrogated the effects of IL-7. In addition, knockdown of the Mcl-1 binding partner and proapoptotic protein Bim protected T cells from these dysfunctional alterations. CONCLUSION: These observations confirm the role for Bcl-2 family members in cytokine signaling and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T-cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells.