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OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.
Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.
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Neoplasias da Mama , Oligodesoxirribonucleotídeos , Receptor ErbB-2 , Trastuzumab , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/uso terapêutico , Trastuzumab/uso terapêutico , Trastuzumab/administração & dosagem , Receptor ErbB-2/metabolismo , Pessoa de Meia-Idade , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , IdosoRESUMO
CTLA-4 is an immune checkpoint expressed on active anticancer T cells. When it combines with its ligand B7 on dendritic cells, it inhibits the activity of the T cells. The Bromo- and Extra-Terminal (BET) protein family includes proteins that regulate the expression of key oncogenes and antiapoptotic proteins. BET inhibitor (BETi) has been shown to reduce the expression of MYC by suppressing its transcription factors and to down-regulate the hypoxic transcriptome response to VEGF-A. This paper develops a mathematical model of the treatment of cancer by combination therapy of BETi and CTLA-4 inhibitor. The model shows that the two drugs are positively correlated in the sense that the tumor volume decreases as the dose of each of the drugs is increased. The model also considers the effect of the combined therapy on levels of myeloid-derived suppressor cells (MDSCs) and the overexpression of TNF-α, which may predict gastrointestinal side effects of the combination.
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Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Antígeno CTLA-4/antagonistas & inibidores , Modelos Teóricos , Proteínas/antagonistas & inibidores , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , CamundongosRESUMO
BACKGROUND: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. METHODS: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test. RESULTS: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. CONCLUSIONS: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. TRIAL REGISTRATION: NCT04022616.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante/métodos , Receptor de Morte Celular Programada 1/metabolismo , Adulto , Idoso , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Quimioterapia Adjuvante , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/imunologia , Microambiente TumoralRESUMO
Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.
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GTP Fosfo-Hidrolases/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Indóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Neoplasias Cutâneas/metabolismo , Sulfonamidas/uso terapêutico , Regulação para Cima , VemurafenibRESUMO
The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.
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Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Macrófagos/metabolismo , Monócitos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de IgG/metabolismo , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Macrófagos/patologia , Camundongos , Monócitos/patologia , Piperidinas , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/genéticaRESUMO
This study sought to evaluate whether myeloid-derived suppressor cells (MDSC) could be affected by chemotherapy and correlate with pathologic complete response (pCR) in breast cancer patients receiving neo-adjuvant chemotherapy. Peripheral blood levels of granulocytic (G-MDSC) and monocytic (M-MDSC) MDSC were measured by flow cytometry prior to cycle 1 and 2 of doxorubicin and cyclophosphamide and 1st and last administration of paclitaxel or paclitaxel/anti-HER2 therapy. Of 24 patients, 11, 6 and 7 patients were triple negative, HER2+ and hormone receptor+, respectively. 45.8% had pCR. Mean M-MDSC% were <1. Mean G-MDSC% and 95% confidence intervals were 0.88 (0.23-1.54), 5.07 (2.45-7.69), 9.32 (4.02-14.61) and 1.97 (0.53-3.41) at draws 1-4. The increase in G-MDSC by draw 3 was significant (p < 0.0001) in all breast cancer types. G-MDSC levels at the last draw were numerically lower in patients with pCR (1.15; 95% CI 0.14-2.16) versus patients with no pCR (2.71; 95% CI 0-5.47). There was no significant rise in G-MDSC from draw 1 to 3 in African American patients, and at draw 3 G-MDSC levels were significantly lower in African Americans versus Caucasians (p < 0.05). It was concluded that G-MDSC% increased during doxorubicin and cyclophosphamide therapy, but did not significantly differ between patients based on pathologic complete response.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Adulto , Negro ou Afro-Americano , Idoso , Neoplasias da Mama/etnologia , Contagem de Células , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Citocinas/sangue , Doxorrubicina/administração & dosagem , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Humanos , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/patologia , Células Supressoras Mieloides/patologia , Terapia Neoadjuvante , Paclitaxel/administração & dosagem , Projetos Piloto , Resultado do Tratamento , População BrancaRESUMO
Myeloid-derived suppressor cell (MDSC) levels are elevated in patients with cancer and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton's tyrosine kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the antitumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of fluorescence-activated cell sorting-enriched MDSC from patients with different cancer types [breast, melanoma, head and neck squamous cell cancer (HNSCC)]. Melanoma patient MDSC were treated in vitro for 4 hours with 5 µmol/L ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10× Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from patients with breast, melanoma, and HNSCC cancer revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from patients with melanoma resulted in significant changes in gene expression. GBP1, IL-1ß, and CXCL8 were among the top downregulated genes whereas RGS2 and ABHD5 were among the top upregulated genes (P < 0.001). Double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes compared with early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL-6 signaling (P < 0.004). IMPLICATIONS: scRNA-seq revealed characteristic gene expression patterns for MDSC from different patients with cancer and BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration.
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Neoplasias de Cabeça e Pescoço , Melanoma , Células Supressoras Mieloides , Animais , Humanos , Camundongos , 1-Acilglicerol-3-Fosfato O-Aciltransferase , Tirosina Quinase da Agamaglobulinemia , Análise da Expressão Gênica de Célula Única , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Epigenetic therapy is an emerging field in the treatment of human cancer, including hematologic malignancies. This class of therapeutic agents approved by the US Food and Drug Administration for cancer treatment includes DNA hypomethylating agents, histone deacetylase inhibitors, IDH1/2 inhibitors, EZH2 inhibitors, and numerous preclinical targets/agents. Most studies measuring the biological effects of epigenetic therapy focus their attention on either their direct cytotoxic effects on malignant cells or their effects on modifying tumor cell antigen expression, exposing them to immune surveillance mechanisms. However, a growing body of evidence suggests that epigenetic therapy also has effects on the development and function of the immune system, including natural killer cells, which can alter their response to cancer cells. In this review, we summarize the body of literature studying the effects of different classes of epigenetic therapy on the development and/or function of natural killer cells.
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Antineoplásicos , Neoplasias Hematológicas , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/metabolismo , Células Matadoras Naturais , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Epigênese GenéticaRESUMO
Myelodysplastic syndromes (MDS) are hematopoietic malignancies characterized by the clonal expansion of hematopoietic stem cells, bone marrow failure manifested by cytopenias, and increased risk for evolving to acute myeloid leukemia. Despite the fact that the acquisition of somatic mutations is considered key for the initiation of the disease, the bone marrow microenvironment also plays significant roles in MDS by providing the right niche and even shaping the malignant clone. Aberrant immune responses are frequent in MDS and are implicated in many aspects of MDS pathogenesis. Recently, myeloid-derived suppressor cells (MDSCs) have gained attention for their possible implication in the immune dysregulation associated with MDS. Here, we summarize the key findings regarding the expansion of MDSCs in MDS, their role in MDS pathogenesis and immune dysregulation, as well their potential as a new therapeutic target for MDS.
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BACKGROUND: Breast cancer is one of the most common causes of brain metastases. However, the presence of isolated central nervous system (CNS) metastatic disease early in the course of disease relapse is a rare event in cases of hormone receptor positive, human epidermal growth factor receptor 2 (HER2) negative breast cancer. CASE PRESENTATION: We summarize the clinical course of a pre-menopausal, 39-year old Caucasian female with history of operable, hormone receptor positive, HER2 negative breast cancer who was initially treated with curative-intend therapy but who unfortunately developed solitary metastatic lesion in the left thalamus. A biopsy of the lesion confirmed the presence of hormone receptor positive, HER2 negative metastatic breast cancer. Patient's CNS metastases continued to progress without any evidence of metastatic disease outside of the central nervous system and she eventually passed away about 5 years after the date of her initial diagnosis and 18 months following the diagnosis with brain metastasis. CONCLUSION: Based on our case, although rare, patients with treated, operable, hormone receptor positive, HER2 negative breast cancer can present with solitary brain metastasis as the only sign of disease recurrence.
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Neoplasias Encefálicas , Neoplasias da Mama , Neoplasias do Sistema Nervoso Central , Adulto , Feminino , Humanos , Metástase Neoplásica , Recidiva Local de Neoplasia , PrognósticoRESUMO
Introduction: Myeloid-derived suppressor cells (MDSC) are a subset of immature myeloid cells that inhibit anti-tumor immunity and contribute to immune therapy resistance. MDSC populations were measured in melanoma patients receiving immune checkpoint inhibitors (ICI). Methods: Patients with melanoma (n=128) provided blood samples at baseline (BL), and before cycles 2 and 3 (BC2, BC3). Peripheral blood mononuclear cells (PBMC) were analyzed for MDSC (CD33+/CD11b+/HLA- DRlo/-) and MDSC subsets, monocytic (CD14+, M-MDSC), granulocytic (CD15+, PMN-MDSC), and early (CD14-/CD15-, E-MDSC) via flow cytometry. Statistical analysis employed unpaired and paired t-tests across and within patient cohorts. Results: Levels of MDSC as a percentage of PBMC increased during ICI (BL: 9.2 ± 1.0% to BC3: 23.6 ± 1.9%, p<0.0001), and patients who developed progressive disease (PD) had higher baseline MDSC. In patients who had a complete or partial response (CR, PR), total MDSC levels rose dramatically and plateaued (BL: 6.4 ± 1.4%, BC2: 26.2 ± 4.2%, BC3: 27.5 ± 4.4%; p<0.0001), whereas MDSC rose less sharply in PD patients (BL: 11.7 ± 2.1%, BC2: 18.3 ± 3.1%, BC3: 19.0 ± 3.2%; p=0.1952). Subset analysis showed that within the expanding MDSC population, PMN-MDSC and E-MDSC levels decreased, while the proportion of M-MDSC remained constant during ICI. In PD patients, the proportion of PMN-MDSC (as a percentage of total MDSC) decreased (BL: 25.1 ± 4.7%, BC2: 16.1 ± 5.2%, BC3: 8.6 ± 1.8%; p=0.0105), whereas a heretofore under-characterized CD14+/CD15+ double positive MDSC subpopulation increased significantly (BL: 8.7 ± 1.4% to BC3: 26.9 ± 4.9%; p=0.0425). Conclusions: MDSC levels initially increased significantly in responders. PMN-MDSC decreased and CD14+CD15+ MDSC increased significantly in PD patients. Changes in MDSC levels may have prognostic value in ICI.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Ipilimumab/uso terapêutico , Melanoma/tratamento farmacológico , Células Supressoras Mieloides/imunologia , Nivolumabe/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Contagem de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
An inflammatory microenvironment has been shown to play an important role in the growth and metastasis of tumors. The NLRP3 inflammasome is a multi-protein complex of the innate immune system that is responsible for the production of the potent inflammatory cytokine IL-1ß. Tumor- associated macrophages (TAM) are an expanded population of immune cells found in the tumor microenvironment that can promote the initiation and metastasis of tumor cells. Their presence has been correlated with disease burden, highlighting the therapeutic potential of targeting this population. However, to date clinically relevant pharmacologic strategies to target TAM remain elusive. Here, we show that in vitro generated TAM harbor NLRP3 inflammasome components and produce IL-1ß. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), is in clinical use for the treatment of B- cell malignancies. We report that BTK is expressed by human in vitro generated TAM and murine macrophages and that it physically associates with the NLRP3 inflammasome. Furthermore, ibrutinib is able to inhibit BTK phosphorylation in TAM generated in vitro. Treatment of TAM with ibrutinib significantly impaired the ability of these cells to produce IL-1ß. The present study provides evidence that BTK physically associates with the NLRP3 inflammasome and that inhibition of BTK with ibrutinib can impair the production of IL-1ß by in vitro generated TAM. Thus, ibrutinib could potentially be of clinical use in abrogating inflammation-associated cancer progression and the immune-suppressive effects of myeloid cells within the tumor microenvironment.
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Neuroblastoma RAS viral oncogene homolog is a commonly mutated oncogene in melanoma, and therapeutic targeting of neuroblastoma RAS viral oncogene homolog has proven difficult. We characterized the expression and phenotypic functions of five recently discovered splice isoforms of neuroblastoma RAS viral oncogene homolog in melanoma. Canonical neuroblastoma RAS viral oncogene homolog (isoform-1) was expressed to the highest degree and its expression was significantly increased in melanoma metastases compared to primary lesions. Isoform-5 expression in metastases showed a significant, positive correlation with survival and tumours over-expressing isoform-5 had significantly decreased growth in a xenograft model. In contrast, over-expression of any isoform resulted in enhanced proliferation, and invasiveness was increased with over-expression of isoform-1 or isoform-2. Downstream signalling analysis indicated that the isoforms signalled differentially through the mitogen-activated protein kinase and PI3K pathways and A375 cells over-expressing isoform-2 or isoform-5 showed resistance to vemurafenib treatment in vitro. The neuroblastoma RAS viral oncogene homolog isoforms appear to play varying roles in melanoma phenotype and could potentially serve as biomarkers for therapeutic response and disease prognosis.
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Processamento Alternativo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Melanoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/terapia , Camundongos , Camundongos Nus , Mutação , Metástase Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Neoplasias Cutâneas/terapia , Vemurafenib/uso terapêuticoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Purpose: mAbs are used to treat solid and hematologic malignancies and work in part through Fc receptors (FcRs) on natural killer cells (NK). However, FcR-mediated functions of NK cells from patients with cancer are significantly impaired. Identifying the mechanisms of this dysfunction and impaired response to mAb therapy could lead to combination therapies and enhance mAb therapy.Experimental Design: Cocultures of autologous NK cells and MDSC from patients with cancer were used to study the effect of myeloid-derived suppressor cells (MDSCs) on NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in vitro Mouse breast cancer models were utilized to study the effect of MDSCs on antibody therapy in vivo and test the efficacy of combination therapies including a mAb and an MDSC-targeting agent.Results: MDSCs from patients with cancer were found to significantly inhibit NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in a contact-independent manner. In addition, adoptive transfer of MDSCs abolished the efficacy of mAb therapy in a mouse model of pancreatic cancer. Inhibition of iNOS restored NK-cell functions and signal transduction. Finally, nonspecific elimination of MDSCs or inhibition of iNOS in vivo significantly improved the efficacy of mAb therapy in a mouse model of breast cancer.Conclusions: MDSCs antagonize NK-cell FcR-mediated function and signal transduction leading to impaired response to mAb therapy in part through nitric oxide production. Thus, elimination of MDSCs or inhibition of nitric oxide production offers a strategy to improve mAb therapy. Clin Cancer Res; 24(8); 1891-904. ©2018 AACR.
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Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Óxido Nítrico/biossíntese , Receptores Fc/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro. RESULTS: Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis. CONCLUSION: Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Substituição de Aminoácidos , Aurora Quinase B , Aurora Quinases , Benzamidas/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Sequência Consenso , Cicloeximida/farmacologia , Fase G2 , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Proteínas Inibidoras de Apoptose , Interfase , Metáfase , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Nocodazol/farmacologia , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Quinazolinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Fluoreto de Sódio/farmacologia , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Especificidade por Substrato , Survivina , TransfecçãoRESUMO
Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4+ T cells through Jak-STAT signal transduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhibit DC antigen presentation. Antigen presentation from DCs to CD4+ T cells (T cell receptor transgenic OT-II) was measured via a [3H]-thymidine incorporation proliferation assay. MDSC from melanoma tumor models decreased the levels of proliferation more than pancreatic cancer derived MDSC. T cell proliferation was restored when MDSC were treated with inhibitors of inducible nitric oxide synthase (L-NAME and NCX-4016). A NO donor inhibited OT II T cell receptor recognition of OT II specific tetramers, thus serving as a direct measure of NO inhibition of antigen presentation. Our group has previously demonstrated that STAT1 nitration also mediates MDSC inhibitory effects on immune cells. Therefore, a novel liquid chromatography-tandem mass spectrometry assay demonstrated that nitration of the STAT1-Tyr701 occurs in PBMC derived from both pancreatic cancer and melanoma patients.
Assuntos
Melanoma Experimental/imunologia , Células Supressoras Mieloides/metabolismo , Óxido Nítrico/metabolismo , Neoplasias Pancreáticas/imunologia , Fator de Transcrição STAT1/metabolismo , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Células Supressoras Mieloides/imunologia , Óxido Nítrico/imunologia , Doadores de Óxido Nítrico/metabolismo , Neoplasias Pancreáticas/sangue , Fator de Transcrição STAT1/análise , Espectrometria de Massas em TandemRESUMO
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that accumulate in the blood and tumors of patients with cancer. MDSC play a critical role during tumor evasion and promote immune suppression through variety of mechanisms, such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and cytokines. AMPactivated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. However, the role of AMPK in the regulation of MDSC function remains largely unexplored. This study was designed to investigate whether treatment of MDSC with OSU-53, a PPAR-inactive derivative that stimulates AMPK kinase, can modulate MDSC function. Our results demonstrate that OSU-53 treatment increases the phosphorylation of AMPK, significantly reduces nitric oxide production, inhibits MDSC migration, and reduces the levels of IL-6 in murine MDSC cell line (MSC2 cells). OSU53 treatment mitigated the immune suppressive functions of murine MDSC, promoting T-cell proliferation. Although OSU-53 had a modest effect on tumor growth in mice inoculated with EMT-6 cells, importantly, administration of OSU53 significantly (p < 0.05) reduced the levels of MDSC in the spleens and tumors. Furthermore, mouse MDSC from EMT-6 tumor-bearing mice and human MDSC isolated from melanoma patients treated with OSU-53 showed a significant reduction in the expression of immune suppressive genes iNOS and arginase. In summary, these results demonstrate a novel role of AMPK in the regulation of MDSC functions and provide a rationale of combining OSU-53 with immune checkpoint inhibitors to augment their response in cancer patients.
RESUMO
It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy.