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1.
Mol Ther ; 31(9): 2561-2565, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37595584

RESUMO

There has been rapid growth in gene therapy development with an expanding list of approved clinical products. Several therapies are particularly relevant to patients in low- and middle-income countries. Moreover, investing in research and manufacturing presents an opportunity for economic development. To increase awareness of gene therapy, the American Society of Gene and Cell Therapy partnered with the Muhimbili University of Health and Allied Sciences, Tanzania, to create a certificate-bearing course. The goal was to provide faculty teaching in graduate and medical schools with the tools needed to add gene therapy to the university curriculum. The first virtual course was held in October of 2022, and 45 individuals from 9 countries in Africa completed the training. The content was new to approximately two-thirds of participants, with the remaining third indicating that the course increased their knowledge base. The program was well received and will be adapted for other under-resourced regions.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Humanos
2.
Proc Natl Acad Sci U S A ; 114(52): 13679-13684, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29229836

RESUMO

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/genética , Melanoma/metabolismo , Transdução de Sinais , Análise de Célula Única , Adaptação Fisiológica , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Cadeias de Markov , Melanoma/tratamento farmacológico , Melanoma/patologia , NF-kappa B/metabolismo , Fenótipo , Proteoma , Proteômica/métodos , Proteínas Proto-Oncogênicas B-raf/genética
3.
J Pharmacol Exp Ther ; 368(3): 503-513, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30622170

RESUMO

Here we have investigated whole-body pharmacokinetics (PK) of exogenously administered T cells in a mouse model of melanoma and have developed a physiologically based pharmacokinetic (PBPK) model to quantitatively characterize the data. T cells were isolated from the spleen of tumor-bearing mice, activated, and labeled with chromium-51 to facilitate the quantification. Labeled T cells were injected in the tumor-bearing mice, and PK was measured in 19 different tissues. It was found that T cells disappear from the blood rapidly after administration and accumulate in the tissues to various extents. Spleen, liver, lung, kidney, bone, and lymph nodes accounted for more than 90% of T cells in the body. The distribution of T cells in solid tumors was found to be very low, hovering below 1%ID/g (percent of injected dose per gram of tissue) during the entire study. However, this observation may differ for targeted TCR-T and CAR-T cells. Observed PK profiles also suggest that T-cell-based therapies may be more successful in treating cancers of the lymphatic system and bone marrow metastases compared to solid tumors. A PBPK model was developed to characterize the whole-body PK of T cells, which incorporated key processes such as extravasation, elimination, and recirculation of T cells via lymph flow. Retention factors were incorporated into the spleen, liver, and kidney compartment to adequately capture the PK profiles. The model was able to characterize observed PK profiles reasonably well, and parameters were estimated with good confidence. The PK data and PBPK model presented here provide unprecedented insight into the biodistribution of exogenously administered T cells.


Assuntos
Melanoma Experimental/metabolismo , Modelos Biológicos , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Tecidual/fisiologia
4.
Adv Exp Med Biol ; 1143: 217-229, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338822

RESUMO

Cancer immunotherapy has been shown to be an efficacious therapeutic approach in the treatment of cancers including hematopoietic malignancies. Induction of T cell cytotoxicity against tumors by adoptive cell therapies (ACT), cancer vaccines, gene therapies, and monoclonal antibody therapies has been intensively studied. In particular, immune checkpoint blockade and chimeric antigen receptor T (CAR-T) cell therapies are the recent clinical successes in cancer immunotherapy. This article introduces the main concepts and addresses the most relevant clinical modalities of cellular immunotherapies for hematological malignancies: antigen non-specific T cell therapy, genetically modified T cell receptor (TCR) T cell therapy, chimeric antigen receptor (CAR) T cell therapy, and CAR-T cell clinical trials in leukemia, lymphoma, and multiple myeloma. Clinical trials have shown encouraging results, but future studies may need to incorporate novel CAR constructs or targets with enhanced safety and efficacy to ensure long-term benefits.


Assuntos
Neoplasias Hematológicas , Imunoterapia Adotiva , Neoplasias Hematológicas/terapia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T
5.
Nature ; 468(7326): 973-7, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21107323

RESUMO

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ∼7% of human malignancies and ∼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRß (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRß RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRß-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRß or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRß or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Vemurafenib
6.
N Engl J Med ; 366(3): 207-15, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22256804

RESUMO

BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).


Assuntos
Carcinoma de Células Escamosas/genética , Genes ras , Indóis/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/genética , Sulfonamidas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/administração & dosagem , Masculino , Camundongos , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Sulfonamidas/administração & dosagem , Vemurafenib
7.
BMC Cancer ; 15: 356, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25939769

RESUMO

BACKGROUND: Malignant melanoma is an aggressive tumor type that often develops drug resistance to targeted therapeutics. The production of colony stimulating factor 1 (CSF-1) in tumors recruits myeloid cells such as M2-polarized macrophages and myeloid derived suppressor cells (MDSC), leading to an immune suppressive tumor milieu. METHODS: We used the syngeneic mouse model of BRAF (V600E) -driven melanoma SM1, which secretes CSF-1, to evaluate the ability of the CSF-1 receptor (CSF-1R) inhibitor PLX3397 to improve the antitumor efficacy of the oncogenic BRAF inhibitor vemurafenib. RESULTS: Combined BRAF and CSF-1R inhibition resulted in superior antitumor responses compared with either therapy alone. In mice receiving PLX3397 treatment, a dramatic reduction of tumor-infiltrating myeloid cells (TIM) was observed. In this model, we could not detect a direct effect of TIMs or pro-survival cytokines produced by TIMs that could confer resistance to PLX4032 (vemurafenib). However, the macrophage inhibitory effects of PLX3397 treatment in combination with the paradoxical activation of wild type BRAF-expressing immune cells mediated by PLX4032 resulted in more tumor-infiltrating lymphocytes (TIL). Depletion of CD8+ T-cells abrogated the antitumor response to the combination therapy. Furthermore, TILs isolated from SM1 tumors treated with PLX3397 and PLX4032 displayed higher immune potentiating activity. CONCLUSIONS: The combination of BRAF-targeted therapy with CSF-1R blockade resulted in increased CD8 T-cell responses in the SM1 melanoma model, supporting the ongoing evaluation of this therapeutic combination in patients with BRAF (V600) mutant metastatic melanoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Melanoma Experimental/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Aminopiridinas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Indóis/administração & dosagem , Ativação Linfocitária , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Pirróis/administração & dosagem , Sulfonamidas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vemurafenib
8.
Immunol Invest ; 44(8): 817-36, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26575466

RESUMO

Clinical progress in the field of cancer immunotherapy has been slow for many years but within the last 5 years, breakthrough successes have brought immunotherapy to the forefront in cancer therapy. Promising results have been observed in a variety of cancers including solid tumors and hematological malignancies with adoptive cell therapy using natural host tumor infiltrating lymphocytes, host cells that have been genetically engineered with antitumor T-cell receptors or chimeric antigen receptors, immune checkpoint inhibitors like anti-CTLA-4, anti-PD-1 or PD-L1 monoclonal antibodies and oncolytic virus-based immunotherapy. However, most treatment modalities have shown limited efficacy with single therapy. The complex nature of cancer with intra- and inter-tumor antigen and genomic heterogeneity coupled with the immune suppressive microenvironment emphasizes the prospect of personalized targeted immunotherapy to manipulate the patient's own immune system against cancer. For successful, robust and long-lasting cure of cancer, a multi-modal approach is essential, combining anti-tumor cell therapy with manipulation of multiple pathways in the tumor microenvironment to ameliorate tumor-induced immunosuppression.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Vacinas Anticâncer , Imunoterapia Ativa , Linfócitos do Interstício Tumoral/transplante , Neoplasias/terapia , Linfócitos T/transplante , Animais , Ciclo Celular/efeitos dos fármacos , Terapia Combinada , Terapia Genética , Humanos , Neoplasias/imunologia , Medicina de Precisão , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia
9.
Proc Natl Acad Sci U S A ; 108(51): E1408-16, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22123951

RESUMO

The goal of cancer immunotherapy is the generation of an effective, stable, and self-renewing antitumor T-cell population. One such approach involves the use of high-affinity cancer-specific T-cell receptors in gene-therapy protocols. Here, we present the generation of functional tumor-specific human T cells in vivo from genetically modified human hematopoietic stem cells (hHSC) using a human/mouse chimera model. Transduced hHSC expressing an HLA-A*0201-restricted melanoma-specific T-cell receptor were introduced into humanized mice, resulting in the generation of a sizeable melanoma-specific naïve CD8(+) T-cell population. Following tumor challenge, these transgenic CD8(+) T cells, in the absence of additional manipulation, limited and cleared human melanoma tumors in vivo. Furthermore, the genetically enhanced T cells underwent proper thymic selection, because we did not observe any responses against non-HLA-matched tumors, and no killing of any kind occurred in the absence of a human thymus. Finally, the transduced hHSC established long-term bone marrow engraftment. These studies present a potential therapeutic approach and an important tool to understand better and to optimize the human immune response to melanoma and, potentially, to other types of cancer.


Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Engenharia Genética/métodos , Humanos , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/citologia , Camundongos , Camundongos SCID , Modelos Genéticos , Transplante de Neoplasias , Células-Tronco/citologia , Timo/metabolismo , Transgenes
10.
Proc Natl Acad Sci U S A ; 107(32): 14286-91, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20624956

RESUMO

A key issue in advancing the use of adoptive cell transfer (ACT) of T cell receptor (TCR) engineered lymphocytes for cancer therapy is demonstrating how TCR transgenic cells repopulate lymphopenic hosts and target tumors in an antigen-specific fashion. ACT of splenocytes from fully immunocompetent HLA-A2.1/K(b) mice transduced with a chimeric murine/human TCR specific for tyrosinase, together with lymphodepletion conditioning, dendritic cell (DC)-based vaccination, and high-dose interleukin-2 (IL-2), had profound antitumor activity against large established MHC- and antigen-matched tumors. Genetic labeling with bioluminescence imaging (BLI) and positron emitting tomography (PET) reporter genes allowed visualization of the distribution and antigen-specific tumor homing of TCR transgenic T cells, with trafficking correlated with antitumor efficacy. After an initial brief stage of systemic distribution, TCR-redirected and genetically labeled T cells demonstrated an early pattern of specific distribution to antigen-matched tumors and locoregional lymph nodes, followed by a more promiscuous distribution 1 wk later with additional accumulation in antigen-mismatched tumors. This approach of TCR engineering and molecular imaging reporter gene labeling is directly translatable to humans and provides useful information on how to clinically develop this mode of therapy.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Quimiotaxia de Leucócito , Genes Reporter , Humanos , Cinética , Camundongos , Monofenol Mono-Oxigenase/imunologia , Neoplasias/terapia , Linfócitos T/transplante , Distribuição Tecidual
11.
Mol Cancer ; 11: 22, 2012 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-22515704

RESUMO

BACKGROUND: TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2. METHODS: The growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733. RESULTS: Fourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays. CONCLUSIONS: The MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.


Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Cutâneas/metabolismo , Neoplasias Uveais/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Concentração Inibidora 50 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traçadores Radioativos , Transdução de Sinais/efeitos dos fármacos
12.
J Transl Med ; 10: 127, 2012 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-22713761

RESUMO

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFß), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFß by transduction with a TGFß dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.


Assuntos
Transferência Adotiva , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Western Blotting , Linfócitos T CD8-Positivos/citologia , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Microambiente Tumoral
13.
Cancer Cell ; 40(10): 1145-1160.e9, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36150390

RESUMO

Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61ß-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.


Assuntos
Interferon Tipo I , Neoplasias , Animais , Estresse do Retículo Endoplasmático , Interferon Tipo I/metabolismo , Camundongos , Transdução de Sinais , Linfócitos T/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
14.
J Transl Med ; 9: 192, 2011 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-22060015

RESUMO

BACKGROUND: The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies. METHODS: We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells. RESULTS AND DISCUSSION: Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells. CONCLUSIONS: Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis.


Assuntos
Antígenos de Neoplasias/imunologia , Azacitidina/análogos & derivados , Proteína Ligante Fas/metabolismo , Glioma/tratamento farmacológico , Glioma/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/efeitos dos fármacos , Receptor fas/metabolismo , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Decitabina , Glioma/patologia , Glioma/cirurgia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Terapia de Alvo Molecular , Retroviridae/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética , Regulação para Cima/efeitos dos fármacos
15.
J Immunother Cancer ; 9(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33589522

RESUMO

BACKGROUND: Immunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate, but the regulatory mechanisms that govern tumor infiltration are still poorly understood. To address this gap, we investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase enzyme that targets dimethyl and trimethyl H3K36. WHSC1 is known to promote malignant growth and progression in multiple tumors, but its role in the interface between PCa and immune system is unknown. METHODS: RNA Sequencing (RNASeq) data from patients with PCa from The Cancer Genome Atlas (TCGA) were collected and divided into top/bottom 30% based on the expression of WHSC1 and disease-free survival was calculated. Publicly available chromatin immunoprecipitation (ChIPSeq) data were obtained from Cistrome and integrated with the available RNASeq data. RNASeq, ATACSeq and methylomic were analyzed using R Bioconductor packages comparing C42 cells with or without stable knockdown on WHSC1. Flow cytometry was used to measure Major Histocompatibility complex (MHC) levels, MHC-bound ovalbumin and tumor infiltration. C57B6 and NOD scid gamma (NSG) mice were subcutaneously grafted with TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) C2 cells and treated with MCTP39 (10 mg/kg); tumor size was monitored over time and curves were compared using permutation analyses. All analyses used a significance threshold of 0.05. RESULTS: Leveraging TCGA data, we demonstrated that elevated WHSC1 levels positively correlate with the presence of an immunosuppressive microenvironment. We validated those results in vitro, demonstrating that genetic and pharmacological inhibition of WHSC1 restores antigen presentation. This occurs via an elegant epigenetic regulation of gene expression at the chromatin and DNA methylation levels. In vivo studies in immunocompetent mice also show an increased frequency of CD8+ T cells in tumors from mice treated with WHSC1 inhibitor, supporting the hypothesis that the antitumor effect following WHSC1 inhibition requires a fully functional immune system. CONCLUSIONS: This study demonstrates a novel role for WHSC1 in defining immune infiltration in PCa, with significant future implications for the use of immunotherapies in prostate malignancies.


Assuntos
Perfilação da Expressão Gênica/métodos , Histona-Lisina N-Metiltransferase/genética , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Quinoxalinas/administração & dosagem , Proteínas Repressoras/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Quinoxalinas/farmacologia , Análise de Sequência de RNA , Análise de Sobrevida , Microambiente Tumoral
16.
J Med Chem ; 64(1): 741-767, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33400524

RESUMO

Erlotinib was covalently linked to 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) and structurally related chlorins and bacteriochlorins at different positions of the tetrapyrrole ring. The functional consequence of each modification was determined by quantifying the uptake and subcellular deposition of the erlotinib conjugates, cellular response to therapeutic light treatment in tissue cultures, and in eliminating of corresponding tumors grown as a xenograft in SCID mice. The experimental human cancer models the established cell lines UMUC3 (bladder), FaDu (hypopharynx), and primary cultures of head and neck tumor cells. The effectiveness of the compounds was compared to that of HPPH. Furthermore, specific functional contribution of the carboxylic acid side group at position 172 and the chiral methyl group at 3(1') to the overall activity of the chimeric compounds was assessed. Among the conjugates investigated, the PS 10 was identified as the most effective candidate for achieving tumor cell-specific accumulation and yielding improved long-term tumor control.


Assuntos
Cloridrato de Erlotinib/química , Fármacos Fotossensibilizantes/síntese química , Porfirinas/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos SCID , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Estereoisomerismo , Relação Estrutura-Atividade , Taxa de Sobrevida
17.
Clin Immunol ; 136(3): 338-47, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20547105

RESUMO

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Engenharia Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia Ativa , Imunoterapia Adotiva , Técnicas In Vitro , Ativação Linfocitária , Melanoma/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Auxiliares-Indutores/classificação , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Transdução Genética
18.
J Transl Med ; 8: 39, 2010 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-20406486

RESUMO

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 muM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 muM, and three were moderately sensitive with IC50 values between 1 and 10 muM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.


Assuntos
Indóis/farmacologia , Melanoma/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Substituição de Aminoácidos/genética , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diagnóstico por Imagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genoma Humano/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vemurafenib
19.
J Am Chem Soc ; 131(28): 9695-703, 2009 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-19552409

RESUMO

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.


Assuntos
Separação Celular/métodos , Técnicas Citológicas/métodos , DNA de Cadeia Simples/metabolismo , Antígenos de Histocompatibilidade/imunologia , Análise Serial de Proteínas/métodos , Linfócitos T/imunologia , Animais , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA/metabolismo , DNA Complementar/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Vidro/química , Antígenos de Histocompatibilidade/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Proteínas Imobilizadas/metabolismo , Camundongos , Hibridização de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Reprodutibilidade dos Testes , Estreptavidina/química , Estreptavidina/metabolismo , Especificidade por Substrato
20.
Cancer Immunol Immunother ; 58(5): 699-708, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-18807035

RESUMO

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.


Assuntos
Compostos de Bifenilo/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Neoplasias do Colo/terapia , Imunoterapia/métodos , Melanoma Experimental/terapia , Proteínas de Neoplasias/antagonistas & inibidores , Nitrofenóis/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Animais , Antígenos de Neoplasias/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/imunologia , Neoplasias do Colo/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoterapia Adotiva , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Células Matadoras Ativadas por Linfocina/transplante , Listeria monocytogenes/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Piperazinas/uso terapêutico , Receptores de Morte Celular/agonistas , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/farmacologia
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