RESUMEN
It is estimated that ~50% of patients with melanoma harbour BRaf (BRAF)V600 driver mutations, with the most common of these being BRAFV600E, which leads to the activation of mitogenactivated protein kinase proliferative and survival pathways. BRAF inhibitors are used extensively to treat BRAFmutated metastatic melanoma; however, acquired resistance occurs in the majority of patients. The effects of longterm treatment with PLX4032 (BRAFV600 inhibitor) were studied in vitro on sensitive V600E BRAFmutated melanoma cell lines. After several weeks of treatment with PLX4032, the majority of the melanoma cells died; however, a proportion of cells remained viable and quiescent, presenting senescent cancer stem celllike characteristics. This surviving population was termed SUR cells, as discontinuing treatment allowed the population to regrow while retaining equal drug sensitivity to that of parental cells. RNA sequencing analysis revealed that SUR cells exhibit changes in the expression of 1,415 genes (P<0.05) compared with parental cells. Changes in the expression levels of a number of epigenetic regulators were also observed. These changes and the reversible nature of the senescence state were consistent with epigenetic regulation; thus, it was investigated as to whether the senescent state could be reversed by epigenetic inhibitors. It was found that both parental and SUR cells were sensitive to different histone deacetylase (HDAC) inhibitors, such as SAHA and MGCD0103, and to the cyclindependent kinase (CDK)9 inhibitor, CDKI73, which induced apoptosis and reduced proliferation both in the parental and SUR populations. The results suggested that the combination of PLX4032 with HDAC and CDK9 inhibitors may achieve complete elimination of SUR cells that persist after BRAF inhibitor treatment, and reduce the development of resistance to BRAF inhibitors.
Asunto(s)
Redes Reguladoras de Genes/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/farmacología , Sulfonamidas/farmacología , Vemurafenib/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Mutación , Análisis de Secuencia de ARNRESUMEN
In a previous work, we showed that CSF-470 vaccine plus bacillus Calmette-Guerin (BCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) as adjuvants resulted in a significant benefit in the distant metastasis-free survival when comparing vaccinated vs. IFN-α2b-treated high-risk cutaneous melanoma patients in a Phase II study. Immune monitoring demonstrated an increase in anti-tumor innate and adaptive immunities of vaccinated patients, with a striking increase in IFN-γ secreting lymphocytes specific for melanoma antigens (Ags). In an effort to dissect the first steps of the immune response elicited by CSF-470 vaccine plus adjuvants, we evaluated, in an in vitro model, leukocyte migration, cytokine production, and monocyte phagocytosis of vaccine cells. Our results demonstrate that leukocytes recruitment, mostly from the innate immune system, is an early event after CSF-470 vaccine plus BCG plus GM-CSF interaction with immune cells, possibly explained by the high expression of CCL2/MCP-1 and other chemokines by vaccine cells. Early release of TNF-α and IL-1ß pro-inflammatory cytokines and efficient tumor Ags phagocytosis by monocytes take place and would probably create a favorable context for Ag processing and presentation. Although the presence of the vaccine cells hampered cytokines production stimulated by BCG in a mechanism partially mediated by TGF-ß and IL-10, still significant levels of TNF-α and IL-1ß could be detected. Thus, BCG was required to induce local inflammation in the presence of CSF-470 vaccine cells.
RESUMEN
BACKGROUND: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. METHODS: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. RESULTS: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed. CONCLUSION: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis. TRIAL REGISTRATION: Clinicaltrials.gov (NHI) NCT00515983.
Asunto(s)
Vacunas contra el Cáncer/toxicidad , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Interleucina-10/genética , Melanoma/inmunología , Melanoma/patología , Adolescente , Adulto , Apoptosis , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Hipersensibilidad Tardía , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Necrosis , Estadificación de Neoplasias , Selección de Paciente , Fagocitosis , Polimorfismo Genético , Valor Predictivo de las Pruebas , Regiones Promotoras Genéticas , Trasplante AutólogoRESUMEN
BACKGROUND: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells). METHODS: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. RESULTS: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. CONCLUSION: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
Asunto(s)
Antígenos de Neoplasias/inmunología , Apoptosis , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Melanoma/patología , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de la radiación , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Quimiocina CCL19/farmacología , Técnicas de Cocultivo , Reactividad Cruzada/efectos de los fármacos , Reactividad Cruzada/efectos de la radiación , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/ultraestructura , Epítopos/inmunología , Rayos gamma , Humanos , Interleucina-10/inmunología , Interleucina-12/inmunología , Antígeno MART-1 , Melanoma/inmunología , Melanoma/ultraestructura , Monocitos/citología , Monocitos/inmunología , Necrosis , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Fagocitosis/efectos de la radiación , Receptores CCR7/inmunología , Factores de Tiempo , Antígeno gp100 del MelanomaRESUMEN
We investigated whether recombinant human granulocyte-monocyte-colony-stimulating factor (rhGM-CSF) increased the immunogenicity of VACCIMEL, a vaccine consisting of 3 irradiated allogeneic melanoma cell lines. A phase I clinical trial was performed on 20 melanoma patients in stages IIB (n=2), III (n=10), and IV (n=8), who were disease free after surgery (n=16) or had minimal disease (n=4). Cohorts of 4 patients were vaccinated 4 times with VACCIMEL and bacillus Calmette Guerin (BCG) as adjuvant. Besides, the patients received placebo (group 1) or GM-CSF: 150 microg (group 2), 300 microg (group 3), 400 microg (group 4), and 600 microg (group 5) per vaccine. The combination of VACCIMEL and GM-CSF had low toxicity. Only in group 5, grade 2 thoracic pain (3/4 patients) and abdominal cramps (2/4 patients) were observed. Delayed-type hypersensitivity increased after vaccination and it was highest in group 4. Phytohemagglutinin stimulation of peripheral blood lymphocytes was analyzed in 9 patients: 4/9 had normal stimulation; 3/9 had low basal stimulation, which recovered after vaccination; and 2/9 were not stimulated. Antimelanoma antibodies preexisted in 9/19 patients; in 3/19 patients, antibodies anti-33 kd, 90 kd, and 100 kd antigens were induced by vaccination. IgG2 but not IgG1 antibodies were detected. Anti-BCG antibodies, mostly IgG2, reached the highest post/prevaccination ratio in group 4. Median serum interleukin-12 was lower in progressing patients (61.6 pg/mL) than in those without evident disease (89 pg/mL). Thus, its low toxicity and the induction of a predominantly cellular immune response suggest that the addition of 300 to 400 microg GM-CSF to VACCIMEL is useful in increasing the immune response.