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1.
J Immunother Cancer ; 11(12)2023 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-38056899

RESUMO

BACKGROUND: Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. METHODS: We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination. RESULTS: The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. CONCLUSIONS: Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.


Assuntos
Neoplasias Encefálicas , Melanoma Experimental , Animais , Humanos , Linfócitos T CD4-Positivos , Antígeno CTLA-4 , Linfócitos T Citotóxicos/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo
3.
Clin Cancer Res ; 28(10): 2131-2146, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35247927

RESUMO

PURPOSE: Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets. EXPERIMENTAL DESIGN: We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell-cell interactions were inferred and compared with those in nonacral cutaneous melanoma. RESULTS: Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells. CONCLUSIONS: Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.


Assuntos
Melanoma , Segunda Neoplasia Primária , Neoplasias Cutâneas , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Melanoma/tratamento farmacológico , Melanoma/terapia , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/terapia , Melanoma Maligno Cutâneo
4.
Cancer Immunol Res ; 9(5): 554-567, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33653716

RESUMO

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.


Assuntos
Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/química , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Imidazóis/química , Imunoterapia , Melanoma/genética , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Oximas/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Piridonas/química , Pirimidinas/química , Pirimidinonas/química , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Sulfonas/química , Linfócitos T Reguladores/imunologia
5.
Mol Cancer Ther ; 14(6): 1354-64, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25873592

RESUMO

Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1.


Assuntos
Compostos de Anilina/farmacologia , Aurora Quinases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Compostos de Anilina/química , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Aurora Quinases/genética , Aurora Quinases/metabolismo , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/química , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estrutura Molecular , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Pharmacol Exp Ther ; 331(1): 77-86, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19628630

RESUMO

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that acts at the intersection of energy metabolism and stress response in tumor cells. To elucidate the role of GAPDH in chemotherapy-induced stress, we analyzed its activity, protein level, intracellular distribution, and intranuclear mobility in human carcinoma cells A549 and UO31 after treatment with cytarabine, doxorubicin, and mercaptopurine. After treatment with cytosine arabinoside (araC), enzymatically inactive GAPDH accumulated in the nucleus. Experiments on fluorescence recovery after photobleaching with green fluorescent protein-GAPDH fusion protein in the live cells treated with araC demonstrated reduced mobility of green fluorescent protein-GAPDH inside the nucleus, indicative of interactions with nuclear macromolecular components after genotoxic stress. Depletion of GAPDH with RNA interference stopped cell proliferation, and induced cell cycle arrest in G(1) phase via p53 stabilization, and accumulation of p53-inducible CDK inhibitor p21. Neither p21 accumulation nor cell cycle arrest was detected in GAPDH-depleted p53-null NCI-H358 cells. GAPDH-depleted A549 cells were 50-fold more resistant to treatment with cytarabine (1.68 +/- 0.182 microM versus 0.03 +/- 0.015 microM in control). Depletion of GAPDH did not significantly alter cellular sensitivity to doxorubicin (0.05 +/- 0.023 microM versus 0.035 +/- 0.0154 microM in control). Induction of cell cycle arrest in p53-proficient carcinoma cells via GAPDH abrogation suggests that GAPDH-depleting agents may have a cytostatic effect in cancer cells. Our results define GAPDH as an important determinant of cellular sensitivity to antimetabolite chemotherapy because of its regulatory functions.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma/enzimologia , Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes/métodos , Gliceraldeído-3-Fosfato Desidrogenases/deficiência , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia
7.
Mol Cancer Ther ; 8(4): 864-72, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19372559

RESUMO

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 micromol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no gamma-H2AX accumulated after 24-72 hours of incubation with 1 micromol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/genética , Proteína HMGB2/antagonistas & inibidores , Proteína HMGB2/genética , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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