RESUMO
CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.
Assuntos
Anticorpos Monoclonais , Sarcoma , Animais , Camundongos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Imunossupressores , Adenosina , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Microambiente TumoralRESUMO
Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.
RESUMO
In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Metabolismo Energético/efeitos dos fármacos , Complexos Multiproteicos/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Trifosfato de Adenosina/metabolismo , Anilidas/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Regulação para Baixo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 1/genética , Glicólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitocôndrias/metabolismo , Complexos Multiproteicos/fisiologia , Niacinamida/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Sorafenibe , Serina-Treonina Quinases TOR/fisiologiaRESUMO
In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1h) and late (8h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5-6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.
Assuntos
Receptores ErbB/antagonistas & inibidores , Quinazolinas , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Compostos de Anilina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Química Farmacêutica , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Fatores de TempoRESUMO
BACKGROUND: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. RESULTS: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. CONCLUSION: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Análise de Variância , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estabilidade Proteica/efeitos dos fármacos , Quinazolinas/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Conventional chemotherapeutic regimens have limited impact against most solid tumors and deal with significant toxicity. Over the last 10 years, novel anticancer treatments targeting specific molecules or genes involved in cancer progression have been developed to improve outcome and reduce side effects. In particular, the tyrosine kinase inhibitors gefitinib and erlotinib have been approved for the treatment of non-small-cell lung cancer. Their clinical activity has been related to different clinical and biological parameters, such as EGFR-activating mutations. However, not all clinical outcomes, including tolerability, are explained, and the identification/validation of novel biomarkers is a viable area of research. Germline polymorphisms can be easily assessed in blood samples, and candidate polymorphisms in EGFR and ABCG2 have been correlated with outcome and toxicity in non-small-cell lung cancer patients treated with gefitinib or erlotinib. However, differences in study population and design resulted in several controversial findings, while the prognostic versus predictive role of the polymorphisms still needs to be validated within larger prospective studies. More studies on the relationship of the genotype with drug pharmacokinetics and mechanism of action are also warranted. These future studies, as well as further development and application of novel technologies to decipher genetic alterations, might contribute to the validation of selected polymorphisms as molecular markers predictive of drug activity and help in the selection of tyrosine kinase inhibitors best suited to the individual patient.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Mutação em Linhagem Germinativa , Polimorfismo Genético , Inibidores de Proteínas Quinases/uso terapêutico , Antineoplásicos/efeitos adversos , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Gefitinibe , Humanos , Inibidores de Proteínas Quinases/efeitos adversos , Quinazolinas/efeitos adversos , Quinazolinas/uso terapêutico , Resultado do TratamentoRESUMO
BACKGROUND: Precision immuno-oncology approaches are needed to improve cancer care. We recently demonstrated that in patients with metastatic melanoma, an increase of clonality or diversity of the T cell receptor (TCR) repertoire of peripheral T cells following one cycle of immunotherapy is coincident with response to immune-checkpoint blockade (ICB). We also identified a subset of peripheral CD8+ immune-effector memory T cells (TIE cells) whose expansion was associated with response to ICB and increased overall survival. To improve our understanding of peripheral T cell dynamics, we examined the clinical correlates associated with these immune signatures. METHODS: Fifty patients with metastatic melanoma treated with first-line anti-PD-1 ICB were included. We analysed TCR repertoire and peripheral TIE cell dynamics by age before treatment (T0) and after the first cycle of treatment at week 3 (W3). RESULTS: We observed a correlation between TIE abundance and age at T0 (r = 0.40), which reduced following treatment at W3 (r = 0.07). However, at W3, we observed two significantly opposing patterns (p = 0.03) of TCR repertoire rearrangement in patients who responded to treatment, with patients ≥70 years of age showing an increase in TCR clonality and patients <70 years of age showing an increase in TCR diversity. CONCLUSIONS: We demonstrate that immunotherapy-induced immune-awakening patterns in patients with melanoma are age-related and may impact patient response to ICB, and thus have implications for biomarker development and planning of personalised therapeutic strategies.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Idoso , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Recém-Nascido , Melanoma/tratamento farmacológico , Receptores de Antígenos de Linfócitos TRESUMO
BACKGROUND: Immune cell-driven anti-cancer activity is paramount for effective responses to checkpoint inhibitors (ICB). However, the contribution of the different immune cell subsets in the circulation and within the tumour is poorly understood. MATERIALS AND METHODS: To elucidate the role of the different cell subsets in anti-tumour responses elicited by ICB, we performed single-cell analysis of the transcriptome and surface proteome of paired pre- and early on-treatment metastatic melanoma tumour biopsies and matched peripheral blood mononuclear cell samples. We next compared the survival of metastatic melanoma patients treated with ICB according to the abundance of pre-treatment tumour-infiltrating B cell clonotypes. RESULTS: We identified cell clusters associated with disease control or progression, defined differential expression of biological pathways likely involved in the immune awakening against the tumour and examined how cell-cell communication patterns between the tumour cell subsets change during treatment. Furthermore, we discovered that B cells (immunoglobulin expression and abundance of B cell clonotypes) discriminate the clinical response after ICB and propose that B cells likely contribute to anti-tumour immunity by antigen presentation through major histocompatibility complex molecules. Finally, we demonstrated that the abundance of tumour-infiltrating B cell clonotypes at baseline identifies two distinct risk groups, a finding that we confirmed in an independent cohort. CONCLUSIONS: Our exploratory translational study provides new insights on the mechanistic role of B cells in anti-melanoma immunity during treatment with ICB. Additionally, we support pre-treatment B cell tumour infiltration as a promising prognostic biomarker to be further validated as a tool for clinical risk stratification.
Assuntos
Leucócitos Mononucleares , Melanoma , Humanos , Melanoma/patologia , Linfócitos B , Transcriptoma , Estudos de Coortes , ImunoterapiaRESUMO
BACKGROUND: Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment.The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness. RESULTS: In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation. CONCLUSION: Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC cells lacking activating mutations. On the other hand, in metabolizing cells, the inhibition of CYP1A1 might lead to increased local exposure to the active drug and thus increase gefitinib potency.
Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Receptores ErbB/antagonistas & inibidores , Quinazolinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares , Mutação , FosforilaçãoRESUMO
Tumor infiltration by T cells is paramount for effective anti-cancer immune responses. We hypothesized that the T cell receptor (TCR) repertoire of tumor infiltrating T lymphocytes could therefore be indicative of the functional state of these cells and determine disease course at different stages in cancer progression. Here we show that the diversity of the TCR of tumor infiltrating T cell at baseline is prognostic in various cancers, whereas the TCR clonality of T cell infiltrating metastatic melanoma pre-treatment is predictive for activity and efficacy of PD1 blockade immunotherapy.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Biópsia , Estudos de Coortes , Feminino , Humanos , Imunoterapia , Masculino , Melanoma/patologia , Melanoma/terapia , Pessoa de Meia-Idade , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Taxa de SobrevidaRESUMO
BACKGROUND: Immuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is a critical need to identify biomarkers that can predict or enrich for treatment response. So far, the majority of putative biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure, obviating the ability to directly link potential biomarkers to long-term treatment outcomes. METHODS: To address this, we developed and validated a novel non-terminal tumor sampling method to enable biopsy of the TME in mouse models based on fine needle aspiration. RESULTS: We show that this technique enables repeated in-life sampling of subcutaneous flank tumors and yields sufficient material to support downstream analyses of tumor-infiltrating immune cells using methods such as flow cytometry and single-cell transcriptomics. Moreover, using this technique we demonstrate that we can link TME biomarkers to treatment response outcomes, which is not possible using the current method of terminal tumor sampling. CONCLUSION: Thus, this minimally invasive technique is an important refinement for the pharmacodynamic analysis of the TME facilitating paired evaluation of treatment response biomarkers with outcomes and reducing the number of animals used in preclinical research.
Assuntos
Biomarcadores Tumorais/metabolismo , Biópsia por Agulha Fina/métodos , Imunoterapia/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
Our understanding of how checkpoint inhibitors (CPI) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs. Here we analyzed peripheral T cell populations after one cycle of CPI and identified a dynamic awakening of the immune system revealed by T cell evolution in response to treatment. We sequenced T cell receptors (TCR) in plasma cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMC) and performed phenotypic analysis of peripheral T cell subsets from metastatic melanoma patients treated with CPI. We found that early peripheral T cell turnover and TCR repertoire dynamics identified which patients would respond to treatment. Additionally, the expansion of a subset of immune-effector peripheral T cells we call TIE cells correlated with response. These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients responses using minimally invasive liquid biopsies."
Assuntos
Leucócitos Mononucleares , Melanoma , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
BACKGROUND: Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies. METHODS: We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. RESULTS: Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. CONCLUSION: Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments.
Assuntos
Melanoma , Neoplasias Cutâneas , Encéfalo , Humanos , Imunoterapia , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Microambiente TumoralRESUMO
Although immune checkpoint inhibitors (ICIs) have achieved unprecedented results in melanoma, the biological features of the durable responses initiated by these drugs remain unknown. Here we show the genetic and phenotypic changes induced by treatment with programmed cell death-1 (PD-1) blockade in a genetically engineered mouse model of melanoma driven by oncogenic BRAF. In this controlled system anti-PD-1 treatment yields responses in ~35% of the tumors, and prolongs survival in ~27% of the animals. We identify increased stroma remodeling and reduced expression of proliferation markers as features associated with prolonged response. These traits are corroborated in two independent early on-treatment anti-PD-1 melanoma patient cohorts. These insights into the biological responses of tumors to ICI provide a strategy for identification of durable response early during the course of treatment and could improve patient stratification for checkpoint inhibitory drugs.
Assuntos
Divisão Celular/fisiologia , Melanoma/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Células Estromais/metabolismo , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Proliferação de Células , Modelos Animais de Doenças , Exoma/genética , Feminino , Humanos , Imunoterapia , CamundongosRESUMO
UNLABELLED: Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from â¼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. SIGNIFICANCE: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.
Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Melanoma/diagnóstico , Melanoma/tratamento farmacológico , Medicina de Precisão , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biópsia , Análise por Conglomerados , Gerenciamento Clínico , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Terapia de Alvo Molecular , Mutação , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.
Assuntos
Acrilamidas/farmacologia , Azetidinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , RNA Interferente Pequeno , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The disappointing results in long-term survival of patients affected by non-small cell lung cancer (NSCLC) may be attributed, at least in part, to the lack of knowledge on the way by which genetic characteristics in normal and neoplastic cells affect responsiveness as well as metabolism of chemotherapy and new targeted agents. This issue deserves further pharmacogenetics studies, in order to identify patients who are most likely to benefit from specific therapies selected on the base of the individual and tumor genetic features, thus improving the efficacy/toxicity profile of the treatment strategy. Even if most meta-analyses in NSCLC yielded contradictory results, a number of candidate biomarkers for response/resistance to conventional chemotherapeutic agents such as gemcitabine, platinum-compounds, pemetrexed and taxanes have been proposed. Similarly, recent studies suggested the key role of polymorphisms in the prediction of toxicity to EGFR-targeted agents. However, larger prospective randomized trials of personalized therapy to validate these biomarkers are still needed. The unification of the technical procedures, as well as additional investigation to unravel pivotal factors influencing genotype-phenotype relationships, represent other crucial issues. From this perspective, functional studies aiming at unravel eventual pharmacokinetics/pharmacodynamics interactions are critical for the pharmacogenetic optimization of anti-cancer regimens. With the development of high-throughput technologies, including whole exome analyses, the traditional pharmacogenetic approach that till present has relied only on candidate genes suspected of influencing drug response/metabolism can be fulfilled with further lists of potential predictive alleles. The clinical implementation of such pharmacogenetics/genomics studies as well as of therapeutic drug monitoring could enable clinicians to personalize treatment to enhance efficacy and/or limit toxicity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Farmacogenética , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismoRESUMO
Non-small cell lung cancer (NSCLC) is one of the deadliest types of cancer. One explanation for this poor prognosis is the failure of most chemotherapeutic regimens, which prompted the development of new, rationally designed, targeted antitumor agents, such as inhibitors of the epidermal growth factor receptor (EGFR) and downstream pathways. However, most of these targeted therapies also fail, and studies on the mechanisms underlying resistance toward targeted agents might provide critical findings for NSCLC research and treatment. Some of these studies showed that drug resistance can emerge not only from genetic aberrations, but also from epigenetic changes, including regulation of different signaling pathways by microRNAs (miRNAs), which act as key post-transcriptional regulators of gene expression. There is accumulating evidence that specific miRNAs correlated with drug sensitivity and can be used as prognostic markers in NSCLC. However, a greater knowledge of miRNAs might also provide novel insights in several drug-resistance mechanisms; hence, suggesting their potential in novel therapeutic interventions, by sensitizing tumor cells to drug-induced apoptosis as well as by inhibiting tumor proliferation and invasive capabilities. Therefore, this review highlights several recent and clinically relevant aspects of the regulation of drug resistance by miRNAs from the perspective of current anti-EGFR-targeted therapies in NSCLC.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/terapia , MicroRNAs/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genéticaRESUMO
Overcoming the emergence of acquired resistance to clinically approved epidermal growth factor receptor (EGFR) inhibitors is a major challenge in the treatment of advanced non-small cell lung cancer (NSCLC). The aim of this study was to investigate the effects of a series of novel compounds affecting viability of NSCLC NCI-H1975 cells (carrying the EGFR T790M mutation). The inhibition of the autophosphorylation of EGFR occurred at nanomolar concentrations and both UPR1282 and UPR1268 caused a significant induction of apoptosis. Targeting of EGFR and downstream pathways was confirmed by a peptide substrate array, which highlighted the inhibition of other kinases involved in NSCLC cell aggressive behavior. Accordingly, the drugs inhibited migration (about 30% vs. control), which could be, in part, explained also by the increase of E-cadherin expression. Additionally, we observed a contraction of the volume of H1975 spheroids, associated with the reduction of the cancer stem-like cell hallmark CD133. The activity of UPR1282 was retained in H1975 xenograft models where it determined tumor shrinkage (P < .05) and resulted well tolerated compared to canertinib. Of note, the kinase activity profile of UPR1282 on xenograft tumor tissues showed overlapping results with respect to the activity in H1975 cells, unraveling the inhibition of kinases involved in pivotal proliferation and invasive signaling pathways. In conclusion, UPR1282 and UPR1268 are effective against various processes involved in malignancy transformation and progression and may be promising compounds for the future treatment of gefitinib-resistant NSCLCs.