Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma

Metabolic regulation of anoikis and implications in melanoma metastasis

La tutora de la tesis es Cristina Solano GoñiLas células tumorales circulantes son el vínculo clave entre un tumor primario y las metástasis distales, pero una vez en el torrente sanguíneo, la pérdida de adherencia induce la muerte celular (anoikis). Para identificar mecanismos relevantes en la supervivencia de las células tumorales circulantes de melanoma, realizamos análisis de secuenciación del ARN y descubrimos que tanto las células de melanoma en suspensión como las células tumorales circulantes de melanoma reconfiguran el metabolismo lipídico mediante la inducción del transporte de ácidos grasos (AG) y los genes relacionados con la beta-oxidación de AG. En pacientes de melanoma, la elevada expresión de transportadores de AG y enzimas de beta-oxidación de AG correlacionan significativamente con una menor supervivencia global y menor supervivencia libre de progresión. Entre los reguladores más expresados en las células tumorales circulantes de melanoma se encuentran las carnitina transferasas carnitina O-octanoiltransferasa y carnitina acetiltransferasa. Estas proteínas controlan el transporte de los AG de cadena media producidos en el peroxisoma hacia la mitocondria para alimentar la beta-oxidación mitocondrial. La inhibición de la carnitina O-octanoiltransferasa o la carnitina acetiltransferasa y el tratamiento a corto plazo con Aoridazina o ranolazina (inhibidores de la beta-oxidación de AG peroxisomal y mitocondrial, respectivamente), suprimieron la formación metástasis de melanoma en ratones. El efecto de la inhibición de la carnitina O-octanoiltransferasa y la carnitina acetiltransferasa pudo ser rescatada mediante la suplementación de AG de cadena media, lo que indica que el suministro peroxisomal de AGs es crucial para la supervivencia de las células de melanoma no adherentes. Además, mediante un estudio con CRISPR-cas9 identificamos la cadena transportadora de electrones mitocondrial y el metabolismo del colesterol como posibles procesos mediadores de la resistencia a anoikis en melanoma. Nuestro estudio pone de manifiesto la gran importancia de la modulación metabólica en el proceso metastásico, identificando por primera vez la oxidación de AG peroxisomal como posible diana terapéutica para bloquear la progresión del melanoma. Además, proponemos explotar la interacción entre peroxisomas y mitocondrias, basada en el catabolismo de los AG, como posible oportunidad terapéutica gracias al descubrimiento de la actividad antimetastásica de los fármacos Aorizadina y ranolazina. Ambos fármacos están aprobados por la Administración de Alimentos y Medicamentos (Food and Drug Administration, FDA). Por ello, este estudio conlleva un gran potencial traslacional.Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death known as anoikis. In order to identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating faay acid (FA) transport and FA beta- oxidation related genes. In melanoma patients, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knock down of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of non-adherent melanoma cells. Besides, using CRISPR-cas9 technology we identified the mitochondrial electron transport chain and cholesterol metabolism as possible mediators of anoikis resistance in melanoma. Our study highlights the great importance of metabolic rewiring in the metastatic process, identifying for the first time peroxisomal FA oxidation as a potential therapeutic target to challenge melanoma progression. Moreover, we propose to exploit the interaction between peroxisomes and mitochondria based on FA catabolism as a possible therapeutic opportunity thanks to the discovery of the antimetastatic activity of the drugs thiorizadine and ranolazine. These drugs have been approved by the Food and Drug Administration (FDA); therefore, our results have great translational potential.Programa de Doctorado en Ciencias de la Salud (RD 99/2011)Osasun Zientzietako Doktoretza Programa (ED 99/2011

Identification of a dexamethasone mediated radioprotection mechanism reveals new therapeutic vulnerabilities in glioblastoma

(1) Background: Despite the indisputable effectiveness of dexamethasone (DEXA) to reduce inflammation in glioblastoma (GBM) patients, its influence on tumour progression and radiotherapy response remains controversial. (2) Methods: We analysed patient data and used expression and cell biological analyses to assess effects of DEXA on GBM cells. We tested the efficacy of tyrosine kinase inhibitors in vitro and in vivo. (3) Results: We confirm in our patient cohort that administration of DEXA correlates with worse overall survival and shorter time to relapse. In GBM cells and glioma stem-like cells (GSCs) DEXA down-regulates genes controlling G2/M and mitotic-spindle checkpoints, and it enables cells to override the spindle assembly checkpoint (SAC). Concurrently, DEXA up-regulates Platelet Derived Growth Factor Receptor (PDGFR) signalling, which stimulates expression of anti-apoptotic regulators BCL2L1 and MCL1, required for survival during extended mitosis. Importantly, the protective potential of DEXA is dependent on intact tyrosine kinase signalling and ponatinib, sunitinib and dasatinib, all effectively overcome the radio-protective and pro-proliferative activity of DEXA. Moreover, we discovered that DEXA-induced signalling creates a therapeutic vulnerability for sunitinib in GSCs and GBM cells in vitro and in vivo. (4) Conclusions: Our results reveal a novel DEXA-induced mechanism in GBM cells and provide a rationale for revisiting the use of tyrosine kinase inhibitors for the treatment of GBM