RESUMEN
CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.
Asunto(s)
Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Epigénesis Genética , Mutación de Línea Germinal , Neoplasias/genética , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Autofagia , Línea Celular Tumoral , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunoterapia , Ipilimumab/farmacología , Masculino , Melanoma/genética , Melanoma/inmunología , Antígenos Específicos del Melanoma/genética , Antígenos Específicos del Melanoma/inmunología , Ratones , Ratones Transgénicos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunologíaRESUMEN
An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.