RESUMEN
Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.
Asunto(s)
Melanoma , Monofenol Monooxigenasa , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Melanocitos/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patologíaRESUMEN
Proximity-dependent labeling techniques such as BioID and APEX2 allow the biotinylation of proteins proximal to a protein of interest in living cells. Following streptavidin pulldown and mass spectrometry analysis, this enables the identification of native protein-protein interactions. Here we describe split-BioID, a protein-fragment complementation assay that increases the resolution of BioID. Using this technique, context-specific protein complexes can be resolved.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteoma , Proteómica/métodos , Animales , Biotinilación , Línea Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Espectrometría de Masas , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Coloración y Etiquetado , Estreptavidina/metabolismoRESUMEN
Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.
