RESUMEN
Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Fosfolipasa C gamma/genética , Síndrome de Sézary/genética , Transducción de Señal/genética , Neoplasias Cutáneas/genética , Animales , Células COS , Chlorocebus aethiops , Mutación con Ganancia de Función , Células HEK293 , Humanos , Mutación INDEL , Células Jurkat , Modelos Moleculares , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación/genética , Dominios Proteicos/genética , Síndrome de Sézary/patología , Neoplasias Cutáneas/patología , Factor de Transcripción AP-1/metabolismoRESUMEN
FK228 (romidepsin) and suberoylanilide hydroxamic acid (vorinostat) are histone deacetylase inhibitors (HDACi) approved by the US Food and Drug Administration for cutaneous T-cell lymphoma (CTCL), including the leukemic subtype Sézary syndrome. This study investigates RAD23B and STAT3 gene perturbations in a large cohort of primary Sézary cells and the effect of FK228 treatment on tyrosine phosphorylation of STAT3 (pYSTAT3) and RAD23B expression. We report RAD23B copy number variation in 10% (12/119, P ≤ 0.01) of SS patients, associated with reduced mRNA expression (P = 0.04). RAD23B knockdown in a CTCL cell line led to a reduction in FK228-induced apoptosis. Histone deacetylase inhibitor treatment significantly reduced pYSTAT3 in primary Sézary cells and was partially mediated by RAD23B. A distinct pattern of RAD23B-pYSTAT3 co-expression in primary Sézary cells was detected. Critically, Sézary cells harboring the common STAT3 Y640F variant were less sensitive to FK228-induced apoptosis and exogenous expression of STAT3 Y640F, and D661Y conferred partial resistance to STAT3 transcriptional inhibition by FK228 (P ≤ 0.0024). These findings suggest that RAD23B and STAT3 gene perturbations could reduce sensitivity to histone deacetylase inhibitors in SS patients.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Depsipéptidos/farmacología , Resistencia a Antineoplásicos/genética , Inhibidores de Histona Desacetilasas/farmacología , Factor de Transcripción STAT3/genética , Síndrome de Sézary/genética , Neoplasias Cutáneas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Linfocitos T CD4-Positivos , Variaciones en el Número de Copia de ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Depsipéptidos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Células Neoplásicas Circulantes , Fosforilación/efectos de los fármacos , Polimorfismo de Nucleótido Simple , Cultivo Primario de Células , Factor de Transcripción STAT3/metabolismo , Síndrome de Sézary/sangre , Síndrome de Sézary/tratamiento farmacológico , Síndrome de Sézary/patología , Piel/citología , Piel/patología , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Consumption of unhealthy diets is exacerbating the burden of age-related ill health in aging populations. Such diets can program mammalian physiology to cause long-term, detrimental effects. Here, we show that, in Drosophila melanogaster, an unhealthy, high-sugar diet in early adulthood programs lifespan to curtail later-life survival despite subsequent dietary improvement. Excess dietary sugar promotes insulin-like signaling, inhibits dFOXO-the Drosophila homolog of forkhead box O (FOXO) transcription factors-and represses expression of dFOXO target genes encoding epigenetic regulators. Crucially, dfoxo is required both for transcriptional changes that mark the fly's dietary history and for nutritional programming of lifespan by excess dietary sugar, and this mechanism is conserved in Caenorhabditis elegans. Our study implicates FOXO factors, the evolutionarily conserved determinants of animal longevity, in the mechanisms of nutritional programming of animal lifespan.