RESUMEN
To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 × 105-fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 × 103-fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents "signal 1," CD137 represents "signal 2," and cytokines represent "signal 3." The precise nature of signal 1 remains to be defined.
Asunto(s)
Ligando 4-1BB/metabolismo , Interleucina-15/inmunología , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Anticuerpos/inmunología , Línea Celular Tumoral , Proliferación Celular , Humanos , Inmunoterapia , Activación de Linfocitos/inmunología , Transducción de Señal/inmunologíaRESUMEN
Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimidopyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Pirimidinonas/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Área Bajo la Curva , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Células CACO-2 , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Células HeLa , Humanos , Masculino , Ratones SCID , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Pirimidinonas/química , Pirimidinonas/farmacocinética , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Termodinámica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.