RESUMEN
CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.
Asunto(s)
Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/química , Péptidos Cíclicos/farmacología , Investigación Biomédica Traslacional , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de ProteínaRESUMEN
We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Neovascularización Patológica/prevención & control , Péptidos Cíclicos/farmacología , Animales , Biomarcadores/metabolismo , Western Blotting , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Membrana Corioalantoides/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Análisis de Secuencia por Matrices de Oligonucleótidos , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CIGB-300, formerly known as P15-tat, is a proapoptotic peptide with established antiproliferative activity in vitro and antitumoral activity in vivo. This hypothesis-driven peptide was initially selected for its ability to impair the in vitro CK2-mediated phosphorylation in one of its substrates through direct binding to the conserved acidic phosphoaceptor domain. However, the actual in vivo target(s) on human cancer cells among the hundreds of CK2 substrates as well as the subsequent events that lead to apoptosis on tumor cells remains to be determined. In this work, we identified the multifunctional oncoprotein nucleophosmin/B23 as a major target for CIGB-300. In vivo, the CIGB-300-B23 interaction was shown by pull-down experiments and confirmed by the early in situ colocalization of both molecules in the cell nucleolus. Moreover, CIGB-300 inhibits the CK2-mediated phosphorylation of B23 in a dose-dependent fashion both in vitro and in vivo as shown using the recombinant GST fusion protein and the metabolic labeling approach, respectively. Such phosphorylation impairment was correlated with the ability of CIGB-300 to induce nucleolar disassembly as documented by the use of established markers for nucleolar structure. Finally, we showed that such a sequence of events leads to the rapid and massive onset of apoptosis both at the molecular and cellular levels. Collectively, these findings provide important clues by which the CIGB-300 peptide exerts its proapoptotic effect on tumor cells and highlights the suitability of the B23/CK2 pathway for cancer-targeted therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Péptidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , Biomarcadores de Tumor , Línea Celular Tumoral , Humanos , Espacio Intracelular , Terapia Molecular Dirigida , Nucleofosmina , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Transporte de Proteínas , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties in vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates in vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.