Modulación de la actividad de EGFR en gioblastomas: nuevas aproximaciones terapéuticas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lecdtura: 23-01-2015Los glioblastomas (GBMs) son tumores cerebrales primarios muy agresivos y resistentes al tratamiento convencional con quimio y radioterapia. Dado que EGFR (del inglés Epidermal growth factor receptor) se encuentra alterado en el 50 % de los GBMs, representa actualmente una de las dianas terapéuticas más prometedoras en este tipo de tumores. Sin embargo, los inhibidores de la actividad quinasa de EGFR han generado escasos resultados en ensayos clínicos con pacientes de GBM, sin que exista una clara explicación para esta resistencia a dicha terapia. En este trabajo se ha evaluado la eficacia antitumoral de un inhibidor de EGFR de segunda generación denominado dacomitinib (PF299804, Pfizer), que se une de manera irreversible al receptor. Los resultados obtenidos confirman que dacomitinib es capaz de reducir la viabilidad celular, la autorenovación y la proliferación de las células de GBM con EGFR amplificado, con independencia de la expresión de la forma mutante EGFRvIII. Además, se ha observado una disminución notable de la tasa de crecimiento tumoral in vivo de dichas líneas de GBM tras la administración sistémica de dacomitinib a los ratones, coincidiendo con la reducción de los niveles de expresión de marcadores celulares característicos de célula madre. Sin embargo, nuestros resultados indican también que el efecto del inhibidor es reversible y que los tumores vuelven a crecer cuando se retira el tratamiento. Además, aunque en presencia de dacomitinib se produce una clara inhibición de la cascada de señalización inducida por EGFR, también se observa la acumulación del mismo, lo que podría provocar la activación de otras señales independientes de su actividad quinasa. Es por ello que el presente trabajo explora una estrategia novedosa y alternativa a dacomitinib. Nuestros resultados describen el papel fundamental de DYRK1A (del inglés Dual-specificity tyrosine-phosphorylation-regulated kinase 1A) en la regulación de la estabilidad de EGFR en las células de GBM. Así hemos comprobado que la inhibición de DYRK1A (ya sea farmacológicamente o genéticamente) es capaz de promover la degradación de EGFR en cultivos primarios de GBM, con una reducción notable de la capacidad de autorenovación de las células tumorales. Además, los resultados obtenidos sugieren que la supervivencia celular de un subgrupo de GBMs depende de la presencia de elevados niveles de EGFR en la superficie celular, ya que la inhibición de DYRK1A provoca un importante descenso de la carga tumoral. A la luz de los resultados obtenidos, se podría postular que la inhibición de EGFR con dacomitinib y el bloqueo de de DYRK1A representan una aproximación terapéutica prometedora en aquellos GBMs dependientes de EGFR, ya sea de manera individual o combinada, bloqueando toda la señalización activada por el receptor, tanto dependiente como independiente de su actividad quinasa.Glioblastomas (GBMs) are very aggressive primary brain tumors, which are resistant to conventional chemo and radiotherapy. Since EGFR (epidermal growth factor receptor) is altered in almost 50% of GBM, it currently represents one of the most promising therapeutic targets. However EGFR kinase activity inhibitors have produced poor results in clinical trials with GBM patients, with no clear explanation for the therapy resistance observed. Here it has been tested the antitumoral efficacy of a second-generation inhibitor: dacomitinib (PF299804, Pfizer) that binds in an irreversible way to the receptor. The results obtained confirm that dacomitinib is able to reduce the cell viability, the self-renewal and the proliferation of EGFR amplified GBM cells, independently of the EGFRvIII mutant form expression. Moreover we have observed a considerable decrease of the in vivo tumor growth rate of these EGFR amplified cell lines after systemic administration of dacomitinib to the mice, which provokes also a decrease in the expression of stem-cell-related markers. Nevertheless our results point out that the inhibitor effect is reversible and the tumors grow again when the treatment is interrupted. Moreover despite the clear inhibition of EGFR phosphorilation in the presence of the drug we observe also a clear accumulation of the receptor, which could provoke the activation of other signals that are independent of its kinase activity. For that reason we decided to analyze an alternative strategy to dacominib. Our results describe the basic role of DYRK1A (dual-specificity tyrosinephosphorylation- regulated kinase) in regulating the EGFR stability in GBM cells. We have observed that the inhibition of DYRK1A (pharmacologically or genetically) is able to promote the EGFR degradation in the GBM primary cell cultures, reducing the self-renewal capacity of tumorigenic cells. Moreover, the obtained results suggest that the cell survival of a subset of GBMs depends on the presence of high levels of EGFR on the cell surface, as the DYRK1A inhibition causes a profound decrease in tumor burden. In light of the obtained results it could be postulated that the inhibition of EGFR with dacomitinib and the blockade of DYRK1A (individually or combined) represent a promising therapeutic approach in those EGFR-dependent GBMs, blocking all the signals activated by the receptor, both dependent and independent of its kinase activity

DYRK1A: the double-edged kinase as a protagonist in cell growth and tumorigenesis

DYRK1A (dual-specificity tyrosine-regulated kinase 1A) is a kinase with multiple implications for embryonic development, especially in the nervous system where it regulates the balance between proliferation and differentiation of neural progenitors. The DYRK1A gene is located in the Down syndrome critical region and may play a significant role in the developmental brain defects, early neurodegeneration, and cancer susceptibility of individuals with this syndrome. DYRK1A is also expressed in adults, where it might participate in the regulation of cell cycle, survival, and tumorigenesis, thus representing a potential therapeutic target for certain types of cancer. However, the final readout of DYRK1A overexpression or inhibition depends strongly on the cellular context, as it has both tumor suppressor and oncogenic activities. Here, we will discuss the functions and substrates of DYRK1A associated with the control of cell growth and tumorigenesis with a focus on the potential use of DYRK1A inhibitors in cancer therapy.This study was supported by grants from Ministerio de Economía y Competitividad, Fondo de Investigación Sanitaria, PI12/00775 and from Ministerio de Economía y Competitividad, Red Temática de Investigación Cooperativa en Cancer (RD12/0036/0027) to PSG.S

Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma

Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133(+), Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy.This study was supported by grants from: Ministerio de Economía y Competitividad, Fondo de Investigación Sanitaria (PI12/101 to HM; PI12/00775 to PSG; PS09/1786 to MGF and PI13/01258 to AHL), Comunidad de Madrid (S2010/BMD-2336 to HM), Xunta de Galicia (EM2013/042 to MGF), Fundación BBVA (2014-PO010 to MGF) and Ministerio de Economía y Competitividad, Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0027 to PSG and AHL). PGG was recipient of a “Sara Borell” postdoctoral fellowship, and MdF of a “Miguel Servet” contract from Ministerio de Economía y CompetitividadS

Preclinical Test of Dacomitinib, an Irreversible EGFR Inhibitor, Confirms Its Effectiveness for Glioblastoma.

Glioblastomas (GBM) are devastating tumors in which there has been little clinical improvement in the last decades. New molecularly directed therapies are under development. EGFR is one of the most promising targets, as this receptor is mutated and/or overexpressed in nearly half of the GBMs. However, the results obtained with first-generation tyrosine-kinase inhibitors have been disappointing with no clear predictive markers of tumor response. Here, we have tested the antitumoral efficacy of a second-generation inhibitor, dacomitinib (PF299804, Pfizer), that binds in an irreversible way to the receptor. Our results confirm that dacomitinib has an effect on cell viability, self-renewal, and proliferation in EGFR-amplified ± EGFRvIII GBM cells. Moreover, systemic administration of dacomitinib strongly impaired the in vivo tumor growth rate of these EGFR-amplified cell lines, with a decrease in the expression of stem cell-related markers. However, continuous administration of the compound was required to maintain the antitumor effect. The data presented here confirm that dacomitinib clearly affects receptor signaling in vivo and that its strong antitumoral effect is independent of the presence of mutant receptor isoforms although it could be affected by the PTEN status (as it is less effective in a PTEN-deleted GBM line). Dacomitinib is being tested in second line for EGFR-amplified GBMs. We hope that our results could help to select retrospectively molecular determinants of this response and to implement future trials with dacomitinib (alone or in combination with other inhibitors) in newly diagnosed GBMs.This work was supported by grants from the Fundación Mutua-madrileña (FMM2011/89) to J.M. Sepúlveda and from Ministerio de Economía y Competitividad, Fondo de Investigación Sanitaria (FIS): PI12/00775 to P. Sánchez-Gómez and PI13/01258 to A. Hernández-Laín, and from Ministerio de Economía y Competitividad, Red Temática de Investigación Cooperativa en Cancer (RTICC) (RD12/0036/0027) to J.M. Sepúlveda, P. Sánchez-Gómez, A. Pérez-Núñez and A. Hernández-Laín.S

Inhibition of DYRK1A destabilizes EGFR and reduces EGFR-dependent glioblastoma growth

Glioblastomas (GBMs) are very aggressive tumors that are resistant to conventional chemo- and radiotherapy. New molecular therapeutic strategies are required to effectively eliminate the subpopulation of GBM tumor-initiating cells that are responsible for relapse. Since EGFR is altered in 50% of GBMs, it represents one of the most promising targets; however, EGFR kinase inhibitors have produced poor results in clinical assays, with no clear explanation for the observed resistance. We uncovered a fundamental role for the dual-specificity tyrosine phosphorylation-regulated kinase, DYRK1A, in regulating EGFR in GBMs. We found that DYRK1A was highly expressed in these tumors and that its expression was correlated with that of EGFR. Moreover, DYRK1A inhibition promoted EGFR degradation in primary GBM cell lines and neural progenitor cells, sharply reducing the self-renewal capacity of normal and tumorigenic cells. Most importantly, our data suggest that a subset of GBMs depends on high surface EGFR levels, as DYRK1A inhibition compromised their survival and produced a profound decrease in tumor burden. We propose that the recovery of EGFR stability is a key oncogenic event in a large proportion of gliomas and that pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for EGFR-dependent GBMs.This work was supported by grants from the Ministerio de Educación y Ciencia (MEC; SAF2008-04531), the Ministerio de Ciencia e Innovación (MICINN, PLE2009-0115), and the Ministerio de Asuntos Exteriores y Cooperación (MAEC-AECID A/023963/09; to P. Sánchez-Gómez), as well as by grants from the Fondo de Investigación Sanitaria (FIS-PS09-01977) and Fundación Mutua-madrileña grants (FMM 2007/057, to J.R. Ricoy; and FMM2011/89, to J.M. Sepúlveda).S

Contrapicados y puntos de fuga. Las otras historias de la historia de la filosofía

Depto. de Lógica y Filosofía TeóricaFac. de FilosofíaFALSEsubmitte

Precariedad, exclusión social y diversidad funcional (discapacidad): lógicas y efectos subjetivos del sufrimiento social contemporáneo (II). Innovación docente en Filosofía

El PIMCD "Precariedad, exclusión social y diversidad funcional (discapacidad): lógicas y efectos subjetivos del sufrimiento social contemporáneo (II). Innovación docente en Filosofía" se ocupa de conceptos generalmente eludidos por la tradición teórica (contando como núcleos aglutinantes los de la precariedad laboral, la exclusión social y diversidad funcional o discapacidad), cuyo análisis propicia nuevas prácticas en la enseñanza universitaria de filosofía, adoptando como meta principal el aprendizaje centrado en el estudiantado, el diseño de nuevas herramientas de enseñanza y el fomento de una universidad inclusiva. El proyecto cuenta con 26 docentes de la UCM y otros 28 docentes de otras 17 universidades españolas (UV, UNED, UGR, UNIZAR, UAH, UC3M, UCA, UNIOVI, ULL, EHU/UPV, UA, UAM, Deusto, IFS/CSIC, UCJC, URJC y Univ. Pontificia de Comillas), que permitirán dotar a las actividades programadas de un alcance idóneo para consolidar la adquisición de competencias argumentativas y dialécticas por parte de lxs estudiantes implicados en el marco de los seminarios previstos. Se integrarán en el PIMCD, aparte de PDI, al menos 26 estudiantes de máster y doctorado de la Facultad de Filosofía, a lxs que acompañarán durante el desarrollo del PIMCD 4 Alumni de la Facultad de Filosofía de la UCM, actualmente investigadores post-doc y profesorxs de IES, cuya experiencia será beneficiosa para su introducción en la investigación. Asimismo, el equipo cuenta con el apoyo de varixs profesorxs asociadxs, que en algunos casos son también profesores de IES. Varixs docentes externos a la UCM participantes en el PIMCD poseen una dilatada experiencia en la coordinación de proyectos de innovación de otras universidades, lo que redundará en beneficio de las actividades a desarrollar. La coordinadora y otrxs miembros del PIMCD pertenecen a la Red de Innovación Docente en Filosofia (RIEF), puesta en marcha desde la Universitat de València (http://rief.blogs.uv.es/encuentros-de-la-rief/), a la que mantendremos informada de las actividades realizadas en el proyecto. Asimismo, lxs 6 miembros del PAS permitirán difundir debidamente las actividades realizadas en el PIMCD entre lxs estudiantes Erasmus IN del curso 2019/20 en la Facultad de Filosofía, de la misma manera que orientar en las tareas de maquetación y edición que puedan ser necesarias de cara a la publicación de lxs resultados del PIMCD y en las tareas de pesquisa bibliográfica necesarias para el desarrollo de los objetivos propuestos. Han manifestado su interés en los resultados derivados del PIMCD editoriales especializadas en la difusión de investigaciones predoctorales como Ápeiron y CTK E-Books

EGFR-dependent mechanisms in glioblastoma: towards a better therapeutic strategy.

Glioblastoma is a particularly resilient cancer, and while therapies may be able to reach the brain by crossing the blood-brain barrier, they then have to deal with a highly invasive tumor that is very resistant to DNA damage. It seems clear that in order to kill aggressive glioma cells more efficiently and with fewer side effects on normal tissue, there must be a shift from classical cytotoxic chemotherapy to more targeted therapies. Since the epidermal growth factor receptor (EGFR) is altered in almost 50% of glioblastomas, it currently represents one of the most promising therapeutic targets. In fact, it has been associated with several distinct steps in tumorigenesis, from tumor initiation to tumor growth and survival, and also with the regulation of cell migration and angiogenesis. However, inhibitors of the EGFR kinase have produced poor results with this type of cancer in clinical trials, with no clear explanation for the tumor resistance observed. Here we will review what we know about the expression and function of EGFR in cancer and in particular in gliomas. We will also evaluate which are the possible molecular and cellular escape mechanisms. As a result, we hope that this review will help improve the design of future EGFR-targeted therapies for glioblastomas.The work in the authors ‘laboratory is funded by Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) (PI12/00775 and RD12/0036/0027). We thank Angel Ayuso-Sacido and Juan Sepúlveda-Sánchez for critical review of the manuscript.S