Mar Orzáez

MCL1, an anti-apoptotic BCL-2 family member, is frequently overexpressed in multiple tumour types and correlates with poor prognosis; however, targeting its cytosolic domain to release pro-apoptotic effectors has been limited by cardiotoxicity in clinical trials. In this study, we describe a chemically mediated disruption of the MCL1/BOK transmembrane interaction that induces tumour cell death without affecting viability in 3D cardiomyocyte cultures. Molecular dynamics simulations and LUV-based assays converged to show that the MBoIN179 disrupts transmembrane dimerization, thereby restoring BOK pore formation inhibited by MCL1. In cells, interference with the MCL1/BOK transmembrane interaction promoted relocalization of BOK from the endoplasmic reticulum to mitochondria, where it engaged a BOK-dependent cell death program in both 2D and 3D breast cancer models, leading to reduced tumour growth and metastasis in vivo. Analysis of patient tumour microarrays further showed that BOK is overexpressed in aggressive breast cancer subtypes and correlates with poor prognosis. Together, these findings identify the MCL1/BOK transmembrane interaction as a tumour-selective vulnerability that can be engaged to promote antitumour responses with cardiac safety, highlighting transmembrane interactions as viable molecular targets.

Entosis is a cell-in-cell (CIC) invasion process in which one living cell actively invades into another, with important implications for tumor evolution, cell competition, and responses to cellular stress. Both the initiation and progression of entosis require extensive actin cytoskeletal remodeling, as one cell must physically accommodate the complete internalization of another viable cell, which can subsequently migrate, divide, undergo degradation, or escape. Nearly two decades after its discovery, mechanistic insight into how cytoskeleton organization is regulated during entosis remains poorly understood. The most consistent observation is that inhibition of Rho–ROCK signaling blocks entosis, indicating a central role for actomyosin contractility. Consistently, increased phosphorylation of myosin light chain and cortical actomyosin accumulation have been reported in invading cells, yet the underlying mechanisms remain largely unknown. Here, we systematically interrogated ROCK activation as a driver of the entotic internalization program. Constitutively active mutant ROCK1 or pharmacological ROCK1 activation using narciclasine rapidly triggered canonical entosis pathway through MLC2 phosphorylation. Mass spectrometry identified Plastin-3 (PLS3, T-plastin), a crucial calcium-sensitive actin-bundling protein, which was recruited to the plasma membrane to trigger entosis upon ROCK1 activation. Our study uncovers a key cytoskeletal remodeling step which may support cortical tension required for CIC invasion during entosis.

Esta comunicación presenta la evolución e impacto de diferentes proyectos de innovación docente desarrollados entre los cursos 2022 y 2025 en la asignatura de Bioquímica y Biología Molecular (BBM), del primer curso del Grado en Medicina. La iniciativa surgió como respuesta a la baja motivación y rendimiento académico observado por el profesorado y manifestado por el propio alumnado. A lo largo de tres cursos, se han aplicado metodologías activas como el aprendizaje basado en problemas (ABP), centrado en casos clínicos bioquímicos, y estrategias de gamificación como el Trivial bioquímico y la Ruleta de palabras. Estas herramientas han permitido transformar el aula en un espacio participativo, dinámico y clínicamente contextualizado. Los resultados indican una mejora sostenida en el rendimiento académico, un mayor compromiso del estudiantado y una percepción positiva sobre la utilidad y aplicabilidad de la asignatura. Este trabajo resume las fases de diseño, implementación, evaluación y mejora continua del proyecto, así como sus perspectivas de sostenibilidad y transferencia a otras asignaturas del área biomédica.

The BCL2 family of proteins controls cell death by modulating the permeabilization of the mitochondrial outer membrane through a fine-tuned equilibrium of interactions among anti- and pro-apoptotic members. The upregulation of anti-apoptotic BCL2 proteins represents an unfavorable prognostic factor in many tumor types due to their ability to shift the equilibrium toward cancer cell survival. Furthermore, cancer-associated somatic mutations in BCL2 genes interfere with the protein interaction network, thereby promoting cell survival. A range of studies have documented how these mutations affect the interactions between the cytosolic domains of BCL2 and evaluate the impact on cell death; however, as the BCL2 transmembrane interaction network remains poorly understood, somatic mutations affecting transmembrane regions have been classified as pathogenic-based solely on prediction algorithms. We comprehensively investigated cancer-associated somatic mutations affecting the transmembrane domain of BCL2 proteins and elucidated their effect on membrane insertion, hetero-interactions with the pro-apoptotic protein BAX, and modulation of cell death in cancer cells. Our findings reveal how specific mutations disrupt switchable interactions, alter the modulation of apoptosis, and contribute to cancer cell survival. These results provide experimental evidence to distinguish BCL2 transmembrane driver mutations from passenger mutations and provide new insight regarding selecting precision anti-tumor treatments.

Accumulation of senescent cells with age leads to tissue dysfunction and related diseases. Their detection in vivo still constitutes a challenge in aging research. We describe the generation of a fluorogenic probe (sulfonic-Cy7Gal) based on a galactose derivative, to serve as substrate for β-galactosidase, conjugated to a Cy7 fluorophore modified with sulfonic groups to enhance its ability to diffuse. When administered to male or female mice, β-galactosidase cleaves the O-glycosidic bond, releasing the fluorophore that is ultimately excreted by the kidneys and can be measured in urine. The intensity of the recovered fluorophore reliably reflects an experimentally controlled load of cellular senescence and correlates with age-associated anxiety during aging and senolytic treatment. Interestingly, our findings with the probe indicate that the effects of senolysis are temporary if the treatment is discontinued. Our strategy may serve as a basis for developing fluorogenic platforms designed for easy longitudinal monitoring of enzymatic activities in biofluids.

The main cause of subretinal neovascularisation in wet age-related macular degeneration (AMD) is an abnormal expression in the retinal pigment epithelium (RPE) of the vascular endothelial growth factor (VEGF). Current approaches for the treatment of AMD present considerable issues that could be overcome by encapsulating anti-VEGF drugs in suitable nanocarriers, thus providing better penetration, higher retention times, and sustained release. In this work, the ability of large pore mesoporous silica nanoparticles (LP-MSNs) to transport and protect nucleic acid molecules is exploited to develop an innovative LP-MSN-based nanosystem for the topical administration of anti-VEGF siRNA molecules to RPE cells. siRNA is loaded into LP-MSN mesopores, while the external surface of the nanodevices is functionalised with polyethylenimine (PEI) chains that allow the controlled release of siRNA and promote endosomal escape to facilitate cytosolic delivery of the cargo. The successful results obtained for VEGF silencing in ARPE-19 RPE cells demonstrate that the designed nanodevice is suitable as an siRNA transporter.

In this article, we report one of the few examples of nanoparticles capable of simultaneously delivering CRISPR-Cas9 gene-editing machinery and releasing drugs for one-shot treatments. Considering the complexity of inflammation in diseases, the synergistic effect of nanoparticles for gene-editing/drug therapy is evaluated in an in vitro inflammatory model as proof of concept. Mesoporous silica nanoparticles (MSNs), able to deliver the CRISPR/Cas9 machinery to edit gasdermin D (GSDMD), a key protein involved in inflammatory cell death, and the anti-inflammatory drug VX-765 (^GSDMD45CRISPR-VX-MSNs), were prepared. Nanoparticles allow high cargo loading and CRISPR-Cas9 plasmid protection and, thus, achieve the controlled codelivery of CRISPR-Cas9 and the drug in cells. Nanoparticles exhibit GSDMD gene editing by downregulating inflammatory cell death and achieving a combined effect on decreasing the inflammatory response by the codelivery of VX-765. Taken together, our results show the potential of MSNs as a versatile platform by allowing multiple combinations for gene editing and drug therapy to prepare advanced nanodevices to meet possible biomedical needs.