Vascular anomalies are rare soft tissue tumours and malformations formed by abnormal vascular elements of various types, and mainly affect infants, children and young adults. These lesions are painful, many lead to bleeding, infections, organ dysfunction, and they can damage and metastasise to other tissues. Current treatment strategies are invasive, not fully efficient, and there is a high risk of recurrence. To date no specific targeted therapies have been developed. Most of the studies on vascular anomalies are descriptive and focused on their clinical aspects; at the moment, there is a lack of molecular understanding and pre-clinical studies on the field of vascular anomalies. Thus, identifying the causative molecular alterations of vascular anomalies and understanding their biology will lead to more refined diagnoses and will provide better and more directed therapies. Recent published and unpublished observations from our laboratory show that activation of phosphoinositide 3-kinase (PI3K) signalling pathway drive to the developments of a fraction of vascular anomalies; however, the underlying molecular and cellular mechanisms remain enigmatic. In this proposal we aim to (i) unveil alterations in the PI3K signalling pathway components that are involved in the pathogenesis of vascular anomalies, (ii) understand the role of PI3K pathway activation in the development and maintenance of these lesions, and (iii) explore the potential of repurposing PI3K pathway inhibitors for these conditions. For this, we have set up collaboration with clinicians who will provide a large collection of vascular anomalies from patients. Also, in collaboration with Pharma, we aim to use PI3K inhibitors for these diseases. Importantly, these collaborations will allow us to translate our findings into the clinic for future clinical trials. This project proposal seeks to broad our understanding of PI3K activation in vascular biology and to ultimately improve patients’ quality of life.

Precise regulation of gene expression is achieved through the coordinated action of genomic cis-regulatory modules (CRMs). The identification of CRMs has long been a goal of functional genomics as CRM dysregulation can have devastating consequences for health and development such as autism. For example, mutations in the MeCP2 gene, which encodes protein that binds methylated DNA and regulates gene expression in neurons, results an Autism Spectrum Disorder termed Rett Syndrome. While thought to modulate CRM activity as a repressor, MeCP2’s function remains ambiguous and would benefit from a functional genomics characterization. To identify CRMs I recently developed a novel approach called FIREWACh. Here, I propose to utilize FIREWACh first to identify active CRMs within a homogenous population of neurons, identifying genomic loci of CRMs whose function may be compromised by MeCP2 mutations. I will adapt FIREWACh to allow the quantification of CRM output in a new method I propose to call ROQ-WACh (regulatory output quantification within accessible chromatin) by addition of barcodes to reporter mRNAs. This will allow high-throughput readout of CRM activity globally in vivo and will be broadly applicable to many biological fields. Lastly, I aim to combine the above approaches to quantify the changes in CRM output in response to pathological mutations in MeCP2. This Marie Sklodowska-Curie Action will allow me (the Experienced Researcher, Matthew Murtha) mobility to Spain to join the laboratory of Dr. Manel Esteller (Host Supervisor), renowned epigeneticist and expert in DNA-methylation biology to perform the research. Together the approaches and data generated by this MSCA action will provide key insights into the relationship between methylated DNA, precise control of gene expression, and high-order phenotypes such as cognitive behaviors.

Controlling cell cycle, process by which cells progress and divide, is at the heart of cancer research therapies. Tumor cells signature often lays in high proliferation rate, alterations of multiple intracellular signaling networks and hijacking of programmed-cell death pathways. Implication of autophagy and lysosomal biology in cancer is still controversial. Lysosomes are catabolic vesicles involved in the degradation of biological material and need to be tightly regulated. Lysosomes may influence cancer formation and progression by regulating many complex processes of cell biology, such as cell signaling, cell death, nutrient sensing and cell metabolism. Lysosome function depends on lysosome acidification capacity modulated by proton pump v-ATPase activity and on its intracellular localization driven by motor proteins, mainly by motor protein kinesin KIF5B. We recently discover an innovative mechanism involving lysosomes impairment to induce mitotic error that could potentially lead tumor cells to cell death.

Implementation of next generation sequencing to genetic diagnosis and precision medicine has revolutionized the fields of hereditary cancer and oncology, increasing exponencially the identification of genetic variants of uncertain significance. Their classification is one of the most relevant and urgent challenges we face, since decision-making in the clinics depends on it. Evidence obtained from empirical functional studies is essential to reach a definitive classification; however, clinical relevance of such studies is currently low due to lack of standardized tests, use of inadequate models, and tediousness and cost- and time-inefficiency of available assays. Organ-VIP aims at surpassing the barriers we face when using and implementing functional assays for the interpretation of genetic variants in colorectal cancer (CRC) genes. State-of-the-art methodological and conceptual developments will facilitate the development of a screening platform to interpret the pathogenicity of variants in any CRC gene. To do so, we aim to: i) Use a model that faithfully represents the target tissue and genetic context (CRISPR/Cas9-edited human normal colon organoids); ii) Optimize end-point assay(s) to maximize performance, implementation, robustness and agreement with clinical evidence; iii) Assess genetic variants in hereditary CRC and polyposis genes; and iv) Calibrate the results for implementation in variant classifiers. Organ-VIP integrates clinical aspects, molecular and cell biology, next generation sequencing, and advanced bioinformatics analysis. The platform will not only be useful for variant interpretation in germline and somatic testing, but also for functional evaluation of new candidate CRC genes. In the longer term, Organ-VIP will become high-throughput by the implementation of saturation genome editing, high-throughput organoid culture, automation of sampling, and implementation of artificial intelligence.