LABORATORY OF NEURAL TUMORS Principal Investigator (Name, MD/PhD) Miguel F. Segura   Research Team Adrià Molero Valencuela, PhD Researcher Ainara Magdaleno Cazón, Tehcnician Ariadna Boloix Amenós, Postdoctoral Researcher Carlos Jiménez Jiménez, Postdoctoral Researcher María José Pérez García, Postdoctoral Researcher & Project Manager Roberta Antonelli, Postdoctoral Researcher Clinical Associated Researchers Lucas Moreno, MD PhD. Expertise in drug development and clinical trials. Anna Llort, MD PhD. Expertise in pediatric brain tumors Constantino Sábado, MD. Expertise in Hepatoblastoma BACKGROUND Pediatric cancer has specific features that define it as an entity that in the majority of cases is certainly different than adult tumors; i.e. etiology, biology, response to treatment and prognosis are really different. The implementation of multimodal treatments (i.e. combinations of chemotherapies, radiotherapies, biological agents…) in the lasts 20 years has enabled the raise patient survival around 80%. Although the global survival of pediatric cancer is around 80%, patients with some types of tumors present a much lower survival. Moreover, almost two-thirds of patients who survive suffer severe side effects due to the treatment. Therefore, the development of therapies to treat 20% of patients who do not survive and to reduce toxicity of current treatments avoiding long-term side effects is extremely necessary. Pediatric tumors of the nervous system are the most common solid malignant neoplasms of childhood cancer and the leading cause of cancer-related death in children. The main objective of my laboratory is to implement the use of epigenetic therapies, that is, therapies against gene expression modulators that, in turn, regulate several genes, pathways or cellular processes, without affecting the DNA sequence. The laboratory focus on neuroblastoma, a pediatric solid tumor of the peripheral nervous system, and on pediatric brain tumor of the central nervous system pediatric such as ependymoma, medulloblastoma or high-grade glioma among others. RESEARCH STRATEGY AND SCOPE The research strategy of the Neural Tumors Lab is based on the following research lines: Development of RNA-based therapies for pediatric tumors RNA-based therapies may represent a paradigm shift for the treatment of neuroblastoma, or cancer in general, because they have the potential to act on the entire transcriptome, thus expanding the number of drug targets, since they could modulate both RNA coding (2% of the total genome that is transcribed) and non-coding RNAs (80% of the genome that is transcribed). Within RNA-based therapies, the most advanced molecules from a clinical development point of view are small RNAs (e.g. siRNA or microRNA). Moreover, they will be safer therapies, since their mechanism of action will not induce permanent effects, especially in healthy cells. One of the working hypothesis of this line is that the microRNAs (miRNAs) deregulation is contributing to the metastatic and chemoresistant behavior of high-risk neuroblastoma and miRNA(s) levels restoration represents an attractive novel therapeutic approach. Exploiting epigenetic vulnerabilities in metastatic neuroblastoma Metastatic relapse is the major cause of death in neuroblastoma. The liver, bone and bone marrow and lymph nodes are among the most common metastatic sites of neuroblastoma patients, which constitute a “reservoir” of tumor cells that persistently reside in patients following local and systemic cancer therapy. Their elimination continues to represent the most difficult challenges for neuroblastoma patients. Our hypothesis is that metastatic cells can colonize distant organs thanks to the re-organization of their epigenetic landscape. Results of the last five years of the laboratory clearly shows that chromatin remodelers may play significant role in the aggressiveness of neuroblastoma. Therefore, in this cell line we are i) Characterizing the epigenetic landscape of metastatic neuroblastoma; ii) Characterizing the transcriptomic map of chromatin remodelers (i.e. SWI/SNF)-regulated genes in neuroblastoma metastasis; iii) Developing of an epigenetic therapy based on the inhibition of chromatin remodeling complexes; iv) Developing more sensitive tools to diagnose and target neuroblastoma metastasis. Participation in drug development in pediatric solid tumors (e.g. Neuroblastoma, Hepatoblastoma, Brain Tumors) We are interested in testing the therapeutic potential of new synthetic or natural compounds which can represent clear advantages (i.e. increased effectiveness, reduced toxicities) compared to traditional chemotherapy. We have a clinically representative panel of pediatric solid tumor cell lines and preclinical mouse models to test the efficacy and safety of new drugs aimed to improve the efficacy and safety of currently available treatments. Currently, we are evaluating the therapeutic potential of ABTL0812 in pediatric tumors, both preclinically but also in a “first in child” Phase I clinical trial. Additionally, we are also pursuing the evaluation of the therapeutic potential of the KIF11 inhibitor 4SC-205 in pediatric tumors. Identification of new treatments for pediatric brain tumors (i.e. Ependymoma) Pediatric tumors of the central nervous system (CNS) are the most common solid malignancies in children, second only to hematologic malignancies, and are the leading cause of cancer-related deaths. Epigenetic therapies aimed at reversing the oncogenic alterations in chromatin structure and function are an emerging alternative against aggressive tumors that are or will become resistant to conventional treatments. One of the most advanced epigenetic therapies, only second to DNA methyl-transferase inhibitors, is the use of histone deacetylase inhibitors (HDACi). However, while shown to be effective in certain tumor types, their use in brain tumors did not yielded the expected results, probably because these drugs were not specifically designed to reach sealed tissues such as the brain. This research line constitutes a truly translational approach to further enhance the possibility of using HDACi in the treatment of brain tumors such as, but not restricted to, Ependymoma. Here, we are also elaborating a roadmap for the implementation of immunotherapies in pediatric brain tumors and characterizing the role of the tumor microenvironment in tumor growth and aggressiveness. ONGOING COMPETITIVE PROJECTS: -Ref. PI20/00530 (AES ISCiii 2021-2023). PI: Miguel F. Segura. Nanotools for the treatment of metastatic neuroblastoma. Summary: Metastatic relapse is the major cause of death in NB, where there remains a lack of therapies to target this stage of the disease. Therefore, understanding how NB cells invade, arrive at the metastatic niche and adapt to the new environment at the metastatic site, will help in designing more effective therapies to control the development and metastasis in NB. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that interfere with the translation of coding messenger RNAs (mRNAs) in a sequence-specific manner. In NB, over- or under-expression of specific miRNAs correlates with stage, progression (i.e. relapse / metastasis) and patient outcome. Particularly, miR-323a-5p was shown to be downregulated in NB metastasis and its restoration impaired tumor growth in vitro and in vivo. MiRNA-based therapies face a major challenge, i.e. the limitations associated with their clinical administration, such as inefficient biodistribution and specific targeting of tissues. Conjugation of miRNA with nanostructured materials can improve targeting to specific tissues and reduce potential side-effects. The aim of this project is to test a new miRNA-based nanoformulation for the treatment of metastatic neuroblastoma. Ref. DTS20/00018 (AES ISCiii 2021-2022). PI: Miguel F. Segura. Quatsomes as a novel nanomaterial platform for nucleic acids delivery. Summary: RNA-based therapies against cancer have proved to show great potential since they are able to target all the transcriptome (coding and non-coding mRNAs), thereby expanding significantly the number of therapeutic targets. The most therapeutically-advanced molecules in RNA-based therapies are microRNA (miRNA), endogenous noncoding small RNA that interfere with the translation and stability of RNA (coding and non-coding) in a sequence-specific manner. However, the application of these small molecules for treating human disease faces a major challenge, i.e. the limitations associated with their clinical administration such as inefficient biodistribution and specific targeting of tissues. The aim of this project is to optimize our QS-miRNA nanoformulation for the treatment of high-risk neuroblastoma, an incurable pediatric tumor that demands new therapeutic approaches. Ours will consist on functionalizing QS-miRNA complexes with stealth polymers and specific targeting peptides which will reduce QSmiRNA complex aggregation, enhance blood circulation time and increase specificity towards neuroblastoma tumors. Ref. ICI21/00076 (AES ISCiii 2022-2025). PI: Lucas Moreno. Scientific Officer: Miguel F. Segura. A phase I trial of ABTL0812 in paediatric patients with advanced cancer including neuroblastoma. Summary: Childhood cancer is the first cause of death due to disease in children. New drugs are needed to increase cure rates and reduce long-term sequelae of survivors. More than half of children with high-risk tumors such as metastatic neuroblastoma or sarcomas, or those with refractory or relapsed disease will not achieve a long-term cure. Most current therapies target cell division and proliferation by inducing DNA damage and programmed cell death. However, aggressive tumors often present alterations of these processes and are resistant to therapy. Therefore, exploring alternative pathways to induce tumor cell death, particularly in combination, will provide new therapeutic opportunities for these patients. ABTL0812 is a first-in-class oral targeted anticancer compound that produces autophagy-mediated cytotoxicity selectively in cancer cells. It induces endoplasmic reticular stress (ER-stress) and blocks the AKT/mTOR pathway, resulting in cancer cell death. Our preclinical data indicate that ABTL0812 is effective in models of neuroblastoma (NB), even in cells that are resistant to multiple drugs. In NB, ABTL0812 further induces the associated unfolded protein response (UPR), which results in autophagy-dependent apoptosis and decreases the expression of MYCN, a key oncogenic driver of NB. Additionally, our preclinical studies suggest that ABTL0812 can potentiate the antitumor activity of chemotherapies and differentiating agents. In conclusion, ABTL0812 distinctive mechanism of action makes it standout to be used alone or in combination in high-risk neuroblastoma patients. Based on our group's preclinical work, ABTL0812 received orphan drug designation (ODD) by the FDA (15-4893) and EMA (EU/3/15/1485) agencies for neuroblastoma in 2015. ABTL0812 is currently in Phase II clinical development stage for adult tumors (pancreatic cancer) after successfully completing phase I, demonstrating safety and activity. This project arising from our group's preclinical work has the following aims: 1) to conduct a “first- in-child” clinical trial with ABTL0812, 2) to demonstrate the added value of combining ABTL0812 with immunotherapeutics and conventional chemotherapeutics3) to conduct an ambitious translational study to identify pharmacodynamic, predictive, prognostic and radiomic biomarkers. The clinical trial will evaluate ABTL0812 as single agent, in combination with irinotecan-temozolomide chemotherapy and with immunotherapy, i.e. anti-PD1, anti-GD2 or antiangiogenic therapies, to be decided based on preclinical work embedded into this project. The project addresses several objectives of the Strategic Action for Health of this call and priority actions of AES2021 such as development of new drugs for treatment of rare and severe diseases and drugs with ODD. Furthermore, this study is aligned with the neuroblastoma drug development strategy aimed at finding to target MYCN, one of the key drivers of aggressive pediatric tumors such as neuroblastoma. SELECTED PUBLICATIONS (Top 10): 1.- Segura MF, Soriano A, Roma J, Piskareva O, Jiménez C, Boloix A, Fletcher JI, Haber M, Gray JC, Cerdá-Alberich L, Martínez de Las Heras B, Cañete A, Gallego S, Moreno L. Methodological advances in the discovery of novel neuroblastoma therapeutics. Expert Opin Drug Discov. 2022 Feb;17(2):167-179. doi: 10.1080/17460441.2022.2002297. Epub 2021 Nov 22. PMID: 34807782. 2.- Boloix A, Feiner-Gracia N, Köber M, Repetto J, Pascarella R, Soriano A, Masanas M, Segovia N, Vargas-Nadal G, Merlo-Mas J, Danino D, Abutbul-Ionita I, Foradada L, Roma J, Córdoba A, Sala S, de Toledo JS, Gallego S, Veciana J, Albertazzi L, Segura MF*, Ventosa N*. Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics. Small. 2022 Jan;18(3):e2101959. doi: 10.1002/smll.202101959. Epub 2021 Nov 16. PMID: 34786859. (*corresponding authors). 3.- Masanas M, Masiá N, Suárez-Cabrera L, Olivan M, Soriano A, Majem B, Devis-Jauregui L, Burgos-Panadero R, Jiménez C, Rodriguez-Sodupe P, Boloix A, Toledano I, Guillén G, Navarro A, Llobet-Navas D, Villanueva A, Sánchez de Toledo J, Roma J, Noguera R, Moreno L, Krauss R, Gallego S, Santamaria A*, Segura MF*. The oral KIF11 inhibitor 4SC-205 exhibits antitumor activity and potentiates standard and targeted therapies in primary and metastatic neuroblastoma models. Clin Transl Med. 2021 Oct;11(10):e533. doi: 10.1002/ctm2.533. PMID: 34709738; PMCID: PMC8516339. (*corresponding authors). 4.- Muñoz-Guardiola P, Casas J, Megías-Roda E, Solé S, Perez-Montoyo H, Yeste-Velasco M, Erazo T, Diéguez-Martínez N, Espinosa-Gil S, Muñoz-Pinedo C, Yoldi G, Abad JL, Segura MF, Moran T, Romeo M, Bosch-Barrera J, Oaknin A, Alfón J, Domènech C, Fabriàs G, Velasco G, Lizcano JM. The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells. Autophagy. 2021 Jun;17(6):1349-1366. doi: 10.1080/15548627.2020.1761651. Epub 2020 May 25. PMID: 32397857; PMCID: PMC8204958. 5.- París-Coderch L, Soriano A, Jiménez C, Erazo T, Muñoz-Guardiola P, Masanas M, Antonelli R, Boloix A, Alfón J, Pérez-Montoyo H, Yeste-Velasco M, Domènech C, Roma J, Sánchez de Toledo J, Moreno L, Lizcano JM, Gallego S, Segura MF. The antitumour drug ABTL0812 impairs neuroblastoma growth through endoplasmic reticulum stress-mediated autophagy and apoptosis. Cell Death Dis. 2020 Sep 17;11(9):773. doi: 10.1038/s41419-020-02986-w. PMID: 32943619; PMCID: PMC7498451. 6.- Antonelli R, Jiménez C, Riley M, Servidei T, Riccardi R, Soriano A, Roma J, Martínez-Saez E, Martini M, Ruggiero A, Moreno L, Sánchez de Toledo J, Gallego S, Bové J, Hooker JM, Segura MF. CN133, a Novel Brain-Penetrating Histone Deacetylase Inhibitor, Hampers Tumor Growth in Patient-Derived Pediatric Posterior Fossa Ependymoma Models. Cancers (Basel). 2020 Jul 16;12(7):1922. doi: 10.3390/cancers12071922. PMID: 32708733; PMCID: PMC7409080. 7.- Qadeer ZA, Valle-Garcia D, Hasson D, Sun Z, Cook A, Nguyen C, Soriano A, Ma A, Griffiths LM, Zeineldin M, Filipescu D, Jubierre L, Chowdhury A, Deevy O, Chen X, Finkelstein DB, Bahrami A, Stewart E, Federico S, Gallego S, Dekio F, Fowkes M, Meni D, Maris JM, Weiss WA, Roberts SS, Cheung NV, Jin J, Segura MF, Dyer MA, Bernstein E. ATRX In-Frame Fusion Neuroblastoma Is Sensitive to EZH2 Inhibition via Modulation of Neuronal Gene Signatures. Cancer Cell. 2019 Nov 11;36(5):512-527.e9. doi: 10.1016/j.ccell.2019.09.002. Epub 2019 Oct 17. PMID: 31631027; PMCID: PMC6851493. 8.- Soriano A, Masanas M, Boloix A, Masiá N, París-Coderch L, Piskareva O, Jiménez C, Henrich KO, Roma J, Westermann F, Stallings RL, Sábado C, de Toledo JS, Santamaria A, Gallego S, Segura MF. Functional high-throughput screening reveals miR-323a-5p and miR-342-5p as new tumor-suppressive microRNA for neuroblastoma. Cell Mol Life Sci. 2019 Jun;76(11):2231-2243. doi: 10.1007/s00018-019-03041-4. Epub 2019 Feb 15. PMID: 30770954; PMCID: PMC6502783. 9.- Jubierre L, Jiménez C, Rovira E, Soriano A, Sábado C, Gros L, Llort A, Hladun R, Roma J, Toledo JS, Gallego S, Segura MF. Targeting of epigenetic regulators in neuroblastoma. Exp Mol Med. 2018 Apr 27;50(4):51. doi: 10.1038/s12276-018-0077-2. PMID: 29700278; PMCID: PMC5938021. 10.- Jubierre L, Soriano A, Planells-Ferrer L, París-Coderch L, Tenbaum SP, Romero OA, Moubarak RS, Almazán-Moga A, Molist C, Roma J, Navarro S, Noguera R, Sánchez-Céspedes M, Comella JX, Palmer HG, Sánchez de Toledo J, Gallego S, Segura MF. BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways. Oncogene. 2016 Sep 29;35(39):5179-90. doi: 10.1038/onc.2016.50. Epub 2016 Mar 21. PMID: 26996667. ACTIVE COLLABORATIONS WITH INDUSTRY: (Name, country) Nanomol Technologies S.L. (Barcelona, Spain) Ability Pharma S.L. (Barcelona, Spain) 4SC (Münich, Germany) PAST MEMBERS: (Name, actual position) Dra. Luz Jubierre Zapater. Currently postdoctoral researcher at MSKCC, NY, USA. Dra. Laia París-Coderch. Currently working as Validation and GMP Junior Consultant at TDV SL. Dra. Aroa Soriano. Currently leading the Personalized Medicine Program at Vall Hebron Hospital. Dr. Marc Masanas. Currently enrolled in a Master Program.

RESEARCH LINE: RARE ANEMIA DISORDERS Principal Investigator María del Mar Mañú Pereira, PhD   Research Team Amira Idrizovic, MSc, PhD Researcher Valeria Rizzuto, MSc, PhD Researcher Anna Collado Gimbert, MD, Postgraduate Researcher Victoria Gutierrez Valle, MSc, Postgraduate Researcher María de los Ángeles Rodríguez, PhD, Project Manager Claire Diot Lefebvre, MA, Project Manager BACKGROUND Rare anemia disorders (RADs) embrace a highly heterogeneous group of red blood cell and erythropoiesis defects characterized by presenting anemia of variable degree, from mild to life threatening chronic blood-transfusion dependence conditions. RADs include mainly sickle cell disorders (SCD), Thalassemia disorders, Rare constitutional hemolytic anemia due to a red cell membrane anomaly or to an enzyme disorder, Constitutional dyserythropoietic anemia and Constitutional anemia due to iron metabolism disorders. Despite the rapid developments in genetic testing and the subsequent increased knowledge of molecular defects underlying RADs, disease pathophysiology and complex genotype-phenotype correlations are poorly understood and often unexplained. The recent availability of new therapeutic options for RADs makes even more crucial the development of innovative diagnostic strategies for patients’ genetic and phenotypic characterization for development of predictive scores and personalized medicine. Thus, novel diagnostic, exploratory and functional tests are needed in order to identify new disease mechanisms, to discover novel biomarkers, to improve disease classification, and to investigate differences in response to therapy. Genetic and genome-wide association studies have found that two factors influence the SCD clinical expression: the ability of the patient to produce HbF and the co-existence with alpha-thalassemia but, it is unlikely they are the only ones. Through the new techniques of massive sequencing and association studies, genes with potentially effects to the pathogenesis of SCD or modulators of the phenotype disease have become candidates for study. These genetics modulators can be divided in; Primary: responsible of HbS polymerization and RBCs sickling and Secondary: modulators of sub phenotypes and complications of the disease. Predicting the phenotype of SCD in the first months of life, or even in the adult life, would allow a precise prognosis, individualized treatment and avoid unnecessary interventions. However, it is necessary to correlate these new variants with the physiological behavior of the RBCs and certain clinical manifestations allowing the definition of markers that prevent the appearance of acute events or predict the response to treatments. The Lorrca® Maxsis is a unique instrument which combines RBC deformability by ektacytometry, osmoscan, oxygenscan and aggregometry; all temperature controlled. It is capable of fully automated measurement and calculation of various phenomena of RBC’s by analysis of their rheological behavior. The technique accurately detects deformability as a function of shear stress, as a function of oxygen tension (pO2) and aggregation of the RBCs respectively. Additionally, the future of medicine aims to bioengineer devices to mimic human systems to predict efficiently for different patients (personalized medicine) in a fast way. They also may provide new approaches to develop novel dynamic disease models. A step forward in this field concerns "organ-on-chip" technologies. By integrating living cells cultures in microfluidic devices, the most relevant biological and mechanical properties of organic functional units can be reproduced. However, as for other rare diseases, basic, translational and clinical research in RADs is hampered by the existing fragmentation or unavailability of comparable data at the EU level. A European approach for the standardised collection of data regarding the main clinical complications of RADs is fundamental to establish the need and the priorities in the development of research projects, clinical trials, guidelines and health policies that allow the better provision of healthcare to RADs patients. RESEARCH STRATEGY AND SCOPE Our research strategy is focused on the development and validation of innovative methodological approaches for better characterization of patients affected by RADs according to their individual features to allow personal risk profiles and personalized medicine. Specific objectives include: 1) To investigate and / or validate genetic modifiers of RADs both new and previously described by GWAS as markers for prognosis and clinical course based on massive sequencing approaches. 2) To assess the RBC rheological properties by use of ektacytometer LoRRca and the two available modules: Osmoscan and Oxygenscan. The results will provide information of RADs patients risk profile and response to treatment. 3) In collaboration with IBEC, to model the progression of RADs in a spleen-like filtering unit using microfluidic technologies to develop a novel diagnostic device for prognosis and patients’ stratification. This device will be used for the characterization under flow of rheological and mechanical properties of single RBCs. 4) To develop AI-algorithms combining different –OMICs data (genomics, metabolomics) with other laboratory and clinical data for personalized medicine in RADs. In parallel, we are one of the three coordinating hubs of the European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet), established in 2017 to contribute to innovative, efficient and sustainable health systems and facilitate access to better and safer healthcare for EU citizens while decreasing the cross-border health barriers existing for information and patient mobility in rare hematological diseases (RHDs). On the specific area of the epidemiological surveillance, we are coordinating the establishment of the European Rare Blood Disorders Platform (ENROL), conceived in the core of ERN-EuroBloodNet and with the endorsement of European Hematology Association, as an umbrella for both new and already existing registries on RHDs. ENROL aims at avoiding fragmentation of data by promoting the standards for patient registries' interoperability released by the EU RD platform, combining the exhaustiveness of data collection at EU level for health planning and epidemiological porpoises, with a higher level of data granularity for identification of patients’ cohorts. In this context, we are also coordinating the implementation of the Rare Anaemia Disorders European Epidemiological Platform (RADeep), contributing to ENROL on the field of RADs. RADeep was endorsed by ERN-EuroBloodNet for the standardized collection of data of patients affected by any RADs at the European level. RADeep is built in line with the EU RD Platform, and is open to any national registry and medical center willing to actively collaborate as data providers in EU, starting with a pilot in 7 European countries: Belgium, Denmark, France, Germany, Italy, Spain and Sweden. ONGOING COMPETITIVE PROJECTS ERN-EuroBloodNet – CHAFEA - EU4H-2022-ERN-IBA. ‘European Reference Network on Rare Hematological Disorders’. Coordinador: Pierre Fenaux (AP-HP, Paris). PI: María del Mar Mañú Pereira. Duration: March 2022 – September 2023. Summary: The European Reference Network in Rare Hematological Disorders, ERN-EuroBloodNet, was officially established by the European Commission in March 2017 as one of the 24 ERNs in rare disorders. ERN-EuroBloodNet is conceived to contribute to innovative, efficient and sustainable health systems and facilitate access to better and safer healthcare for EU citizens while decreasing the cross-border health barriers existing for information, samples and patient mobility in Rare Hematological Diseases (RHD). www.eurobloodnet.eu GA 964908 — ERICA — H2020-SC1-BHC-2018-2020. ‘European Rare dIsease research Coordination and support Action’. Coordinator: Franz Schaefer (University of Heidelberg, Germany). PI: María del Mar Mañú Pereira. Duration: March 2021 – February 2025 Summary: ERICA consortium aims to build on the strength of the 24 individual European Reference Networks (ERNs) in Rare Diseases and create a platform that integrates all ERN’s research and innovation capacity. Through knowledge sharing, engagement with stakeholders in the rare disease domain and assembly of transdisciplinary research groups working across the global health spectrum ERICA strives to reach the following goals: new intra- and inter-ERN rare disease competitive networks; effective data collection strategies; better patient involvement; enhanced quality and impact of clinical trials; increased awareness of ERN’s innovation potential; Through integration of ERN research activities, outreach to European research infrastructures to synergistically increase impact and innovation ERICA will strengthen the research and innovation capacity of the ERNs. This will result in safe, accessible and efficient access of therapies for the benefit of patients suffering from rare diseases and conditions. GA 101017549 – GENOMED4ALL - H2020-SC1-FA-DTS-2020-1. ‘Genomics and Personalized Medicine for all though Artificial Intelligence in Haematological Diseases’. Coordinator: Federico Álvarez (Universidad Politécnica de Madrid, Spain). PI: María del Mar Mañú Pereira. Duration: January 2021 – December 2024 Summary: GENOMED4ALL will support the pooling of genomic, clinical data and other “-omics” health data through a secure and privacy respectful cross-border data sharing platform based on the novel Federated Learning scheme, to advance research in personalised medicine in haematological diseases thanks to novel AI models. GENOMED4ALL will make use of the existing infrastructures, including powerful High Performance Computing facilities, hospital registries, data processing tools, and pre-existing repositories, starting from 10 clinical partners repositories to be enlarged especially by the resources provided by ERN-EuroBloodNet where GENOMED4ALL clinical partners have a leading position. GENOMED4ALL will demonstrate the potential and benefits of trustable and explainable AI technologies, with a novel approach to AI models and algorithms to exploit the powerful set of "-omics" data which will be at researchers' disposal leading to more reliable and meaningful outcomes for advancing research and personalised medicine, with 3 use cases covering oncological and non-oncological Haematological Diseases, including: Myelodysplastic syndromes, Multiple Myeloma, and Sickle Cell Disease. PI20/01454 – INTEGRA - AES – ISCiii. ‘Enabling personalized medicine of sickle cell disease patients based on integrative diagnosis of new generation methodologies’. PI: María del Mar Mañú Pereira. Duration: January 2021 – December 2023. Summary: Sickle cell disease (SCD) is a rare life threating condition with an increasing health burden in Spain. Hematologic stem cells transplantation is the only curative treatment, usually indicated before the severity of the clinical picture is still unclear; meanwhile gene therapy is still on clinical research. New therapeutic options (i.e Crizanlizumab, Voxelotor) arise a new challenge in SCD; prognosis and personalized medicine become increasingly more important. INTEGRA aims to develop an integrative diagnostic approach based on deep phenotypic and genetic characterization through combining new generation methodologies. Red blood cells sickling behavior will be evaluated by the newly invented Oxygenscan ektacytometry to assess changes in parameters linked to SCD physiological and genetic modulators evaluated through GWAS approach. INTEGRA-SCD will translate into clinical practice the results of genomics, phenotypic characterization and clinical data combined in a single approach resulting on a reliable stratification of SCD patients for personalized medicine. GA947670 – ENROL - CHAFEA — 3HP- HP-PJ-2019. ‘European Rare Blood Disorders Platform’. Coordinator: María del Mar Mañú Pereira. Duration: June 2020 – May 2023. Summary: ENROL has been conceived in the core of ERN-EuroBloodNet as an umbrella for both new and already existing registries on rare hematological disorders (RHDs). ENROL aims at avoiding fragmentation of data by promoting the standards for patient registries’ interoperability released by the EU RD platform. ENROL’s principle is to maximize public benefit from data on RHDs opened-up through the platform with the only restriction needed to guarantee patient rights and confidentiality, in agreement with EU regulations for crossborder sharing of personal data. Accordingly, ENROL will map at the EU level demographics, survival rates, diagnosis methods, genetic information, main clinical manifestations and treatments in order to obtain epidemiological figures and identify trial cohorts for basic and clinical research. Moreover, it will allow promoting research especially for those issues that remain unanswered or sub optimally addressed by the scientific community and promoting clinical trials for new drugs. ENROL will enable the generation of evidence for better healthcare for RHD patients in EU as ultimate goal. GA 860436 – EVIDENCE - H2020-MSCA-ITN-2019. ‘Erythrocytes Properties And Viability In Dependence Of Flow And Extra-Cellular Environment’. Coordinador: Lars Kaestner (Universität des Saarlandes, Germany). Principal Investigator: María del Mar Mañú Pereira. Duration: October 2019 – September 2023. Summary: The objective of EVIDENCE is the exploration of the properties and behaviour of RBCs under flow conditions and in vivo to understand pathophysiology and to design novel diagnostic devices. Theoretical models will help to understand these RBC properties and will enable the transfer of the gained knowledge into diagnostic devises in general and into the development of a spleen-on-the-chip in particular. Furthermore, we aim to understand the effect of the flow in bioreactors, allowing the efficient production of RBCs in vitro with the goal to produce RBC for transfusion. SELECTED PUBLICATIONS Moraleda C, Aguilar R, Quintó L, Nhampossa T, Renom M, Nhabomba A, Ruperez M, Aponte JJ, Achtman AH, Mañú Pereira MDM, Schofield L, Alonso PL, Macete E, Menéndez C. Pathophysiology of Anemia in HIV-Infected Children Exposed to Malaria. Am J Trop Med Hyg. 2021 Jan 18;104(3):1003-1012 Rizzuto V, Mencattini A, Álvarez-González B, Di Giuseppe D, Martinelli E, Beneitez-Pastor D, Mañú-Pereira MDM, Lopez-Martinez MJ, Samitier J. Combining microfluidics with machine learning algorithms for RBC classification in rare hereditary hemolytic anemia. Sci Rep. 2021 Jun 30;11(1):13553. Kountouris P, Stephanou C, Archer N, Bonifazi F, Giannuzzi V, Kuo KHM, Maggio A, Makani J, Mañú-Pereira MDM, Michailidou K, Nkya S, Nnodu OE, Trompeter S, Tshilolo L, Wonkam A, Zilfalil BA, Inusa BPD, Kleanthous M; on behalf of the International Hemoglobinopathy Research Network (INHERENT). The International Hemoglobinopathy Research Network (INHERENT): An international initiative to study the role of genetic modifiers in hemoglobinopathies. Am J Hematol. 2021 Nov 1;96(11):E416-E420. Rizzuto V, Koopmann TT, Blanco-Álvarez A, Tazón-Vega B, Idrizovic A, Díaz de Heredia C, Del Orbe R, Pampliega MV, Velasco P, Beneitez D, Santen GWE, Waisfisz Q, Elting M, Smiers FJW, de Pagter AJ, Kerkhoffs J-LH, Harteveld CL and Mañú-Pereira MdM (2021) Usefulness of NGS for Diagnosis of Dominant Beta-Thalassemia and Unstable Hemoglobinopathies in Five Clinical Cases. Front. Physiol. 2021 Volume 12. Hanny Al-Samkari, M.D, Eduard J. van Beers, M.D., Ph.D, Kevin H.M. Kuo, M.D., Wilma Barcellini, M.D, Paola Bianchi, Ph.D, Andreas Glenthøj, M.D., Ph.D, María del Mar Mañú Pereira, Ph.D., Richard van Wijk, Ph.D., Bertil Glader, M.D., Ph.D, and Rachael F. Grace, M.D. The Manifestations of Disease in Pyruvate Kinase Deficiency: Report from the Pyruvate Kinase Deficiency Burden of Disease International Working Group. Haematologica 2020 Volume 105(9):2229-2239 Huisjes R, Makhro A, Llaudet-Planas E, Hertz L, Petkova-Kirova P, Verhagen LP, Pignatelli S, Rab MA, Schiffelers RM, Seiler E, van Solinge WW, Vives Corrons JL, Mañú-Pereira M, Kaestner L, Bogdanova A, van Wijk R. Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis. Haematologica. 2020 Jan 31;105(2):338-347 Bianchi P, Fermo E, Glader B, Kanno H, Agarwal A, Barcellini W, Eber S, Hoyer JD, Kuter DJ, Maia TM, Mañu-Pereira MDM, Kalfa TA, Pissard S, Segovia JC, van Beers, Gallagher PG, Rees DC, van Wijk R; with the endorsement of EuroBloodNet, the European Reference Network in Rare Hematological Diseases. Addressing the diagnostic gaps in pyruvate kinase deficiency: Consensus recommendations on the diagnosis of pyruvate kinase deficiency. Am J Hematol. 2019 Jan;94(1):149-161. Llaudet-Planas E, Vives-Corrons JL, Rizzuto V, Gómez-Ramírez P, Sevilla Navarro J, Coll Sibina MT, García-Bernal M, Ruiz Llobet A, Badell I, Velasco-Puyó P, Dapena JL, Mañú-Pereira MM. Osmotic gradient ektacytometry: A valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders. Int J Lab Hematol. 2018 Feb;40(1):94-102. Mañú Pereira M, Ropero P, Loureiro C, Vives Corrons JL. Low affinity haemoglobinopathy (Hb Vigo) due to a new mutation of beta globin gene (c200 A>T; Lys>Ile). A cause of rare anaemia misdiagnosis. Am J Hematol. 2017 Jan 9. Maria Garcia-Gomez, Andrea Calabria, Maria Garcia-Bravo, Fabrizio Benedicenti, Penelope Kosinski, Sergio López-Manzaneda, Collin Hill, María del Mar Mañú-Pereira, Miguel A. Martín, Israel Orman, Joan-Lluis Vives-Corrons, Charles Kung, Axel Schambach, Shengfang Jin, Juan A. Bueren, Eugenio Montini, Susana Navarro, Jose C. Segovia. “Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency”. Molecular Therapy 2016 Aug;24(7):1187-98 ACTIVE COLLABORATIONS WITH BIOTECH COMPANIES Agios Pharmaceuticals, Inc (Cambridge, USA) Novartis Pharma AG (Basel, Switzerland) Celgene International II Sàrl (Couvet, Switzerland) Novartis Farmacéutica S.A. (Barcelona, Spain)

Sarcoma Laboratory (Human Team) Head of Sarcoma Laboratory Josep Roma, PhD Head of clinical service and scientific coordinator: Lucas Moreno, MD, PhD Post-doctoral Researchers Gabriel Gallo-Oller, PhD Pre-doctoral Researchers Patricia Zarzosa Natàlia Navarro Guillem Pons Julia Sansa Lorena Valero Technicians Ainara Magdaleno Research strategy and scope Sarcomas originate in cells of mesodermal origin which, in normal conditions, end up forming some of the supporting tissues of the body, such as muscles, bones, tendons, fat, lymphatic vessels, blood vessels, nerves and joints. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, being the third most common extracranial solid tumour in children. This sarcoma accounts for approximately 5% of all malignant tumours in children and adolescents, with an annual incidence of 5.3 cases per million children under the age of 15. Approximately 65% ??of RMS cases are diagnosed in children under 6 years of age, with a lower incidence between 10 and 14 years of age and an increase in the number of cases in adolescents between 15 and 19. Currently, the survival rate of RMS has improved to around 70%. However, patients with metastatic RMS have 5-year disease-free survival as low as 30%. Therefore, mortality rates for RMS are still improvable, particularly in metastatic patients. For this reason, new therapeutic modalities are needed, especially for cases that still fail to respond adequately to conventional treatments and eventually die due to disease progression. Therefore, it is vital to strengthen research in this field, especially for cases refractory to conventional therapies. Our research is focused on finding new molecular therapeutic targets and biomarkers based on knowledge of the biology of paediatric sarcomas. Our group has identified and continues to identify new molecular targets in soft tissue sarcomas (Notch, Hedgehog, miRNAs, pro-metastatic proteins, etc…) and the identification of biomarkers (tumour-specific cfDNAs in liquid biopsies). The experience of the group in recent years has permitted the identification of new potential targets that have entered the phase of drug development. In this stage, we have active collaborations with biotechnological industries in order to develop novel small molecules able to inhibit pro-oncogenic processes such as invasion and proliferation with the aim of providing patients with alternative and more specific biological evidence-based therapies. We are convinced that well-directed research not only has to spawn scientific publications but should also lead to the development of new therapeutic compounds or biomarkers that may directly benefit patients and the launch of new biotechnological products on the market. Main competitive projects granted in the last years: COMRDI15-1-0014 (ACCIÓ/FEDER) - IP: Josep Roma. AMMIC NEXTHEALTH COMMUNITY – Acceleradora de malalties minoritàries de Catalunya. From 2016 to 2019. Amount granted: 281,000€ (Total consortium 2.4M €). Summary: This project consists of a network of high-technology companies and academic research centres, who act as an accelerator in the development of therapeutic solutions from early discovery to clinical stages. This integration of organisations will lead to more effective development of this type of solution and overcome difficulties and barriers of the environment thanks to collaboration, knowledge exchange and sharing of resources. This project is divided into two lines of research: • New target development: Discovery and development of new therapeutic drugs and solutions for the treatment of rare diseases (such as paediatric cancer), from the stages of discovery of new molecules to the early stages of efficacy in the clinical setting. • Personalised medicine and liquid biopsy: The development of genetic and epigenetic analysis tools as well as new screening platforms, which aid the research of the specific DNA alterations to be applied in the clinical setting for the benefit of pediatric cancer patients via selection of the best option for each patient (personalised medicine). This line of research includes also the follow-up of specific cell-free DNAs to evaluate the tumour burden in liquid biopsies with the aim of evaluating response to therapies and predicting relapses. RTC-2017-6037 (Ministerio de ciencia e Innovación) – IP: Josep Roma. Desarrollo pre-clínico regulatorio de nuevos candidatos a fármacos oncológicos From 2018 to 2020. Amount granted: 60,000€. El proyecto PRECLINONCO persigue como principal objetivo seleccionar entre 1 y 3 nuevos candidatos clínicos a fármaco oncológico de entre un grupo de aproximadamente 20 nuevas moléculas que ya han demostrado eficacia in vitro en las indicaciones tumorales sólidas de pulmón, páncreas y riñón en adultos, y en rabdomiosarcoma pediátrico. Para ello, será imprescindible explorar en profundidad su eficacia, toxicidad, farmacocinética, farmacodinamia y manufacturabilidad necesarios para priorizar las moléculas de partida y situar en el estadio preclínico regulatorio al candidato mejor posicionado. CFP Kick cancer (ITCC2017) - IP: Soledad Gallego and Josep Roma. Pilot study to evaluate use of liquid biopsies in clinical and preclinical assessment of rhabdomyosarcoma disease burden. From 2018 to 2020. Amount granted: 180,000€. Summary: There is an unmet clinical need to improve the assessment of disease burden and response to treatment in rhabdomyosarcoma to improve patient management and outcome. This pilot study aims to demonstrate the ability to detect and quantify cell-free tumour DNA in liquid biopsies from RMS patients. For this purpose, tumour-specific mutations and translocation breakpoints are defined for each patient and analysed in blood samples via established and co-ordinated processes to permit patient follow-up. The main two goals of this project are: Quantify circulating cell-free tumour cfDNA levels in plasma and in bone marrow. Results will be compared with clinical outcome and imaging results. Compare results of plasma analyses in treated and untreated RMS mouse models, potentially capitalising on amplified tumour genes for preclinical monitoring. Results are expected to justify sample collection and more extensive assessment in the next international clinical trial for Frontline and Relapse RMS (FaR-RMS) through the European paediatric and Soft tissue Study Group (EpSSG) in partnership with ITCC (Innovative Therapies for Children with Cancer). PI18/00398 (AES ISCIII) - IP: Josep Roma. Innovative strategies for Hedgehog pathway inhibition as therapeutic target in rhabdomyosarcoma. From 2019 to 2021. Amount granted: 87,000€. Summary: In this project we propose the study of some of the components of the Hedgehog pathway as a starting point for developing new molecular-specific therapies, with the strategy of inhibiting ligand binding with its classical Patched receptor and the much less studied Hedgehog co-receptors. The general philosophy of the project is to benefit from the knowledge generated from our previous basic research in order to translate it – with the indispensable help of the biotechnological Industry – into more effective therapies by attacking a pathway (Hedgehog) that plays a central role in rhabdomyosarcoma progression given its capability to activate proliferation and metastasis and, on the other hand, evade apoptosis. The proposed inhibition strategies and drugs are totally innovative, as there are no available therapies directed at these specific targets, which confers on them high potential as a complement to the current therapies. 54/032/201937 (Fundació La Marató de TV3) – IP and coordinator: Josep Roma. Liquid biopsy in pediatric sarcomas: deciphering the predictive potential of circulating tumor DNA and tumor-derived exosomes for early relapse detection. From 2021 to 2023. Amount granted: 300,000€ Summary: Sarcomas constitute a wide family of cancers, including rhabdomyosarcoma (RMS), non-RMS soft-tissue sarcomas, osteosarcoma and Ewing’s sarcoma. Despite their high degree of malignancy and given their high complexity and biological variability, sarcomas have historically been poorly studied compared to the most common cancers. Currently, only assessment by imaging techniques (NMR, PET, etc.) is accepted as sufficient evidence for starting a second-line therapy. The main limitation of these techniques is that they require a relatively large tumour size and knowledge of its location. No molecular methods that could be incorporated into clinical practice to advance the diagnosis of relapses in these diseases are currently available. Being able to do so in the future would be a breakthrough for patients who are not cured with first-line therapy. Thanks to the great progress made in massive sequencing in recent years, infinitesimal amounts of tumour-derived DNA circulating in blood (ctDNA) can be detected, and therefore, the presence of these fragments can be used as very early and sensitive biomarkers to allow very early detection of relapses. The same strategy can be attempted using exosomes, small structures released by cells that can travel through blood. If we are able to detect and measure specifically the exosomes emitted by tumours, we can assess whether a cancer is in progression long before image analysis can do so. Early detection of ctDNA and/or tumour-derived exosomes could trigger alarms some time before, thus allowing intensification of follow-up and even initiating a second-line treatment, with the obvious impact this may have on survival rates in the patients. PI21/00640 (AES ISCIII) - IP: Josep Roma. Development of a new therapeutic target with high anti-oncogenic potential in childhood sarcomas: the pharmacological blockade of the Hedgehog co-receptor CDO. From 2022 to 2024. Amount granted: 120.000€ Summary: Survival rates in paediatric neoplasms have improved considerably in recent decades with an overall survival close to 80% in developed countries. However, some of the most common solid tumors, such as soft tissue or bone sarcomas, still show an adverse prognosis in a high percentage (close to 40%) of patients. Given the particularities of childhood cancers, identifying the molecules responsible for cancer progression may contribute to the advancement of new targeted therapies specially designed for these tumors. The present project stem from previous work by our group on the oncogenicity of the Hedgehog pathway in rhabdomyosarcoma (RMS) and explores one of its most interesting and innovative targets: the CDO co-receptor. After studying this co-receptor in recent years, we have been able to show that it is a target with exceptional anti-oncogenic potential. For this reason, we plan to deepen its study at the molecular level, address structural improvements of the inhibitor compound and assess its possible applicability to the treatment of childhood sarcomas with the generation of several animal models. The philosophy of the project is to start from basic biological knowledge to be able to translate it - with the indispensable help of the Biotechnology Company - into an improvement of future therapies by attacking a pathway that plays a central role in tumor progression in sarcomas, such as it is the Hedgehog pathway. The inhibition strategy proposed here has a high translational potential and is highly innovative, since there are no therapies directed against this specific target, not even in adult cancers. PMP21/00073 (Ministerio de ciencia e innovación/NextGenerationEU - IP: Lucas Moreno. SEHOP-PENCIL study- Personalised medicine for Cancer in Children in Spain. From 2022 to 2026. Amount granted: 1.101.000€ Summary: Childhood cancer remains a challenge for our society with an unacceptable number of children dying from disease recurrence or suffering sequelae from intensive therapy. Incorporating high level Personalised Medicine (PerMed) in standard treatment of childhood cancer offers a unique opportunity to improve survival and reduce morbidities for all children. The PENCIL project by the Spanish Society of Paediatric Haematology and Oncology has four major objectives: 1) To implement a nation-wide sequencing program offering access to next generation sequencing (NGS) panels at the time of diagnosis for high-risk cancers, whole exome/whole-genome sequencing (WES/WGS) and RNASeq at relapse, germline NGS panel or WGS to identify cancer predisposition syndromes and DNA methylation profiling for CNS tumours and sarcomas. 2) To develop tools to facilitate access to PerMed to all patients across Spain, by creating a network of clinicians and genomic hubs and molecular tumour boards. 3) To evaluate the implementation of PerMed into routine care for childhood cancer including cost-effectiveness and clinical outcomes. 4) To develop novel technologies that will overcome current limitations of diagnostic and surveillance approaches, such as liquid biopsy. The incorporation of PerMed into routine care will lead to improved diagnostic and prognostic information at diagnosis, increased access to novel targeted therapies at relapse and early identification and intervention on cancer predisposition syndromes for patients in all autonomous regions of Spain, resulting in major benefits for our society. 10 selected publications on Paediatric Sarcomas: 1 - Dickkopf-1 Inhibition Reactivates Wnt/â-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo. Giralt I, Gallo-Oller G, Navarro N, Zarzosa P, Pons G, Magdaleno A, Segura MF, Sábado C, Hladun R, Arango D, Sánchez de Toledo J, Moreno L, Gallego S, Roma J. Int J Mol Sci. 2021;22(23):12921. PMID: 34884726. 2 - Dickkopf Proteins and Their Role in Cancer: A Family of Wnt Antagonists with a Dual Role. Giralt I, Gallo-Oller G, Navarro N, Zarzosa P, Pons G, Magdaleno A, Segura MF, Sánchez de Toledo J, Moreno L, Gallego S, Roma J. Pharmaceuticals. 2021;14(8):810. PMID: 34451907. 3 - Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance. Alcon C, Manzano-Muñoz A, Prada E, Mora J, Soriano A, Guillén G, Gallego S, Roma J, Samitier J, Villanueva A, Montero J. Cell Death Dis. 2020;15;11(8):634. PMID: 32801295. 4 - miRNA-7 and miRNA-324-5p regulate alpha9-Integrin expression and exert anti-oncogenic effects in rhabdomyosarcoma. Molist C, Navarro N, Giralt I, Zarzosa P, Gallo-Oller G, Pons G, Magdaleno A, Moreno L, Guillén G, Hladun R, Garrido M, Soriano A, Segura MF, Sánchez de Toledo J, Gallego S, Roma J. Cancer Lett. 2020;477:49-59. PMID: 32142919. 5 - Clonal dynamics in osteosarcoma defined by RGB marking. Gambera S, Abarrategi A, González-Camacho F, Morales-Molina Á, Roma J, Alfranca A, García-Castro J. Nat Commun. 2018;9(1):3994. PMID: 30266933. 6 - Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma: the oncogenic role of the ligands. Almazán-Moga A, Zarzosa P, Molist C, Velasco P, Pyczek J, Simon-Keller K, Giralt I, Vidal I, Navarro N, Segura MF, Soriano A, Navarro S, Tirado OM, Ferreres JC, Santamaria A, Rota R, Hahn H, Sánchez de Toledo J, Roma J, Gallego S. Br J Cancer. 2017;117(9):1314-1325. PMID: 28881358. 7 - Hedgehog pathway inhibition hampers sphere and holoclone formation in rhabdomyosarcoma. Almazán-Moga A, Zarzosa P, Vidal I, Molist C, Giralt I, Navarro N, Soriano A, Segura MF, Alfranca A, Garcia-Castro J, Sánchez de Toledo J, Roma J, Gallego S. Stem Cells Int. 2017;2017:7507380. PMID: 28243259. 8 - Embryonic Signaling Pathways as Potential Targets for the Treatment of Rhabdomyosarcoma (Editorial Thematic Issue). Gallego S, Roma J. Curr Drug Targets. 2016. Jul 29;17(11):1226-7. PMID: 26947582. 9 - Notch-mediated induction of N-cadherin and á9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells. Masià A, Almazán-Moga A, Velasco P, Reventós J, Torán N, Sánchez de Toledo J, Roma J, Gallego S. Br J Cancer. 2012;107(8):1374-83. PMID: 22976797. 10 - Notch pathway inhibition significantly reduces rhabdomyosarcoma invasiveness and mobility in vitro. Roma J, Masià A, Reventós J, Sánchez de Toledo J, Gallego S. Clin Cancer Res. 2011;17(3):505-13. PMID: 21177409. Active collaborations with Biotech companies: BCN peptides – Development of anti-metastatic compounds. Leitat/Selabtec – Development of anti-cancer compounds. QGenomics – Liquid biopsy development for detection of cfDNA in sarcomas. Oncoheroes Biosciences – Development of anti-cancer compounds. Flomics – Liquid biopsy for histiocytosis follow up.