Periodic Reporting for period 3 - PedSarc (Targeting genetic and epigenetic mechanisms in pediatric sarcomas.) Reporting period: 2022-01-01 to 2023-06-30 Summary of the context and overall objectives of the project Sarcomas are mesenchymal tumors arising from the bone or soft tissues and are relatively more common in the pediatric age group being the cause of 13% of cancer related deaths in patients 0–19 years of age. They currently encompass many separate morphologic entities, with many being rare; therefore they pose a challenge to clinicians both in terms of classifications and therapeutic strategies. Gene fusions resulting from chromosomal translocations and epigenetic de-regulation are hallmarks of several pediatric sarcomas, however, their impact on tumor development and cancer therapy is currently understudied due to the lack of adequate model systems. As a consequence, the treatment of soft tissue sarcomas affecting children and young adults remains a challenge. My laboratory aims to overcome these issues by combining state-of-the-art functional genomics tools and new technologies to model cancer in mice. This proposal is comprehensively studying the molecular function of sarcoma-related gene fusions, their downstream targets and associated cancer dependencies, as well as generating versatile mouse models to study them in vivo. The results obtained here will help to better understand epigenetic de-regulation in the pathogenesis of pediatric sarcomas and identify novel vulnerabilities in these diseases, which could be exploited to develop more specific therapies. Although we use in this proposal sarcomas as a model system, we anticipate that our findings could also have implications in other cancer entities, and may more broadly help to understand fundamental aspects of epigenetic de-regulation in cancer and developmental diseases. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Since the project started we have worked on characterizing the oncogenic activity of SS18-SSX, the oncoprotein driving synovial sarcoma. We first identified the most important domains in the oncofusion and characterized the proteins able to interact with the SSX portion. This work revealed how SS18-SSX is able to bind chromatin and therefore regulate specific genes. In addition, we used CRISPR/Cas9 screens to identify signaling pathways important in synovial sarcoma. These results have been validated and will be further explored. Given the role of a class of epigenetic regulators – the polycomb repressive complex 1 (PRC1) - in cancer and development, we also studied the effect of PRC1 mutations in sarcoma. Here, and given the lack of suitable models to study tumors driven by these alterations, we took advantage of genome engineering tools and our recently developed mouse models to address the impact of these mutations in mesenchymal cells. Finally, we established a new mouse model based on muscle electroporation of transposon vectors for stable delivery of different gene fusions and other genetic alterations to study sarcoma development and future use for pre-clinical analyses. This method was successfully established and tested in some of the most commonly found genes fusions in pediatric sarcomas. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our studies have already identified new mechanisms of function of sarcoma gene fusions which provide progress beyond the state of the art. In particular we have identified interactors of SS18-SSX that mediate its localization as well as new signaling pathways that promote synovial sarcoma. We further expanded our knowledge on the role of epigenetic de-regulation by polycomb repressive complexes in synovial sarcoma and in other sarcoma types. Importantly, we were able to establish an efficient and flexible mouse model platform to study the role of gene fusions and other sarcoma-related genetic alterations. These models will allow to study sarcoma biology but also perform pre-clinical studies in an immunocompetent background. Therefore they will fill a gap in sarcoma research that is the lack of models systems that reflect the diverse and heterogeneous nature of sarcoma subtypes. Until the end of the project we will study in depth specific dependencies identified in our screens, further understand the molecular regulation by polycomb complexes in sarcoma and apply the sarcoma mouse models to study new identified dependencies with potential clinical translation. General overview of PedSarc main aims
