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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - EwiSarc (A novel in vivo platform to study and target undruggable Ewing onco-chimera.)

Teaser

Summary

Cancer represents the second major cause of death and morbidity after cardiovascular diseases, with more than 3 Million new cases and 1.7 Million deaths each year (WHO data). While most of the new cancer diagnosis is made in adults, childhood cancers make up less than 1% of all cases diagnosed every year, but the rate has been rising slightly for the past few decades (American Cancer Society). Cancer in children is relatively rare, yet it is the leading cause of death by disease in developed countries. About 3% of childhood cancers are represented by primary bone tumors, of which 2 main types have been described: Osteosarcoma and Ewing sarcoma. While osteosarcoma is the most common type in teens, Ewing sarcoma represents 1% of all childhood cancers (incidence approximately 3 cases/million/year). However, given its aggressiveness, while the current 5-years survival rate for young patients with localized ES is about 70%, this prognosis value drops to 15-30% for patients with metastatic tumors at diagnosis. Chemotherapy and radiotherapy have drastically reduced the lethality of leukemia and lymphoma, however, they have only slightly improved sarcomas outcome in the past 30 years, and Ewing sarcoma treatment is still only based on the use of generic chemo agents such as doxorubicin, vincristine, cyclophosphamide, and dactinomycin.
In many different forms of sarcoma, the initial oncogenic event is a balanced chromosomal translocation originating a chimeric oncoprotein. Even though much is known regarding the oncogenic functions of different chimeras, the success rate at which this advanced knowledge has been translated into effective therapies is pitifully low. Chimeric oncoproteins are exclusive hallmarks of tumor cells and should represent the most valuable therapeutic target to identify novel precision\'s medicine approaches to flank and support the current chemotherapy protocols against Ewing sarcoma.
The identification of novel drugs through preclinical studies is however heavily influenced by the model enrolled in the studies. Faithfull preclinical models able to recapitulate most of the biological characteristics of the human disease should always be employed to improve the efficiency of the preclinical study and to decrease the percentage of subsequently failing clinical tests. Currently, however, no faithful preclinical models of Ewing Sarcoma are available. Therefore, main objectives of this study will be the development of reliable preclinical models able to faithfully recapitulate Ewing sarcomagenetic process to study the pathobiology basis of the disease (Work Package 1) and the identification of molecular mechanisms whose pharmacological tuning will tear down Ewing sarcoma lethality (Work Package 2).
Thanks to the work carried out in this project, our understanding of Ewing sarcomaâ€™s pathobiology has further improved. Indeed, while previous scientific knowledge suggested that Ewing sarcoma development is driven uniquely by specific chromosomal translocations, results from this projects strongly support the idea that Ewing oncochimeras are not sufficient to induce cell transformation, and other currently unknown events are required to unleash their full transforming potential.

Work performed

The project started in May 2017. Extensive initial work has been performed to establish/optimize all the protocols necessary to achieve our objectives, first of all the development of faithful preclinical models of Ewing sarcoma. Initially, we developed the tools necessary to induce the expression of the oncoproteins responsible for Ewing sarcoma development. Thus, extensive molecular biology and virology experiments were performed to develop viral particles at high purity and concentration and able to efficiently integrate into the genome of mesenchymal stem cells inducing the expression of Ewing oncoproteins. In addition to the generation of such viral tools, fundamental for the development of preclinical models was the identification, isolation and culture of the mesenchymal stem cells, which are considered to be the cells of origin of the disease. However, classical methods to isolate mesenchymal stem cells lead to the expansion of highly heterogeneous cell cultures, which unlikely represent the cells of origin of Ewing sarcoma. Therefore, I have spent several months trying to establish and optimize in the laboratory the best protocols to identify, isolate and functionally characterize a highly pure and homogeneous bone-derived mesenchymal stem cell culture.
To induce the expression of Ewing oncoproteins in the isolated mesenchymal stem cells, we have transduced the latter with the viral particles described above. However, integration of the viral genome in the host genome, and consequent expression of the oncoproteins, caused the death of the cells. This unpredicted result suggests that the expression of Ewing oncoproteins is itself toxic for the hosting mesenchymal stem cells and not sufficient to transform them in tumor cells, and that other currently unknown mechanisms are required to induce full cell transformation. Therefore, given our results, we temporarily focused our project on the identification of those molecular events/altered pathways that are required to support Ewing oncochimeras promoting the sarcomagenetic process. Given the massive establishment and optimization work required to develop the preclinical models of Ewing sarcoma and the unforeseen obtained results, two years project were unfortunately not sufficient to complete all the estimated objectives. However, I will continue to work with the Host laboratory on the project towards the overall objectives.
Equally important, this fellowship allowed me to resume my scientific career after two maternity leaves, and to acquire technical skills and scientific knowledge in oncology field that will be of fundamental importance to progress towards more independent positions.
Several outreach activities allowed me also to illustrate and explain the project to the non-scientific community. I have indeed participated to â€œla Notte del Ricercatoreâ€ at the Museum of Science of Trento (MUSE), to the TYSC (Trentino Young Scientist Challenge) initiative, organized for middle school students, and an article has been published on the local magazine Lâ€™Adige7 on my research activities.

Final results

The results obtained within the framework of this project belong to the basic/translational research field, and represent a first step towards the potential development of more efficient therapeutic protocols to be used in the future in the clinic.
Ewing Sarcoma is an aggressive type of bone cancer with poor prognosis, and current therapeutic protocols only rely on the use of generic chemo-agents. To develop novel and precise therapeutic strategies, preclinical screenings need to be performed on reliable models of the disease, which are currently missing. Therefore, thanks to this project we can develop faithful preclinical models of Ewing Sarcoma which can be used by the scientific community not only to better understand the biology of the disease, but also to identify novel promising therapeutic strategies which could both potentiate the efficacy of current clinical protocols and decrease the morbidity associated with the aggressive chemotherapy regimen currently used.