(P 434) MECHANISMS OF CHEMORESISTANCE IN CIC-DUX4 SARCOMA AS DETERMINED BY IN VIVO FUNCTIONAL GENOMICS

Objective: Sarcomas are malignant tumors of mesenchymal origin. Although considered rare, they affect many children and young adults accounting for 20% of pediatric malignant neoplasms. A recently described fusion-driven sarcoma, Capicua (CIC) rearranged sarcoma, is similar in appearance to Ewing family tumors (EFTs) but their biology and clinical course are different. CIC-DUX4 is the most common fusion in this type of sarcoma. Because of their previous classification as EFTs and their rare incidence, studies on these tumors have been sparse and the molecular pathways that cooperate with CIC-DUX4 in tumorigenesis, therapy failure, and metastatic dissemination are poorly described.

Methods: To understand which genes and pathways cooperate with CIC-DUX4, we have applied a high-throughput functional genomics screening, based on random insertional mutagenesis with transposons, in two cell lines with CIC-DUX4 sarcoma. In our system, engineered transposons are delivered to the target cells by lentiviral transduction and mobilized to create gain-of-function and loss-of-function mutations by the action of an inducible transposase. Engineered CIC-DUX4 tumor cells are implanted in immunocompromised mice, subcutaneously and orthotopically. Tumor progression and metastasis are monitored using bioluminescence imaging, primary tumors, relapses, and metastasis will be harvested, followed by next-generation sequencing molecular profiling. To discover drivers of chemo resistance in this context, we developed a fractionated high-dose chemotherapy protocol to use on engineered CIC-DUX4 implanted mice. This regimen is based on the same genotoxic chemotherapy that is currently in use for CIC-DUX4 patients: VDC-IE (vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide).

Results: Two different breakpoints for the CIC-DUX4 fusions were detected in the two cell lines and the fusion transcripts have been confirmed. The cells were not metastatic or low metastatic at baseline, and three cycles of chemotherapy treatment significantly improved the overall survival of the mice. Our human cell based, functional genomics model with transposons resulted in CIC-DUX4 tumors with a significantly higher proclivity to metastasize. For instance, the metastatic rate went from 10 to 60% metastases to the lungs or liver in the mice that received three cycles of chemotherapy. Transcriptomic profiling of the resistant tumors showed enrichment in pathways regulating pluripotency such as Wnt, TGF-b, and Notch. Increased representation of cancer stem cell markers such as CD44, ALDH1A1, and Sox2 was a significant feature of tumors chemotherapy-resistant tumors.

Conclusion: Functional genomics can be used in vivo in rare cancers to model cancer evolution during therapy. A somatic mutagenesis-based approach to model recurrent CIC-DUX4 tumors showed that genetic driver events support the evolution of CIC-DUX4 tumors to a more aggressive phenotype under chemotherapy-induced selective pressure. The genetic basis of this phenotype is being mined to identify actionable candidates to prevent or treat CIC-DUX4 sarcoma progression.