(P 232) DECIPHERING METASTASIS MAINTENANCE AND CHEMO RESISTANCE EVENTS IN CIC-DUX4 SARCOMA

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 is driven by Capicua (CIC) gene rearrangements, while these tumors are similar in appearance to Ewing family tumors (EFTs), their biology and clinical course are different. The most common rearrangements of CIC involve a DNA fusion with NUTM1, FOXO4, and DUX4. CIC-DUX4 is the most common fusion in this type of sarcoma and is the result of a translocation between chromosomes 4 and 19, t(4;19) (q35;q13) or less commonly 10 and 19, t(10;19) (q26.3;q13). 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, to 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 is monitored using bioluminescence imaging, primary tumors, relapses, and metastasis will be harvested, followed by next-generation sequencing molecular profiling.
To discover drivers of chemoresistance 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 four cycles of chemotherapy treatment significantly improved the overall survival of the mice.
Engineering of CIC-DUX4 cells with gain of function (GOF) transposons, increased the rate of metastatic dissemination to 45% and we observed metastases in the lungs and/or liver. Metastases in the lymph nodes were also observed in 25% of the cases. Transcriptomic profiling of primary vs relapsed tumors showed 243 genes significantly differentially expressed in VDC-IE group in comparison to the vehicle group, with 137 genes upregulated and 106 downregulated. Principal components analysis identified two distinct clusters in the data which indicates that the chemotherapy has induced enough selective pressure on tumor cells, to either select the resistant subpopulation in the primary tumor or make the cells change their expression to cope with the pressure.

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 primary and relapsed CIC-DUX4 sarcomas are different, and 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.