(P 303) INVESTIGATING THE THERAPEUTIC POTENTIAL OF TRIPTOLIDE PRODRUG, MINNELIDE, AS A TREATMENT ALTERNATIVE FOR CIC-DUX4 SARCOMA

Objective: CIC::DUX4 sarcoma (CDS) is driven by a t(4;19) or t(10;19) chromosomal translocation and is the most common genetic abnormality detected in EWSR1 fusion-negative small round blue cell tumors. Until recently, CDS was generalized as “Ewing-like sarcoma” due to having partial morphological overlap and similar immunobiological features to Ewing sarcoma. However, unlike Ewing sarcoma, CDS 1) rarely arises in the bone, 2) lacks the Ewing-defining ETS gene fusion, and 3) is associated with a more aggressive clinical course. Although the World Health Organization recently classified CDS as its own entity, patients are routinely treated with Ewing-based chemotherapies which are largely ineffective resulting in high rates of metastasis and poor clinical outcomes. In this study, we evaluated the therapeutic efficacy of 160 epigenetic compounds on human CDS cells, with Triptolide emerging as a top candidate. Minnelide, the water-soluble prodrug of Triptolide, is currently being evaluated in phase I/II clinical trials for adenosquamous carcinoma of the pancreas, gastric cancer, breast cancer, and non-small cell carcinoma.

Methods: We conducted a viability drug screen on human CDS cells (Kitra-SRS, NCC-CDS2, NCC-CDS-X1C1) using the Tocriscreen Epigenetics 3.0 compound library. In technical triplicate, cells were incubated in drug at four different concentrations (1, 10, 100, and 1000nM) for 72 hours and then assayed using Cell Titer Glo luminescence to asses cell viability. The top hit, Triptolide, was subsequently validated using a water-soluble prodrug called Minnelide which replicated the screen results. To investigate mechanism of action, cells were treated for 72hrs at IC50 (5nM) and processed for RNA-sequencing. Finally, we modeled CDS by generating subcutaneous xenograft tumors in immunodeficient NOD-scid IL2Rgammanull and NU/J mice. In total, 8 mice were transplanted with CDS tumor derived Kitra-SRS cells resuspended in a media/Matrigel mixture. After tumor masses were detected at the site of injection, the animals were treated with Minnelide (or vehicle control) once daily by intraperitoneal injection for 20 consecutive days. The drug dose (0.42mg/kg) was selected based on bioavailability data and maximum tolerability observed in previous pre-clinical studies. Tumor growth was measured weekly using digital calipers and the animals were monitored closely for adverse effects. At 20 days, or pre-determined humane endpoint, the animals were euthanized, and tumors were collected for additional studies.

Results: The Triptolide prodrug, Minnelide, demonstrated potent antitumor activity both in vitro and in vivo. Minnelide treatment decreased CDS cell viability, and significantly reduced the size of CIC::DUX4 sarcoma xenografts within 20 days. The normalized tumor volume at completion of the study was used as criterion of efficacy. The average normalized tumor volume of the Minnelide treatment group was significantly (two-way ANOVA, P= 0.0284) different than the average normalized tumor volume of the control group at day 20 post treatment initiation. Moreover, in our hands, drug treatment was not associated with any adverse side effects. RNA sequencing of CDS cells treated with Minnelide revealed many CIC::DUX4 target genes such as ETV1/4/5, DUSP6 and SPRED3 are downregulated. These results suggest Minnelide alters the CDS transcriptional program and could represent an efficacious treatment alternative.

Conclusion: To date, therapeutic advancements for CIC::DUX4 sarcoma have been limited. Utilizing a high-throughput approach, we screened the effects of 160 epigenetic compounds on human CDS cells and identified triptolide as a top candidate. Our studies demonstrate that treatment with the Triptolide prodrug, Minnelide, significantly reduces CDS cell viability and CDS xenograft growth. Additionally, Minnelide treatment appears to downregulate many CIC::DUX4 target genes. Given that the CIC::DUX4 oncoprotein acts as a potent transcriptional activator, it is hypothesized that malignant cell transformation occurs through the aberrant activation of these target genes. Minnelide likely disrupts this activation, presenting a promising alternative for efficacious treatment.