Abstract A063 Selective targeting of BET family epigenetic regulators in metastatic alveolar rhabdomyosarcoma

Abstract Objective: Alveolar rhabdomyosarcoma (ARMS) is a soft tissue cancer driven by a PAX3/7-FOXO1 (P3F) fusion and is the most aggressive pediatric RMS subtype. P3F transcriptional activity is dependent on the bromodomain-containing protein 4 (BRD4) epigenetic protein. Given that >70% of high-risk patients develop resistance to chemotherapy, we focus on novel BET-inhibitors (BETi) as a therapeutic option for metastatic ARMS. Methods: To establish metastatic models of the axilla/lymph node (AX), liver (LV) and retroperitoneum (RP), NSG mice (n=10) were injected with human ARMS cells expressing luciferase (Luc) and the tumor-bearing hindlimb was amputated. Metastases (mets) were detected using bioluminescent imaging (BLI) followed by necropsy. Histology was evaluated by pathologist Dr. Brendan Dickson, and P3F fusion was assessed using RT-PCR. The parent cell lines and cells derived from metastatic models (n=3; LV, AX, RP) were treated with BETi (n=2) using a 12-point concentration (2.6nM-10µM). Half maximal inhibitory concentrations (IC50) were calculated after an ATPlite assay. Caspase-3 apoptotic marker was assessed via flow cytometry. On-target efficacy was confirmed with IncuCyte after siRNA-depletion of BETi targets BRD2/3/4. ARMS cells expressing Luc controlled by a constitutively active or P3F dependent reporter were treated with BETi (1nM-1µM) and relative Luc activity was detected. An ex vivo pulmonary lung metastasis assay (PuMA) was established by injecting GFP expressing ARMS cells into the mouse tail vein (n=4), followed by euthanasia. The lungs were insufflated with an agarose mix and allowed to solidify. Lung slices were placed on gelatin sponges and incubated in media. PuMA sections were treated with IC50 values of BETi and lung metastatic burden was detected over time with confocal fluorescent microscopy and analyzed via ImageJ. Results: Mets in surgical models occurred at sites relevant to human disease (AX, LV, RP). AX mets, one of the most aggressive sites in humans, occurred in 90% of models. Mets displayed ARMS histopathology and the P3F fusion present in parent cells was preserved. BLI detected AX mets with high sensitivity ∼1.5 weeks prior to palpability. The in vitro IC50 of BETi compounds was in the micromolar range in both the parent and met-derived cell lines, and increased apoptosis >3-fold compared to vehicle. Depletion of BRD4 hindered cell proliferation, ensuring target specificity of the BETi. BETi selectively suppressed the activity of the P3F dependent reporter, indicating an impairment of P3F activity. Given that lungs are the most common metastatic site, we developed an ARMS PuMA model which showed significant increase in metastatic burden over time (p<0.01). Treatment of PuMA sections with IC50 doses of BETi reduced lung metastatic burden (p<0.01). Conclusion: Our metastatic models demonstrate clinical relevance and BETi sensitivity. Next, we aim to compare the transcriptomes of these models with patient data and test the BETi compounds in our surgical models in vivo. Citation Format: Anna Mandel, Yael Babichev, Claire Wunker, Jennifer Dorsey, Abha A. Gupta, Elizabeth Demicco, Richard Marcellus, Brendan Dickson, Rebecca Gladdy. Selective targeting of BET family epigenetic regulators in metastatic alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A063.