(P 265) SELECTIVE TARGETING OF BET FAMILY EPIGENETIC REGULATORS IN ADVANCED AND METASTATIC ALVEOLAR RHABDOMYOSARCOMA

Objective: Standard of care drugs for rhabdomyosarcoma (RMS), such as vincristine and doxorubicin, have a high systemic toxicity and >70% of high-risk patients develop resistance. Epigenetic modulation has shown promise in predicting fusion-positive (FP) alveolar RMS (ARMS) outcomes. Therefore, our studies focus on this subtype and BET inhibition (BETi) as a potential therapy. Additionally, we are working on metastatic ARMS mouse models, which better represent high-grade disease compared to primary xenografts. Our goal is to determine what effect BETi has on localized and metastatic ARMS, and whether BETi could be a viable treatment option for these patients.

Methods: ARMS cell lines (RH4, RH30) were treated with BETi compounds (BMS-986158, PLX51107) using a 12-point serial dilution (2.6 nM-10 M). Half maximal inhibitory concentration (IC50) values were calculated using a sigmoidal curve fit with three parameters following ATPlite viability assay. Caspase-3 activation assay was used to evaluate apoptosis via flow cytometry. The downstream impact of BETi on MYC expression was analyzed using qPCR, and on-target effect was confirmed through IncuCyte live cell imaging after siRNA knockdowns of BETi target proteins BRD2/BRD3/BRD4 were created in the ARMS cell lines. To establish metastatic models, NSG mice were injected with ARMS (RH4, RH30) cells and after tumor growth, amputation was performed on the tumor-bearing hindlimb. Mice were monitored for systemic illness and palpable metastatic or recurrent tumor growth until reaching endpoint, followed by euthanasia and necropsy to assess for metastases. Metastases were stored as frozen sections for transcriptome analysis and in formalin for immunohistochemistry staining. Histological characterization of metastatic tumors was performed by sarcoma pathologist Dr. Brendan Dickson, and PAX3-FOXO1 (P3F) fusion was assessed using RT-PCR. Metastatic tumor-derived cells were tested for BETi sensitivity using a 12-point serial dilution (2.6 nM-10 M) followed by ATPlite assay.

Results: BETi reduced ARMS proliferation and induced apoptosis in vitro. The oncogenic driver MYC is a known downstream transcriptional target of BET proteins. MYC mRNA levels significantly decreased in ARMS cells treated with BETi, confirming dose efficacy. Depletion of BRD4 hindered proliferation, ensuring target specificity of the compounds (Fig. 1). Metastatic models exhibited metastasis in sites relevant to human disease (liver, lymph nodes, lungs, retroperitoneum) (Fig. 2). Metastatic and primary tumors shared RMS histopathological features. To ensure that the ARMS metastatic tumors reflected the molecular characteristics of the original P3F FP cells, we evaluated the P3F fusion status of the tumors and found that the fusion was preserved, confirming the origin of the metastases (Fig. 3). BETi sensitivity of metastasis-derived cell lines (n=3) was comparable to the original cell lines. This suggests that metastatic RMS might respond well to BETi treatment, indicating the need for further in vivo studies using BETi in our metastatic models.

Conclusion: Functional validation confirmed BETi’s potential for in vivo ARMS studies. Ongoing clinical trials of the primary drug candidate, BMS-986158, in childhood cancers increases the likelihood of clinical trial opportunities in RMS. Our metastatic models demonstrate clinical relevance and BETi sensitivity, crucial for addressing the lack of established metastatic ARMS studies. Next, we will compare transcriptomes of metastatic tumors and primary xenografts. With the help of our collaborators, we aim to cluster metastatic tumors with large-scale patient data, potentially informing patient study stratification. Ultimately, our in vivo studies will assess BETi’s impact on preventing metastasis and reducing metastatic burden.