(P 190) SELECTIVELY TARGETING THE EPIGENOME IN EMBRYONAL RHABDOMYOSARCOMA

Objective: Rhabdomyosarcoma (RMS) is a tumor originating from the skeletal muscle lineage and is the most common type of soft tissue sarcoma in children and adolescents. Current treatment includes chemotherapy, radiation therapy, and/or surgery. Vincristine, the primary standard of care drug, has high systemic toxicity and >70% of high-risk RMS patients develop resistance to this therapy over time. Epigenetic modulation has been recently described as a powerful predictor of outcome in RMS. Targetable epigenetic factors include bromodomain and extra-terminal domain (BET) and histone deacetylase (HDAC) family proteins. Thus, the goal of this study is to explore the molecular mechanisms behind promising epigenetic inhibitors in RMS, focusing on embryonal RMS, and to test these drugs in vivo in combination with vincristine.

Methods: Our laboratory obtained a selection of epigenetic compounds from the Ontario Institute of Cancer Research (OICR) and Structural Genomics Consortium (SGC) that mainly target BET (BMS-986158, I-BET151, PLX51107) or HDAC (Apicidin, Domatinostat, Fimepinostat) proteins. We conducted a two-step hit compound validation with EC50 curves using ATPlite cell viability assay to confirm the sensitivity of a panel of human ERMS cell lines (RD, RMS-YM, RMS559) and a murine ERMS tumor-derived cell line (M25-FV24c) to the inhibitors (Figure 1). The effect of the compounds on apoptosis was assessed by PARP cleavage and caspase-3 activation assays using immunoblot and flow cytometry. To investigate endogenous target expression and the mechanism of action of the inhibitors we performed immunoblots and qPCR. To investigate the kinetics of the compounds, we used IncuCyte live cell imaging.

Results: Selected BETi and HDACi compounds decreased cell proliferation in ERMS in vitro. The cells were treated with the epigenetic inhibitors using a 12-point serial dilution (2.6 nM-10 uM) and EC50 values were calculated using a sigmoidal curve fit with three parameters. Endogenous protein expression assay showed that both BRD4 and HDAC1, the respective primary targets of these compounds, were overexpressed in RD and RMS-YM when compared to control myoblasts (AB1190) and the overexpression of BRD4 was significant in both cell lines used in the assay (RD, p=0.002; RMS-YM, p=0.007) (Figure 2). Treatment with the EC50 concentrations of the BETi and HDACi compounds induced apoptosis in RD and RMS-YM, with the BETi compounds inducing a higher level of apoptosis compared to HDACi. When comparing the BETi compounds, PLX51107 induced the highest level of apoptosis in the MYC-amplified RD cell line. Furthermore, the level of apoptosis in this cell line was higher after treatment with PLX51107 than after treatment with the positive control, doxorubicin (Figure 3). Doxorubicin was used as positive control as vincristine failed to induce apoptosis in these cell lines. Finally, the kinetics study suggested that the BETi compounds inhibited cell proliferation early on within 24 hours, which will be important to consider for future studies and designing in vivo experiments (Figure 4).

Conclusion: Our data suggest that BET inhibitors may exploit an epigenetic weakness of ERMS, perhaps related to MYC amplification. In the future, we plan to conduct transcriptome assays to explore the mechanism of action of BET inhibitors in ERMS, and in vivo drug combination studies in an ERMS mosaic mouse model and patient-derived xenografts (PDXs).

Figure 1.pdf

Figure 2.pdf

Figure 3.pdf

Figure 4.pdf