Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas and is a highly aggressive, malignant solid tumor that primarily affects children and young adults. RMS is thought to arise as a consequence of regulatory disruption of the differentiation program of the skeletal muscle cells. Current knowledge of the molecular mechanisms responsible for this disruption in RMS tumors, however, is limited. The most aggressive form of this muscle cancer is alveolar rhabdomyosarcoma (ARMS), which has a poor prognosis and a high frequency of metastasis. Current aggressive chemotherapeutic approaches have improved the outcome of ARMS treatment; however, the cure rate for metastatic ARMS is still only 20% to 30%. Previously, our laboratory has reported that histone methyltransferase Suv39h (mouse homologue of human SUV39H)-mediated epigenetic mechanism controls the growth and differentiation of murine skeletal muscle progenitor cells. We demonstrated that Suv39h blocks MyoD, which acts as a key transcriptional regulator of the muscle differentiation program. In our present study, we have found increased expression of SUV39H in ARMS cells when they are cultured under differentiation-permissible conditions. Moreover, SUV39H-depleted ARMS cells showed MyoD-mediated transcriptional activation, MyoD-dependent growth arrest, reduced anchorage-independent growth, replacement of a repressive mark with an active mark on the muscle-specific gene promoter, and induction of differentiation-associated gene expression. These results suggest that SUV39H overexpression blocks myogenic differentiation program of ARMS cells. Altogether, our results from the current study indicate that SUV39H negatively regulates MyoD in ARMS cells in the failure of muscle differentiation.
Based on these results on ARMS cells, we aimed to isolate the pharmacological compound(s) that target the SUV39H-associated mechanism and restore the differentiation program in ARMS cells. To achieve this aim, we generated a Suv39h-overexpressing myoblast reporter cell line (C2-Suv39h-4RE-Luc), where MyoD-mediated transactivation is suppressed by Suv39h overexpression. In search of new molecular therapeutic targets for this disease, we carried out small molecule library screens using C2-Suv39h-4RE-Luc reporter cells in order to target the restoration of an abortive myogenic differentiation program in ARMS cells as a novel and safe anti-ARMS chemotherapeutic approach.
Among screened compounds, we found that camptothecin, a topoisomerase I inhibitor, shows the restoration of MyoD-mediated transactivation in both C2-Suv39h-4RE-Luc cells and ARMS reporter cells. Moreover, camptothecin treatment reactivated the terminal differentiation marker gene expression in ARMS cells. This seems to be caused by the inhibition of SUV39H, as demonstrated by an in vitro histone methyltransferase (HMTase) activity assay. Taken together, our evidence shows the feasibility of this new approach to identify prospective bioactive candidates targeting SUV39H to develop new anti-ARMS pharmaceuticals.