Identifying and Overcoming Mechanisms of Impaired Myogenic Differentiation in Alveolar Rhabdomyosarcoma
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Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and adolescents, and is thought to represent abortive myogenic differentiation due to histology and expression of skeletal muscle-specific factors. The most aggressive RMS subtype is alveolar rhabdomyosarcoma (aRMS) expressing the fusion PAX3-FOXO1 oncoprotein, which results from a recurrent chromosome translocation. PAX3-FOXO1 drives tumor initiation and progression, and confers a dismal prognosis for patients presenting with PAX3-FOXO1-positive tumors. Thus, there is an urgent need for new therapeutic strategies targeting specific mechanisms driving aggressive aRMS behavior. Importantly, a key feature of this fusion protein is its suppression of terminal myogenic differentiation. PAX3-FOXO1 is known to suppress differentiation through various mechanisms, including by inhibiting the myogenic function of MyoD, a master differentiation-promoting factor. In this context, our laboratory has previously demonstrated that histone methyltransferase KMT1A strongly suppresses MyoD activity in aRMS cells. Accordingly, loss of KMT1A expression leads to terminal differentiation and suppression of tumor phenotype in these cells. Chapter 1 describes efforts to identify compounds which alleviate KMT1A-mediated suppression of MyoD in cells. A cell-based chemical screen of pharmacological compounds ultimately led to identification of camptothecin (CPT) as one such compound. Surprisingly, we uncovered that CPT suppresses KMT1A in cells independently of its known function, Topoisomerase 1 poisoning. As it was previously reported that CPT treatment suppressed PAX3-FOXO1 in aRMS cells, our study also led us to ask whether KMT1A has any role in regulation PAX3-FOXO1 in aRMS. In Chapter 2, experiments are described which demonstrate that KMT1A is necessary for high PAX3-FOXO1 expression in aRMS cells. Mechanistic studies further showed that MyoD upregulates PAX3-FOXO1 mRNA expression and binds to a known intronic regulatory element of PAX3-FOXO1 in aRMS cells. Accordingly, the data suggests that KMT1A promotes MyoD binding to this element by influencing modifications of the MyoD protein, as loss of KMT1A leads to decreased MyoD enrichment at this regulatory element and increased acetylation of MyoD, which is known to influence its binding to myogenic target genes. Collectively, these data uncover new roles for KMT1A and MyoD in the regulation of PAX3-FOXO1, and provide additional rationale for the further evaluation of KMT1A as a potential drug target in aRMS. In Chapter 3, we asked whether methyltransferase SET7/9, which is known to promote MyoD activity in differentiating myoblast cells, would antagonize KMT1A to increase MyoD activity and lead to differentiation of aRMS cells. We found that ectopic SET7/9 overexpression indeed induces MyoD activity and expression of its critical downstream target MyoG in aRMS cells. Surprisingly, SET7/9-induced MyoG fails to induce terminal differentiation or suppress growth. Mechanistically, we found that, unlike KMT1A depletion, overexpression of SET7/9 did not suppress PAX3-FOXO1 or increase MyoD acetylation. Accordingly, loss of PAX3-FOXO1 permitted SET7/9 to promote differentiation of aRMS cells. This finding highlights the importance of PAX3-FOXO1 suppression in potential aRMS pro-differentiation therapeutic approaches, as it argues that MyoD activation and MyoG induction are insufficient to suppress malignant phenotypes.