Rene L. Galindo

Rhabdomyosarcoma (RMS) is an aggressive soft tissue malignancy comprised histologically of skeletal muscle lineage precursors that fail to exit the cell cycle and fuse into differentiated syncytial muscle—for which the underlying pathogenetic mechanisms remain unclear. In contrast to myogenic transcription factor signaling, the molecular machinery that orchestrates the discrete process of myoblast fusion in mammals is poorly understood and unexplored in RMS. The fusogenic machinery in Drosophila, however, is understood in much greater detail, where myoblasts are divided into two distinct pools, founder cells (FC) and fusion competent myoblasts (fcm). Fusion is heterotypic and only occurs between FCs and fcms. Here, we interrogated a comprehensive RNA-sequencing database and found that human RMS diffusely demonstrates an FC lineage gene signature, revealing that RMS is a disease of FC lineage rhabdomyoblasts. We next exploited our Drosophila RMS-related model to isolate druggable FC-specific fusogenic elements underlying RMS, which uncovered the EGFR pathway. Using RMS cells, we showed that EGFR inhibitors successfully antagonized RMS RD cells, whereas other cell lines were resistant. EGFR inhibitor–sensitive cells exhibited decreased activation of the EGFR intracellular effector Akt, whereas Akt activity remained unchanged in inhibitor-resistant cells. We then demonstrated that Akt inhibition antagonizes RMS—including RMS resistant to EGFR inhibition—and that sustained activity of the Akt1 isoform preferentially blocks rhabdomyoblast differentiation potential in cell culture and in vivo. These findings point towards selective targeting of fusion- and differentiation-arrest via Akt as a broad RMS therapeutic vulnerability. Significance: EGFR and its downstream signaling mediator AKT1 play a role in the fusion and differentiation processes of rhabdomyosarcoma cells, representing a therapeutic vulnerability of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a soft tissue malignancy composed of neoplastic cells that morphologically resemble skeletal muscle-lineage precursor cells. High-risk RMS has a dismal prognosis, and treatments for this condition have not improved for three decades now. RMS is a heterogeneous disease composed of four subtypes. Embryonal RMS (ERMS), the most common subtype, associates with various tumour-promoting signalling pathways (for example, RAS and Hedgehog) and/or loss of tumour surveillance (for example, TP53 mutations). Pleomorphic RMS is very aggressive and occurs during the sixth and seventh decades of life. Spindle cell/sclerosing RMS affects both children and adults, with favourable and unfavourable prognoses, respectively. Alveolar RMS (ARMS), which is notoriously aggressive, is a genetically distinct disease driven by the PAX3–FOXO1 and PAX7–FOXO1 chimeric oncoproteins that are unique to ARMS. Understanding the cell (or cells) of origin for RMS is complex, reflecting the fact that multiple precursor cells can give rise to RMS in mouse models. To relieve a bottleneck that has existed in the design of new RMS therapies, an impressive array of new RMS model systems, spanning the evolutionary spectrum from flies to fish to mammals, have recently been developed; these new models have proven successful in uncovering new pathogenic mechanisms of RMS. Moreover, RMS gene discovery prompted by these diverse yet remarkably complementary model systems is inspiring new possibilities for targeted RMS therapy. These models also point to viable approaches for the genetic and molecular dissection of other clinically problematic non-RMS sarcomas.

Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, Alveolar-RMS (A-RMS), is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene to FOXO1. The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. Here, we report a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, we have performed a comprehensive deletion screen against the Drosophila autosomes, and demonstrate that mutation of Mef2, a myogenesis lynchpin in both flies and mammals, dominantly suppresses PAX7-FOXO1 pathogenicity and acts as a PAX7-FOXO1 gene target. Additionally, we reveal that mutation of mastermind, a gene encoding a MEF2 transcriptional co-activator, similarly suppresses PAX7-FOXO1, further pointing towards MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F₁) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease.