LAUSR

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry. Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins. Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously “undruggable” PAX3-FOXO1 oncogenic driver of aggressive FP-RMS. Citation Format: Robert G. Hawley, Girma M. Woldemichael, Berkley E. Gryder, Young Song, Sivasish Sindiri, Vineela Gangalapudi, Jun S. Wei, James B. McMahon, Jack F. Shern, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5867.