1 2 Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer in need for novel 3 therapies. Here we show that the PROX1 transcription factor, which is essential for normal 4 myoblast differentiation, is highly expressed in RMS tumors. We demonstrate that PROX1 5 is needed for RMS cell stemness and growth in vitro, and for RMS tumor formation in mouse 6 xenograft models. In addition, we unveil that PROX1 is an essential for myogenic properties 7 in RMS. PROX1 depletion reprogrammed the RMS transcriptome to resemble benign 8 mesenchymal stem cells and repressed many of the previously identified RMS effector 9 transcripts and myogenic genes. By using proximity labeling and mass spectrometry, we 10 found that PROX1 interacts with the NuRD and CoREST complexes containing class I 11HDACs. Our studies reveal a major role of PROX1-HDAC interaction in RMS and give 12 insights that inhibiting this interaction could be a promising therapeutic approach. 13 14

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.