Highlights d Inhibition of SDMA or ADMA preferentially kills splicing factor (SF)-mutant cells d Combined inhibition of PRMT5 and type I PRMTs has synergistic effects d RNA-binding proteins are the most enriched cellular substrates of PRMTs d Inhibition of RNA splicing underlies the cytotoxic effects of PRMT inhibition

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation-sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activitydependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9adeficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. Significance: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.