Abstract A09: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. While outcomes for patients with localized disease have improved, patients with metastatic or recurrent disease continue to do poorly; therefore, new treatments are needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single-agent dasatinib (Src family kinase inhibitor) have not shown clinical activity in sarcomas. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES-YAP interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling with cell fractionation, immunoblots, methylation-specific PCR, qPCR, RNAseq, and genetic loss of function. Results: We demonstrate that DNMTi treatment decreases RMS cell growth and increases apoptosis and differentiation. DNMTi treatment activates canonical Hippo signaling through LATS, shown by a LATS activity biosensor, and increases YAP1 phosphorylation. DNMTi treatment also induces RASSF1 re-expression by promoter demethylation, and the DNMTi-induced growth inhibition can be partially rescued by expression of constitutively active YAPS127A. RNAseq analysis after DNMTi treatment also revealed upregulation of RASSF family members RASSF5 and RASSF6 and downregulation of RASSF4. RASSF4 was previously shown to inhibit MST1 and promote tumorigenesis in the alveolar subtype of RMS. These data suggest the effects of DNMTi treatment are in part due to inhibition of YAP1 in a Hippo-dependent manner. DNMTi treatment also decreases YES1 expression and activation, suggesting DNMTis impact both YES1 and YAP1 activity. Furthermore, YES1 and YAP1 interact in the nucleus of RMS cells and shRNA knockdown of YES1 results in YAP1 cytoplasmic retention and decreased expression of YAP1 target genes, suggesting a role for YES1 in regulating YAP1 cellular localization in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulation of YAP1 we combine DNMTi treatm