Abstract 3892: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Background: Patients with metastatic or recurrent rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, continue to do poorly and new treatments are urgently 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 failed in sarcomas. YAP1 (YES-associated protein) is also highly expressed in RMS, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced. 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 YES1-YAP1 interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling using cell fractionation, immunoblots, qPCR, RNAseq, and genetic loss-of-function. We evaluated changes in DNA methylation by methylation-specific PCR and the Illumina EPIC methylation profiling microarray. To evaluate drug treatments in vivo we utilized orthotopic xenografts in immunocompromised mice. Results: We demonstrate that DNMTi treatment decreases RMS cell growth, and increases apoptosis and myogenic differentiation. DNMTi treatment upregulates Hippo-activators RASSF1 and RASSF5 by promoter demethylation, activates canonical Hippo signaling through LATS1/2, and increases inactivation of YAP1 by phosphorylation. The DNMTi-induced growth inhibition is partially rescued by expression of constitutively active YAPS127A, suggesting the effects of DNMTi treatment are in part due to Hippo-dependent inhibition of YAP1. Additionally, YES1 and YAP1 interact in the nucleus of RMS cells and YES1 shRNA knockdown or pharmacologic inhibition results in YAP1 cytoplasmic retention and decreased YAP1 target gene expression, suggesting YES1 can regulate YAP1 in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulations of YAP1 we combined DNMTi treatment with Dasatinib. Combination treatment in vitro ablates RMS cell growth and induces more apoptosis than either drug alone. Combination treatment in orthotopic xenografts in mice results in decreased tumor weight, a trend towards decreased tumor growth, and