Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the Treatment of Childhood Rhabdomyosarcoma.

Katherine K Slemmons,Choh Yeung,Lee J Helman,Jhazeel O Martinez Juarez,Joshua T Baumgart,Amy Mccalla

doi:10.1158/0008-5472.can-19-3853

Abstract

Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations. SIGNIFICANCE: This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.

Highlights

Rhabdomyosarcoma is the most common soft-tissue sarcoma of childhood
We analyzed the effect of treatment with a DNA methyltransferase inhibitor (DNMTi), SGI-110 known as guadecitabine, on the growth of rhabdomyosarcoma cell lines using live cell imaging
The alveolar rhabdomyosarcoma cells were more sensitive to DNMTi treatment exhibiting a 5Â increase in the percentage of cleaved caspase-3/7 positive cells compared with control while the embryonal rhabdomyosarcoma cells only had a 2Â increase in apoptosis

Summary

Introduction

Rhabdomyosarcoma 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, new treatments are needed [1, 2]. The two most common subtypes of rhabdomyosarcoma, embryonal and alveolar, are phenotypically and molecularly distinct but often treated with the same modalities. Standard-of-care treatment includes a combination of surgery, radiation, and a backbone of cytotoxic chemotherapies made up of vincristine, actinomycin D, and cyclophosphamide but does not include any targeted therapies. Embryonal rhabdomyosarcoma is characterized by genomic instability with high rates of copynumber alternations, single-nucleotide variations and up to one-third have alterations in the RAS signaling pathway [3, 4].

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