Receptor tyrosine kinases as therapeutic targets in rhabdomyosarcoma.

Rhabdomyosarcomas (RMSs) are the most common soft tissue sarcomas of childhood and adolescence. To date, there are no effective treatments that target the genetic abnormalities in RMS, and current treatment options for high-risk groups are not adequate. Over the past two decades, research into the molecular mechanisms of RMS has identified key genes and signaling pathways involved in disease pathogenesis. In these studies, members of the receptor tyrosine kinase (RTK) family of cell surface receptors have been characterized as druggable targets for RMS. Through small molecule inhibitors, ligand-neutralizing agents, and monoclonal receptor-blocking antibodies, RTK activity can be manipulated to block oncogenic properties associated with RMS. Herein, we review the members of the RTK family that are implicated in RMS tumorigenesis and discuss both the problems and promise of targeting RTKs in RMS.

The Transcriptional Coactivator TAZ Is a Potent Mediator of Alveolar Rhabdomyosarcoma Tumorigenesis.

Purpose: Alveolar rhabdomyosarcoma (aRMS) is a childhood soft tissue sarcoma driven by the signature PAX3-FOXO1 (P3F) fusion gene. Five-year survival for aRMS is <50%, with no improvement in over 4 decades. Although the transcriptional coactivator TAZ is oncogenic in carcinomas, the role of TAZ in sarcomas is poorly understood. The aim of this study was to investigate the role of TAZ in P3F-aRMS tumorigenesis.Experimental Design: After determining from publicly available datasets that TAZ is upregulated in human aRMS transcriptomes, we evaluated whether TAZ is also upregulated in our myoblast-based model of P3F-initiated tumorigenesis, and performed IHC staining of 63 human aRMS samples from tissue microarrays. Using constitutive and inducible RNAi, we examined the impact of TAZ loss of function on aRMS oncogenic phenotypes in vitro and tumorigenesis in vivo Finally, we performed pharmacologic studies in aRMS cell lines using porphyrin compounds, which interfere with TAZ-TEAD transcriptional activity.Results: TAZ is upregulated in our P3F-initiated aRMS model, and aRMS cells and tumors have high nuclear TAZ expression. In vitro, TAZ suppression inhibits aRMS cell proliferation, induces apoptosis, supports myogenic differentiation, and reduces aRMS cell stemness. TAZ-deficient aRMS cells are enriched in G2-M phase of the cell cycle. In vivo, TAZ suppression attenuates aRMS xenograft tumor growth. Preclinical studies show decreased aRMS xenograft tumor growth with porphyrin compounds alone and in combination with vincristine.Conclusions: TAZ is oncogenic in aRMS sarcomagenesis. While P3F is currently not therapeutically tractable, targeting TAZ could be a promising novel approach in aRMS. Clin Cancer Res; 24(11); 2616-30. ©2018 AACR.

Loss of MST/Hippo Signaling in a Genetically Engineered Mouse Model of Fusion-Positive Rhabdomyosarcoma Accelerates Tumorigenesis.

A hallmark of fusion-positive alveolar rhabdomyosarcoma (aRMS) is the presence of a chromosomal translocation encoding the PAX3-FOXO1 fusion oncogene. Primary cell-based modeling experiments have shown that PAX3-FOXO1 is necessary, but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously, we showed that PAX3-FOXO1-positive aRMS is promoted by dysregulated Hippo pathway signaling, as demonstrated by increased YAP1 expression and decreased MST activity. We hypothesized that ablating MST/Hippo signaling in a genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis. To this end, MST1/2-floxed (Stk3F/F;Stk4F/F ) mice were crossed with a previously established aRMS GEMM driven by conditional expression of Pax3:Foxo1 from the endogenous Pax3 locus and conditional loss of Cdkn2a in Myf6 (myogenic factor 6)-expressing cells. Compared with Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ controls, Stk3F/F;Stk4F/F;Pax3PF/PF;Cdkn2aF/F;Myf6ICN/+ animals displayed accelerated tumorigenesis (P < 0.0001) and increased tumor penetrance (88% vs. 27%). GEMM tumors were histologically consistent with aRMS. GEMM tumor-derived cell lines showed increased proliferation and invasion and decreased senescence and myogenic differentiation. These data suggest that loss of MST/Hippo signaling acts with Pax3:Foxo1 expression and Cdkn2a loss to promote tumorigenesis. The rapid onset and increased penetrance of tumorigenesis in this model provide a powerful tool for interrogating aRMS biology and screening novel therapeutics.Significance: A novel mouse model sheds light on the critical role of Hippo/MST downregulation in PAX3-FOXO1-positive rhabdomyosarcoma tumorigenesis. Cancer Res; 78(19); 5513-20. ©2018 AACR.

A Novel Notch-YAP Circuit Drives Stemness and Tumorigenesis in Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by skeletal muscle features, is the most common soft-tissue sarcoma of childhood. While low- and intermediate-risk groups have seen improved outcomes, high-risk patients still face a 5-year survival rate of <30%, a statistic that has not changed in over 40 years. Understanding the biologic underpinnings of RMS is critical. The developmental pathways of Notch and YAP have been identified as potent but independent oncogenic signals that support the embryonal variant of RMS (eRMS). Here, the cross-talk between these pathways and the impact on eRMS tumorigenesis is reported. Using human eRMS cells grown as three-dimensional (3D) rhabdospheres, which enriches in stem cells, it was found that Notch signaling transcriptionally upregulates YAP1 gene expression and YAP activity. Reciprocally, YAP transcriptionally upregulates the Notch ligand genes JAG1 and DLL1 and the core Notch transcription factor RBPJ This bidirectional circuit boosts expression of key stem cell genes, including SOX2, which is functionally required for eRMS spheres. Silencing this circuit for therapeutic purposes may be challenging, because the inhibition of one node (e.g., pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Finally, supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases Notch signaling and SOX2 expression.Implications: This study identifies a novel oncogenic signaling circuit driving eRMS stemness and tumorigenesis, and provides evidence and rationale for combination therapies co-targeting Notch and YAP. Mol Cancer Res; 15(12); 1777-91. ©2017 AACR.

A Review: Molecular Aberrations within Hippo Signaling in Bone and Soft-Tissue Sarcomas.

The Hippo signaling pathway is an evolutionarily conserved developmental network vital for the regulation of organ size, tissue homeostasis, repair and regeneration, and cell fate. The Hippo pathway has also been shown to have tumor suppressor properties. Hippo transduction involves a series of kinases and scaffolding proteins that are intricately connected to proteins in developmental cascades and in the tissue microenvironment. This network governs the downstream Hippo transcriptional co-activators, YAP and TAZ, which bind to and activate the output of TEADs, as well as other transcription factors responsible for cellular proliferation, self-renewal, differentiation, and survival. Surprisingly, there are few oncogenic mutations within the core components of the Hippo pathway. Instead, dysregulated Hippo signaling is a versatile accomplice to commonly mutated cancer pathways. For example, YAP and TAZ can be activated by oncogenic signaling from other pathways, or serve as co-activators for classical oncogenes. Emerging evidence suggests that Hippo signaling couples cell density and cytoskeletal structural changes to morphogenic signals and conveys a mesenchymal phenotype. While much of Hippo biology has been described in epithelial cell systems, it is clear that dysregulated Hippo signaling also contributes to malignancies of mesenchymal origin. This review will summarize the known molecular alterations within the Hippo pathway in sarcomas and highlight how several pharmacologic compounds have shown activity in modulating Hippo components, providing proof-of-principle that Hippo signaling may be harnessed for therapeutic application in sarcomas.

Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

Secreted Frizzled-Related Protein 3 (SFRP3) Is Required for Tumorigenesis of PAX3-FOXO1-Positive Alveolar Rhabdomyosarcoma.

PURPOSE: Rhabdomyosarcoma (RMS) is a soft tissue sarcoma associated with the skeletal muscle lineage. Of the two predominant subtypes, known as embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a five-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3-FOXO1. As PAX3-FOXO1 has proven chemically intractable, this study aims to identify targetable proteins that are downstream from or cooperate with PAX3-FOXO1 to support tumorigenesis. EXPERIMENTAL DESIGN: Microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PAX3-FOXO1 revealed alteration of several Wnt pathway gene members, including secreted frizzled related protein 3 (SFRP3), a secreted Wnt pathway inhibitor. Loss-of-function using shRNAs against SFRP3 was used to interrogate the role of SFRP3 in human aRMS cell lines in vitro and conditional murine xenograft systems in vivo. The combination of SFRP3 genetic suppression and the chemotherapeutic agent vincristine was also examined. RESULTS: In vitro, suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. In vivo, doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold, in addition to increasing myogenic differentiation and ß-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. CONCLUSIONS: SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.

Alveolar rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression.

Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncogene. Despite its discovery nearly two decades ago, the mechanisms by which PAX3-FOXO1 drives tumor development are not well characterized. Previously, we reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence checkpoints. We have now found that this bypass occurs in part through PAX3-FOXO1-mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member. RASSF4 expression was upregulated in PAX3-FOXO1-positive aRMS cell lines and tumors. Enhanced RASSF4 expression promoted cell cycle progression, senescence evasion, and tumorigenesis through inhibition of the Hippo pathway tumor suppressor MST1. We also found that the downstream Hippo pathway target Yes-associated protein 1 (YAP), which is ordinarily restrained by Hippo signaling, was upregulated in RMS tumors. These data suggest that Hippo pathway dysfunction promotes RMS. This work provides evidence for Hippo pathway suppression in aRMS and demonstrates a progrowth role for RASSF4. Additionally, we identify a mechanism used by PAX3-FOXO1 to inhibit MST1 signaling and promote tumorigenesis in aRMS.

Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: utility and pitfalls.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

FGFR4 blockade exerts distinct antitumorigenic effects in human embryonal versus alveolar rhabdomyosarcoma.

PURPOSE: Rhabdomyosarcoma (RMS) is a malignancy with features of skeletal muscle, and the most common soft tissue sarcoma of childhood. Survival for high-risk groups is approximately 30% at 5 years and there are no durable therapies tailored to its genetic aberrations. During genetic modeling of the common RMS variants, embryonal RMS (eRMS) and alveolar RMS (aRMS), we noted that the receptor tyrosine kinase (RTK) fibroblast growth factor receptor 4 (FGFR4) was upregulated as an early event in aRMS. Herein, we evaluated the expression of FGFR4 in eRMS compared with aRMS, and whether FGFR4 had similar or distinct roles in their tumorigenesis. EXPERIMENTAL DESIGN: Human RMS cell lines and tumor tissue were analyzed for FGFR4 expression by immunoblot and immunohistochemistry. Genetic and pharmacologic loss-of-function of FGFR4 using virally transduced short hairpin RNA (shRNA) and the FGFR small-molecule inhibitor PD173074, respectively, were used to study the role of FGFR4 in RMS cell lines in vitro and xenografts in vivo. Expression of the antiapoptotic protein BCL2L1 was also examined. RESULTS: FGFR4 is expressed in both RMS subtypes, but protein expression is higher in aRMS. The signature aRMS gene fusion product, PAX3-FOXO1, induced FGFR4 expression in primary human myoblasts. In eRMS, FGFR4 loss-of-function reduced cell proliferation in vitro and xenograft formation in vivo. In aRMS, it diminished cell survival in vitro. In myoblasts and aRMS, FGFR4 was necessary and sufficient for expression of BCL2L1 whereas in eRMS, this induction was not observed, suggesting differential FGFR4 signaling. CONCLUSION: These studies define dichotomous roles for FGFR4 in RMS subtypes, and support further study of FGFR4 as a therapeutic target.

Cerebral cavernous malformation 2 protein promotes smad ubiquitin regulatory factor 1-mediated RhoA degradation in endothelial cells.

Mutation of CCM2 predisposes individuals to cerebral cavernous malformations, vascular abnormalities that cause seizures and hemorrhagic stroke. CCM2 has been proposed to regulate the activity of RhoA for maintenance of vascular integrity. Herein, we define a novel mechanism where the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3) Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown of CCM2 have increased RhoA protein and display impaired directed cell migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to localize Smurf1 for RhoA degradation. These findings provide a molecular mechanism for the pathogenesis of cerebral cavernous malformations (CCM) resulting from loss of CCM2-mediated localization of Smurf1, which controls RhoA degradation required for maintenance of normal endothelial cell physiology.