Browsing by Author "Linardic, Corinne M"

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents, is characterized by skeletal muscle features. The Ras-driven subset, which includes the embryonal (eRMS) and pleomorphic (pRMS) histologic subtypes, is an aggressive high risk subgroup with a 5-year survival rate of <30%. Recently the YAP oncoprotein, which is ordinarily silenced by the Hippo tumor suppressor pathway, was found to be highly upregulated in RMS tumors. However, the role of YAP in the Ras-driven subset was unknown.

In patient-derived Ras-driven eRMS cell lines, we suppressed YAP genetically via shRNAs. YAP suppression decreased cell proliferation, increased myogenic differentiation, and promoted apoptosis in vitro and in vivo in subcutaneous xenografts. Pharmacologic inhibition by the YAP-TEAD inhibitor verteporfin also decreased cell proliferation and tumor growth. In a genetically defined model of Ras-driven RMS, constitutively active YAPS127A can serve as the initial oncogenic alteration whereby YAPS127A is sufficient for senescence bypass in primary skeletal muscle myoblasts, but requires expression of hTERT and oncogenic Ras for tumorigenesis in vivo. Importantly these tumors are histologically consistent with human Ras-driven RMS.

To understand the impact of YAP signaling on cell stemness, we cultured eRMS cells as 3D spheres. These spheres are enriched in stem cell genes, as well as in YAP and Notch signaling. The Notch pathway is another developmental pathway that is also highly upregulated in eRMS and contributes to tumorigenesis. Using the spheres as a model, we uncovered a bidirectional signaling circuit between YAP and Notch that regulates stemness. Active Notch signaling upregulates YAP, and YAP in turn upregulates the Notch ligands JAG1 and DLL1 and the transcription factor RBPJ. This circuit controls expression of several stem cell genes including SOX2, which is functionally required for eRMS cell stemness. Silencing this circuit for therapeutic purposes may be challenging, since the inhibition of one node (for example pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. 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 both Notch signaling and SOX2 expression. In preliminary studies, we also analyzed the differential effects of the three Ras isoforms on eRMS tumorigenesis, Ras-Notch, and Ras-YAP signaling, and developed a method to culture the alveolar RMS subtype as spheres. In conclusion, the YAP oncoprotein drives Ras-driven tumorigenesis by promoting cell growth, survival, and stemness, and through signaling interactions with the Notch pathway. This study also provides rationale for combination therapies targeting YAP and Notch for the treatment of Ras-driven RMS.

Childhood cancer persists to be the leading cause of death by disease in children in the United States. While advances in survival have been made in recent decades for certain subtypes, others have seen little to no improvement in survival outcomes. One such example is cancer of the soft tissues, called soft tissue sarcoma. As compared to adults, the incidence of sarcoma is considerably higher in children. Soft tissue sarcomas are diagnosed and treated based on a variety of histological and molecular features, such as the presence or absence of a fusion oncoprotein, or mutations in oncogenic signaling pathways. Understanding the relationship between molecular signatures of soft tissue sarcoma and clinical outcomes such as metastasis and refractory disease is necessary to develop new and effective treatments. Fusion-positive rhabdomyosarcoma (FP-RMS) is an example of a subtype of soft tissue sarcoma in children and young adults for which advances in standard of care have been limited, and outcomes remain poor. Despite decades of work aimed at understanding the significance of the chromosomal translocation that produces the pathognomonic fusion oncoprotein PAX3-FOXO1 (PF), efforts to directly target and inhibit the fusion protein have failed, in part due to the nature of the PAX3-FOXO1 as a fusion of two essential transcription factors. Therefore, identification of downstream targets of PF may reveal more actionable therapeutic opportunities. In this study, we identify an axis in which PAX3-FOXO1 transcriptionally upregulates RASSF4, which in turn binds to and inhibits MST kinase. Using a characterized genetically engineered mouse model of FP-RMS, we crossed in animals with conditional loss of MST1 and MST2 and monitored for tumor development. Animals with additive loss of MST1 and MST2 developed tumors with increased penetrance and decreased latency. Using tumor-derived cell lines, we found that loss of MST increased invasiveness and decreased the cells’ ability to differentiate down the myogenic lineage. Investigation of signaling downstream of the MST kinase in the Hippo pathway revealed that phosphorylation of LATS is unchanged as a result of this MST loss, and YAP1 and WWTR1 remain active. Instead, we identified MOB1 as a candidate effector of this MST-mediated phenotype. In preliminary studies, we confirmed that loss of phospho-MOB1 is observed in xenografts of human patient-derived FP-RMS cells. In a cell-based model of FP-RMS, in which intrinsic stemness characteristics of these cells are supported, we discuss how aberrant Notch- driven developmental signaling contributes to chemoresistance and increased tumorigenic potential. In additional studies, we developed a model of STS metastasis and in an unbiased manner, identified genes that contribute to metastatic clonal outgrowth. Understanding the common drivers of metastasis in human patients is essential to developing new interventions that slow or prevent refractory and metastatic disease. Together, these studies provide rationale for using specific model systems to investigate the features of STS that are most difficult to treat in the clinical setting. Finally, in a fourth system, we show that inhibition of developmental pathways at a defined node is an effective approach to slow growth of STS. In these fusion-negative RMS (FN-RMS) studies, we show that genetic and pharmacologic inhibition of HES1, downstream of both Hippo and Notch, impedes tumor growth and encourages myogenic differentiation. In summary, aberrant non-canonical Hippo signaling is an effector of FP-RMS driven by the PAX3-FOXO1 fusion oncoprotein. Hippo signaling, like Notch and other developmental pathways, is required for both RMS tumorigenesis and normal human development. Using sophisticated modeling systems, we can identify nodes at which these critical pathways can be pharmacologically inhibited to develop therapeutics that are effective against refractory and metastatic disease.

Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.

First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression.

Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-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. In conclusion, 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.