Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

Abstract

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Selfrenewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/ β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/ β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced selfrenewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/ β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.

New Strategies in Sarcoma: Linking Genomic and Immunotherapy Approaches to Molecular Subtype

“…A potential solution is to assess the dependence of particular sarcomas on more common targetable cancer pathways. In this regard, one recent development is the identification of aberrant Wnt/b-catenin signaling in synovial sarcoma models (68) and rhabdomyosarcoma (69). Combination targeted therapy again may have merit: a phase II trial of sorafenib and everolimus combination therapy achieved 45% 6-month progression-free survival (PFS; ref.…”

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

“…This approach was useful for expansion of double transgenic primary tumors without the need for generating tumors within a syngeneic transgenic line. Our recent work has shown that cell transplantation approaches provide novel experimental models to assess ERMS drug sensitivity in vivo, where a single tumor can be expanded into thousands of animals and assessed for effects on growth, self-renewal, and neovascularization 15 . Moreover, we have successfully engrafted a wide range of tumors into rag2 homozygous mutant fish including T cell acute lymphoblastic leukemia, melanoma, and ERMS 23 .…”

“…7. Utilize engrafted fish for downstream applications including Fluorescence Activated Cell Sorting (FACS) to assess differentiation status (Figure 3H), standard histological analysis (Figure 3F), imaging therapy responses 15 , and/or serial transplantation approaches including limiting dilution analysis 11 .…”

“…In the setting of cancer, cell transplantation approaches have become widely adopted and permit the dynamic assessment of important cancer processes including self-renewal 11 , functional heterogeneity 12,13 , neovascularization 14 , proliferation, therapy responses 15 , and invasion 16,17 . However, engrafted cells are often rejected from recipient fish due to host immune defenses that attack and kill the graft 18 .…”

Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis

“…Although most chemical screens described to date are using embryos less than 72 hpf, there are examples of chemical screens performed in older embryos up to 20 dpf with soaking technique. 34 Thousands of wildtype or genetically modified embryos can be obtained from a large mating, ideal for chemical screens, both genetic and phenotypic. 35 In a given genetic or disease model, a chemical suppressor screen can be performed in an attempt to reverse the phenotype or the genetic lesion, identifying new pathways of regulation in the pathophysiology of disease.…”