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Abe et al. J Cancer Metastasis Treat 2020;6:51 I http://dx.doi.org/10.20517/2394-4722.2020.117 Page 5 of 15

Rhabdomyosarcoma
Rhabdomyosarcoma is the most prevalent pediatric sarcoma and is characterized by tumor cells with a
skeletal myoblast-like phenotype, possibly arising from primitive mesenchymal cells [52,53] . The two major
subtypes are embryonal (approximately 60% of cases) and alveolar (20%) rhabdomyosarcoma, with the less
[54]
prevalent subtypes being pleomorphic (10%) and spindle/sclerosing (10%) . While rhabdomyosarcomas
can arise at any anatomical site, the embryonal subtype preferentially arises in the head and neck region
and in the genitourinary tract of children and young adolescents. This tumor subtype frequently shows
loss of heterozygosity at the 11p15 locus that includes the insulin-like growth factor-II gene. The alveolar
subtype is notoriously aggressive and affects the trunk (in particular, the perineal and paraspinal areas)
and extremities in adolescents and young adults. This subtype is characterized genetically by gene
rearrangement of the forkhead box O-subfamily 1 (FOXO1) resulting in t(1;13)(p36;q14) translocation
generating the paired box (PAX)3-FOXO1 fusion or t(2;13)(q35;q14) translocation generating the PAX7-
FOXO1 fusion proto-oncogene [52-55] .

The treatment strategy for rhabdomyosarcoma is based on a risk stratification (low-, intermediate-,
and high-risk) of the disease that consists of tumor histological subtype, the tumor stage (equivalent to
TNM classification) prior to treatment, and the post-surgery clinical grouping (e.g., extent of residual
tumor, presence of lymph node metastasis and of distant metastasis) [52,53,55] . Most rhabdomyosarcoma
patients require a multimodal combination of chemotherapy, surgery, and/or radiation therapy. The
two standard chemotherapy regimens include the combination of vincristine and actinomycin D with
either cyclophosphamide or ifosfamide. Implementation of combined multi-agent chemotherapy has
significantly improved patient outcomes. However, the efficacy of treatment for patients with high-risk
rhabdomyosarcoma (defined as the presence of distant metastasis [52,55] ) has not improved for the past three
decades [53,55] . Several clinical trials for rhabdomyosarcoma conducted over the last decade have evaluated
molecular targeted agents, immune checkpoints-blocking agents, and cellular immunotherapeutics. Only
[56]
pazopanib, a multi-kinase inhibitor that targets PDGFR-α, VEGFRs, and c-Kit , has been tested in a
phase III clinical trial for patients with metastatic soft tissue sarcomas including rhabdomyosarcoma. All
[57]
the remaining trials have been either phase I or II . Comprehensive whole genome analyses for embryonal
and alveolar rhabdomyosarcomas has failed to identify any actionable therapeutic targets [55,58,59] , hence the
urgent need to identify new therapeutic targets.

A previous study showed that a liposome-protamine-siRNA (LPR) nanoparticle that targets the PAX3-
FOXO1 fusion proto-oncogene transcript inhibited the proliferation of alveolar rhabdomyosarcoma cells
[60]
and their xenograft tumors . Another study demonstrated that entinostat, a class-I histone deacetylase
inhibitor, reduced the expression of PAX3-FOXO1 in alveolar rhabdomyosarcoma cells, thereby sensitizing
them to chemotherapeutic agents . These studies suggest that PAX3-FOXO1 fusion proto-oncogene and
[61]
its product are potentially actionable targets in the treatment of alveolar rhabdomyosarcoma. Consistent
[62]
with this suggestion is an earlier study that screened 160 different kinase inhibitors against alveolar
[63]
rhabdomyosarcoma cell lines and identified GSK3b inhibitors including TWS119 as tumor type-selective
inhibitors. This study found that GSK3b phosphorylated the PAX3-FOXO1 fusion protein in tumor cells
and that inhibition of GSK3b attenuated the transcriptional activity of this oncoprotein, suggesting a
role for GSK3b in sustaining alveolar rhabdomyosarcoma [Figure 1]. Subsequently, a large chemical
[62]
screen directed against self-renewing, tumor-propagating cells (TPCs) in embryonal rhabdomyosarcoma
identified GSK3(b) inhibitors (e.g., BIO, CHIR 98014, and CHIR 99021) as potent suppressors of this
tumor type via the inhibition of proliferation and the induction of terminal myogenic differentiation of
TPCs . The tumor-suppressive effect of GSK3(b) inhibitors was associated with induction of the canonical
[64]
Wnt/b-catenin pathway, which was underpinned by the finding that recombinant Wnt3A and stabilized
b-catenin enhanced the terminal differentiation of rhabdomyosarcoma TPCs [Figure 1]. Collectively, these
studies [62,64] suggest that GSK3b is a potential therapeutic target that covers the major subtypes (embryonal