Rhabdomyosarcoma (RMS), derived from primitive mesenchymal cells in the striated skeletal muscle lineage and myogenic progenitors, is the most common soft tissue sarcoma in children.

For localized RMS cases, gross surgical resection of the primary tumor plus radiation therapy and chemotherapy is considered standard-of-care, although long-term toxicities from RT is a concern in younger patients. Over 90% of low-risk localized RMS patients have relapse-free survival with cytotoxic multi-agent chemotherapy. Most recently vinorelbine and low-dose cyclophosphamide maintenance therapy was trialed in high-risk localized RMS patients and demonstrated improvement in overall survival (Chen et al., 2019). However, overall survival for metastatic and recurrent RMS remains low at 21 and 30%, respectively, indicating the need for novel targeted therapies to improve survival outcomes (Chen et al., 2019).

Alveolar RMS (ARMS), harboring an oncofusion, is the most aggressive subtype due to its high metastasis and recurrence rates (van Erp et al., 2018). ARMS tumors are characterized by the presence of a chromosomal translocation, either the PAX3-FOXO1 fusion gene or the PAX7-FOXO1 fusion gene. The PAX3-FOXO1 rearrangement fuses the DNA-binding domain of paired box gene 3 (PAX3) on chromosome 2 to the transactivation domain of forkhead box protein O1 (FOXO1) on chromosome 13 (van Erp et al., 2018). The PAX-FOXO1 fusion protein is an aberrant oncogenic transcription factor, resulting in downstream transcription and translation of proteins involved in oncogenic transformation (Chen et al., 2019).

The PAX-FOXO1 transcription factor, which is both upstream to many of the signal cascades that promote RMS tumorigenesis, is a direct and promising target, although inhibitors that bind to PAX-FOXO1 with good specificity and affinity have yet to be designed. Another way to suppress PAX-FOXO1 transcription factor activity is through epigenetic targets, with therapy aimed at inhibiting the co-regulators and chromatin-remodeling complexes involved in transcription. Small molecule inhibitors such as JQ1 that target the BET bromodomain-containing protein (BRD4), an epigenetic reader that mediates transcription, can disrupt BRD4 and PAX3-FOXO1 interaction, leading to degradation of PAX3-FOXO1 and reduced transcription of the oncogenic fusion protein (Gryder et al., 2017). Histone deacetylase (HDAC) inhibitors such as entinostat, panobinostat, and vorinostat have also been shown to delay tumor growth in xenograft RMS models (Hedrick et al., 2015).

Receptor tyrosine kinases (RTKs), such as IGFR1, VEGFR, EGFR, FGFR4, and PDGFRα become constitutively activated and cause downstream tumorigenic effects in the presence of PAX-FOXO1 gene fusions and have also been identified as potential targets in RMS. Recently completed and currently ongoing clinical trials that target these RTKS are summarized in Table 3.

A phase II clinical trial compared the IGF-1R inhibitor cixutumumab to conventional multi-agent chemotherapy in pediatric and adult patients with metastatic alveolar and embryonal RMS. Event-free survival (EFS) at 18 months was initially higher in patients receiving cixutumumab treatment (68%) compared to combinational chemotherapy (39%), but at 3-year follow-up, cixutumumab therapy had an event-free survival rate of 16% (Malempati et al., 2015; van Erp et al., 2018; Malempati et al., 2019). A phase II trial of the IGF-1R inhibitor ganitumab in combination with the Src Family Kinase (SFK) inhibitor dasatinib is ongoing in patients with relapsed or refractory RMS (clinicaltrials.gov, NCT03041701).

In a phase II clinical trial comparing conventional chemotherapy against an experimental arm adding the VEGFR inhibitor bevacizumab to cytotoxic chemotherapy in pediatric and adolescent patients with metastatic RMS and soft-tissue sarcomas, median EFS was higher in patients treated with bevacizumab (20.6 months, compared to 14.9 months for chemotherapy only). Higher objective response with bevacizumab was also observed at 54%, compared to 36% in the chemotherapy cohort, but findings were not statistically significant (Chisholm et al., 2017). In another phase I clinical trial evaluating the efficacy of bevacizumab with sorafenib and low-dose cyclophosphamide, 1 of 2 patients with refractory/recurrent RMS experienced a partial response (Navid et al., 2013).

EGFR is over-expressed in about 15–20% of ARMS tumors. Although no in vivo experiments targeting EGFR have been conducted yet, in vitro experiments in ARMS cell lines found that combination therapy of the anti-EGFR antibody cetuximab and the standard chemotherapy dactinomycin had synergistic effects in inhibiting cell growth and inducing apoptosis, with greater anticancer toxicity than dactinomycin alone (Yamamoto et al., 2013).

PAX3-FOXO1 also amplifies FGFR4 expression in ARMS, and FGFR4 has been implicated in resistance to apoptosis in tumors treated with therapeutics targeting the IGF1R-PI3K-mTOR pathway. However, the FGFR4 inhibitor BGJ398 showed synergy with IGF-1R inhibitor AEW54 in an ARMS cell line, suggesting that FGFR4 may be a promising target in treatment resistance prevention (Wachtel et al., 2014). Relapsed ARMS has also been shown to be responsive to pazopanib, a multi-kinase inhibitor currently in use for soft tissue sarcomas (Heske and Mascarenhas, 2021).

In the Children’s Oncology Group phase II clinical trial ARST0921, treatment of first-relapse RMS patients with a vinorelbine and cyclophosphamide chemotherapy backbone in combination with either the mTOR inhibitor temsirolimus or the VEGFR inhibitor bevacizumab resulted in 6-month event-free survival rates of 69 and 55%, respectively, with a greater proportion of partial responses in patients treated with temsirolimus (Heske and Mascarenhas, 2021). This data was promising enough to lead to further investigation of temsirolimus in the upfront setting for patients with newly diagnosed intermediate-risk RMS. This randomized phase III trial compares conventional cytotoxic chemotherapy with VAC/VI (alternating vincristine, dactinomycin, cyclophosphamide, and vincristine, irinotecan), with the addition of temsirolimus to the conventional VAC/VI chemotherapy backbone (Heske and Mascarenhas, 2021).

Developmental pathways may also provide therapeutic targets in RMS. GLI transcription factor activation through the constitutively active Hedgehog signaling pathway in RMS is involved in tumorigenesis. ERMS and ARMS xenograft models treated with GLI1/2 inhibitor GANT-61 combined with either temsirolimus or vincristine showed inhibition of proliferation via cell cycle arrest at the G0/G1 phase and significant reductions in tumor growth (Srivastava et al., 2014). Further research is needed to establish the potential of GLI inhibitors in RMS treatment.

Therapeutic targets may also lie in the apoptosis pathway. The Bcl-2 family of apoptotic proteins is involved in cancer cell survival and proliferation, and combination of Bcl-2 inhibitor ABT-737 and mTOR inhibitor AZD8055 was highly synergistic in inducing caspase-dependent apoptosis in ARMS and ERMS cells (Preuss et al., 2013).

Numerous targets for RMS are being investigated, yet there are currently no clinically impactful novel agents for the treatment of RMS. Inhibitors of the PAX-FOXO1 fusion protein have yet to be developed as well, despite the discovery of this oncogenic driver since 1993 (Galili et al., 1993). As prognosis for recurrent and metastatic RMS remains poor, this is an area of research that greatly needs further investigation and analysis of both in vivo and in vitro studies and clinical trials. The targeted therapies discussed above for RMS are summarized in Figure 2.

Infantile fibrosarcoma (IFS) is a soft tissue sarcoma that presents as a localized, but large and rapidly growing tumor in the neonatal setting. IFS accounts for 24.5% of all soft tissue sarcomas found in children under the age of one. IFS has a very good overall prognosis, with studies demonstrating a 5-year survival rate of approximately 90%.

The optimal form of treatment for IFS is surgical resection, but wide local excision often results in mutilating surgery. In particular cases, neoadjuvant chemotherapy may be used to improve the probability of achieving a complete surgical resection; radiotherapy is used when surgical resection is deemed impossible (Parida et al., 2013). Thus there is a need for novel therapies in those IFS patients with extensive or unresectable disease, as well as those with metastatic IFS.

Nearly all IFS tumors harbor a neurotrophic tyrosine receptor kinase (NTRK) fusion, with 70% of IFS cases containing the ETS Variant Transcription Factor 6 (ETV6)-NTRK3 gene rearrangement (Knezevich et al., 1998). In NTRK rearrangements, the 5’ fusion partner induces ligand-independent constitutive activation of the tropomyosin receptor kinase (TRK). This leads to uninterrupted downstream signaling of the RAS/RAF/MEK/ERK and PI3K/AKT pathways, which promote cancer cell survival, invasion, and proliferation (Amatu et al., 2019).

Larotrectinib is a TRKA, TRKB, and TRKC inhibitor that prevents neurotrophin-TRK interaction and activation, inducing apoptosis and inhibition of tumor growth. Larotrectinib has been approved by the FDA for use in children and adults with NTRK-fusion-positive tumors that are metastatic and/or will result in severe morbidity upon surgical removal and do not have any resistance mutations (Dunn, 2020).

Various clinical trials have shown efficacy of larotrectinib against ETV6-NTRK3 fusion-positive IFS with negligible toxicity. In a phase I clinical trial, a 16-month-old female patient with refractory IFS was treated with larotrectinib. One month later, MRI scans revealed a reduction in tumor size by over 90%, and continued clinical response was also observed in later cycles of therapy (Nagasubramanian et al., 2016). Multiple phase 1 studies and case reports show reduction in tumor size, facilitating complete surgical resection, and remarkable reduction in size in other cases without surgical intervention (Caldwell et al., 2020).

Larotrectinib can also be used as a pre-surgical therapy to reduce tumor size, preventing radical and disfiguring surgeries. Larotrectinib treatment resulted in rapid and durable tumor regression and allowed for limb-sparing surgeries in patients who otherwise would have undergone amputations, confirming larotrectinib as a promising therapy for ETV6-NTRK3 fusion-positive IFS (DuBois et al., 2018). In a multi-center, phase I clinical study enrolling eight NTRK-fusion-positive IFS patients between 1 month and 21 years of age, larotrectinib was well-tolerated and demonstrated antitumor activity in all eight patients. Four patients avoided disfiguring surgery and instead underwent R0 (negative resection margins with no tumor at marked resection region) and R1 (microscopic residual tumor at resection margin) surgical resection following larotrectinib treatment, indicating that larotrectinib may represent a new standard of care in controlling disease without morbid surgery (Laetsch et al., 2018).

Another gene fusion identified in IFS is the non-classical LMNA-NTRK1 fusion. Crizotinib, an ALK inhibitor, has been shown to be effective in treating these tumors. Case reports show patients with refractory and metastatic IFS on crizotinib therapy had responses ranging from stable disease to complete and durable responses (Wong et al., 2016; Bender et al., 2019). The targeted therapies discussed above for IFS are summarized in Figure 3.

Alveolar soft part sarcoma (ASPS) is a rare, slow-growing but highly-angiogenic soft tissue sarcoma that often remains undetected until metastasis. ASPS is characterized by the TFE3-ASPSCR1 fusion gene, which induces overexpression of the MET receptor tyrosine kinase. This leads to downstream activation of MEK1/2 and AKT, which promote tumor cell proliferation and angiogenesis (Mitton and Federman, 2012).

The 5-year survival rates of localized and metastasized ASPS are 80% and 10–40%, respectively. The main form of treatment for ASPS is surgical removal; radiation therapy may also be used to prevent regrowth following surgery. For unresectable tumors, however, ASPS is resistant to conventional chemotherapies, indicating the need for novel therapeutic targets such as receptor tyrosine kinase inhibitors and other anti-angiogenic agents in the treatment of ASPS (Mitton and Federman, 2012).

Tivantinib (ARQ 197), a selective inhibitor of MET receptor tyrosine kinase, has been investigated in a Phase II clinical trial involving 27 patients ≥13 years with metastatic and/or surgically unresectable ASPS. After 10 cycles of tivantinib, 78% of ASPS patients had stable disease for at least 4 months, and median PFS was 6 months (Wagner et al., 2012).

Sunitinib, a PDGFR and VEGFR tyrosine kinase inhibitor that reduces tumor angiogenesis and triggers cancer cell apoptosis, has demonstrated efficacy in controlling progressive metastatic ASPS in adults. In a case series of 10 adult patients, with a median age of 24, treated with daily sunitinib, treatment was well-tolerated and induced long-lasting responses. Partial response and stable disease were confirmed in 9 cases after 6 months, and median PFS was 17 months (Stacchiotti et al., 2011). A further case study reporting a patient with refractory metastatic ASPS treated with sunitinib described complete primary tumor regression and stabilization of the metastases (Ghose et al., 2012). These anecdotal reports of sunitinib efficacy in adult ASPS tumors suggest a potential therapeutic role of sunitinib in pediatric ASPS patients as well.

Cediranib, a potent inhibitor of VEGFR tyrosine kinase, has also been evaluated in treating adult ASPS. In a phase II clinical trial involving 46 patients, with a median age of 27, with unresectable and metastatic ASPS, 35% had partial response and 60% had stable disease after 24 weeks of daily cediranib treatment (Kummar et al., 2013).

Inflammatory myofibroblastic tumors (IMT) are rare soft tissue neoplasms made up of myofibroblastic spindle cells, typically characterized by benign local recurrence and rare metastasis. The standard treatment for IMT is complete surgical resection, which has favorable prognosis with most patients surviving past 10 years after complete tumor removal. However, therapy remains lacking for malignant unresectable IMT, especially since chemotherapy and radiation are not particularly effective (Maruyama et al., 2017).

About half of all IMTs are anaplastic lymphoma kinase (ALK) fusion-positive, consisting of cells that express an ALK gene rearranged with over 30 different identified 5’ fusion partners (Coffin et al., 2001). Rearrangement of ALK over-activates ALK receptor tyrosine kinase, which leads to downstream activation of the PI3K/AKT, JAK/STAT, and Ras/ERK pathways, increasing cancer cell proliferation and survival.

Crizotinib, a receptor tyrosine kinase inhibitor, inhibits ALK phosphorylation and its downstream signaling pathways, leading to G1/S cell cycle arrest and apoptosis. Crizotinib is currently used to treat ALK-rearranged cancers, such as anaplastic large-cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC), and crizotinib has demonstrated efficacy in treating ALK-positive IMT as well.

In a 2017 study involving 14 unresectable, ALK-positive IMT pediatric patients who were given two doses of oral crizotinib daily, complete and partial responses were observed in five and seven patients, respectively (Mossé et al., 2017). In a cohort of eight ALK-positive IMT patients diagnosed with IMT between 2009-2016 and treated with crizotinib, four achieved a complete response, 3 a partial response, and one patient had stable disease. When preoperative crizotinib was used to decrease tumor size prior to surgical resection, 2 of the 3 partial responses achieved complete response. Furthermore, seven of eight patients were still alive at long-term follow-up, with no evidence of disease in six patients. Crizotinib was also found to be well-tolerated. As such, crizotinib combined with surgical resection appears to be effective in long-term disease control of ALK-positive IMT (Trahair et al., 2019).

For patients with RANBP2-ALK fusion positive epithelioid inflammatory myofibroblastic sarcoma (eIMS), a malignant variant of ALK-fusion-positive IMT, CD30 and ALK combination therapy may have high therapeutic potency. RANBP2-ALK eIMS xenografts treated with brentuximab-vedotin, targeting CD30 ⁺ tumor cells, and crizotinib resulted in tumor shrinkage and prolonged disease-free survival. In the diagnosis eIMS model, the majority of mice were confirmed as tumor-free 180 days past the study end date. However, disease recurrence was observed in all mice in the relapsed eIMS model, indicating that CD30 and ALK combination therapy may be most effective as early treatment for eIMS (Fordham et al., 2020).

Ceritinib, another ALK receptor tyrosine kinase inhibitor, has demonstrated efficacy against crizotinib-resistant tumors, with stronger potency in both crizotinib-naïve and crizotinib-refractory patients. Ceritinib is currently approved for use in ALK-positive NSCLC, but it has also shown excellent disease control in various ALK-positive IMT case studies (Santarpia et al., 2017). Recent case reports also show response in stage IV IMT or unresectable IMTs with ceritinib therapy and tumor response, allowing for surgical resection or complete response with ceritinib therapy alone (Mittal et al., 2021).

Alectinib is another ALK-inhibitor that has demonstrated efficacy against ALK-fusion-positive IMT. A case study in which a 26-year-old male with hyper-progressive ALK-fusion-positive IMT experienced a significant and durable response to treatment with alectinib was reported in 2017 (Saiki et al., 2017).

Entrectinib is an inhibitor of TRK, ROS1, and ALK, and has been shown to induce rapid and durable anti-cancer responses in NTRK, ROS1, and ALK-fusion tumors. A 16-year-old female patient with unresectable DCTN1-ALK-fusion-positive IMT had a complete response to entrectinib therapy, which controlled disease with low toxicity (Ambati et al., 2018).

Other gene translocations observed in IMT include proto-oncogene tyrosine-protein kinase (ROS1) fusions, which lead to tumorigenesis in mechanisms similar to ALK fusions. In a case study reported in 2021, the ALK- and ROS1-inhibitor lorlatinib was reported successful in treating a patient with refractory TFG-ROS1 fusion-positive IMT (Carcamo et al., 2021).

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare form of hepatocellular carcinoma (HCC), affecting adolescents and young adults with no prior history of liver disease or risk factors for live cancer. In FL-HCC, a ∼400 kilobase deletion on chromosome 19 gives rise to the DnaJ Heat Shock Protein Family (HSP40) Member B1 (DNAJB1)-Protein Kinase CAMP-Activated Catalytic Subunit Alpha (PRKACA) fusion gene, and the corresponding DNAJB1-PRKACA-fusion protein has upregulated protein kinase activity to promote tumorigenesis (Honeyman et al., 2014).

Current treatments for FL-HCC remain in development. Chemotherapy for unresectable hepatocellular carcinoma (HCC) is a combination of atezolizumab and bevacizumab, but this therapy showed no clinical benefits in case studies in two patients with advanced FL-HCC (Al Zahrani and Alfakeeh, 2021). Sorafenib is another multi-tyrosine kinase inhibitor in use against advanced HCC; however, sorafenib has limited efficacy against FL-HCC, with only delayed progression of disease as the best response (Ang et al., 2013).

The only potentially curative treatment for FL-HCC is liver resection or liver transplantation; if the tumor is not completely removed, likelihood of recurrence is high. For patients with metastatic and/or unresectable disease, there are no effective treatments available and FL-HCC is progressive and fatal; median survival time is less than 12 months (Kassahun, 2016).

However, novel combination and targeted therapies are being investigated in the treatment of FL-HCC. In a phase II trial of continuous IV fluorouracil and thrice weekly recombinant interferon alfa-2b administered to eight FL-HCC patients, one patient had complete response while four had partial responses, and overall median survival was 23.1 months (Patt et al., 2003). In a case study involving a 27-year-old woman with FL-HCC, 10 cycles of gemcitabine and oxaliplatin were administered, and 5 years after the regimen had been completed, the woman remained in complete remission (Gras et al., 2012).

The PRKACA kinase domain was also found to play an essential role in FL-HCC tumor formation, suggesting the potential for small molecule PRKACA-inhibitors as a therapy for FL-HCC (Kastenhuber et al., 2017). However, further research is needed to investigate the efficacy and viability of PRKACA inhibitors against FL-HCC.

Pediatric translocation renal cell carcinoma (tRCC) is a rare kidney cancer found in children, accounting for 2–5% of all childhood renal neoplasms. Less-advanced tRCC tumors can be treated via surgical resection, whereas more-advanced tRCC is treated with targeted therapies.

The most common mutation associated with RCC is the TFE3-fusion gene, where the TFE3 gene on chromosome Xp11.12 is rearranged with a fusion partner (i.e., PRCC, ASPSCR1, NONO, CLTC, SFPQ, etc.) from a different chromosome. The TFE3-fusion gene is a constitutively active promoter, causing dysregulated transcriptional TFE3 activity that leads to tumorigenesis through downstream upregulation of the PI3K/AKT/mTOR signaling pathway (Damayanti et al., 2018).

Cabozantinib is a small-molecule VEGFR2 and MET tyrosine kinase inhibitor that has already been approved for use in medullary thyroid cancer and renal cell carcinoma. For tRCC tumors that express MET, cabozantinib has been shown to regress disease with tolerable toxicity. In a 16-year-old female patient presenting with refractory, locally recurrent TFE3-fusion-positive tRCC with lung metastases, treatment with cabozantinib led to prompt and durable disease control. The patient was also noted to have reduction in pain, weight gain of 90% of weight previously lost on chemotherapy, and improved quality of life. In a 12-year-old male patient with stage IV TFE3-fusion-positive tRCC, treatment with cabozantinib demonstrated excellent disease control, including reduced pain just after 3 months and halted disease progression even 18 months later (Wedekind et al., 2017).

VEGFR tyrosine kinase and mTOR inhibitors, which are used in metastatic RCC therapies, have also demonstrated efficacy in disease control of tRCC. In a case study of 53 patients with tRCC, 21 patients who were TFE3-fusion-positive received targeted therapy. Fourteen patients treated with sunitinib had a median PFS of 8.2 months, with seven patients achieving either a partial or complete response. Eight patients treated with sorafenib had a median PFS of more than 6 months. Two pediatric patients had partial responses with single agent therapy of either sunitinib or sorafenib, and both patients continued with stable disease after 29 months. For patients that experienced disease progression while being treated with VEGFR inhibitors, switching to mTOR inhibitors temsirolimus and everolimus resulted in stable disease (Malouf et al., 2010).

Synovial sarcoma (SS) is a rare soft tissue sarcoma that occurs in adolescents and young adults, with a pathognomonic t (X; 18) (p11.2; q11.2) chromosomal translocation, fusing the SS18 (formerly known as SYT) gene with the SSX1, SSX2, or SSX4 gene (Clark et al., 1994; Shipley et al., 1994). Therapeutic approaches involve systemic chemotherapy with doxorubicin and ifosfamide combined with radiation therapy and/or surgery for local control.

The SS18-SSX fusion protein acts as an epigenetic modifier, driving tumorigenesis in SS cells (Kadoch and Crabtree, 2013). SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, containing the BAF (BRG1 or BRM associated factors) complex, are disrupted in SS cells when the SS18-SSX fusion protein competitively replaces the wild-type SS18 in the BAF complex (Kadoch and Crabtree, 2013). The fusion SS18-SSX protein is found to co-localize with Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) (Soulez et al., 1999; Lubieniecka et al., 2008). PRC2 silences chromatin with its catalytic subunit, the histone methyltransferase Enhancer of Zeste 2 (EZH2) (Wilson et al., 2010). The relocalization of oncogenic BAF complexes to PRC repressed domains, recruitment of RNA Polymerase II, and initiation of transcription leads to epigenetic reprogramming and drives tumorigenesis (McBride et al., 2018).

Preclinical studies using EZH2 inhibitors in SS cell lines were promising, showing a decrease in cellular proliferation and migration in vitro, and with decreased tumor burden in xenograft models (Kawano et al., 2016; Shen et al., 2016). However, in a phase II clinical trial in adult patients with synovial sarcoma, the selective EZH2 inhibitor tazemetostat exhibited less promising preliminary results. Tazemetostat was well tolerated by patients with few adverse events, but the best response observed was stable disease, which occurred in 33% of patients (Schoffski P et al., 2017).

Various strategies have been employed to promote the immune response against pediatric solid tumors. These include oncolytic virus-based therapy, antibody-dependent cellular cytotoxicity (ADCC), bispecific antibodies, immune checkpoint inhibitors, tumor microenvironment targeted therapies (cancer-associated fibroblasts, macrophages), cytokines, growth factors and CAR-T cells (Marayati et al., 2019).

TRAIL-Inducing Compound #10 (TIC10)/ONC201 is a novel agent that activates a potent innate immune pro-apoptotic anti-cancer response through the integrated stress response (Allen et al., 2013; Kline et al., 2016; Wagner et al., 2018; Prabhu et al., 2020). ONC201 has potential for further development in pediatric solid tumors including in combination with epigenetic modulators (Chang et al., 2020; Chang et al., 2021; Honeyman et al., 2021; Zhang et al., 2021).