Frontline and Relapsed Rhabdomyosarcoma (FAR-RMS) Clinical Trial: A Report from the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG).

The Frontline and Relapsed Rhabdomyosarcoma (FaR-RMS) clinical trial is an overarching, multinational study for children and adults with rhabdomyosarcoma (RMS). The trial, developed by the European Soft Tissue Sarcoma Study Group (EpSSG), incorporates multiple different research questions within a multistage design with a focus on (i) novel regimens for poor prognostic subgroups, (ii) optimal duration of maintenance chemotherapy, and (iii) optimal use of radiotherapy for local control and widespread metastatic disease. Additional sub-studies focusing on biological risk stratification, use of imaging modalities, including [18F]FDG PET-CT and diffusion-weighted MRI imaging (DWI) as prognostic markers, and impact of therapy on quality of life are described. This paper forms part of a Special Issue on rhabdomyosarcoma and outlines the study background, rationale for randomisations and sub-studies, design, and plans for utilisation and dissemination of results.

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting.

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

Mini-Review on Targeted Treatment of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a devastating disease which most commonly affects adolescents, with a male predominance. Despite the best multimodality treatment efforts, most patients will ultimately not survive more than 3-5 years after diagnosis. Some research trials in soft-tissue sarcoma and Ewing sarcoma include DSRCT patients but few studies have been tailored to the specific clinical needs and underlying cytogenetic abnormalities characterizing this disease such as the typical EWSR1-WT1 gene fusion. Downstream activation of EWSR1-WT1 gene fusion includes signaling pathways of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin growth factor (IGF)-1. Other biological pathways that are activated and expressed in DSRCT cells include endothelial growth factor receptor (EGFR), androgen receptor pathway, c-KIT, MET, and transforming growth factor (TGF) beta. Investigation of somatic mutations, copy number alterations (CNA), and chromosomes in DSRCT samples suggests that deregulation of mesenchymal-epithelial reverse transition (MErT)/epithelial-mesenchymal transition (EMT) and DNA damage repair (DDR) may be important in DSRCT. This mini review looks at known druggable targets in DSRCT and existing clinical evidence for targeted treatments, particularly multityrosine kinase inhibitors such as pazopanib, imatinib, and sorafenib alone or in combination with other agents such as mTOR (mammalian target of rapamycin) inhibitors. The aim is to increase shared knowledge about current available treatments and identify gaps in research to further efforts toward clinical development of targeted agents.

A tailored molecular profiling programme for children with cancer to identify clinically actionable genetic alterations.

BACKGROUND: For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed. METHODS: We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings. RESULTS: A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma. CONCLUSION: We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients.

Metabolic plasticity maintains proliferation in pyruvate dehydrogenase deficient cells.

BACKGROUND: Pyruvate dehydrogenase (PDH) occupies a central node of intermediary metabolism, converting pyruvate to acetyl-CoA, thus committing carbon derived from glucose to an aerobic fate rather than an anaerobic one. Rapidly proliferating tissues, including human tumors, use PDH to generate energy and macromolecular precursors. However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth. Although PDH is one of the most widely studied enzyme complexes in mammals, its requirement for cell growth is unknown. In this study, we directly addressed whether PDH is required for mammalian cells to proliferate. RESULTS: We genetically suppressed expression of the PDHA1 gene encoding an essential subunit of the PDH complex and characterized the effects on intermediary metabolism and cell proliferation using a combination of stable isotope tracing and growth assays. Surprisingly, rapidly dividing cells tolerated loss of PDH activity without major effects on proliferative rates in complete medium. PDH suppression increased reliance on extracellular lipids, and in some cell lines, reducing lipid availability uncovered a modest growth defect that could be completely reversed by providing exogenous-free fatty acids. PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids. By deleting the cytosolic isoform of IDH (IDH1), the enhanced contribution of glutamine to the lipogenic acetyl-CoA pool during PDHA1 suppression was eliminated, and growth was modestly suppressed. CONCLUSIONS: Although PDH suppression substantially alters central carbon metabolism, the data indicate that rapid cell proliferation occurs independently of PDH activity. Our findings reveal that this central enzyme is essentially dispensable for growth and proliferation of both primary cells and established cell lines. We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Glutamine and cancer: cell biology, physiology, and clinical opportunities.

Glutamine is an abundant and versatile nutrient that participates in energy formation, redox homeostasis, macromolecular synthesis, and signaling in cancer cells. These characteristics make glutamine metabolism an appealing target for new clinical strategies to detect, monitor, and treat cancer. Here we review the metabolic functions of glutamine as a super nutrient and the surprising roles of glutamine in supporting the biological hallmarks of malignancy. We also review recent efforts in imaging and therapeutics to exploit tumor cell glutamine dependence, discuss some of the challenges in this arena, and suggest a disease-focused paradigm to deploy these emerging approaches.