Preclinical testing of the glycogen synthase kinase-3β inhibitor tideglusib for rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3β is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3β inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3β-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3β inhibitors may not be a viable treatment for aRMS or eRMS

YAPping About Differentiation Therapy in Muscle Cancer

Overcoming a presumed differentiation block in the childhood muscle cancer embryonal rhabdomyosarcoma is often thought to hold promise as an approach to replace cytotoxic chemotherapy with molecularly-targeted differentiation therapies. In this issue of Cancer Cell, Tremblay and colleagues implicate YAP1 and the Hippo signaling pathway in the maintenance of differentiation-arrested and proliferative phenotypes for embryonal rhabdomyosarcoma

NFκB signaling in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB–YY1–miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKβ, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKβ exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKβ wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial

EphB4/EphrinB2 therapeutics in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma affecting children and is often diagnosed with concurrent metastases. Unfortunately, few effective therapies have been discovered that improve the long-term survival rate for children with metastatic disease. Here we determined effectiveness of targeting the receptor tyrosine kinase, EphB4, in both alveolar and embryonal RMS either directly through the inhibitory antibody, VasG3, or indirectly by blocking both forward and reverse signaling of EphB4 binding to EphrinB2, cognate ligand of EphB4. Clinically, EphB4 expression in eRMS was correlated with longer survival. Experimentally, inhibition of EphB4 with VasG3 in both aRMS and eRMS orthotopic xenograft and allograft models failed to alter tumor progression. Inhibition of EphB4 forward signaling using soluble EphB4 protein fused with murine serum albumin failed to affect eRMS model tumor progression, but did moderately slow progression in murine aRMS. We conclude that inhibition of EphB4 signaling with these agents is not a viable monotherapy for rhabdomyosarcoma

Exogenous expression of the glycosyltransferase LARGE1 restores α-dystroglycan matriglycan and laminin binding in rhabdomyosarcoma

α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-] called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS. A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor. Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan. Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor

IL-13 receptors as possible therapeutic targets in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal childhood cancer of the brain. Despite the introduction of conventional chemotherapy and radiotherapy, improvements in survival have been marginal and long-term survivorship is uncommon. Thus, new targets for therapeutics are critically needed. Early phase clinical trials exploring molecularly-targeted therapies against the epidermal growth factor receptor (EGFR) and novel immunotherapies targeting interleukin receptor-13α2 (IL-13Rα2) have demonstrated activity in this disease. To identify additional therapeutic markers for cell surface receptors, we performed exome sequencing (16 new samples, 22 previously published samples, total 38 with 26 matched normal DNA samples), RNA deep sequencing (17 new samples, 11 previously published samples, total 28 with 18 matched normal RNA samples), and immunohistochemistry (17 DIPG tissue samples) to examine the expression of the interleukin-4 (IL-4) signaling axis components (IL-4, interleukin 13 (IL-13), and their respective receptors IL-4Rα, IL-13Rα1, and IL-13Rα2). In addition, we correlated cytokine and receptor expression with expression of the oncogenes EGFR and c-MET. In DIPG tissues, transcript-level analysis found significant expression of IL-4, IL-13, and IL-13Rα1/2, with strong differential expression of IL-13Rα1/2 in tumor versus normal brain. At the protein level, immunohistochemical studies revealed high content of IL-4 and IL-13Rα1/2 but notably low expression of IL-13. Additionally, a strong positive correlation was observed between c-Met and IL-4Rα. The genomic and transcriptional landscape across all samples was also summarized. These data create a foundation for the design of potential new immunotherapies targeting IL-13 cell surface receptors in DIPG

EphB4 and EphrinB2 expression in model systems of human eRMS.

<p><i>A</i>. Patient survival improves with high EphB4 expression determined using the. p = 0.0033, n = 72. <i>B</i>. Human eRMS expression of EphB4 and EphrinB2, respectively, of PCB82 tissue sections from a human eRMS tissue microarray. Control skeletal muscle from the same TMA is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183161#pone.0183161.g001" target="_blank">Fig 1B</a>. <i>C</i>. Human eRMS expression of EphB4 and EphrinB2 in Rh18 xenograft sections. Mag bar = 50 μm. <i>D</i>. Representative western blot demonstrating EphB4 and EphrinB2 protein expression in human eRMS xenograft models, PCB82 and Rh18.</p

VasG3 treatment of PCB82 eRMS xenografts.

<p><i>A</i>. Kaplan-Meier curve demonstrating percent event-free survival based on day post-treatment. Isotype control (black line) versus VasG3 (red line). p = 0.351, n = 6 female mice per cohort. <i>B</i>. Western blot demonstrating loss of EphB4 protein levels following VasG3 treatment. <i>C</i>. Differences in EphB4 protein levels following VasG3 treatment were quantified with densitometry. **p = 0.006.</p