Marco Wachtel

Gene Expression Signatures Identify Rhabdomyosarcoma Subtypes and Detect a Novel t(2;2)(q35;p23) Translocation Fusing PAX3 to NCOA1

Rhabdomyosarcoma is a pediatric tumor type, which is classified based on histological criteria into two major subgroups, namely embryonal rhabdomyosarcoma and alveolar rhabdomyosarcoma. The majority, but not all, alveolar rhabdomyosarcoma carry the specific PAX3(7)/FKHR-translocation, whereas there is no consistent genetic abnormality recognized in embryonal rhabdomyosarcoma. To gain additional insight into the genetic characteristics of these subtypes, we used oligonucleotide microarrays to measure the expression profiles of a group of 29 rhabdomyosarcoma biopsy samples (15 embryonal rhabdomyosarcoma, and 10 translocation-positive and 4 translocation-negative alveolar rhabdomyosarcoma). Hierarchical clustering revealed expression signatures clearly discriminating all three of the subgroups. Differentially expressed genes included several tyrosine kinases and G protein-coupled receptors, which might be amenable to pharmacological intervention. In addition, the alveolar rhabdomyosarcoma signature was used to classify an additional alveolar rhabdomyosarcoma case lacking any known PAX3 or PAX7 fusion as belonging to the translocation-positive group, leading to the identification of a novel translocation t(2;2)(q35;p23), which generates a fusion protein composed of PAX3 and the nuclear receptor coactivator NCOA1, having similar transactivation properties as PAX3/FKHR. These experiments demonstrate for the first time that gene expression profiling is capable of identifying novel chromosomal translocations.

Subtype and Prognostic Classification of Rhabdomyosarcoma by Immunohistochemistry

PURPOSE Rhabdomyosarcoma (RMS) is classified into two main subgroups: the embryonal (ERMS) and the alveolar (ARMS) form. The majority of the ARMSs are associated with specific chromosomal translocations (pARMS). Because ARMS is much more aggressive than ERMS, RMS subclassification has clinical relevance. However, diagnosis of RMS subgroups on the basis of histology or molecular biology can be difficult, and supplementing diagnostic methods would be desirable. The aim of this study was to establish a panel of markers for RMS subgroup classification by immunohistochemistry. MATERIALS AND METHODS Gene expression data were used for selection of subgroup-specific markers. Single sections of RMS with available expression data were used for establishment of the immunohistochemistry. Evaluation of the sensitivity and specificity of the markers was carried out using a tissue array representing 252 RMSs. Kaplan-Meier survival curves were calculated for determination of differences in overall survival of the different staining subgroups. RESULTS AP2beta and P-cadherin were selected as markers for pARMS, and epidermal growth factor receptor (EGFR) and fibrillin-2 as markers for ERMS. EGFR + fibrillin-2 detected ERMS with a specificity of 90% and with a sensitivity of 60%. AP2beta + P-cadherin detected pARMS with a specificity of 98% and a sensitivity of 64%, and allowed the detection of several misclassified tumors. The EGFR + fibrillin-2-positive group is associated with a favorable outcome, and the AP2beta + P-cadherin-positive group is associated with an unfavorable outcome. CONCLUSION The presented set of marker proteins detects RMS subgroups with high specificity and may be useful in routine subtype classification of RMS.

Targets for cancer therapy in childhood sarcomas

Development of chemotherapeutic treatment modalities resulted in a dramatic increase in the survival of children with many types of cancer. Still, in case of some pediatric cancer entities including rhabdomyosarcoma, osteosarcoma and Ewings sarcoma, survival of patients remains dismal and novel treatment approaches are urgently needed. Therefore, based on the concept of targeted therapy, numerous potential targets for the treatment of these cancers have been evaluated pre-clinically or in some cases even clinically during the last decade. This review gives an overview over many different potential therapeutic targets for treatment of these childhood sarcomas, including receptor tyrosine kinases, intracellular signaling molecules, cell cycle and apoptosis regulators, proteasome, hsp90, histone deacetylases, angiogenesis regulators and sarcoma specific fusion proteins. The large number of potential therapeutic targets suggests that improved comparability of pre-clinical models might be necessary to prioritize the most effective ones for future clinical trials.

Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities.

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.

Small-molecule screen identifies modulators of EWS/FLI1 target gene expression and cell survival in Ewing's sarcoma

Ewings sarcoma family of tumors (EFT) is characterized by the presence of chromosomal translocations leading to the expression of oncogenic transcription factors such as, in the majority of cases, EWS/FLI1. Because of its key role in Ewings sarcoma development and maintenance, EWS/FLI1 represents an attractive therapeutic target. Here, we characterize PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Using this gene and additional specific well‐characterized target genes such as NROB1, NKX2.2 and CAV1, all activated by EWS/FLI1, as a read‐out system, we screened a small‐molecule compound library enriched for FDA‐approved drugs that modulated the expression of EWS/FLI1 target genes. Among a hit‐list of nine well‐known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of six Ewings sarcoma cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against Ewings cells, which might act by modulating the expression of EWS/FLI1 target genes.

Cannabinoid receptor 1 is a potential drug target for treatment of translocation-positive rhabdomyosarcoma

Gene expression profiling has revealed that the gene coding for cannabinoid receptor 1 (CB1) is highly up-regulated in rhabdomyosarcoma biopsies bearing the typical chromosomal translocations PAX3/FKHR or PAX7/FKHR. Because cannabinoid receptor agonists are capable of reducing proliferation and inducing apoptosis in diverse cancer cells such as glioma, breast cancer, and melanoma, we evaluated whether CB1 is a potential drug target in rhabdomyosarcoma. Our study shows that treatment with the cannabinoid receptor agonists HU210 and Δ9-tetrahydrocannabinol lowers the viability of translocation-positive rhabdomyosarcoma cells through the induction of apoptosis. This effect relies on inhibition of AKT signaling and induction of the stress-associated transcription factor p8 because small interfering RNA–mediated down-regulation of p8 rescued cell viability upon cannabinoid treatment. Finally, treatment of xenografts with HU210 led to a significant suppression of tumor growth in vivo. These results support the notion that cannabinoid receptor agonists could represent a novel targeted approach for treatment of translocation-positive rhabdomyosarcoma. [Mol Cancer Ther 2009;8(7):1838–45]

Immunohistochemical detection of EGFR, fibrillin‐2, P‐cadherin and AP2β as biomarkers for rhabdomyosarcoma diagnostics

Aims:  Subclassification of rhabdomyosarcoma (RMS) has clinical relevance, as the two major subclasses embryonal (ERMS) and alveolar (ARMS) rhabdomyosarcoma differ greatly in terms of aggressiveness and prognosis. However, histological analysis is not always sufficient for an unequivocal subclassification of RMS. Furthermore, clinical presentation of ARMS has been reported to mimic other tumour types, specifically lymphoma. The aim was to determine the role of four biomarkers in the diagnosis of rhabdomyosarcoma.

PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

PLK1 phosphorylates PAX3-FOXO1, the inhibition of which triggers regression of alveolar Rhabdomyosarcoma.

Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1-mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1-PAX3-FOXO1 axis as a rational target to treat aRMS.

FGFR4 signaling couples to Bim and not Bmf to discriminate subsets of alveolar rhabdomyosarcoma cells

Biological heterogeneity represents a major obstacle for cancer treatment. Therefore, characterization of treatment‐relevant tumor heterogeneity is necessary to develop more effective therapies in the future. Here, we uncovered population heterogeneity among PAX/FOXO1‐positive alveolar rhabdomyosarcoma by characterizing prosurvival networks initiated by FGFR4 signaling. We found that FGFR4 signaling rescues only subgroups of alveolar rhabdomyosarcoma cells from apoptosis induced by compounds targeting the IGF1R‐PI3K‐mTOR pathway. Differences in both proapoptotic machinery and FGFR4‐activated signaling are involved in the different behavior of the phenotypes. Proapoptotic stress induced by the kinase inhibitors is sensed by Bim/Bad in rescue cells and by Bmf in nonrescue cells. Anti‐apoptotic ERK1/2 signaling downstream of FGFR4 is long‐lasting in rescue and short‐termed in most non‐rescue cells. Gene expression analysis detected signatures specific for these two groups also in biopsy samples. The different cell phenotypes are present in different ratios in alveolar rhabdomyosarcoma tumors and can be identified by AP2β expression levels. Hence, inhibiting FGFR signaling might represent an important strategy to enhance efficacy of current RMS treatments.