High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

Toosendanin induces the apoptosis of human Ewing's sarcoma cells via the mitochondrial apoptotic pathway.

Toosendanin, a triterpenoid extracted from the root bark of Melia toosendan, has its origin from traditional Chinese medicine and has been used as a non­polluting and pesticide­free plant insecticide in China for fruit and vegetable production. In recent years, toosendanin has been found to inhibit tumor cell proliferation and promote tumor cell apoptosis. Ewing's sarcoma (ES) is the second most common primary malignant bone and soft tissue tumor in children and adolescents. Although the overall prognosis of ES has improved, the 5­year survival rate has not significantly increased. To analyze the role of toosendanin on ES progression, CCK­8 viability assay, flow cytometry, Hoechst 33258 staining and western blotting were performed. The present results suggested that toosendanin suppressed cell viability and induced apoptosis in human SK­ES­1 cells compared with DMSO treatment. In addition, in the present study, toosendanin was found to upregulate the expression of Bax and downregulate the expression of Bcl­2, altering the Bax/Bcl­2 ratio. Additionally, toosendanin promoted the release of cytochrome c, resulting in the activation of the mitochondrial apoptotic pathway, thus inducing the activation of caspase­9 and caspase­3, and the cleavage of PARP. Our results demonstrated that toosendanin inhibited the growth of ES cells in a dose­dependent manner and triggered mitochondrial apoptotic pathway to induce apoptosis. Therefore, toosendanin can potentially be utilized as an anticancer botanical drug for the treatment of ES.

Establishment of a novel patient-derived Ewing's sarcoma cell line, NCC-ES1-C1.

Ewing's sarcoma is an aggressive mesenchymal tumor characterized by the presence of a unique EWSR1-FLI1 translocation. Ewing's sarcoma primarily occurs in the bone and soft tissues. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of genetic alterations in relevant cellular contexts. Here, we report the establishment and characterization of a novel Ewing's sarcoma cell line following primary Ewing's sarcoma tumor tissue culture. The established cell line was authenticated by DNA microsatellite short tandem repeat analysis, characterized by in vitro assays, and named NCC-ES1-C1. The NCC-ES1-C1 cell line grew well for 15 mo and was subcultured more than 50 times during this period. Characterization of the cells revealed that they were not adherent and showed floating features. In conclusion, we successfully established a novel Ewing's sarcoma cell line, NCC-ES1-C1, from primary tumor tissue. The cell line has the characteristic EWSR1-FLI1 gene fusion and exhibits aggressive growth in vitro. Thus, the NCC-ES1-C1 cell line will be a useful tool for investigating the mechanisms of disease and the biological role of the EWSR1-FLI1 fusion gene.

Abemaciclib Is Active in Preclinical Models of Ewing Sarcoma via Multipronged Regulation of Cell Cycle, DNA Methylation, and Interferon Pathway Signaling.

PURPOSE: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. EXPERIMENTAL DESIGN: Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. RESULTS: Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. CONCLUSIONS: Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm.

In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma.

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.

Exenteration and Custom Implant Brachytherapy as a Treatment for Recurrent Primary Extraskeletal Orbital Ewing Sarcoma.

A 6-year-old boy initially presented to an outside hospital with a right orbital mass with biopsy positive for translocation involving EWS RNA-binding protein 1 gene and imaging consistent with primary extraskeletal Ewing sarcoma (ES). There was no evidence of metastatic disease. Patient underwent gross tumor resection and adjuvant chemotherapy (VAdriaC/IE) followed by postoperative 45-Gy proton beam radiation. After 19 months, a solitary in-field local recurrence occurred, which was unsuccessfully surgically resected. Thereafter, treatment commenced with irinotecan and temozolomide, and the patient presented to the center of the authors. MRI showed locally recurrent disease without evidence of metastatic disease. Right orbital exenteration was performed, and an orbital mold was fashioned to deliver brachytherapy. There were no complications. The patient had no evidence of recurrent disease at 37-month follow up. This is the first report of orbital implant brachytherapy for recurrent primary ES of the orbit, and an additional report of primary extraskeletal ES of the orbit, which is a rare primary orbital tumor.

Initial testing (stage 1) of the PARP inhibitor BMN 673 by the pediatric preclinical testing program: PALB2 mutation predicts exceptional in vivo response to BMN 673.

BACKGROUND: BMN 673 is a potent inhibitor of poly-ADP ribose polymerase (PARP) that is in clinical testing with a primary focus on BRCA-mutated cancers. BMN 673 is active both through inhibiting PARP catalytic activity and by tightly trapping PARP to DNA at sites of single strand breaks. PROCEDURE: BMN 673 was tested in vitro at concentrations ranging from 0.1 nM to 1 µM and in vivo at a daily dose of 0.33 mg/kg administered orally twice daily (Mon-Fri) and once daily on weekends (solid tumors) for 28 days. RESULTS: The median relative IC50 (rIC50 ) concentration against the PPTP cell lines was 25.8 nM. The median rIC50 for the Ewing cell lines was lower than for the remaining cell lines (6.4 vs. 31.1 nM, respectively). In vivo BMN 673 induced statistically significant differences in EFS distribution in 17/43 (39.5%) xenograft models. Three objective regressions were observed: a complete response (CR) in a medulloblastoma line (BT-45), a maintained CR in a Wilms tumor line (KT-10), and a maintained CR in an ependymoma line (BT-41). BMN 673 maintained its high level of activity against KT-10 with a threefold reduction in dose. KT-10 possesses a truncating mutation in PALB2 analogous to PALB2 mutations associated with hereditary breast and ovarian cancer that abrogate homologous recombination (HR) repair. CONCLUSIONS: The PPTP results suggest that single agent BMN 673 may have limited clinical activity against pediatric cancers. Single agent activity is more likely for patients whose tumors have defects in HR repair.

Molecular mechanisms and gene regulation of melphalan- and hyperthermia-induced apoptosis in Ewing sarcoma cells.

The prognosis of high-risk Ewing tumours (HR-ET) remains poor. Melphalan-containing chemotherapy regimens are commonly applied for HR-ET patients. Moreover, melphalan (Mel) is a promising agent in thermochemotherapy. Therefore, we investigated the single effects, the synergism and the gene regulation of Mel and hyperthermia (HT) in an ET cell line (RD-ES). Dose-dependent cytotoxicity by Mel was demonstrated, which was enhanced by the concomitant application of HT (42 degrees C for 2 h). Mel, HT and their combination caused a significant activation of caspase-3. Using the pan-caspase inhibitor z-VAD-fmk, we demonstrated that both stimuli mediated predominantly caspase-dependent cytotoxicity. With cDNA array analysis, 20 out of 198 apoptosis-related genes were identified to be differentially expressed by Mel and/or HT. Although a significant enhancement of three selected genes could not be proven at the protein level in subsequent experiments, this study gives insight into the complex molecular and genetic response of tumour cells to cytotoxic stimulation.

Molecular heterogeneity and function of EWS-WT1 fusion transcripts in desmoplastic small round cell tumors.

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma with a consistent cytogenetic abnormality, t(11;22)(p13;q12). This chromosomal translocation generates a chimeric transcript that is formed by fusion of the 5' region of the Ewing's sarcoma gene, EWS, with the 3' DNA-binding segment of WT1, the Wilms' tumor suppressor gene. We collected 14 DSRCT tumor samples and examined the hybrid transcripts. We identified: (a) combinatorial heterogeneity of EWS exons fused to WT1 including use of EWS exons 7, 8, and 9; (b) subpopulations of variant transcripts in 6 of 14 tumors characterized by aberrant splicing resulting in loss of EWS exon 6 or WT1 exon 9; (c) multiple cDNA products with large internal deletions; and (d) insertion of small stretches of heterologous DNA at the fusion site or exon splice region in transcripts from two tumors. Most of the splice variants were in-frame, and in vitro translated fusion proteins with intact DNA-binding motifs formed complexes with a WT1 response element in gel mobility assays. Each of the chimeric proteins retains the ability to bind to the GC and TC elements of the early transcription factor EGR-1 as well as WT1 consensus sequences. We present evidence that various EWS-WT1 proteins up-regulated EGR-1 promoter activity and that this up-regulation is specifically dependent upon the absence of the exon 9 KTS domain of WT1. The molecular diversity and functionality exhibited by these fusion transcripts may have significant biological implications for their transactivating and tumorigenic potential.

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Molecular analysis of the fusion of EWS to an orphan nuclear receptor gene in extraskeletal myxoid chondrosarcoma.

The pathogenesis of myxoid chondrosarcoma (CS) is poorly understood. A recurrent translocation, t(9;22) (q22;q12), has been recognized in CS, specifically in extraskeletal myxoid CS. Recently, this translocation has been shown to represent a rearrangement of the EWS gene at 22q12 with a novel gene at 9q22 designated CHN (or TEC). Sequence analysis suggests that CHN encodes a novel orphan nuclear receptor with a zinc finger DNA-binding domain. The structure of this gene fusion has been characterized in only a limited number of extraskeletal myxoid CSs and its presence in other types of CS has not been extensively examined. We studied 46 cases of CS (8 extraskeletal myxoid, 4 skeletal myxoid, 4 mesenchymal, and 30 other) for the EWS/CHN gene fusion by reverse transcriptase polymerase chain reaction, Southern blotting, and long-range DNA polymerase chain reaction. The EWS/CHN gene fusion was present in 6 of 8 extraskeletal myxoid CSs and was not detected in any of the remaining cases, including the 4 skeletal myxoid CSs. The negative findings in the latter cases suggest that skeletal myxoid CS is pathogenetically distinct from its extraskeletal counterpart. Notably, 2 cases of extraskeletal myxoid CS showed neither an EWS/CHN fusion transcript nor EWS/CHN genomic fusion nor EWS or CHN genomic rearrangement, suggesting genetic heterogeneity within extraskeletal myxoid CS. Finally, we also provide evidence for alternative splicing of the 3' end of the fusion transcript. Extraskeletal myxoid CS thus represents yet another sarcoma type containing a gene fusion involving EWS.

EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro.

Among primitive neuroectodermal tumors, Ewing tumors are characterized by a gene rearrangement recombining the 5'-EWS gene portion with one of two ETS-related proto-oncogenes, FLI-1 or ERG, in roughly 90 and 10% of cases, respectively. We attempted to antagonize EWS/FLI-1 function in Ewing tumor cell lines. As a control, a cell line derived from another small round cell tumor of neuroectodermal origin, neuroblastoma, was used. This cell line was found to express almost identical patterns of ETS proteins except for EWS/FLI-1 and a novel, ELF-related gene product. Stable expression of antisense EWS/FLI-1 cDNA resulted in decreased endogenous EWS/FLI-1 RNA levels and growth reduction of ET cells but not of neuroblastoma cells. DNA-binding FLI-1 derivatives localizing to the nucleus in which the 5'-EWS regulatory domain was replaced by bacterial beta-galactosidase dominantly modulated transcriptional transactivation from a degenerate ETS-binding motif. Transient transfection of these dominant-negative recombinants resulted in a significant decrease in the relative number of mitoses in four Ewing tumor cell lines tested but not in the neuroblastoma cell line. The presented evidence for modulation of tumor cell proliferation by EWS/FLI-1 antagonists suggests a causal role for EWS/FLI-1-mediated gene activation in the malignant transformation of the enigmatic Ewing tumor-precursor cell.