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The EWS-FLI1 translocation is the hallmark genomic alteration in the Ewing Family of Tumors (EWFT) a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five year survival rate. EWS-FLI is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. It is well known that the EWS-FLI1 translocation induces a variety of epigenetic changes which impact tumorigenesis and disease progression. Using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to the H3K27 demethylase inhibitor GSK-J4 (GlaxoSmithKline). Subsequent treatment with GSK-J4 leads to a decrease in H3K27 acetylation and ultimately silencing of EWS-FLI1 gene targets. We sought to fully characterize the hypersensitivity of EWFT to GSK-J4 and to elucidate the mechanisms of histone modifications caused by the EWS-FLI1 translocation in EWFT. We are examining key shifts in histone modifications at FLI1 target gene enhancer elements in the presence of GSK-J4 as a means of directly regulating FLI1 tumorigenesis. Additionally, we seek to determine the efficacy of GSK-J4 in combination with a CDK7 inhibitor (THZ1), which have shown synergy in vitro, through the utilization of patient derived xenograft models (PDX) and the identification of biomarkers of sensitivity to rationalize the use of this combination for clinical trials. Citation Format: Daniel A. Heisey, Timothy Lochmann, Marissa Calbert, Maninderjit Ghotra, Yuki Kato Maves, Sosipatros Boikos, Cyril Benes, C. Patrick Reynolds, Anthony Faber. Pharmaceutical means of targeting the fusion oncogene EWS-FLI1 in the Ewing family of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4329.

Ewing Family of tumors (EFT) comprises the fourth most common highly malignant childhood cancer. Although sustained event-free survival (EFS) can be achieved with intensive chemo-radiation therapy for patients with local-regional disease, this therapy is relatively ineffective in the treatment of metastatic disease with EFS of 12% at 5 years. Ewing sarcoma is characterized by a reciprocal translocation between chromosomes 11 and 22 that encodes a chimeric oncoprotein resulting from the fusion of EWSR1 to the FLI1 transcription factor in ~85% of tumors. Therapy for patients with EFT comprises surgery, intensive use of cytotoxic agents and radiation therapy. Dose intensification and dose compression has resulted in some improvement in outcome, but patents with advanced or metastatic disease at diagnosis still represent a challenge. Further, patients alive at 5 years from diagnosis still have a high probability of subsequent relapse. Further, long-term consequences of treatment included cardiac dysfunction, and secondary malignancies. Thus, more effective and less toxic therapies are required to treat patients with advanced disease. Our studies, as part of the Pediatric Preclinical Testing Program (PPTP), identified the combination of the PARP inhibitor, talazoparib, with the DNA damaging agent temozolomide, as being highly synergistic in xenograft models of Ewing sarcoma, but not against other tumor types. In this study 5 of 10 Ewing tumor xenografts models showed dramatic regressions to the combination, while administered as single agents neither talazoparib or temozolomide were active. We have studied the talazoparib-temozolomide synergy in vitro, and results indicate that in models where there is no synergy as xenografts, the cell lines have either intrinsic resistance to talazoparib, temozolomide or both drugs. In mice, and in the clinical trial (NCT02116777), the talazoparib-temozolomide combination is toxic requiring a reduction in temozolomide dose to ~15% of its single maximum-tolerated dose. We are exploring the use of nanoparticle-formulated talazoparib (npTLZ) developed by Nanomaterials Synthesis Laboratory at Northeastern University without tumor targeting or with antibody-mediated targeting to increase the tumor-drug delivery, reduce normal tissue toxicity (mainly thrombocytopenia), and potentially allow escalation of temozolomide dose. In the PPTP study, the MTD for temozolomide combined with free talazoparib (0.25 mg/kg PO BID daily x 5) was 12 mg/kg. Our recent data showed no toxicity of temozolomide at 66 mg/kg (PO daily x 5) in mice treated with npTLZ (0.5 mg/kg IV daily x 5), suggesting that npTLZ does not potentiate TMZ toxicity to normal mouse tissues. We anticipate that nanoparticle delivery of talazoparib combined with temozolomide will allow reduced toxicity while increasing the response rate for this combination in preclinical models of Ewing sarcoma. Citation Format: Nabeela Baig, Rostislav Likhotvorik, Paige Baldwin, Srinivas Sridhar, Raushan Kurmasheva. Nanoformulation of talazoparib to increase efficacy when combined with temozolomide for the treatment of Ewing sarcoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5054. doi:10.1158/1538-7445.AM2017-5054

EWS-ETS genetic fusion appears to be the initiating mechanism behind Ewing Family of Tumors (EFT) pathogenesis; however, progression of the disease is dependent on additional genetic alterations. Using the Progenetix database, we identified mainly alterations at 1q, and at 16q, as the most recurrent genetic alterations in EFT without consistency as markers of relapse. Clinic features of a large genotype cohort of 135 patients with EFT were retrospectively reviewed from A.C. Camargo hospital records. Tissue microarrays (TMA) were built for multi-color interphase fluorescent in situ hybridization (iFISH) analysis of aberrations at 1q32.1 (KIF14/MDM4/PIK3C2ß) and 16q21 (CDH11) using in-house probes. Gains were defined by the presence of more than three hybridization signals per interphase nucleus with simultaneous intact centrosome signal occurring in more than 5% of tumor nuclei. Losses were indicated by one target signal with the intact centrosome signals in more than 30% of nuclei. The frequencies of gene copy number alterations (CNA) were compared with clinical parameters and follow-ups. Statistical analyses were performed by SPSS program. A p-value of less than 0.05 was considered significant. CNA analysis showed gains at 1q32.1 in 15/128 (11.7%) cases, while abnormalities at 16q21 were seen in 24/118 (20.3%) patients, split into two groups. One group of patients, 11/118 (9.3%), showed only hemizygous deletion, whereas, the other group with 13/118 (11%) patients showed gain. A significant association between 1q32.1 gains and 16q21 losses (p=0.029) was detected, but not a significant clinical synergism when interacting at the same patient. Univariate analyses showed gains at 16q21 (p=0.029) or at 1q32.1 (p=0.005) associated with tumor relapse in the localized disease subgroup. Disease-free survival curves represented these gained regions as unfavorable outcome parameters (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2011-317

Pediatric undifferentiated soft tissue sarcomas (USTS) are a challenging group of tumors to diagnose and accurately categorize. By definition, they do not exhibit consistent immunohistochemical profiles or harbor recurrent chromosomal rearrangements to aid in their recognition. Traditionally, they have been classified with poorly differentiated forms of embryonal rhabdomyosarcoma. More recently, however, this notion has been challenged as such tumors share characteristics with the Ewing family of tumors. Thus, the histogenesis of pediatric USTS remains unclear and optimum therapy remains a challenging decision. The aims were twofold: firstly, to determine whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of pediatric USTSs; and secondly, to determine whether pediatric USTS expressed a unique panel of stem cell-associated proteins and form a distinct sarcoma subcategory. Tumors included 32 Ewing sarcoma/PNETs (EWS), 22 embryonal rhabdomyosarcomas (ERMSs), 8 alveolar rhabdomyosarcomas (ARMSs), 5 synovial sarcomas (SSs), 5 malignant peripheral nerve sheath tumours (MPNSTs) and 16 USTSs. Stem cell antibodies used included a series of three mesenchymal stem cell markers (CD44, CD105 and CD166) and five neural stem cell markers (CD15, CD29, CD56, CD133 and nestin). Antibodies were applied to the sections using standard immunohistochemical techniques and the sections were scored on a six-tiered scoring system using both intensity and distribution components. The scores were then imported into Partek Genomic Suite Software, for statistical analyses, cluster analysis, and visualizations. The datasets were analyzed by comparing the expression scores from the groups using the 1-way ANOVA. Each group was compared to each individual group, as well as the remaining groups combined. The resultant Euclidean clustering divided the tumors into two major groups. EWSs and USTSs formed the majority of the first group, whereas ERMSs, ARMSs, MPNSTs and SSs formed the second group. Negativity for CD56 was strongly associated with the EWS/USTS cluster (p < 0.0001). EWSs and USTSs were further separated by CD166 staining, wherein positivity was associated with EWS and negativity with USTS (p < 0.0001). The second group included some EWS (n=5), but the vast majority (n=27) were included in the first cluster. No consistent separation of the different subgroups was seen in the second cluster of tumors. The current study demonstrates the usefulness of applying stem cell markers to pediatric sarcomas, and suggests that pediatric USTSs and EWSs are closely related and may share a common cell of origin. Furthermore, the majority of USTSs have a unique stem-cell expression profile. The findings have both biologic and diagnostic significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3423.