Xenograft Models Of Ewing Sarcoma Research Articles

TNF-related apoptosis-inducing ligand (TRAIL) for bone sarcoma treatment: Pre-clinical and clinical data

Bone sarcomas are rare, highly malignant mesenchymal tumours that affect teenagers and young adults, as well as older patients. Despite intensive, multimodal therapy, patients with bone sarcomas have poor 5-year survival, close to 50%, with lack of improvement over recent decades. TNF-related apoptosis-inducing ligand (TRAIL), a member of the tumour necrosis factor (TNF) ligand superfamily (TNFLSF), has been found to induce apoptosis in cancer cells while sparing nontransformed cells, and may therefore offer a promising new approach to treatment. We cover the existing preclinical and clinical evidence about the use of TRAIL and other death receptor agonists in bone sarcoma treatment. In vitro studies indicate that TRAIL and other death receptor agonists are generally potent against bone sarcoma cell lines. Ewing's sarcoma cell lines present the highest sensitivity, whereas osteosarcoma and chondrosarcoma cell lines are considered less sensitive. In vivo studies also demonstrate satisfactory results, especially in Ewing's sarcoma xenograft models. However, the few clinical trials in the literature show only low or moderate efficacy of TRAIL in treating bone sarcoma. Potential strategies to overcome the in vivo resistance reported include co-administration with other drugs and the potential to deliver TRAIL on the surface of primed mesenchymal or immune cells and the use of targeted single chain antibodies such as scFv-scTRAIL.

Abstract 1943: High-throughput chemical screening identifies Focal Adhesion Kinase and Aurora Kinase B inhibition as a synergistic treatment combination in Ewing sarcoma

Abstract Ewing sarcoma is the second most common bone malignancy of childhood. Current treatment employs chemotherapy, surgery, and radiation. Although this approach cures approximately 70% of patients with localized disease, treatments are largely ineffective for patients with metastases or relapse. Furthermore, these treatments are associated with an alarming rate of long-term toxicities. New treatment combinations are necessary to improve cures and lower toxicities for these patients. We recently found that Ewing sarcoma is dependent on focal adhesion kinase (FAK) for cell viability and tumor proliferation. In order to identify candidate treatment combinations for Ewing sarcoma, we performed a screen of 1912 compounds to identify those with synergistic anti-Ewing activity when combined with FAK inhibition. The A673 Ewing cell line was treated with PF-562271, a FAK-specific inhibitor, in combination with compounds from the Mechanism Interrogation PlatE (MIPE) 4.0 library. Cell viability was measured after 48 hours of treatment. Multiple computational metrics were utilized to identify and rank all compound combinations for synergistic impairment of cell viability. Multiple Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. Aurora kinases are important in the regulation of mitosis and are highly expressed in Ewing sarcoma tumors and cell lines. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibition in multiple Ewing cell lines. We found that AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 when used in combination induced apoptosis in Ewing cells at concentrations that had minimal effect on cell survival when either drug was used alone. We also found that the combination significantly impaired tumor proliferation in zebrafish xenograft models of Ewing sarcoma and prolonged survival in murine xenografts compared to either single-agent treatment alone. Interestingly, treatment with AZD-1152 alone also significantly impaired tumor proliferation and prolonged survival compared to vehicle treatment in a mouse xenograft model of Ewing sarcoma. Our data demonstrate that FAK and Aurora kinase B inhibition synergistically impair Ewing sarcoma cell viability in vitro and significantly inhibit tumor proliferation in vivo. With multiple FAK and Aurora kinase inhibitors in early phase trials for adult malignancies, these results have the potential to be translated into clinical trials for patients with Ewing sarcoma. Previous studies have also suggested a dependency of Ewing sarcoma on Aurora kinase activity; our data further supports a role for Aurora kinase B inhibitors as therapeutic candidates in this disease. Citation Format: Brian Crompton, Sarah Wang, Elizabeth Hwang, Rajarshi Guha, Matthew Boxer, Crystal McKnight, Min Shen, Nicole Melong, Chansey Veinotte, Amy Conway, Jason Berman, Matthew Hall, Mindy Davis, Kimberly Stegmaier. High-throughput chemical screening identifies Focal Adhesion Kinase and Aurora Kinase B inhibition as a synergistic treatment combination in Ewing sarcoma [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 1943. doi:10.1158/1538-7445.AM2017-1943

Abstract 5054: Nanoformulation of talazoparib to increase efficacy when combined with temozolomide for the treatment of Ewing sarcoma xenografts

Abstract 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

Abstract LB-047: Optimizing the trabectedin-mediated inhibition of EWS-FLI1 activity by schedule and fusion type

Abstract BACKGROUND: Ewing sarcoma (ES) is a pediatric malignancy characterized by the EWS-FLI1 transcription factor. ES tumors require the continuous transcriptional activity of EWS-FLI1 for the oncogenic phenotype, regardless of fusion type, which makes EWS-FLI1 an ideal therapeutic target. To date, no EWS-FLI1 directed therapies have been developed. Interestingly, there have been reports of ES patients as exceptional responders in clinical trials, including a complete response in a refractory, metastatic patient that was administered the natural product trabectedin in a Phase I trial. We have since established trabectedin as an EWS-FLI1 inhibitor that acts through a re-localization mechanism at concentrations that are clinically achievable. Intriguingly, trabectedin failed in the Phase II setting after a change in schedule that lead to a decrease in reported Cmax. In this report, we unify the disparate clinical trial conclusions by examining the schedule-dependency of trabectedin in ES cells. METHODS: Here, we model a broad range of exposures to trabectedin and investigate the cell viability kinetics using live cell imaging analysis software and traditional MTS. In parallel, we present a novel assay to quantitatively measure the kinetics of drug clearance in vitro. In addition, we use qPCR and western blotting to examine EWS-FLI1 target gene expression changes in response to different exposures to trabectedin. Finally, we investigated ES cell lines containing variant EWS-FLI1 fusion types and identify an increased sensitivity of type III/IV ES cell lines to trabectedin treatment. We recapitulated these findings in vivo using ES xenograft models. RESULTS: Sensitivity of ES cells to trabectedin treatment is schedule-dependent. Here, we demonstrate that cells treated with trabectedin at equivalent exposures but different Cmax exhibit profound differences in cell viability. The differences in viability correlate with Cmax that are necessary to inhibit EWS-FLI1 activity in vitro. By measuring the kinetics of drug clearance, we show that the inhibitory effects on EWS-FLI1 are durable even after trabectedin has been washed out of the culture medium. In addition, we examine the sensitivity of variant EWS-FLI1 fusion proteins such as type III/IV fusions both in vitro and in vivo. CONCLUSIONS: In this report, we use clinical trial data to develop a more clinically relevant cell culture system for ES drug treatments. Importantly, we demonstrate that equivalent exposures do not lead to equivalent outcomes. We believe that this observation explains the discordant results of the Phase I and II clinical trials, and suggests that the Cmax achieved in serum dictates whether a patient will respond. In addition, we show that type III/IV EWS-FLI1 fusion proteins are particularly sensitive to the drug. Citation Format: Matt Harlow, Elissa Boguslawski, Anderson Peck, Zachary Madaj, Maria J. Navarro, Pablo Aviles, Carlos Galmarini, Patrick Grohar. Optimizing the trabectedin-mediated inhibition of EWS-FLI1 activity by schedule and fusion type [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 LB-047. doi:10.1158/1538-7445.AM2017-LB-047

Abstract LB-177: PM01183 inactivates the EWS/FLI1 transcription factor by redistributing the protein within the nucleus

Abstract BACKGROUND: Ewing sarcoma is a bone and soft tissue sarcoma characterized by fusion of the wild-type EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. Ewing sarcoma cells are dependent on the continued activity of EWS/FLI1 to block differentiation and promote proliferation. For these reasons, EWS/FLI1 represents an ideal drug target. With this in mind, we have shown that the small molecule trabectedin inhibits EWS/FLI1 activity and synergizes with irinotecan in vivo. In addition, we showed that the gene expression response of Ewing sarcoma cells to trabectedin treatment is identical to keratinocytes exposed to UV light. Interestingly, wild-type EWSR1 accumulates in the nucleolus in response to UV light damage. Therefore, we hypothesize that the response and sensitivity of Ewing sarcoma cells to trabectedin is due to the re-localization of EWS/FLI1 in the nucleus. METHODS: Here, we utilize confocal microscopy and chromatin immunoprecipitation to characterize the localization of EWS/FLI1 and EWS/FLI1 deletion mutants in response to trabectedin. Because trabectedin has a narrow therapeutic index, we characterize the effect of PM01183, a second-generation structural analog with an improved pharmacokinetic profile, on EWS/FLI1 localization. In addition, we use qPCR and western blotting to determine the kinetics of EWS/FLI1 inhibition in response to this class of compounds. W demonstrate the effects of PM01183 treatment on the EWS/FLI1 gene signature genome-wide. Finally, we translate these findings in vivo using two Ewing sarcoma xenograft models. RESULTS: Treatment of Ewing sarcoma cells with Trabectedin and PM01183 causes EWS/FLI1 to accumulate in the nucleolus. This effect is accompanied by a loss of binding at target sequences and a reversal in expression of the gene signature of EWS/FLI1. We found the effect on EWS/FLI1 to be sustained, even after removal of the drug from the cell culture medium. We extended these findings in vivo to demonstrate the efficacy of a previously characterized combination therapy. Interestingly, treatment of Ewing sarcoma xenografts with the combination therapy caused the tumor to be replaced by benign adipocytes. CONCLUSIONS: We characterize a novel method of targeting an oncogenic fusion protein by leveraging a property of the wild-type protein to inactivate the oncogenic transcription factor. We demonstrate that wild-type EWSR1 protein's propensity to accumulate in the nucleolus upon UV light damage is maintained within the oncogenic EWS/FLI1 protein with trabectedin or PM01183 treatment. We believe that this accounts for the heightened sensitivity of Ewing sarcoma cells to this class of compounds and has numerous clinical implications. Citation Format: Matt Harlow, Matthew Easton, Maria Jose Guillén Navarro, Lisa Turner, Galen Hostetter, Carlos Galmarini, Pablo Aviles, Patrick Grohar. PM01183 inactivates the EWS/FLI1 transcription factor by redistributing the protein within the nucleus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-177.

Abstract 2098: Understanding the synergy between temozolomide and PARP inhibition in Ewing sarcoma

Abstract Ewing sarcoma is the fourth most common highly malignant childhood cancer; it is defined by a tumor-specific chromosomal translocation. In approximately 85% of all tumors, the EWSR1 gene on chromosome 22 is fused to a member of E26 transformation-specific sequence family of transcription factors, the FLI1 gene on chromosome 11. DNA damage induced by expression of EWSR1-FLI1 fusion gene is potentiated by PARP1 inhibition in Ewing cells, where EWSR1-FLI1 genes act in a positive feedback loop to maintain the expression of PARP1. As single agents, PARP inhibitors have shown promising activity in vitro, but only modest activity in in vivo models. In our previous work (in PPTP), we showed that low level damage to DNA by temozolomide (TMZ) can be potentiated up to 40-fold through inhibition of PARP by BMN-673, leading to dramatic tumor regressions in 5 of the 10 Ewing sarcoma xenograft models. Therefore, we were aimed to determine the differences between the tumors that respond to treatment with BMN-673 and those intrinsically resistant to it, and to understand the underlying mechanisms of such resistance, with the ultimate goal to develop more effective therapy for Ewing sarcoma. We have used the same cell lines to investigate biochemical differences between those Ewing sarcoma lines where there is synergy in xenograft models in mice, and those where the combination is inactive. Our studies using cell lines show that, 1) in vitro BMN-673 and TMZ are synergistic in all cell lines, irrespective of their response as xenografts; 2) In vivo synergy correlates with intrinsic sensitivity to BMN-673 and TMZ in vitro. For Ewing lines, where there is no in vivo synergy when they are grown as xenografts, they are ∼9-fold resistant to BMN-673, and ∼15-fold resistant to TMZ relative to lines where there is synergy as xenografts. The resistance of the Ewing sarcoma xenografts to the synergy of the combination of BMN-673 and TMZ in mice is a consequence of intrinsic resistance to either or both of the drugs. The combination of BMN-673 and TMZ in Ewing sarcoma xenografts is the most dramatic synergy between two drugs to be reported. The synergy in vivo is restricted to Ewing sarcoma, yet only half of the models respond to combination treatment with the other half completely resistant. Studies proposed in this application will potentially identify biomarkers that will allow identification of patients likely to benefit from such treatment, and spare toxicity for those unlikely to respond. Citation Format: Ruth Carlson, Raushan Kurmasheva. Understanding the synergy between temozolomide and PARP inhibition in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2098.

Abstract B14: Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma

Abstract Long-term survival in patients with metastatic relapsed or recurrent Ewing sarcoma (ES) and rhabdomyosarcoma (RMS) is dismal. Encouraging responses to irinotecan, a topoisomerase 1 (Top1) inhibitor, have been seen in these patients; however, limitations in irinotecan bioavailability, including low conversion rate to the active metabolite (SN-38) and high excretion rate of the inactive form, hinder efficacy. HSP90 is widely expressed in cancer cells, and HSP90 inhibitors (HSP90i) have favorable pharmacokinetics for anticancer use, as they remain in tumors for longer periods of time and at higher steady-state levels compared to normal tissue. This property makes them ideal intracellular delivery vehicles for chemotherapeutic drugs, allowing for high tumor exposure and low systemic toxicity. STA-12-8666 (Synta Pharmaceuticals) is an HSP90i drug conjugate (HDC) consisting of a weak HSP90i attached to SN-38 through a cleavable chemical linker. The purpose of this study was to test this HDC in xenograft models of pediatric sarcoma and to investigate its mechanism of action. To test therapeutic efficacy of this HDC, female SCID mice underwent orthotopic injection of ES or RMS cells from established cell lines or PDX tissue. When tumors reached a desired size, mice were randomized and then treated weekly with HDC, vehicle, ganetespib (a highly potent HSP90i), high dose irinotecan, protracted dose irinotecan, or irinotecan plus ganetespib. Tumors were measured twice per week, and mice were weighed weekly to determine drug tolerability. Tumors were harvested at midpoints and at study endpoint for biology studies. Activity of pharmacodynamic (PD) markers was investigated in tumor tissue. In xenograft models of ES and RMS, treatment with HDC produced superior antitumor efficacy compared to the other arms. When initial treatment began in mice with palpable tumors (between 100 and 500 mm3 (ES) or 50 and 90 mm3 (RMS)), all tumors underwent complete regression after 2 doses of HDC, with total tumor eradication in all ES mice and several RMS mice. In the RMS group, 6/8 mice relapsed by 23 weeks, and all 6 of those responded to retreatment with HDC. When initial treatment was delayed until tumors reached between 800 mm3 and 1000 mm3, complete regressions were again achieved in ES after 2 doses and RMS after 4 doses. Compared with high dose weekly irinotecan, which also induced tumor regression, mice treated with HDC had longer and more persistent remissions. A dose response effect was seen in HDC with cures noted in mice with ES at the 100- and 150-mg/kg doses and longest remissions noted in RMS in the 150-mg/kg group. Tolerability of the HDC was excellent with no toxicity-related deaths or weight loss in any treated mice. Studies using PDX models are ongoing and will be reported. Activity of γH2AX in tumor samples was explored as a PD marker of Top1 inhibitor activity. Mice bearing ES were treated with a single dose of vehicle, irinotecan, or HDC and one mouse per group was sacrificed at serial intervals between 6 hours and 10 days post-treatment. Expression of γH2AX in irinotecan mice began to wane between days 1 and 3, whereas in HDC mice, it was still detectable at day 7, suggesting that HDC results in more persistent inhibition of topoisomerase 1 compared to irinotecan. To look at the potential role of HSP90 inhibition in this HDC, HSP70 activity was investigated as a marker of HSP90 inhibition in samples from the dose finding experiment. In ES, no HSP70 was detected in samples from mice treated with irinotecan or HDC, suggesting the primary mechanism of action is via SN-38. In RMS, HSP70 was slightly induced in mice treated with higher doses of the HDC, perhaps contributing to the higher relapse rate in this model. Preclinical data suggest that this HDC may be a promising anticancer agent for ES and RMS patients. Citation Format: Christine M. Heske, Arnulfo Mendoza, Choh Yeung, David A. Proia, Len Neckers, Lee J. Helman. Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B14.

Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma.

Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease.

Abstract 1612: Identification of mithramycin analogs with improved targeting of the EWS/FLI1 transcription factor

Abstract Background: Ewing sarcoma is a bone a soft tissue sarcoma with a poor overall survival. This tumor absolutely depends on the continued expression of the EWS-FLI1 transcription factor for cell survival. We are therefore focused on developing small molecules that inhibit EWS-FLI1. We have previously completed a high throughput screen that identified mithramycin as an inhibitor of EWS-FLI1 and translated this compound to the clinic in a phase I-II trial. The success of this compound in the clinic has been challenged by drug associated liver toxicity that has necessitated dose reductions. Therefore the goal of this study is to identify less toxic and-or more potent mithramycin analogs. Methods: The less toxic analog, EC8042, was identified by evaluating animal toxicity data and serum pharmacokinetics in mice and rats. In order to identify a more potent compound, a panel of more than 20 mithramycin analogs was screened using an EWS-FLI1 reporter NR0B1 luciferase construct to identify EC8105. EWS-FLI1 suppression was confirmed using quantitative PCR and western blot analysis in vitro. The ability of the drug to block EWS-FLI1 binding to chromatin was evaluated by performing chromatin immunoprecipitation in the presence and absence of drug. The relative hepatotoxicity of the analogs was modeled in vitro by comparing doses that achieve suppression of EWS-FLI1 to toxic doses of the drug in HepG2 cells and confirmed in vivo in xenograft experiments. Finally, we tested the ability of both analogs to suppress tumor growth in xenograft models of Ewing sarcoma and confirmed suppression of EWS-FLI1 using immunofluorescence of formalin fixed tissue from these experiments. Results: EC8042 shows equivalent suppression of EWS-FLI1 activity but is substantially less toxic than the parent compound, allowing higher serum levels of drug in vivo in animal models. In contrast, EC8105 is approximately 8 times more potent than the parent compound and demonstrates improved suppression of the EWS-FLI1 gene signature. Both compounds work to block EWS-FLI1 binding to chromatin. More importantly, in contrast to mithramycin, both analogs suppress EWS-FLI1 activity at concentrations that are non-toxic to HepG2 cells. These effects translate into improved suppression of Ewing sarcoma xenograft growth with a corresponding increase in mouse survival and regression of several tumors in both cohorts. Conclusions: We have identified the mithramycin analogs EC8042 and EC8105 as EWS-FLI1 inhibitors. These compounds are less toxic and more potent than the parent compound and suppress EWS-FLI1 at concentrations that do not appear to cause liver toxicity. Together these results suggest that the clinical development of these analogs is warranted. Citation Format: Christy Osgood, Nichole Maloney, Christopher G. Kidd, Meti Gebregiorgis, Luz E. Nunez, Javier Gonzalez-Sabin, Lee J. Helman, Francisco Moris, Patrick J. Grohar. Identification of mithramycin analogs with improved targeting of the EWS/FLI1 transcription factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1612. doi:10.1158/1538-7445.AM2015-1612

Abstract IA32: Integrating genomic approaches to discover new therapeutic strategies for Ewing sarcoma

Abstract While next-generation sequencing studies are unveiling cancer genomes at an unprecedented pace, for most pediatric cancers, only a minority of the tumor samples have been shown to possess lesions that are immediately targetable by our current drug armamentarium. Quite commonly, the signature cancer-promoting events in childhood malignancies disrupt chromatin architecture and/or transcription (e.g., SMARB1 deletion/mutation in malignant rhabdoid tumor, MLL rearrangements in acute leukemia, and PAX3-FKHR in alveolar rhabdomyosarcoma). In Ewing sarcoma, the second most common type of primary bone cancer in children, the defining translocation fuses the EWSR1 gene to an ETS family member gene, most commonly FLI1, resulting in the oncoprotein EWS-FLI and an aberrant gene expression profile. While this translocation event was discovered approximately 20 years ago, current therapies do not directly target the fusion product. With the exception of nuclear hormone receptors, transcription factors have proven largely refractory to conventional drug discovery approaches. Our laboratory is taking a multi-pronged, integrative approach to discovering new therapeutic strategies for Ewing sarcoma, leveraging next-generation sequencing, functional genomic and proteomic studies, and chemical biology. We have co-led a collaborative effort to characterize the genomic landscape of childhood Ewing sarcoma tumors using next-generation sequencing approaches. One immediate and striking observation from this study is that Ewing sarcoma genomes are among the most quiet sequenced to date, with strikingly few samples demonstrating immediately actionable lesions. These results speak to the critical importance of understanding, and either directly targeting the expressed EWS-ETS fusion, or identifying synthetic lethal vulnerabilities in the presence of these fusion proteins. Toward this end, we have focused on the development and implementation of a panel of screening assays to identify new small-molecule modulators of EWS-FLI. We have completed a primary screen of over 350,000 compounds for those inhibiting the activity of an EWS-FLI reporter and a secondary counter-screen for selective killing of Ewing sarcoma cell lines. We are now focusing our attention on 10 promising hits to be studied in a panel of EWS-FLI-relevant assays. In parallel, we have conducted a near genome-wide shRNA screen in a panel of Ewing sarcoma cell lines to identify Ewing sarcoma cell vulnerabilities and are now integrating this data with our next-generation sequencing study and small-molecule screen. Finally, we took a proteomic approach to identify more easily targetable tyrosine kinase vulnerabilities in Ewing sarcoma tumors. Utilizing a Luminex fluorescent, bead-based technology we profiled activated tyrosine kinases using phosphorylation status as a surrogate for kinase activation. We identified focal adhesion kinase (FAK), a tyrosine kinase critical for cellular adhesion, growth, and survival, as a candidate target in Ewing sarcoma. In this study, we demonstrated that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by shRNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, shRNA-directed against FAK and small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo in two xenograft models of Ewing sarcoma. In collaboration with the NIH, we are now screening a library of clinically relevant compounds to identify highly active compound combinations with FAK inhibitors in Ewing sarcoma. Citation Format: Kimberly Stegmaier. Integrating genomic approaches to discover new therapeutic strategies for Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA32.

Abstract 3422: RNAi screening identifies FGFR4 as a modulator of growth and survival in Ewing sarcoma

Abstract Ewing sarcoma is the second most common cancer of bone and soft tissue arising in children and young adults. Standard treatment of Ewing sarcoma includes surgery, radiation, and chemotherapy largely consisting of combinations of non-targeted cytotoxic agents. Although the survival rate has improved for patients treated for localized disease, the survival rate of patients with metastatic tumor remains lower than 30%. In order to improve therapeutic options for Ewing Sarcoma, we employed a functional genomics approach based on RNA interference (RNAi) screening to identify genes whose silencing affected the proliferation and survival of Ewing sarcoma cells. Four Ewing sarcoma cell lines, TC-32, TC-71, SK-ES-1 and RD-ES, were transfected with a library of siRNA targeting 287 cancer-associated genes. The siRNA screening data were normalized and statistical cut-offs were determined for each cell line. From the list of siRNA ‘hits,’ we identified siRNAs targeting Fibroblast growth factor 4, FGFR4, to be among the most effective in inducing cytotoxicity in Ewing sarcoma cells. Further validation was conducted showing that siRNAs to FGFR4, but not FGFR1, FGFR2, or FGFR3, were able to significantly reduce Ewing Sarcoma cell viability. Furthermore, siRNA targeting FGFR4 were able to induce Caspase 3 activity. FGFR4 protein was shown to be expressed in Ewing sarcoma by western blot analysis, with expression levels lower than FGFR4 expression on rhabdomyosarcoma cells. Targeting FGFR4 activity in Ewing sarcoma cells using a pan-FGFR inhibitor, PD-173074, demonstrated that Ewing sarcoma cells were sensitive to FGFR inhibition. Since FGFR4 in a potential therapeutic target in rhadomyosarcoma, we compared the response of the Ewing sarcoma cells to PD-173074 to that of the rhabdomyosarcoma cell lines, RH-30 and RD. The IC50s of the four Ewing sarcoma cells ranged from 3.8-4.7 µM and were slightly lower than the IC50 for the rhabdomyosarcoma cell lines RH-30 (5.6 µM) and RD (7.9 µM). These results indicate that FGFR4 is a potential therapeutic target in Ewing sarcoma and that FGFR4 targeted therapy should be tested in preclinical xenograft models of Ewing sarcoma. Citation Format: David O. Azorsa, Irma M. Gonzales, Shilpi Arora, R. Tanner Hagelstrom, Tanya H. Little, Robert J. Arceci, Spyro Mousses. RNAi screening identifies FGFR4 as a modulator of growth and survival in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3422. doi:10.1158/1538-7445.AM2014-3422

Abstract 3962: PM01183 shows an improved therapeutic index relative to trabectedin and suppresses EWS/FLI1 activity at clinically achievable concentrations

Abstract BACKGROUND: Ewing sarcoma is a pediatric malignancy characterized by the fusion of the EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. It is widely known that Ewing sarcoma cells depend on the transcriptional program of EWS/FLI1 for cell survival. Therefore, the goal of this research is to develop and clinically translate small molecules that suppress EWS/FLI1 activity. We have previously reported that the natural product trabectedin suppresses EWS/FLI1 activity. In addition, the compound synergizes with irinotecan to suppress Ewing sarcoma tumor growth. However, the compound suffers from a narrow therapeutic index that limits the poisoning of EWS/FLI1 in patients. In this report, we show that the trabectedin analog PM01183 has improved targeting of EWS/FLI1 and preserved synergy with irinotecan. In addition, the compound is known to have a dramatically improved therapeutic index suggesting improved activity of this compound in the clinic. METHODS: In this report, we characterize the mechanism of suppression of EWS/FLI1 by trabectedin using confocal immunocytochemistry and chromatin immunoprecipitation. We show enhanced suppression of EWS/FLI1 by PM01183 using the Fluidigm platform and confirm the results with a luciferase reporter construct and high-content quantitative PCR. Finally, we tested the ability of PM01183 to suppress tumor growth in xenograft models of Ewing sarcoma both in the absence and presence of irinotecan. RESULTS: PM01183 more effectively suppressed the gene signature of EWS/FLI1 than the parent compound trabectedin. In addition, the drug re-localizes EWS/FLI1 away from target genes in the nucleus leading to suppression of these EWS/FLI1 targets. These results are consistent with the activity of the parent compound, trabectedin, that blocks binding of EWS/FLI1 to chromatin. More importantly, the drug causes a regression of a TC32 xenograft and markedly suppresses growth of a TC71 xenograft when combined with irinotecan. Finally, PM01183 has a substantially improved pharmacologic profile in patients in comparison to trabectedin suggesting that these effects are bio-achievable in patients. CONCLUSIONS: In comparison to trabectedin, we have shown that its structural analog, PM01183 shows enhanced suppression of an EWS/FLI1 gene signature and preserves the synergy with irinotecan that we previously reported. We suggest a mechanistic basis for this activity against EWS/FLI1 and show excellent activity against Ewing sarcoma xenografts. Together, these results suggest that the clinical translation of PM01183 as an EWS/FLI1 targeted therapy both alone and in combination with irinotecan is warranted. Citation Format: Matt Harlow, Nichole Maloney, Maria Jose Guillen Navarro, Maurizio D'Incalci, Carlos Galmarini, Pablo Manuel Aviles Marin, Patrick Grohar. PM01183 shows an improved therapeutic index relative to trabectedin and suppresses EWS/FLI1 activity at clinically achievable concentrations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3962. doi:10.1158/1538-7445.AM2014-3962

EWS‐FLI‐1 regulates the neuronal repressor gene REST, which controls Ewing sarcoma growth and vascular morphology

RE1-silencing transcription factor (REST), a neuronal repressor gene, regulates neuronal stem cell differentiation. Ewing sarcoma may originate from neural crest cells. In the current study, the authors investigated whether REST plays a role in the growth of this tumor. REST expression was determined by Western blot analysis and reverse transcription-polymerase chain reaction in 3 human Ewing sarcoma cell lines and 7 patient tumor samples. The role of REST in tumor growth and tumor vascular morphology was determined using a Ewing sarcoma xenograft model. Immunofluorescence staining, Hypoxyprobe, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to investigate the impact of REST on pericyte marker expression, hypoxia, and apoptosis in vivo. High levels of REST were expressed in all 3 human Ewing sarcoma cell lines and in 6 of the 7 patient tumor samples. Overexpression of EWS-FLI-1 in human mesenchymal stem cells and human neural progenitor cells was found to increase REST expression. Inhibition of EWS-FLI-1 using small interfering RNA decreased REST expression in human Ewing sarcoma cells. Inhibition of REST did not affect EWS-FLI-1, but significantly suppressed tumor growth in vivo, reduced the tumor vessel pericyte markers α- smooth muscle actin (SMA) and desmin, increased hypoxia and apoptosis in tumor tissues, and decreased the expression of delta-like ligand 4 (DLL4) and Hes1. Inhibition of REST suppressed tumor growth, inhibited pericyte marker expression, and increased tumor hypoxia and apoptosis. Because tumor vessel function has been linked to tumor growth and metastases, REST may be a new therapeutic target in patients with Ewing sarcoma.

Preclinical evaluation of the PARP inhibitor, olaparib, in combination with cytotoxic chemotherapy in pediatric solid tumors

Poly(ADP-ribose) polymerase (PARP) signals DNA damage and facilitates DNA repair. PARP inhibitors are being evaluated in cancers with defective DNA repair mechanisms or in combination with cytotoxic therapy or radiation. We evaluated the PARP inhibitor, olaparib, in combination with chemotherapy using in vitro and in vivo pediatric solid tumor models. The IC50 of olaparib alone and in combination with cytotoxic agents was determined in 10 pediatric solid tumor cell lines. Synergy was assessed using the combination index of Chou-Talalay. Olaparib alone and in combination with topotecan/cyclophosphamide was evaluated in xenograft models of Ewing sarcoma (RD-ES) and neuroblastoma (NGP). PAR activity was evaluated in cell lines and tumor lysates. Olaparib induced growth inhibition, median (range) IC50 = 3.6 (1-33.8) µM, and inhibited PAR activity in pediatric solid tumor cell lines. The addition of olaparib to DNA damaging agents resulted in additive to synergistic interactions. In RD-ES and NGP xenografts, olaparib inhibited PAR activity by 88-100% as a single agent and 100% when administered with cyclophosphamide/topotecan. Although the addition of olaparib did not antagonize the activity of cyclophosphamide/topotecan, clear evidence of synergy could not be demonstrated. In pediatric solid tumor cell lines, clinically achievable concentrations of single agent olaparib caused growth inhibition. Although the in vitro data demonstrated synergistic efficacy of olaparib when added to the camptothecins and alkylating agents, synergy was not discernible in vivo. Clinical trials of PARP inhibitors in combination DNA damaging agents are necessary to establish the role of PARP inhibitors in childhood cancer.

520 An Oral Formulation of YK-4-279 Effective in Xenograft Models of Ewing Sarcoma

520 An Oral Formulation of YK-4-279 Effective in Xenograft Models of Ewing Sarcoma

EWS/Fli‐1 chimeric fusion gene upregulates vascular endothelial growth factor‐A

Vascular endothelial growth factor (VEGF)-A plays an important role in the pathological angiogenesis that occurs in soft-tissue sarcoma and in about half of Ewing's sarcoma cases, where it is highly overexpressed. EWS/Fli-1 is considered to be a transcriptional activator and to play a significant role in tumorigenesis of Ewing's sarcoma. However, the relationship between EWS/Fli-1 and VEGF-A is still unclear. The aim of this research is to investigate the relationship between EWS/Fli-1 and VEGF-A, and to determine whether small interfering RNA (siRNA)-targeting of VEGF-A can be developed as a novel treatment for Ewing's sarcoma. Knockdown of EWS/Fli-1 using siRNA on a Ewing's sarcoma cell line (A673) suppressed VEGF-A expression, and transfection of EWS/Fli-1 into a human osteosarcoma cell line increased VEGF-A expression. To inhibit VEGF-A secretion from Ewing's sarcoma, we developed a chemically synthesized siRNA that targets VEGF-A. Transfection of the VEGF siRNA into the Ewing's sarcoma cell line significantly suppressed VEGF-A secretion by up to 98% in vitro, compared with a control. In vivo, we established Ewing's sarcoma xenograft models and performed intratumoral injection of the siRNA mixed with atelocollagen. We observed that the inhibition of tumor growth occurs in a dose-dependent manner. Histological examination revealed decreased microvessel density and morphological change around microvessels in the Ewing's sarcoma xenografts treated with the siRNA. It is considered that a combination of chemically synthesized siRNA that targets VEGF-A and atelocollagen might be a novel and effective option for treating Ewing's sarcoma that secretes VEGF-A.