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Supplementary Table S1 Sequences of primers used in the study.

Supplementary Figure S3 Single-cell imaging of HA-tagged TC32 cells treated with 2.5 nM trabectedin or lurbinectedin for 6 h shows EWS-FLI1 (green) localized into the nucleolus (red). DAPI (blue) staining of the nucleus.

Purpose: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients.Experimental Design: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma.Results: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo.Conclusions: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105–18. ©2016 AACR.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657–68. ©2016 AACR.

Table S1, PCR primers used in gene signature panel. Table S2, Evidence for selection of each gene as an EWS-FLI1 target. S3A Mean fold change in expression of NR0B1 as a function of GAPDH (2Î"Î"CT) following treatment with MMA or EC-8105. Data is the average of 3 independent experiments as measured by qPCR. S3B Mean fold change in expression of EWS-FLI1-induced targets as a function of GAPDH (2Î"Î"CT) for treatment with an siRNA targeted at the breakpoint of EWS/FLI1 for 24 h. Data is the average of 3 independent experiments as measured by qPCR S3C Mean fold change in expression of EWS-FLI1-suppressed targets as a function of GAPDH (2Î"Î"CT) for treatment with an siRNA targeted at the breakpoint of EWS-FLI1for 24 h. Data is the average of 3 independent experiments as measured by qPCR S4A: Structures and NSC numbers of mithramycin analogs S4A: Structures and NSC numbers of mithramycin analogs (cont'd). S4C. Allometric scaling estimates for dosing as a function of weight of an organism for mithramycin (MMA) and EC-8042. (see text for reference). S5A Pearson correlation analysis showing similar responses of the PPTP panel of cell lines for EC-8105 and EC-8042 relative to mithramycin. S5B IC50 values for cell lines in PPTP. Columns are all cell lines tested, exclusion of rhabdoid tumor (minus rhabdoid) and exclusion of rhabdoid tumor and acute lymphoblastic leukemia cell lines (minus rhabdoid and ALL). The statistics are shown in boxes at the bottom. S5C Graph of IC50 as a function of p53 status. Cell lines with wild-type p53 (P53 wt) were more sensitive to treatment with mithramycin (MMA) and EC-8105 than lines with mutant p53 (P53 Mut) across the entire PPTP panel of cell lines independent of histology. Note there was no difference with the non-DNA damaging analogy, EC-8042. S6 Prediction plot showing mean tumor volume as a function of days of treatment with EC-8105 (1.0 mg/kg IP, M/W/F schedule, 8 doses). Thick, dotted lines represent mean tumor growth and the individual lines in the back represent the individual tumor growth by cohort. S7 Weight of each mouse in cohort of EC-8105 treated mice (black lines) treated on a 1.5 mg/kg/dose IV Q3D X 8 doses schedule relative to weight change of control mice (grey line). S8 Survival curves for control mice (black) relative to EC-8042-treated mice (grey) treated at 24 mg/kg dose X 8 either intravenously (S8A) or intraperitoneal (S8B). Arrows indicate end of treatment. S9. Response of mice treated with 24 mg/kg/dose EC-8042 IP M, W, F X8 (S9A). Regression of tumors for every mouse mouse in the cohort. Each line represents an individual mouse. Asterisks (*) highlight treatment days. S9B Mean tumor volume (+/- SEM) of every mouse in the control cohort (3177 mm3 +/- 308.2) relative to the EC-8042-treated cohort (422.5 mm3 +/- 48) on day 11 of treatment (P < 0.0001). S10: Suppression of NR0B1 staining in cells treated with 1.5 mg/kg EC-8105 IV, 48 hours after treatment. Three representative sections are shown stained for NR0B1 (red) or DAPI (blue) since the tumors were large and showed a range of staining.

Legends for supplementary figures and tables.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1(−/−)) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1(−/−) neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1(−/−) neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1(−/−) neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1(−/−) neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9-/- and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9-/- mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9-/- mice accumulated significantly more IL-13+IL-5+CD4+ T-helper type 2 cells. S100A9-/- mice also accumulated a significantly lower proportion of CD4+ T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4+ T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4+ Treg cell function.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H(2)O(2) stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Landscape disturbances can alter habitat structure and resource availability, often inducing physiological responses by organisms to cope with the changing conditions. Quantifying the endocrine stress response through measurement of glucocorticoids has become an increasingly common method for determining how organisms physiologically respond to challenges imposed by their environment. We tested the hypothesis that Eastern Fence Lizards cope with fire disturbance effects by modulating their secretion of corticosterone (CORT). We measured the baseline and stress-induced plasma CORT of male Eastern Fence Lizards in a chronosequence of fire-altered habitats (recently burned, recovering from burn, and unburned). Although habitat use by lizards differed among burn treatments, including differences in use of canopy cover, leaf litter, and vegetation composition, we did not detect a significant effect of fire-induced habitat alteration on plasma CORT concentration or on body condition. In addition, we found no effect of blood draw treatment (baseline or stress-induced), body temperature, body condition, or time taken to collect blood samples on concentration of plasma CORT. Low intensity burns, which are typical of prescribed fire, may not be a sufficient stressor to alter CORT secretion in Eastern Fence Lizards (at least during the breeding season). Instead, lizards may avoid allostatic overload using behavioral responses and by selecting microsites within their environment that permit thermoregulatory opportunities necessary for optimal performance and energy assimilation.

Zinc (Zn) is an essential trace metal required for all forms of life, but is toxic at high concentrations. While the toxic effects of high levels of Zn are well documented, the mechanism of cell death appears to vary based on the study and concentration of Zn. Zn has been proposed as an anti-cancer treatment against non-small cell lung cancer (NSCLC). The goal of this analysis was to determine the effects of Zn on metabolism and cell death in A549 cells. Here, high throughput multi-omics analysis identified the molecular effects of Zn intoxication on the proteome, metabolome, and transcriptome of A549 human NSCLC cells after 5 min to 24 h of Zn exposure. Multi-omics analysis combined with additional experimental evidence suggests Zn intoxication induces ferroptosis, an iron and lipid peroxidation-dependent programmed cell death, demonstrating the utility of multi-omics analysis to identify cellular response to intoxicants.

Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.

Purpose: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. Experimental Design: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. Results: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo. Conclusions: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105–18. ©2016 AACR.

Ewing sarcoma is a bone and soft-tissue tumor that depends on the activity of the EWS-FLI1 transcription factor for cell survival. Although a number of compounds have been shown to inhibit EWS-FLI1 in vitro, a clinical EWS-FLI1-directed therapy has not been achieved. One problem plaguing drug development efforts is the lack of a suitable, non-invasive, pharmacodynamic marker of EWS-FLI1 activity. Here we show that 18F-FLT PET (18F- 3′-deoxy-3′-fluorothymidine positron emission tomography) reflects EWS-FLI1 activity in Ewing sarcoma cells both in vitro and in vivo. 18F-FLT is transported into the cell by ENT1 and ENT2, where it is phosphorylated by TK1 and trapped intracellularly. In this report, we show that silencing of EWS-FLI1 with either siRNA or small-molecule EWS-FLI1 inhibitors suppressed the expression of ENT1, ENT2, and TK1 and thus decreased 18F-FLT PET activity. This effect was not through a generalized loss in viability or metabolic suppression, as there was no suppression of 18F-FDG PET activity and no suppression with chemotherapy. These results provide the basis for the clinical translation of 18F-FLT as a companion biomarker of EWS-FLI1 activity and a novel diagnostic imaging approach for Ewing sarcoma.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657–68. ©2016 AACR.

Ewing sarcoma (ES) is a highly aggressive cancer of the bone and soft tissue. In ∼85% of ES tumors the primary oncogenic event is a t(11:22)(q24:q12) translocation that generates a fusion of the 5′ end of EWSR1 and the 3′ end of FLI1 referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. The most common EWS-FLI1 transcripts fuse either exon 7 of EWSR1 to exon 6 of FLI1 (a type I or a 7/6 fusion), or fuse exon 7 of EWSR1 to exon 5 of FLI1 (a type II or 7/5 fusion). In an estimated 40% of EWS-FLI1 driven tumors the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. Using an assay of EWS-FLI1 activity and genome-wide siRNA screening we have identified RNA processing as a therapeutic vulnerability in ES. Parallel genome-wide siRNA-mediated RNAi screens were conducted in ES TC32 cell lines expressing a luciferase (luc) reporter protein driven by either the promoter of the EWS-FLI1 target gene NR0B1 (TC32-NR0B1-luc) or the CMV promoter (TC32-CMV-luc). The top gene ontology terms associated with the 28 priority candidate genes that when silenced induced a differential decrease in the TC32-NR0B1-luc signal versus the TC32-CMV-luc signal were mRNA splicing (p-value = 1.42E-08) and mRNA processing (p-value = 2.32E-07). To investigate the mechanistic basis for the identification of specific RNA processing proteins as required for the activity of EWS-FLI1 we focused on two lead candidate genes, the heterogeneous nuclear ribonucleoprotein H1, HNRNPH1, and the core splicing factor, SF3B1. Using PCR analysis we determined that HNRNPH1 is required for the splicing of EWS-FLI1 fusion transcripts expressed in ES cells in which the chromosome 22 breakpoint retains EWSR1 exon 8, specifically in TC32 and SKNMC ES cells. We also show ES cell lines harboring 7/ 6 (TC32, SKNMC, and TC71) or 7/ 5 (RD-ES) EWS-FLI1 fusions are all sensitive to the loss-of-function of SF3B1. Quantitative RT-PCR, immunoblot, and whole transcriptome analysis show that disrupted splicing of the EWS-FLI1 transcript alters its expression and reverses the expression of a significant proportion of genes that are targets of EWS-FLI1. These observations were confirmed in four ES cell lines using the splicing inhibitor Pladienolide B. Our results provide the basis for a novel strategy to target fusion oncogenes by interfering with RNA processing. This study has implications for the treatment of ES through inhibition of proteins required for expression of the EWS-FLI1 transcript and identifies a candidate lead compound for further clinical development. Our findings may also open up strategies for treatment of other cancers driven by fusion oncogenes. Citation Format: Patrick J. Grohar, Suntae Kim, Sara Haddock, Guillermo Rangel Rivera, Matt Harlow, Nichole K. Maloney, Konrad Huppi, Kristen Gehlhaus, Magdalena Grandin, Carleen Klumpp-Thomas, Eugen Buehler, Lee J. Helman, Scott E. Martin, Natasha J. Caplen. Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma. [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 479. doi:10.1158/1538-7445.AM2015-479

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

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

BACKGROUND: Ewing sarcoma is a pediatric malignancy characterized by the fusion of the EWS and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. The transcriptional program of EWS/FLI1 drives malignant transformation as well as high-risk features such as increased angiogenesis, resistance to apoptosis, and increased metastatic potential. It is well known that perturbing the transcriptional activity of EWS/FLI1 leads to cell death in vitro and should be considered as a possible site of therapeutic intervention in Ewing sarcoma patients. To that end, we identified ET-743 (trabectedin, Yondelis), a natural product, which inhibits EWS/FLI1 activity in vitro. Using ET-743 as a lead compound, we have characterized a number of structural analogs of ET-743 to identify more potent and/or more selective inhibitors of EWS/FLI1 activity. METHODS: We evaluated the potency of the ET-743 structural analogs using MTS viability assays using Ewing sarcoma, alveolar rhabdomyosarcoma, and osteosarcoma cell lines. The specific inhibition of EWS/FLI1 downstream transcriptional activity was measured using a luciferase-based approach and western blotting. We also evaluated each analog's ability to suppress a gene signature of EWS/FLI1, which included more than 20 genes known to be deregulated by EWS/FLI1 using a novel microfluidic based assay and the Fluidigm platform. RESULTS: Several analogs proved to be more potent than ET-743 and selective for transcriptional suppression of EWS/FLI1. All of the analogs induced varying degrees of DNA damage and cell death, which were detected by biochemical and metabolic assays. We also show that the analogs sensitize ES cells to treatment with camptothecins to a variable degree based on analog relative to the parent compound. CONCLUSIONS: In comparison to ET-743, we have shown that its structural analogs confer better pharmacological properties such as increased potency and specificity towards Ewing sarcoma cell lines. We are currently working to further characterize the mechanism of action for these analogs and evaluate the compounds in an in vivo model. The ultimate goal would be to translate these compounds to the clinic and improve patient outcomes with Ewing sarcoma. Citation Format: Matt Harlow, Nichole Maloney, Laura Segars, Girma Woldemichael, Lee J. Helman, Patrick J. Grohar. Identification of ET-743 analogs with improved selectivity and potency for EWS-FLI1 and Ewing sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2013-2760

SummaryEwing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.