TC71 Cells Research Articles

Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

Abstract 2855: ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2855.

Abstract 1468: MYC-C interacts with Aurora kinase A in Ewing sarcoma cells

Abstract EWSR1-ETS fusion genes result in upregulation and high expression of the oncogene MYC-C in Ewing sarcoma (ES). MYC-C is therefore an attractive therapeutic target in these aggressive cancers with limited treatment options. Unsuccessful efforts to target MYC-C proteins directly have resulted in the development of inhibitors to associated stabilizing proteins, such as Aurora kinase A (AURKA), to induce proteasomal degradation1. In this study we test the hypothesis that MYC-C and AURKA interact in ES. Peptide pull-down assays were performed using Strep-Tactin Sepharose beads and N-biotinyl MYC-C peptides. After incubation with AURKA, eluted proteins were analyzed by SDS-PAGE. Intracellular colocalization of proteins was examined by immunofluorescence of TC-32 and SK-N-MC cell lines using confocal microscopy (Zeiss LSM880) and high content imaging (CD7). Cells were stained with anti-MYC-C and anti-AURKA antibodies, and DAPI used to visualize nuclei. DuoLink® proximity ligation assays (PLA; Sigma) were performed on TC-32 and SK-N-MC cell lines. Images were captured on the confocal microscope and the number of nuclear foci quantified.MYC-C binds AURKA through its transcriptional activation domain (residues 1-155). Residues 10-34 of MYC-C are sufficient for binding with AURKA (residues 122-403) in cell-free peptide assays. Colocalization of MYC-C and AURKA was visualized in mitotic SK-N-MC cells, but not in TC-32 cells. The proteins colocalize at the spindle poles. PLA confirmed an interaction between MYC-C and AURKA in TC-32 and SK-N-MC ES cells. The number of foci per nucleus was significantly higher in the SK-N-MC p53-null cells compared to the wild-type p53 TC-32 cells (p<0.0001). In conclusion, MYC-C and AURKA colocalize and interact in the nucleus of ES cells. The frequency of interactions is greater in p53-null cells compared to cells with wild-type p53. Peptide pull-down assays identified residues 10-34 of MYC-C as the minimal AURKA interacting region. Disruption of this interaction by AURKA inhibitors may provide a mechanism for MYC-C degradation in ES, which could be exploited to develop novel therapeutic combinations to improve outcomes. 1Richards MW, Burgess SG, Poon E, Carstensen A, Eilers M, Chesler L, Bayliss R. Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc Natl Acad Sci U S A. 2016 ;113(48):13726-13731. Citation Format: Molly McNae, Selena G. Burgess, Richard Bayliss, Susan A. Burchill. MYC-C interacts with Aurora kinase A in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1468.

Abstract 1469: Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription

Abstract EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and regulates transcription. Biochemical and depletion studies have demonstrated that EWSR1 regulates basal transcription, and that in Ewing sarcoma (EWS) cells expressing the fusion oncoprotein EWSR1::FLI1, EWSR1 may contribute to the deregulation of gene expression observed in this cancer. To further study EWSR1’s function in Ewing sarcoma, we generated reporter cell lines to visualize and quantify its endogenous expression within EWS cells. To generate EWSR1-fluorescent reporter A673 and TC-32 EWS cell lines, we used CRISPR-Cas9 and sgRNAs targeting the 5’ end of the first exon of EWSR1 and a donor plasmid consisting of the mNeonGreen (mNG) reporter gene, a G4S3 linker, and EWSR1 homology arm templates. Upon validation of successfully modified clones (A673 and TC-32), we used confocal microscopy to confirm EWSR1’s localization to the nucleoplasm. Also, consistent with previous analysis of EWSR1’s protein-protein interactions, we confirmed using immunoprecipitation that the mNG-EWSR1 protein retains its interaction with EWSR1::FLI1 and RNA polymerase II (RNA pol II) - total and phosphorylated (pS5 and pS2). FRAP analysis (Zeiss Airyscan) showed that in cells, EWSR1 exists in two fractions, one that rapidly exchanges (A673 T½: 9.47 secs; TC-32 T½: 6.13 secs) and one that remains bound for a longer duration (A673 T½: 92.15 secs; TC-32; T½: 52.63 secs). We next used structured illumination microscopy to interrogate EWSR1’s spatial organization in EWS cells at a high resolution (~100 nm). First, we determined that EWSR1’s organization within the nucleoplasm is regular as measured by a signal-to-signal distance of ~300 nm in both cell lines (A673 cells 297±6.71 nm; TC-32 cells 304±8.59 nm). Analysis of EWSR1’s spatial organization relative to nucleic acids and proteins that function in gene expression indicates that EWSR1 partially co-localizes with DNA (visualized using DAPI), Histone 3, BRD4 and MED1 (visualized using immunofluorescence - IF). In contrast, we observed EWSR1 colocalizes completely with nascent RNA (visualized using Click-IT RNA Alexa Fluor 488, Invitrogen) (Manders overlap coefficient (MOC) A673: 0.96±0.005; TC-32: 0.97±0.005). Furthermore, we detected over 90% colocalization of concentrated EWSR1 signals (defined by specific fluorescent intensities) with RNA pol II (IF) in states associated with transcriptional initiation (pS5) (MOC A673: 0.94±0.011; TC-32: 0.93±0.02) and elongation (pS2) (MOC A673: 0.95±0.02; TC-32: 0.97±0.06). These studies will aid analysis of changes in EWSR1’s localization within the nucleus and relative to proteins that regulate gene expression following inhibition of transcription, including the application of compounds that disrupt EWSR1::FLI1’s transcriptional activity. Citation Format: Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Patrick Pichling, Katelyn R. Ludwig, Raj Chari, Michael Kruhlak, Jadranka Loncarek, Natasha J. Caplen. Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1469.

Abstract 2015: The Aurora kinases are a potential therapeutic target in Ewing sarcoma

Abstract The poor survival rates and limited treatment options for patients with relapsed/metastatic Ewing sarcoma (ES) highlights the need for more personalized targeted therapeutic approaches. In this project, we have investigated the prognostic potential of Aurora kinase expression in ES patients and evaluated the activity of Aurora kinase inhibitors in ES cell lines and patient derived cultures. The prognostic value of Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Aurora kinase C (AURKC) was determined through interrogation of the online GSE17618 dataset using Cox models and Kaplan Meier plots. RNA seq data was obtained from the CCRG dataset for expression analysis (Roundhill et al., 2021, Cell Oncol, 44(5), 1065-85). Viable cell number was determined using the trypan blue exclusion assay on the Vi-CELL XR. Cells were treated with increasing concentrations of inhibitor (Table 1) or DMSO vehicle control for 48h. Protein was extracted after 48h for western blotting. In ES patients, high RNA expression of AURKA and AURKB is associated with a 5- and 3-fold increased risk of death respectively (n=42). Risk of an event was increased 3- or 2-fold with high expression of AURKA or AURKB respectively (n=42). High AURKC expression was not prognostic. Expression of AURKA and AURKB was confirmed at the RNA level in ES cell lines and patient derived cultures, and at the protein level in cell lines. Aurora kinase inhibitors (Table 1) decreased viable cell number of ES cell lines and patient derived cultures. Western blotting confirmed a decrease in AURKA and MYC-C expression with increasing concentration of inhibitor. AMG900 was the most potent inhibitor in TC32 cells (Table 1) and decreased viable cell number of patient derived cultures (n=5, p<0.01). High expression of AURKA or AURKB mRNA predicts poor outcome for ES patients. Inhibitors of these kinases reduce viable cell number of both ES cell lines and patient derived cultures. We are currently investigating the mechanism of action. Table 1. Summary of the effect of Aurora kinase inhibitors in TC32 ES cells. Aurora Kinase Inhibitor Concentration Range Used (nM) Target Kinases(IC50, nM) EC50 TC32 Cells (nM) (n=3) P value (EC50 AMG900 vs EC50 another inhibitor) References AMG900 1.25-50 AurA (5), AurB (4), AurC (1) + 10 kinases 1.57 N/A Payton et al., 2010, Cancer Res, 70(23), 9846-54 MLN8237 5-300 AurA (1.2), AurB (396.5) + 22 kinases 29.48 0.0468 Manfredi et al., 2011, Clin Cancer Res, 17(24), 7614-24 CCT137690 5-300 AurA (15), AurB (25), AurC (19) + 3 kinases 97.56 0.0004 Bavetsias et al., 2010, J Med Chem, 53(14), 5213-28 LY3295668 31.25-1000 AurA (0.8) AurB (1038)AurC (98) + 2 kinases Not able to calculate N/A Gong et al., 2019, Cancer Discov, 9(2), 248-263 EC50 values were calculated by nonlinear regression (GraphPad PRISM). Statistical differences between EC50 of AMG900 and the EC50 of other inhibitors were evaluated using two-way ANOVA and Tukey’s multiple comparisons test. IC50 - half maximal inhibitory concentration, EC50 - half maximal effective concentration Citation Format: Molly McNae, Elizabeth A. Roundhill, Susan A. Burchill, Richard W. Bayliss. The Aurora kinases are a potential therapeutic target in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2015.

Abstract 87: EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma

Abstract Background: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R) has been implicated in EWS tumorigenesis and development of metastatic disease, with anti-IGF1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF1R-driven oncogenesis in EWS could provide avenues for more effective therapy. Intracellular trafficking plays a central role in regulating RTK expression and signaling, and this pathway is frequently deregulated in cancer cells. The EPS15 homology domain-containing (EHD) proteins regulate intracellular traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Study design: Three EHD1-expressing EWS cell lines, TC71, SKES1, and A673, were engineered with doxycycline-inducible EHD1 shRNAs to assess the impact of EHD1 knockdown (KD) on in vitro oncogenic properties. Stable EHD1-Crispr-KO and mouse EHD1-rescue TC71 and A673 cell line models were developed and characterized. Further, non-targeting control or EHD1-KD TC71 cells engineered with RFP-luciferase reporter were implanted in the tibia of nude mice in an orthotopic xenograft model and tumor growth was monitored by IVIS imaging. Results: EHD1-KD led to a significant impairment of EWS cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation in vitro and reduced tumor growth in nude mice. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF1R levels upon EHD1-KD, identifying IGF1R as a potential target of regulation by EHD1. Western blotting showed a reduction in total IGF1R levels, and flow cytometric and immunofluorescence analyses revealed a pronounced decrease in the cell surface IGF1R levels upon EHD1 KD/KO in EWS cell lines. EHD1-KO EWS cell lines also exhibited a defect in IGF1-dependent migration and proliferation, and the phenotypes were restored by mouse EHD1 rescue. IGF1R and EHD1 were found to colocalize intracellularly and to co-immunoprecipitate after IGF1 stimulation. Finally, EHD1 depletion was found to impair the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Conclusion: Our studies indicate a novel requirement of the intracellular traffic regulator EHD1 in sustaining IGF1R mediated oncogenesis in EWS. Future studies will aim to evaluate dual targeting of EHD1 and IGF1R in EWS. Citation Format: Sukanya Chakraborty, Bhopal C. Mohapatra, Sameer Mirza, Aaqib M. Bhat, Matthew D. Storck, Isidro Machado, José Antonio López, Antonio Llomart Bosch, Donald W. Coulter, Gargi Ghosal, Vimla Band, Hamid Band. EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 87.

Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for anti-cancer drug development

AimsCharacterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development. Main methodsCBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively. Key findingsqRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [3H]WIN-55,212-2, but not CB1/CB2R radioligand [3H]CP-55,940. Homologous receptor binding demonstrated that [3H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [3H]WIN-55,212-2, whereas two ligands enhanced [3H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [3H]WIN-55,212-2 displacement exhibited high nanomolar affinity (Ki) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity. SignificanceCannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS.

Abstract 1294: Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma

Abstract Background. PDK1, is a master activator of multiple prosurvival and oncogenic protein kinases such as AKT, PKC, RSK and SGK families in oncology[1]. On the other hand, overexpression and aberrant activation of Aurora-A kinase (AurA) have been functionally linked to oncogenic transformation mainly through development of centrosome amplification and chromosomal instability[2]. In this study, the molecular mechanisms at the simultaneous PDK1/AurA inhibition using OXID-pyridonyl[3] compounds were explored in panel of tumor cell lines carrying different oncogenic mutations (PI3KCA, K-RAS, PTEN, RB-1, EGFR and TP53). Materials and Methods. Established human cancer cell lines from breast, glioblastoma, renal prostate, colon, lung and Ewing sarcoma were studied. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72h-exposure with increasing concentrations (from 0.014 to 30 µM). Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds to investigate the functional crosstalk between the two pathways in sensitive cancer cell lines. Results. We assessed anti-proliferative activity of single-agents SA16, IB35, VI8 and VI18 in a panel of 15 cancer cell lines and observed that three Ewing sarcoma and one colon carcinoma cell lines were sensitive (6647,TC71, SKNMC and HCT116), with IC50s values between 2.6 and 25 µM after 72h-exposure, whereas all the studied cell lines were resistant to IB35 and VI18. These results were confirmed by soft agar clonogenic formation studies. AurA, PDK-1, p-PDK1 ser241, AKT, p-AKT ser473 protein levels were characterized but no differences in their basal levels were observed between OXID-pyridonyl compounds -sensitive or -resistant cell lines. SA16 effect at the protein level was evaluated after 5 to 120 min in sensitive cell lines. In SKNMC, TC71 and HCT116 cells, we didn't observe a modulation of AurA or p-PDK1 ser241 while a decrease of p-AKT was observed in SKNMC and HCT116 cells. In addition, a decrease of the p-p44/42 MAPK was observed after 30 min-exposure in SKNMC cells. On the other hand, p-RB1, p-Cdc2, P21 and C-MYC were not regulated at the protein level with SA16 treatment up to 120 min-exposure. In resistant cell lines, U87MG and PC3, both p-PDK1 ser241 and AurA levels were not modulated up to 2h of treatment with SA16. Conclusion. Here we report the characterization of two potent first-in-class dual PDK1-AurA inhibitors, SA16 and VI8. Our findings suggest that innovative PDK1/AurA dual-target molecules which could represent an attractive lead scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma. [1] Nagashima K et al. J Biol Chem. 2011. [2] Yan M et al. Med Res Rev. 2016. [3] Sestito, S et al. Proceedings 2019. Citation Format: Maria Eugenia Riveiro, Simona Rapposelli, Samuel Huguet, Ramiro Vazquez, Mohamed Bekradda, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai. Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1294.

Natural and Synthetic Cannabinoids Reduce Cell Viability of Ewing Sarcoma TC‐71 Cells Potentially via Non‐canonical CB receptors

Ewing Sarcoma (EWS) is the second most common bone tumor in children, characterized by rapid tumor growth and extensive bone destruction. Treatment for EWS is multimodal: surgery, radiation, and chemotherapy. However, these approaches are effective in only 70% of the patients with localized disease and thus new drugs are needed. WIN-55,212-2 (WIN-2), a synthetic cannabinoid in the aminoalkyindole (AI) class, has been shown to slow tumor growth in several types of cancer and human Kaposi sarcoma via cannabinoid (CB) and non-CB mechanisms. Therefore, we hypothesize that WIN-2 and other synthetic CBs will exhibit anti-proliferative effects in EWS TC-71 cells via CB receptors. Competitive binding screens using TC-71 membranes showed that binding of the nonselective CB1/CB2 radiolabeled agonist [3H]CP-55,940 could not be displaced by selective CB1 or CB2 ligands, or even non-radioactive CP-55,940 itself. However, binding of a second nonselective CB1/CB2 radiolabeled agonist [3H]WIN-2 could be displaced with a CB1 but not CB2 antagonist, and non-radioactive WIN-2 itself. This suggests that TC-71 cells express a novel non-canonical CB binding site (WIN-2 site). Subsequent homologous receptor binding studies with [3H]WIN-2 showed that TC-71 cells do indeed express a WIN-2 site at a density (Bmax) of 3.7 +/- 1.6 pmole/mg, to which WIN-2 binds with an affinity (Ki) of 4.5 +/- 0.6 nM. Next, thirty-eight different synthetic CBs were screened for potential binding to WIN-2 receptors in TC-71 cells. Only four compounds exhibited a high affinity for the WIN-2 receptor in the low to the mid-nanomolar range. Interestingly, four other compounds increased [3H]WIN-2 binding, indicating that these ligands might act as positive allosteric modulators of the WIN-2 site. When compounds with the highest affinity were examined in G-protein activation assays for functional activity, two of them reduced [35S]GTPgS binding below basal levels indicating that they may act as inverse agonists at the novel WIN-2 site. Most importantly, WIN-2 and all four compounds with high affinity for the WIN-2 site produced a significant time-dependent reduction in viability of EWS TC-71 cells as measured by trypan blue exclusion and lactate dehydrogenase (LDH) release. In conclusion, WIN-2 and other synthetic CBs reduce EWS cell viability potentially via a non-canonical CB binding site. These results suggest that the WIN-2 site might be a novel therapeutic target for drug development to treat EWS and perhaps other types of cancer as well.

MiR-145 suppresses epithelial-mesenchymal transition by targeting stem cells in Ewing sarcoma cells.

We aimed to examine the performance of stem cell markers and epithelial-mesenchymal transition (EMT) process in miR-145 transfected EWS cells (TC71, TC106). EWS cells were utilized for functional analysis of mir-145. Proliferation, migration, invasion and soft agar colony assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay was used to investigate the underlying reasons of proliferative inhibition in cells in whichmiR-145 is overexpressed. QRT-PCR was used to determine the role of miR-145 in EMT transcription markers and mir-145 targeted genes, KLF4, SOX2 and OCT4 expression levels. The miR-145 expression has been shown to be down-regulated in EWS. The miR-145 overexpression caused inhibition of proliferation and reduced migration in EWS cells through induction of apoptosis. Mir-145 suppresses EMT capacity and SOX2, KLF4 and OCT4 expression levels. This is the first time in the literature we have shown deregulation of miR-145 inhibits EMT process by targeting stem cell properties leading to the inhibition of tumor growth and metastasis in TC71 and TC106 cells. Based on these results, we propose that miR-145, as an important regulator of SOX2, KLF4 and OCT4 carries crucial roles in EWS tumorigenesis and EMT (Tab. 1, Fig. 4, Ref. 26).

Abstract 6594: Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells

Abstract Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo. Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.

Abstract 6158: The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells

Abstract Background: Ewing sarcoma (ES) is a developmental bone/soft tissue neoplasia, for which mesenchymal stem cell (MSC) is the putative cell of origin. Although the outcome of ES patients bearing primary tumors has improved significantly, patients with metastatic ES still have a poor prognosis. The MSC marker endoglin/CD105 (ENG) and the ENG-shedding protease MMP14 have been, respectively, described as cell migration and invasion regulators. In our attempt to better understand the mechanism of ES progression, we are focusing on the role of ENG and MMP14 in ES cells. Methods: In vitro ENG-knockdown (ENG-KD; RM82 and SKNMC cell lines) and ENG-overexpressing (pENG; TC71 cell lines) models were generated by shRNA technology and pDisplay vectors, respectively, and confirmed by qRT-PCR, western blotting and flow cytometry. In vitro MMP14-knockout (MMP14-KO; RM82 and SKNMC) models were generated by CRISPR-Cas9 and confirmed by western blotting and Sanger sequencing. Transwell migration/invasion, adhesion, clonogenicity and proliferation assays were performed in vitro. Actin filament organization was evaluated by immunofluorescence. Extracellular vesicles (EVs) were isolated by ultracentrifugation and density gradient separation. Transcriptomic profiling was performed by Clarion S Human arrays followed by Gene Set Enrichment Analysis. HiPathia tool was used to recode the transcriptomic data into measurements of cell activity by applying mechanistic models. Reverse Phase Protein Array (RPPA) analysis was evaluated on the ENG-KD RM82 model. ENG-targeting immunoprecipitations (IP) from wild type RM82 protein extracts were analyzed by mass spectrometry (MS). Results: The impaired clonogenic potential of ENG-KD RM82 and SKNMC models suggests a role of ENG on the stemness capacity of ES, without affecting the proliferation rate. The reduced migratory and adhesive phenotype of the ENG-KD RM82 model and the transcriptomic profiling of both ENG-KD RM82 and SKNMC cells, suggest a role of ENG in ES cell migration and adhesion. The altered organization of actin filaments in ENG-KD and pENG models could support a role of ENG in actin filament remodeling. The transcriptomic profiling of ENG-KD models and the RPPA results underscore the regulation of FAK signaling by ENG. The IP-MS analysis of ENG partners identifies Integrin-Linked Kinase-Associated serine/threonine Phosphatase (ILKAP) as one of the main interactors of ENG in RM82 cells, suggesting its possible association with the focal adhesion member ILK. ENG is enriched in EVs from different ES cell lines. The transcriptomic profiling of MMP14-KO SKNMC cells highlights the involvement of MMP14 activity in the extracellular matrix organization of ES cells. Conclusion: ENG favors a pro-migratory phenotype in ES cells. These findings help to better understand the molecular landscape of tumoral cells from ES patients, and might have potential clinical implications. Citation Format: Pilar Puerto-Camacho, Ana Teresa Amaral, Juan Diaz-Martin, Alfonso Bolado-Carrancio, Carmen Jordan-Perez, Carmen Salguero-Aranda, Marina Esteban-Medina, Inmaculada Alamo-Alvarez, Joaquin Dopazo, Carmelo Bernabeu, Adam Byron, Valerie G. Brunton, Enrique de Álava. The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6158.

Abstract A41: Novel mithramycin analogues with improved pharmacologic profile and efficacy in ETS transcription factor-driven tumors

Abstract Introduction: Chromosomal translocations of ETS family transcription factors (TF) are found in Ewing sarcoma, prostate cancer, and leukemia and lead to expression of aberrant ETS transcription factors driving tumorigenesis. Mithramycin (MTM) inhibits EWS-FLI1, the most common ETS-related TFs in Ewing sarcoma through interference at its DNA binding sites on promoters. However, MTM has a narrow therapeutic window marked by severe liver and hematologic toxicities and poor pharmacokinetic (PK) properties, as demonstrated recently in a pediatric clinical trial with Ewing sarcoma patients. Here, we sought to develop analogues with specificity toward ETS family TFs and improved pharmacologic properties. Methods: Guided by crystal structures of MTM/DNA/ETS, we used semisynthetic approaches to prepare a library of analogs by substitution on the 3-side chain of MTM-SA and MTM (analogs). To select analogs with specific activity toward cells with ETS-related chromosomal translocations, analog cytotoxicity was determined in TC32 cells before and after EWS-FLI1 silencing. To confirm selectivity, analogs were tested in a panel of Ewing sarcoma cell lines (n=8) expressing EWS-ETS translocations and compared to a panel of non-Ewing cell lines (n=9) that do not express these translocations. CETSA, FRET, and luciferase reporter assays were used to evaluate interactions with EWS-FLI1 and SP1 proteins and gene promoter regions they bind. Inhibition of select transcriptionally regulated genes was determined by qRT-PCR and immunoblotting. Select analogs were tested in vivo to assess PK in mice and primates, identify the maximum tolerated dose (MTD), and determine dose-dependent efficacy in mouse xenograft models. Results: MTM analogs had higher GI50 in silenced EWS-FLI1 cells and > 10-fold lower GI50 in ETS-dependent cell lines vs. non-ETS dependent cancer cells, as compared to ~3-fold for MTM. Target engagement was demonstrated in dose-dependent manner using a luciferase reporter TC32 cell line under control of the NR0B1 promoter, which is regulated by EWS-FLI1, and CETSA experiments showed increased physical interaction of the analogs with EWS-FLI1 as compared to MTM. Treatment with MTM and analogs produced comparable dose-dependent displacement of an ETS DNA binding domain from GGAA (microsatellite-like) DNA fragments as demonstrated by FRET. Exposure to analogs led to concentration-dependent downregulation of EWS-FLI1 and Sp1 regulated genes. In vivo studies showed that MTM analogs attained higher exposure at their respective MTD and had lower clearance in mice and primates. Unlike MTM, analogs reversed tumor growth and increased survival at the MTD and 2/3 of MTD. Conclusion: We demonstrated that MTM analogs are selectively more cytotoxic in tumor cell lines dependent on ETS family TFs and have vastly improved PK properties as confirmed in a primate model. Given their effectiveness at 2/3 of the MTD, they are expected to have an improved therapeutic window. Citation Format: Markos Leggas, Joseph Eckenrode, Yang Liu, Abhisek Mandal, Reiya Hayden, Oleg Tsodikov, Jon Thorson, Jurgen Rorh. Novel mithramycin analogues with improved pharmacologic profile and efficacy in ETS transcription factor-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A41.

Abstract 3661: GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells

Abstract Ewing sarcoma (EWS) is characterized by TET-ETS fusions, most commonly the EWSR1-FLI1 fusion. GLI1, a transcription factor in the Hedgehog (HH) signal transduction pathway and target of canonical HH signaling, is a direct transcriptional target of EWSR1-FLI1. A role for the HH signaling pathway and GLI1 in acquisition of a multidrug resistance phenotype has been previously observed in several human cancers. The role of the HH signal transduction pathway and GLI1 in the biology of EWS remains incompletely understood. We hypothesize that the majority of EWS cell lines express GLI1 based on non-canonical up-regulation by EWSR1-FLI1 and that further up-regulation of GLI1 may be associated with the development of drug resistance. Here, we show that EWS cell lines (CHLA-9, CHLA-10, CHLA-258, TC-32 and TC-71) express canonical HH pathway components (PTCH1, SMO, GLI1 and GLI3) and that cells lines established at the time of recurrence have higher GLI1 expression (CHLA-258 and TC-71) than those established at the time of diagnosis (CHLA-9, CHLA-10 and TC-32). Exposure to Sonic HH ligand in order to activate canonical HH signaling did not affect cell viability (MTT assay), proliferation (BrdU assay), or apoptosis (caspase 3/7 assay). However, inhibition of GLI1 activity with GANT61 reduced GLI1 expression (qRT PCR), cell viability (MTT assay) and cell proliferation (BrdU assay) but increased apoptosis (caspase 3/7 assay) and sensitivity of the cells to vincristine (VCR) (MTT assay), suggesting GLI1 plays roles in each of these processes. We then established VCR-resistant EWS cell lines (TC-71 [45-fold increase in the IC-50] and TC-32 [27-fold increase in the IC-50]) by exposing cells to serially increasing concentrations of VCR. We used an 86-gene cancer drug resistance PCR array (Qiagen) to characterize gene expression differences between untreated vs. recurrent EWS cell lines and between parental vs. VCR-resistant EWS cell lines. Among the changes in gene expression, we showed higher GLI1 expression in the recurrent EWS cell lines (CHLA-258 cells [4.8-fold] and TC-71 [3.4-fold]) vs. untreated CHLA-9 cells, and in VCR-resistant TC-71 cells (2.2-fold) vs. parental TC-71 cells. Down-regulation of GLI1 by GANT61 or GLI1 siRNA enhanced sensitivity of the cells to VCR. Our results suggest that GLI1 expression contributes to proliferation, survival and drug-resistance of EWS cell lines. Strategies to inhibit GLI1 may have therapeutic benefit in EWS. Citation Format: Joon Won Yoon, Marilyn Lamm, Philip Iannaccone, David Walterhouse. GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells [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 3661.

Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition

Peritoneal metastasis (PM) is a debilitating consequence of multiple cancers. As cancer cells lose tonic signaling related to attachment dependence, critical morphologic shifts result in alteration of the transcriptome. Identifying key genes associated with this transformation may lead to targeted therapies for this devastating complication. TC71, CHLA9, PANC1, YOU and HEYA8 cell lines were grown as tumor spheroids in polyHEMA coated plates. Temporal profiling of transcriptomic alterations over 72 hrs was used to develop a comprehensive PM model. We identified transcriptomic outliers using Gaussian mixtures model clustering to identify drivers of spheroid formation. Outliers were validated in The Cancer Genome Atlas (TCGA) and an ovarian tissue microarray (TMA) and by modulation in ovarian cancer models in vitro and in peritoneal xenograft models. Outlier analysis of PM genes identified the gene TXNIP and the TORC signaling as central to PM. Ovarian cancer spheroids isolated from patient ascites had significantly higher TXNIP than their attached counterparts (p = 0.047). TXNIP levels predicted progression-free (log-rank p = 0.026) survival in stage 1/2 ovarian cancer and overall survival (log rank p = 0.047) in stage 3/4 ovarian cancer. In vitro, TXNIP silencing was associated with increased mTOR signaling and enhanced spheroid development which could be overcome by TAK228, a TORC1/2 inhibitor. Similarly, in vivo peritoneal xenograft models of carcinomatosis were prevented by TAK228. PM is driven by TXNIP-associated TORC1/2 signaling. This work provides the first evidence that TORC1/2 inhibition may prevent PM.

A k-mer based transcriptomics approach for antisense drug discovery targeting the Ewing's family of tumors.

Ewing’s sarcoma treatment failures are associated with high mortality indicating a need for new therapeutic approaches. We used a k-mer counting approach to identify cancer-specific mRNA transcripts in 3 Ewing’s Family Tumor (EFT) cell lines not found in the normal human transcriptome. Phosphorodiamidate morpholino oligomers targeting six EFT-specific transcripts were evaluated for cytotoxicity in TC-32 and CHLA-10 EFT lines and in HEK293 renal epithelial control cells. Average morpholino efficacy (EC50) was 0.66 ± 0.13 in TC-32, 0.25 ± 0.14 in CHLA-10 and 3.07 ± 5.02 µM in HEK293 control cells (ANOVA p < 0.01). Synergy was observed for a cocktail of 12 morpholinos at low dose (0.3 µM) in TC-32 cells, but not in CHLA-10 cells. Paired synergy was also observed in both EFT cell lines when the PHGDH pre-mRNA transcript was targeted in combination with XAGE1B or CYP4F22 transcripts. Antagonism was observed when CCND1 was targeted with XAGE1B or CYP4F22, or when IGFBP-2 was targeted with CCND1 or RBM11. This transcriptome profiling approach is highly effective for cancer drug discovery, as it identified new EWS-specific target genes (e.g. CYP4F22, RBM11 and IGBP-2), and predicted effective antisense agents (EC50 < 1 µM) that demonstrate both synergy and antagonism in combination therapy.

Abstract 3178: Up-regulation of GLI1 expression in drug resistant rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines

Abstract Outcome for pediatric patients with recurrent sarcomas remains poor. Therefore, understanding mechanisms that lead to drug resistance and ways to overcome drug resistance therapeutically are essential. Recent reports have suggested a role for the Hedgehog signaling pathway and its downstream mediator GLI1 in acquisition of a multidrug resistance phenotype in esophageal adenocarcinoma, glioblastoma, and myeloid leukemia. Since GLI1 is expressed in the majority of RMS and EWS cases, we hypothesized that GLI1 up-regulation occurs as cells become drug resistant in vitro and in vivo. We established vincristine (VCR) resistant RMS cell lines (Embryonal RMS: RD, Ruch-2; Alveolar RMS: Rh30, Rh41) by serial exposure to increasing concentrations of VCR. Drug-resistance was defined as an IC50 of ≥30 fold of the baseline that was established using parental cells. In addition, we obtained EWS cell lines, representing untreated cases (CHLA-9 and TC-32) and cases following recurrence (CHLA-258 and TC-71). We used an 86-gene cancer drug resistance PCR array (Qiagen), to characterize gene expression differences between the parental vs. VCR-resistant RMS cells lines and between the untreated and recurrent EWS cell lines. GLI1 expression was significantly increased (p≤0.05) in VCR-resistant alveolar RMS cell lines (Rh30 [2.3 fold], and Rh41 [10.3 fold]) compared with parental cells. Expression of 2 additional genes (MDR1 [13,307 fold in Rh30 and 1,755 fold in Rh41 cells] and MVP [2.4 fold in Rh30 and 8.8 fold in Rh41]) was also significantly up-regulated in these cells. Gel mobility shifts showed interaction of GLI1 with the MVP promoter, and cotransfection assays showed that GLI1 up-regulates reporter gene expression through the MVP promoter. GLI1 expression was significantly up-regulated in Ruch-2 embryonal RMS cells (2 fold). We also showed higher GLI1 expression in recurrent EWS cell lines (TC-71 [3.4 fold] and CHLA-258 [4.8 fold]) compared with untreated CHLA-9 cells. Expression of 1 additional gene (androgen receptor [11.1 fold in CHLA-258 cells and 274.6 fold in TC-71 cells]) was also significantly up-regulated in CHLA-258 and TC-71 cells. Treatment of VCR-resistant UKF Rhb-1 RMS cells with VCR together with either GANT61 or Cpd#33 (GLI1 inhibitors) significantly decreased cell viability by MTT assay at doses that did not reduce cell viability individually. Our results suggest that GLI1 up-regulation contributes to development of drug-resistance in RMS and EWS and that GLI1-inhibitors may reduce multidrug resistance. Citation Format: Joon Won Yoon, Iris Smith, Sumeed Manzoor, Marilyn Lamm, Philip Iannaccone, David Walterhouse. Up-regulation of GLI1 expression in drug resistant rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3178.

Abstract 3189: Targeting NPM1 for controlling growth of Ewing sarcoma

Abstract Ewing sarcoma is a rare cancer found in children and young adults. Long term survival rates are approximately 70% for patients who present without clinically overt metastases. However, 5 year survival is less than 20% for patients with recurrent tumors or metastasis. Thus, there is a need to improve local tumor control and to treat metastatic disease. Eighty five percent of these cancers are driven by the reciprocal chromosomal translocation t (11;22) (q24;q12), a consequence of a fusion between the 5' portion of Ewing sarcoma breakpoint region 1 on EWSR1 (chromosome 22) and the 3' portion of Friend leukemia virus integration site 1 on FLI1 (chromosome 11). Nucleophosmin1 (NPM1) is a chaperone protein involved in many cellular functions, including DNA repair. NPM1 has been shown to be a novel prognostic biomarker for patients with localized Ewing's sarcoma. Overall survival is significantly lower for patients whose tumors express high levels of NPM1. Besides promoting repair of DNA double strand breaks NPM1 directly interacts with and induces c-Myc-induced hyperproliferation and transformation. Consistent with this knowledge is the observation that relapsed Ewing's patients exhibit high levels of c-Myc expression compared to disease-free patients. Therefore, we wanted to ascertain whether NPM1 represented a molecular target for controlling growth of Ewing's sarcoma. CRISPR/Cas9-mediated targeting of NPM1 exons 3 and 4, as well as lentivirus-mediated shRNA targeting of NPM1 mRNA revealed that targeting of NPM1 expression reduced survival (colony formation) of TC32 and A673 cells (P&amp;lt;0.05). NPM shRNA also reduced TC32 sphere formation under non-adherent conditions. YTR107 and NSC348884 are small molecule inhibitors of NPM1 oligomerization, which is required for NPM1 activity. Both drugs reduced survival of TC32 and A673 Ewing's sarcoma cells in a dose-dependent fashion (P&amp;lt;0.05). We interpret these data to indicate that NPM1 represents a potential molecular target for controlling growth of Ewing's sarcoma. Supported in part by RO1CA140409. Citation Format: Geri Traver, Konjeti R. Sekhar, Narsimha R. Renthala, Peter A. Crooks, Maxwell Ofori, Michael L. Freeman. Targeting NPM1 for controlling growth of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3189.

Abstract 402: Gene editing for functional analysis of Ewing sarcoma target genes

Abstract Gene editing tools like CRISPR, using Cas9 protein and selected guide RNAs, are now the preferred method to study many coding gene functions, but historically have been hampered by low efficiency (typically &amp;lt;20% of cells), which limits their utility in suppressing highly expressed oncogenic drivers of cancer like fusion genes. Ewing sarcoma (ES) is driven by the EWS-FLI1 or equivalent fusion gene and expresses CD99 on its cell surface. CD99 itself is a direct target of the fusion gene, making it a universal target potentially exploited by CD99 targeted methods. We have utilized CD99 targeted nanoparticles containing plasmid constructs (Cas9-EGFP-guide RNA) targeting the EWS, FLI1, or ETS domains of the fusion gene to achieve up to ~70% knockdown after one transfection in TC32 ES cells. We show that targeting the 5' EWSR1 region of the fusion gene results in dramatically lower expression of the fusion gene (30% of control), with lesser effect when the 3' ETS region is targeted (40% of control). We then compared the genes targeted by the CRISPR EWS-FLI1 KO in TC32 cells with those whose expression was altered by knock down of the fusion gene using shRNA directed against the EWS-FLI1 3'UTR in CHLA9 ES cells and found &amp;gt;2,000 targets in common based on total RNA Seq data with a 2-fold or greater delta in expression. Most targets were down regulated after fusion gene suppression (~1,300 vs ~1,000), indicating they are up regulated by the oncoprotein. Importantly, over half of the affected transcripts were non-coding lncRNAs (Up regulated targets: 421 coding, 539 ncRNA; Down regulated targets: 299 coding, 448 ncRNA) Nearly 600 pseudogenes were also dysregulated. The most statistically significant targets were sense and anti-sense intragenic (regulatory) ncRNAs, documenting the likely importance of ncRNAs in the genesis of Ewing sarcoma. Further, while both coding and non-coding targets were enriched for multimeric GGAA repeats within 1 kb of their TSS when compared to a randomly selected control population, the vast majority of both classes of transcripts lacked multimeric GGAA repeats in their promoters or even GGAA repeats in enhancers within 50kb of the targeted transcripts, indicating that regulation of Ewing sarcoma target gene expression is mediated proximally by factors other than direct or indirect binding of the oncoprotein in cis with the target gene. Our results demonstrate that use of Ewing-specific, CD99 targeted nanoparticles allows high-efficiency (~70%) permanent editing and knockdown of the fusion gene. This enables analysis of fusion gene targets in a native, unperturbed tumor setting, free of artifacts introduced by transfection methods or induced fusion gene expression. It also allows functional studies of the fusion gene RNA targets themselves, using the same gene editing methods, which could enable pre-clinical therapeutic proof-of-concept studies directed against the fusion gene, its validated coding and non-coding targets, or both. Citation Format: Timothy J. Triche, Sheetal A. Mitra, Hyung G. Kang, Jonathan D. Buckley. Gene editing for functional analysis of Ewing sarcoma target genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 402.

Abstract 3188: Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells

Abstract EWS/FLI1 is a unique chimeric fusion protein found in approximately 85% of Ewing sarcoma (ES) tumors. Blocking this fusion protein in conjunction with administration of anticancer agents could be an effective strategy for treating ES. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), also known as survivin, is overexpressed in several malignancies, including ES. Survivin is associated with aggressive growth of cancer and resistance to standard chemotherapy and thus serves as a prognostic marker for ES and other cancers. EWS/FLI1 and Specificity protein1 (Sp1) jointly mediate the expression of cyclins to facilitate cell growth. Mithramycin-A (Mit-A) has been identified as a potent inhibitor of EWS-FLI1 and its downstream targets. Preclinical research from our laboratory demonstrated that clotam, a small molecule, exhibits anticancer activity by targeting Sp1 and survivin. The objective of our investigation is to test the efficacy of Mit-A or clotam to induce the antiproliferative activity of standard chemotherapeutic agents, vincristine (Vin) or etoposide (Eto), in ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mit-A/clotam/Vin/Eto. Cellular growth inhibition was evaluated at 48 h post-treatment using CellTiter-Glo kit (Promega). In order to determine the combination response, cell growth inhibition of each chemotherapeutic drug was assessed in the presence or absence of Mit-A or clotam. Further studies were performed to investigate the effect of individual agents and combinations on apoptosis and cell cycle phase distribution. Levels of cleaved poly (ADP-ribose) polymerase (c-PARP) were determined by Western blot analysis and the apoptotic cell population quantitated by flow cytometry analysis of Annexin-V stained cells. Cell cycle phase distribution was also measured by flow cytometry using propidium iodide. Results showed a dose-dependent antiproliferative effect with all agents. The combination of Mit-A or clotam with Vin or Eto showed greater effect on apoptosis and cell cycle arrest compared to the effects of Vin or Eto alone. Overall, these results indicate the potential clinical advantage of combination treatment involving Mit-A or clotam for inhibiting the growth of ES cells. Molecular profiling analysis is under way to elucidate the candidate pathways associated with beneficial effect of our combination treatments and with other chemotherapeutic agents. Citation Format: Anish Ray, Sagar Shelake, Umesh Sankpal, Lina Albeer, Holly Lout, Abigail Hunter, Kathryn Dunlap, Rajasekhar Maram, W. Paul Bowman, Riyaz M. Basha. Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3188.