Ewing's Cell Lines Research Articles

Abstract A009: Synthetic lethality in the context of STAG2-mutant Ewing sarcoma

Abstract Introduction: Ewing sarcoma is an aggressive type of pediatric bone cancer, characterized by a fusion transcription factor (EWSR1-FLI1 in 85% of cases1,2). Recurrent secondary mutations are limited to alterations in TP53, CDKN2A and most frequently STAG2 (15-22%3-6). STAG2-loss-of-function (LOF) mutations correlate with poor prognosis, metastasis and relapse3,5,6, and are shown to induce a metastatic phenotype in preclinical studies7,8. Despite intensive multimodal treatment (surgery, radiation and chemotherapy), the estimated 5-year survival is a mere 65-75% for patients with localized disease, and below 30% for those presenting with metastasis or relapse9. Targeted treatment is currently not available. Our current work aims at identifying synthetic lethal (SL) interactions with STAG2-LOF mutations in the context of Ewing sarcoma, which could represent potential avenues for targeted therapy. Methods: In order to identify STAG2-SL interactions, we have performed genome-wide and subsequent targeted CRISPR knockout (KO) screening in Ewing sarcoma cell lines and their isogenic CRISPR-engineered STAG2-KO clones. To capture Ewing sarcoma inter- and intra-tumoral heterogeneity, we engineered additional bulk STAG2-KO populations across a wide range of cell lines and primary samples, using a lentiviral approach. These isogenic (STAG2 +/-) models are used for high throughput drug screening, validation of screening results and investigation of underlying SL mechanisms. Results: The quality of our genetic screening is validated by the recurrent emergence of (untargetable) paralog STAG1 as the top STAG2-SL hit. Additional preliminary results include a targetable candidate that emerged from both CRISPR- and drug-based screens in most of our tested models. Validation experiments across a broader panel of isogenic cell lines and primary models are currently ongoing. Conclusion: In this work, we employ genetic and drug-based screening approaches across a panel of isogenic (STAG2+/-) Ewing sarcoma cell lines, aiming to identify STAG2-SL interactions. These approaches have yielded potential hits that are currently being investigated across a broader panel of Ewing cell lines and primary models. Ultimately, this work aims to identify novel targeted therapeutic strategies for this aggressive subtype of Ewing sarcoma.

Abstract 2641: Transcriptional reprogramming of Ewing tumors: gains and losses noted when utilizing immunocompetent in vivo models

Abstract Objective: Ewing sarcoma is an aggressive cancer most frequently diagnosed in adolescents and young adults. Currently, there is a limited understanding of the immunobiology of Ewing sarcoma, including limited pre-clinical studies of immunotherapeutic interventions. This, in part, is due to the historical lack of an immuno-competent murine model. Here, we utilize an immunocompetent, humanized mouse model to investigate the Ewing tumor immune microenvironment. Specifically, we sought to determine the transcriptional differences that occur when comparing human Ewing tumors established in a humanized backdrop versus those developed in an NSG (immuno-deficient) model. Methods: Human Ewing cell lines (A673, TC32, TC71) and PDX (PSaRC219) were injected orthotopically into humanized or NSG mice. After ~3 weeks, bulk RNA sequencing was performed on tumors utilizing an Illumina Next Generation Sequencer. Tumor single-cell suspensions underwent immunophenotyping by flow cytometry (CyTek Aurora) for the following human markers: CD45, CD3, CD4, CD8, CD56, CD14, CD16, etc.. Peripheral blood was collected, PBMCs isolated, and immunophenotyping was again performed to determine peripheral blood immune cell populations. Results: Ewing tumors established in humanized mice demonstrate infiltration of human CD45+ cells as well as immune cell sub-populations (CD3+, CD14+, CD56+ cells). Bulk RNA sequencing analysis utilizing unsupervised hierarchal clustering revealed that the transcriptional profile of Ewing tumors generated in humanized vs NSG mice cluster distinctly. Pathway analysis of differentially expressed genes (p <0.05) in Ewing tumors developed in humanized versus NSG mice demonstrates upregulation of pathways including extracellular matrix remodeling, collagen biosynthesis, and Wnt signaling. Notably, transcripts known to be specific to TGFBeta-dependent signaling in Ewing tumors are upregulated in the immune competent model, including TGFB1, FBLN5, and COLEC12. Conclusion: This work demonstrates the Ewing tumor transcriptional remodeling that occurs when tumors are developed in an immunocompetent in vivo backdrop. Upregulation of several pro-metastatic pathways were noted. Ongoing work focuses on testing drug response differences between Ewing tumors developed in humanized versus NSG backdrops and pursuing immunocompetent Ewing models as a component of preclinical agent testing. Citation Format: Jessica D. Daley, Elina Mukherjee, A Carolina Tufino, Riyue Bao, Kelly M. Bailey. Transcriptional reprogramming of Ewing tumors: gains and losses noted when utilizing immunocompetent in vivo models [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 2641.

Abstract B021: Long non-coding RNAs that are required for robust cell growth in ewing Sarcoma

Abstract While long non-coding RNAs (lncRNAs) are upregulated in many cancers, their role in pediatric cancers is unclear. Among pediatric cancers, Ewing sarcoma is unique in that these tumors show upregulation of a large, specific number of lncRNAs. To perform an unbiased screen of lncRNA function in Ewing sarcoma, we used a pooled CRISPRi screen to identify lncRNAs that modify cell growth. We included in the pooled library lncRNAs either highly expressed in Ewing Sarcoma or directly regulated by the driving oncogene in Ewing Sarcoma, EWS-FLI. We identified 49 lncRNAs that when inhibited by CRISPRi led to decreased growth of Ewing cell lines. Among those that scored most significantly were the small nucleolar host genes SNHG1, SNHG12, and SNHG30. To validate the screening results, we individually cloned the two most depleted sgRNAs targeting for each of these three lncRNAs into a lentiviral vector. We expressed these sgRNAs in three different dCas9-KRAB+ Ewing cell lines and used internally controlled growth assays to test whether the observed phenotypes from the screens were reproducible. We confirmed that for all three lncRNAs hits significantly affected Ewing’s growth, and their depletion leads to an increase in apoptosis. To determine how lncRNAs influence the ability of Ewing sarcoma cells to growth and survive in vivo, we performed a subcutaneous xenograft CRISPRi screen using the same pooled library used in vitro. The lncRNAs with the strongest pro-growth phenotype after knock-down were PVT1 and the uncharacterized LINC02688. Ongoing studies are directed at determining the Ewing-specific role of these lncRNAs. Overall, our studies demonstrate the value of studying lncRNA functions in vitro and in vivo, provides a valuable resource of new lncRNAs targets to further research and establishes a framework for systematic discovery of functional lncRNAs in Ewing cells. Citation Format: Marcela C. Briones Martin del Campo, Alex Lee, Max Horlbeck, Truc Dinh, Marta Roman-Moreno, Leanne Sayles, Bokyung Seong, Kimberly Stegmaier, Luke Gilbert, E. Alejandro Sweet-Cordero. Long non-coding RNAs that are required for robust cell growth in ewing Sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B021.

Combination Flavopiridol and SN38 drives toxicity and loss of DNA damage repair in Ewing Sarcoma

EWS-Fli1 is an oncogenic transcription factor that causes Ewing sarcoma. EWS-Fli1 is a fusion protein comprised of an N-terminal low complexity domain from the RNA-binding protein EWSR1 and the C-terminal DNA-binding domain of the ETS transcription factor Fli1. EWS-Fli1 drives expression of oncogenes and decreases expression of tumor suppressor proteins. EWS-Fli1 acts as a transcriptional regulator by recruiting RNA Pol II to promoter regions. Ewing sarcoma is particularly susceptible to DNA damaging chemotherapy compared to other cancers. However, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given the role of EWS-Fli1 in disrupting transcription, we hypothesized that EWS-Fli1 is concurrently disrupting the DNA damage response. During transcription, RNA Pol II is phosphorylated by CDK9 to escape initiation into elongation. We tested the ability of transcription inhibition to affect the DNA damage response by combining treatment a CDK9 inhibitor, flavopiridol, and the DNA damage agent, SN38. This combination drastically diminished cell viability in Ewing cell lines. This sensitivity was not found in non-Ewing cell lines U2OS and HEK293T/17. We also find in Ewing cells that the combination treatment decreases the DNA damage markers gamma-H2A.X and phospho-CHK1 compared to non-Ewing's cells. Together, these results suggest Ewing sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation and suggest combination CDK9 inhibition with DNA damage treatment could provide a therapeutic approach for Ewing Sarcoma.

Abstract A14: Exploiting DNA damage repair defects to enhance PD-L1 expression in Ewing sarcoma

Abstract Objective: The purpose of this work is to determine whether PD-L1 expression can be upregulated by PARP inhibitor treatment in Ewing tumor cells harboring DNA damage repair defects. Further, we aimed to determine whether enhanced PD-L1 expression renders Ewing cells more susceptible to T-cell mediated apoptosis following PD-1 inhibition. Experimental Procedures: Patient-derived tumor organoids and monolayers (named PSaRC-318) were established from a viably cryofrozen metastatic lung lesion from a patient with Ewing sarcoma and a paternally inherited germline BARD1 mutation. A673, CHLA9, and CHLA10 Ewing tumor cell lines were also employed. Sensitivity of PSaRC-318 to the PARP inhibitors olaparib and talazoparib was established by performing IncuCyte apoptosis assays. PD-L1 expression was examined using RT-PCR, Western blot, and flow cytometry. BARD1, BRCA, and RAD51D siRNA were also utilized to test the role of specific DNA damage repair proteins on this effect. T-cell/tumor cell cocultures and tumor-primed T-cells were established. T cell-induced tumor cell apoptosis was monitored in real-time using IncuCyte caspase and annexin assays. Results: PSaRC-318 organoids demonstrate exquisite sensitivity to PARP inhibitors, with an IC50 for talazoparib of 3.54nM. Treatment of PARP inhibitor-resistant Ewing cell lines, such as CHLA10, with BARD1 siRNA rendered cells sensitive to PARP inhibition. Ewing tumor cells were found to upregulate tumor cell surface expression of the checkpoint protein PD-L1 in response to treatment with PARP inhibitors. The impact of BARD1, BRCA1, and RAD51D on PD-L1 expression in Ewing tumor cells was also determined. Next, Ewing tumor cells were pretreated with PARP inhibitors and then cocultured with tumor-primed T-cells. T cell-mediated tumor cell apoptosis was monitored in real time using caspase IncuCyte assays. PD-1 blocking antibody added to T-cell/tumor cell cocultures resulted in an increase in early tumor cell death compared to IgG controls. Conclusion: Single-agent PARP inhibitors have not demonstrated significant clinical benefit in relapsed Ewing sarcoma to date, suggesting that combinatorial therapy with PARP inhibitors may be a more effective approach. Here, we have shown that Ewing tumor cells with germline mutations in DNA-damage repair proteins, such as BARD1, can upregulate PD-L1 expression following treatment with PARP inhibitors. Thus, PARP inhibitor/anti-PD-1 therapy may be a logical combination for the treatment of relapsed Ewing sarcoma. Ongoing preclinical studies are continuing to examine this combination. Citation Format: Lisa M. Maurer, Rose M. Venier, Claire Julian, Kelly M. Bailey. Exploiting DNA damage repair defects to enhance PD-L1 expression in Ewing sarcoma [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 A14.

High-throughput Chemical Screening Identifies Focal Adhesion Kinase and Aurora Kinase B Inhibition as a Synergistic Treatment Combination in Ewing Sarcoma.

Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients. Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma. Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B-selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma. FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Abstract 4401: Implications of STAG2 loss in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive pediatric bone and soft tissue cancer that is driven by a translocation event, predominantly involving the RNA binding protein EWSR1 and the transcription factor FLI1. The resultant fusion transcription factor EWSR1-FLI1 is a potent oncogene and drives dramatic changes in the transcriptional landscape of Ewing sarcoma. Our previous work demonstrated that EWS-FLI1 drives aberrant accumulation of R-loops, increases replication stress and suppresses homologous recombinational repair in Ewing sarcoma. Similar phenotypes were also observed with the loss of wildtype EWSR1. However, surprisingly, Ewing tumors have highly stable genomes. The most common mutation is in the STAG2 gene, a component of the cohesin ring complex. It was recently reported that STAG2-cohesin complexes not only participate in maintaining chromatin architecture but also play a key role in directing tissue-specific gene expression by interacting with transcription factors. In the present study, we evaluate the STAG2 function in R-loop metabolism, replication and repair and attempt to identify the reason for its selective loss in Ewing sarcoma. Using biochemical and biophysical experimental strategies, we observed that STAG2 and EWSR1 both bind R-loop structures with high affinity and are protective against RNaseH-mediated degradation of R-loops to significantly different degrees. We also examine the effect of STAG2 loss on R-loop accumulation and replication stress in Ewing and non-Ewing cell lines. Finally, we evaluate the correlation between STAG2 binding sites and R-loops. We propose that loss of STAG2 alleviates some of the genomic stresses experienced in Ewing sarcoma. Note: This abstract was not presented at the meeting. Citation Format: Aparna Gorthi. Implications of STAG2 loss in Ewing sarcoma [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 4401.

Effects of Transcriptional Dysregulation on the DNA Damage Response in Ewing's Sarcoma

Ewing's sarcoma is a soft‐tissue bone malignancy characterized by a translocation event wherein the N‐terminal low complexity domain of EWSR1 is fused with the DNA binding domain of the ETS transcription factor Fli1. The resulting EWS‐Fli1 fusion protein drives expression of oncogenes and knockdown of tumor suppressor proteins. More specifically, EWS‐Fli1 acts as transcriptional regulator by helping recruit RNA Pol II to promoter regions, which is subsequently phosphorylated by CDK9 to escape initiation into elongation. Interestingly, Ewing's sarcoma is particularly susceptible to chemotherapy compared to other cancers; however, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given EWS‐Fli1's role in disrupting transcription, it was hypothesized EWS‐Fli1 is concurrently disrupting the DNA damage response. Here, a combination of CDK9 inhibition with DNA damage was found to drastically diminish cell viability in Ewing's cell lines but not in non‐Ewing's osteosarcoma or HEK293 cells. Ordinarily, after DNA damage γH2A.X phosphorylation occurs on histones near sites of damaged DNA early during the DNA damage response to signal DNA damage repair initiation. Further work has shown Ewing's cells undergoing the combination treatment above have a marked decrease in this DNA damage marker γH2A.X as compared to non‐Ewing's cells. Together, these results suggest Ewing's sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation.Support or Funding InformationNIH Grant #214660This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Combination CDK4/6 and IGF1R Inhibitor Strategy for Ewing Sarcoma.

Novel targeted therapeutics have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, with simple tumor genomes and infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The cyclin-dependent kinase (CDK)4/6 pathway recently emerged as a dependency in Ewing sarcoma. Given the heightened efficacy of this class with targeted drug combinations in other cancers, as well as the propensity of resistance to emerge with single agents, we aimed to identify genes mediating resistance to CDK4/6 inhibitors and biologically relevant combinations for use with CDK4/6 inhibitors in Ewing. We performed a genome-scale open reading frame (ORF) screen in 2 Ewing cell lines sensitive to CDK4/6 inhibitors to identify genes conferring resistance. Concurrently, we established resistance to a CDK4/6 inhibitor in a Ewing cell line. The ORF screen revealed IGF1R as a gene whose overexpression promoted drug escape. We also found elevated levels of phospho-IGF1R in our resistant Ewing cell line, supporting the relevance of IGF1R signaling to acquired resistance. In a small-molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6 inhibitor treatment. The combination of CDK4/6 inhibitors and IGF1R inhibitors was synergistic in vitro and active in mouse models. Mechanistically, this combination more profoundly repressed cell cycle and PI3K/mTOR signaling than either single drug perturbation. Taken together, these results suggest that IGF1R inhibitors activation is an escape mechanism to CDK4/6 inhibitors in Ewing sarcoma and that dual targeting of CDK4/6 inhibitors and IGF1R inhibitors provides a candidate synergistic combination for clinical application in this disease.

Abstract 1629: Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy

Abstract Novel targeted drug combinations have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, where tumor genomes are typically simple with infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The CDK4/6 pathway, which has been extensively validated as a target in estrogen receptor-positive breast cancer, recently emerged as a dependency in Ewing sarcoma, an aggressive pediatric bone tumor. Given the heightened efficacy of CDK4/6 inhibitors (CDK4/6i) in combination with other targeted drugs in breast cancer, as well as the known propensity of resistance to emerge with single agent targeted cancer therapy, we aimed to identify CDK4/6i resistance mechanisms and biologically relevant drug combinations in Ewing. To identify candidate resistance mechanisms, we performed a genome-wide lentiviral open-reading frame (ORF) rescue screen in two Ewing cell lines sensitive to the CDK4/6 inhibitors palbociclib and ribociclib and looked for genes that confer resistance to these drugs. This revealed IGF1R as a gene whose overexpression promoted drug escape. In parallel to the ORF screen, we established resistance to CDK4/6i in Ewing sarcoma cell lines by chronic ribociclib exposure. We found elevated levels of phospho-IGF1R in resistant cells by immunoblotting, also supporting that this pathway's activation is relevant to acquired resistance. We performed CRISPR knockout of IGF1R in these drug resistant cells and demonstrated rescue of sensitivity to CDK4/6i. Furthermore, resistant lines maintained high sensitivity to an IGF1R inhibitor. Concurrently, in a small molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6i in Ewing cells. We thus tested ribociclib and the IGF1R tool compound AEW541 in a panel of six Ewing cell lines in vitro. We observed strong synergy utilizing the Chou-Talalay method. This finding was next validated in an in vivo cell line xenograft mouse model, where we demonstrated prolonged survival and decreased tumor volumes with combination drug treatment. In order to investigate mechanism of CDK4/6-IGF1R inhibitor synergy, we used proteomic analysis by reverse phase protein array (RPPA) in single and combined drug treated Ewing cells. RPPA revealed enhanced suppression of the PI3K/mTOR pathway in the combination, a central cancer cell signaling axis. Taken together, these results suggest that IGF1R activation is an escape mechanism to CDK4/6i in Ewing and that dual targeting of CDK4/6 and IGF1R provides a candidate synergistic drug combination for this disease. Citation Format: Lillian M. Guenther, Neekesh V. Dharia, Linda Ross, Amy S. Conway, Amanda L. Robichaud, Alanna J. Church, Rajarshi Guha, Mindy I. Davis, Gabriela Alexe, Jaume Mora, Federica Piccioni, Kimberly Stegmaier. Targeting resistance mechanisms to CDK4/6 inhibitors in Ewing sarcoma with an IGF1R inhibitor drug combination strategy [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 1629.

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 2560: Identification and characterization of EWS-FLI1 binding partners in Ewing sarcoma cell lines

Abstract Ewing sarcoma (ES) is characterized by a reciprocal translocation t(11;22) that results in a fusion of the EWSR1 and FLI1 genes (EWS-FLI). The objective of this study is to identify protein binding partners of the purportedly undruggable chimeric transcription factor EWS-FLI1 to serve as alternative pharmacological targets for potential ES-selective drug therapies. The literature identifies the orphan nuclear receptor DAX1 (encoded by NR0B1) as a binding partner of EWS-FLI1 the expression of which is restricted outside ES, thereby providing a target for a potential ES selective drug. Yeast two-hybrid (Y2H) screening using EWS-FLI1 as the bait was employed to identify other potential targets. The importance of the binding partners identified by the Y2H screen was initially assessed by a series of proliferation assays involving the ES cell lines EW8, ES7 and ES6 as well as rhabdomyosarcoma cell line Rh30 (Non-Ewing control). Prior to measuring proliferation, cells were transfected a siRNA designed to target the putative binding partner of EWS-FLI1. These cells were then grown alongside cells transfected with a control siRNA over a period of 96 hr. The change in cell confluence in the wells was measured by a live-cell real-time measurement (Incucyte). Gene knockdown was confirmed by RT-PCR and western blot analysis. The proliferation assays showed that the knockdown of DAX1 produces a notable reduction in cell proliferation compared to siRNA control proliferation in all three of the Ewing cell lines (10% to 25%). No significant DAX1 siRNA based inhibition of cell proliferation was detected in the Rh30 non-Ewing control cell line. Three of the proteins identified by the Y2H screen showed significant reductions in ES cell proliferation. Methyl-CpG-Binding Domain Protein 1 (MBD1) knockdown caused the proliferation of the ES cell lines to level off at 60% to 80% within 2 to 4 days. Proliferation of MBD1 siRNA and control siRNA transfected Rh30 cells was similar for the full 4 days of the assay. Mannosidase-α-class 2B-member 2 (MAN2B2) knockdown caused the proliferation of the ES and Rh30 cell lines to level off at 25% to 65% within 1 to 2 days. Mixed-Lineage Leukemia Protein 3 (MLL3) knockdown caused the proliferation of the EW8 and ES7 cell lines to level off at 52% and 70% respectively. MLL3 knockdown did not inhibit the proliferation of Rh30 cells when compared to siRNA control cells. In contrast, ES6 cell proliferation was also not inhibited by MLL3 knockdown when compared to their siRNA control cells. The knockdowns of DAX1, MLL3 and MBD1 suggest that these EWS-FLI1 binding partners would likely provide potential targets for ES drug development. The effect of knocking down Y2H-identified EWS-FLI interactors on colony formation, migration and tumorigenicity is ongoing. Citation Format: Matthew L. Rotondi, Peter J. Houghton. Identification and characterization of EWS-FLI1 binding partners in Ewing sarcoma cell lines [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 2560. doi:10.1158/1538-7445.AM2017-2560

Abstract A25: Systems biology approach to delineating DNA damage response and R-loop dependencies in Ewing sarcoma

Abstract Ewing sarcoma is a rare, yet highly aggressive family of bone and soft tissue tumors that afflicts children and young adults. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the EWSR1 gene, most frequently EWS-FLI1. Ewing sarcoma is exquisitely sensitive to genotoxic agents, and more recently PARP1 inhibitors, predominantly when detected at an early stage. However, the molecular basis for this sensitivity is relatively under-explored. Further, the long term side effects of these drugs pose a critical barrier to effective treatment. In this study, we proposed to delineate the pathways dictating Ewing sarcoma chemosensitivity and identify alternative avenues for targeted therapy. R-loops are three nucleic acid DNA structures consisting of an RNA:DNA hybrid and a displaced complementary DNA loop. The importance of R-loops in both being causative as well as disruptive in DNA damage as well as DNA damage response has emerged through recent studies. Our previous work has demonstrated that Ewing sarcoma cell lines have high levels of endogenous damage as well as replication stress. We observe that Ewing cell lines have elevated levels of persistent R-loops and impaired homologous recombination repair of double strand breaks. In the present study, we use a multimodal, systems biology approach involving high-throughput RNAi based survival data, time-course RNA-seq in response to two chemotherapeutics, Chromatin immunoprecipitation-seq and DNA:RNA hybrid immunoprecipitation-seq datasets across multiple Ewing sarcoma and control cell lines. Our data led to the identification of a subset of genes whose expression patterns in response to DNA damage is dysregulated in Ewing sarcoma. Predominantly, among these genes is a network of RNA binding proteins that highlights the importance of RNA metabolism in DNA damage response. We also observe a concomitant alteration in damage-dependent transcription. Finally, we identified several compensatory mechanisms that are upregulated in Ewing sarcoma (RNASEH2, FEN1, Fanconi Anemia, etc.) in order to manage the exacerbated genomic stress conditions and targeting any of these pathways leads to severe tumor cell cytotoxicity. We therefore propose the use of these pathways as potential candidates for targeted therapy. Citation Format: Aparna Gorthi, Eva Loranc, Cao Lin, Alexander JR Bishop. Systems biology approach to delineating DNA damage response and R-loop dependencies in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A25.

Abstract 2442: EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma

Abstract GLI1 expression in Ewing tumors has been shown to be enhanced by EWS/FLI1 expression in both model transformation systems and in Ewing cell lines. In Ewing cells, GLI1 has been shown to produce significant phenotypic effects consistent with a role in Ewing tumor formation and propagation. Also consistent with an important role is the demonstrated correspondence between the transcriptional target sets of EWS/FLI1 and of GLI1 in Ewing tumor cells. The principal physiologic developmental regulator of GLI1 is the diffusible Sonic Hedgehog ligand which binds the cell surface tumor suppressor Patched with consequent activation of the transmembrane protein Smoothened. In Ewing tumors, the enhancement of GLI1 expression has been shown to be Hedgehog independent and to occur downstream of Smoothened. Indeed, evidence has been published that EWS/FLI1 directly activates the GLI1 promoter. While there is experimental evidence that GLI1 inhibition may be clinically useful in Ewing tumors, preclinical evidence for one inhibitory approach has been disappointing. Interestingly, direct transcriptional activation of GLI1 has not been demonstrated to be of significance in other most other physiologic or pathologic contexts. We speculated that direct transcriptional activation of GLI1 by EWS/FLI1 might be only part of the story in Ewing tumors. Since GLI2 is the most potent physiologic transcriptional activator of GLI1, we undertook to investigate the role of GLI2 in Hedgehog/GLI (HH-GLI) signaling in Ewing Tumors. We found that GLI2 is broadly expressed in Ewing tumors. Based on GLI2 overexpression and on shRNA knockdown of GLI2, we found that GLI2 expression increases GLI1 expression in Ewing cell lines. Via GLI2 knockdown, we have found that GLI2 has biologic effects similar to or greater than GLI1 in Ewing cells. Finally, we demonstrate that EWS/FLI1 enhances nuclear localization of GLI2, suggesting an additional indirect mechanism by which EWS/FLI1 activates HH-GLI signaling and GLI1 expression in Ewing tumors. These data indicate that GLI2 is an active player in HH-GLI pathway activation in Ewing tumors. They also suggest that measures which target GLI2 may have equal or greater efficacy to those aimed at GLI1. Citation Format: Laura Christensen, William A. May. EWS/FLI1 activates GLI1 indirectly through GLI2 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 2442.

Abstract 2517: Ewing sarcoma associated long non-coding RNA determines neural cell fate of the tumors

Abstract BACKGROUND: Ewing sarcoma is a highly invasive pediatric bone and soft tissue malignancy with a 20% 5-year survival rate for patients with metastatic disease. 85% of Ewing sarcomas express a chimeric oncogene, mainly EWS-FLI1, which drives the gene expression signature in these tumors. The exact cell of origin for these primitive small round cell tumors is unknown. These undifferentiated tumors express some neuroectodermal, mesenchymal, and epithelial markers. Recently published work suggests that these tumors may have risen either from neural crest or mesenchymal stem cells. These genomic and phenotypic features may be responsible for the aggressive nature of this disease and understanding the tumor biology may help better treatment options for this metastatic cancer. RESULTS: Our study on expression analysis of primary Ewing tumors using Affymetrix's Human Exon arrays identified a highly expressed novel long intergenic non-coding RNA (lincRNA), FEZF1-AS1 in these tumors. Its DNA sequence has no homology in most vertebrates. RNA-mediated knockdown of the lincRNA in Ewing cell lines decreased expression of some of the neural lineage genes as well as genes associated with cell adhesion and migration. In vitro matrigel assays detected a decrease in cell invasion capability of the lincRNA-knockdown cells. In the in vivo studies, the lincRNA-overexpressed cells had better engraftment and tumor growth in the liver than the normal controls. On attempt to differentiate the lincRNA-knockdown Ewing cell lines, we noted that a decrease in the lincRNA expression led to a decrease in the expression of neuroectodermal markers after a week of differentiation when compared to the control cells. We further detected temporal expression of this lincRNA during neuronal differentiation from embryonic stem cells. CONCLUSIONS: Our study identified a unique lincRNA in Ewing sarcoma that may be partly responsible for inducing expression of the neural lineage genes in these tumors as its reduced expression impaired differentiation in the Ewing cell lines. Its prometastatic behavior helps the aggressive nature of these tumors. Its expression during neural differentiation along with its involvement in cell migration suggests that FEZF1-AS1 may be responsible for imparting neural stem cell like-characteristics to Ewing sarcoma. Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, Timothy J. Triche. Ewing sarcoma associated long non-coding RNA determines neural cell fate of the tumors. [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 2517.

Abstract 41: XI-006 induces potent p53-independent apoptosis in Ewing sarcoma

Abstract Despite aggressive multi-modal treatment strategies, limited improvement in overall survival rates for patients diagnosed with Ewing sarcoma, the second most prevalent solid bone tumor observed in children and young adolescents, has been achieved over the past thirty years. As such, new targeted therapies are urgently required particularly for those sarcoma patients with recurrent or metastatic disease. Overexpression of MDM4, a major transcriptional regulator of the TP53 tumour suppressor has been documented in a substantial proportion of Ewing, Synovial and Osteosarcomas (>30%). For this reason, we have examined the molecular and cellular responses of cultured Ewing and Osteosarcoma cell lines following treatment with XI-006 (NSC207895), a small molecule 4-nitrobenzofuroxan derivative MDM4 transcriptional inhibitor. XI-0006 treatment of Ewing and osteosarcoma cell lines (n=11) resulted in rapid and potent apoptosis at low nano-molar concentrations specifically in Ewing sarcoma cell lines (48hr IC50: 0.098-1.61µM). Importantly, the viability of normal human fibroblasts (IMR90) remained unaffected at these low nano-molar concentrations (48hr IC50: 6.80µM). Unexpectedly, apoptotic response was not dependent on MDMX mRNA and protein levels or TP53 status. Alkaline/neutral comet and γH2AX immunofluorescence assays revealed that the cytotoxic effects of XI-006 at low nano-molar concentrations could not be attributed to the induction of DNA damage. RNA expression analysis of XI-006 treated cell lines revealed that the mechanism of action of XI-006 could be attributed to the inhibition of cell division and cycle regulators such as KIF20A and GPSM2. XI-006 was also shown to synergise with current Ewing sarcoma chemotherapeutic protocols, Doxorubicin, Etoposide and Actinomycin D in vitro. Furthermore, combinatorial treatment with Olaparib led to a maximum 56.3% increase in apoptosis compared to XI-006 alone, due to the down-regulation of Mre11. Our pre-clinical findings suggest that XI-006 represents a novel therapeutic intervention for the treatment of Ewing sarcoma, and warrants further pharmacokinetic and pharmacodynamic investigations of XI-006 in vivo. Citation Format: Kathleen Pishas, Susan Neuhaus, Mark Clayer, Gelareh Farshid, Alexander Staudacher, David Callen. XI-006 induces potent p53-independent apoptosis in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 41.

Abstract 2628: Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting

Abstract Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [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 2628. doi:10.1158/1538-7445.AM2015-2628

Abstract 4966: Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover in Ewing sarcoma

Abstract Ewing sarcoma belongs to the family of pediatric tumors which arise most commonly in bone. The majority of Ewing sarcoma is characterized by a balanced translocation between chromosomes 11 and 22 which encodes for the uniquely expressed fusion protein EWS/FLI1. Tumor cells are crucially dependent on expression of the fusion protein. Protein degradation is an important and highly regulated process in all cells and novel insights are beginning to be applied for cancer therapy. We aim to investigate the mechanism of turnover with the goal to diminish EWS/FLI1 protein and thereby identify novel targets for Ewing sarcoma treatment. Here, we show first that EWS/FLI1 protein stabilizes upon proteasome inhibition. Co-immunoprecipitation of EWS/FLI1 with ubiquitin shows a characteristic ubiquitination smear indicating that EWS/FLI1 is ubiquitinated and a substrate of the ubiquitin-proteasome system. We characterized the turnover itself as rather slow. Interestingly, the half-life time of the fusion protein is increased by four-fold compared to the full length protein FLI1. This increased stability possibly contributes to the oncogenic and resistant features of the fusion protein. To identify a deubiquitinating enzyme capable of regulating EWS/FLI1 turnover, we selected 20 deubiquitinating enzymes based on their expression in Ewing cell lines and tumors in-silico. Then, we established a screening approach directly measuring EWS/FLI1 protein levels as a read-out by an ELISA-like assay, allowing us to monitor the amount of 3x flag-tagged EWS/FLI1 protein. After transient knocked down of the 20 candidates, the screening revealed ubiquitin specific protease 19 (USP19) as a promising candidate. Depletion of USP19 via siRNA or shRNA was capable to diminish endogenous EWS/FLI1 protein level which led to modulation of its target gene levels. Vice versa, USP19 overexpression stabilized EWS/FLI1 protein level by twofold. Most importantly, stable knockdown of USP19 in Ewing cell lines led to a 40% reduction of cell growth and proliferation. Currently, we further characterize the influence of USP19 on EWS/FLI1 turnover and Ewing cell viability in vivo to identify novel possibilities for therapeutic interventions. Citation Format: Maria E. Gierisch, Laura A. Lopez-Garcia, Franziska Pfistner, Felix K. Niggli, Beat W. Schaefer. Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover 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 4966. doi:10.1158/1538-7445.AM2015-4966

XI-006 induces potent p53-independent apoptosis in Ewing sarcoma.

There is an imperious need for the development of novel therapeutics for the treatment of Ewing sarcoma, the second most prevalent solid bone tumour observed in children and young adolescents. Recently, a 4-nitrobenzofuroxan derivative, XI-006 (NSC207895) was shown to diminish MDM4 promoter activity in breast cancer cell lines. As amplification of MDM4 is frequently observed in sarcomas, this study examined the therapeutic potential of XI-006 for the treatment of Ewing and osteosarcoma. XI-006 treatment of Ewing and osteosarcoma cell lines (n = 11) resulted in rapid and potent apoptosis at low micro-molar concentrations specifically in Ewing sarcoma cell lines (48 hr IC50 0.099–1.61 μM). Unexpectedly, apoptotic response was not dependent on MDM4 mRNA/protein levels or TP53 status. Alkaline/neutral comet and γH2AX immunofluorescence assays revealed that the cytotoxic effects of XI-006 could not be attributed to the induction of DNA damage. RNA expression analysis revealed that the mechanism of action of XI-006 could be accredited to the inhibition of cell division and cycle regulators such as KIF20A and GPSM2. Finally, potent synergy between XI-006 and olaparib (PARP inhibitor) were observed due to the down-regulation of Mre11. Our findings suggest that XI-006 represents a novel therapeutic intervention for the treatment of Ewing sarcoma.

Abstract A30: Integrative genomic analysis of two Ewing's sarcoma cell lines that show differential drug sensitivity

Abstract Background: Ewing's sarcoma is a rare form of highly aggressive bone tumor of children and young adults. Understanding the molecular mechanisms involved in the development of Ewing's sarcoma forms the key step in the design and development of new therapeutic interventions. Previous studies revealed that genomic alterations play an important role in oncogenesis, disease progression and response of tumors to different therapies. The recent advance of next generation sequencing-based methods provide unprecedented capabilities to scan Ewing's sarcoma genomes for changes such as mutations, insertions/deletions, copy number variations, differential gene expression and discovery of new gene fusions. In this study, we performed detailed investigation to identify the genetic basis of difference between two Ewing cell lines, RDES and SKES1, which show differential sensitivity to natural product extracts, by using RNA-seq, small RNA-seq and exome-capture-seq techniques. Methods: Transcriptomes and genomic DNA of both the Ewing's cell lines, RDES and SKES1 were prepared for paired-end sequencing on the Illumina HiSeq2000 platform using the manufacturer's protocols, for RNA-seq and exome-capture-seq respectively. The reads were aligned to the human hg19 genome assembly and the abundance of mRNAs as well as small RNAs for all annotated genes from the UCSC annotation of human genome was calculated. The count data from abundance estimation was used to compute differentially expressed genes and small RNAs between these two cell lines. Furthermore, we used deFuse for the gene fusion identification and GATK pipeline for the DNA variant analysis. Results: We studied the gene expression profiles from RNA-seq and small RNA-seq reads and identified approximately 270 and 114 differentially expressed genes and microRNAs, respectively between drug-sensitive and insensitive cell lines. Interestingly, we found that two of ABC transporters are significantly overexpressed in the resistant cell line. Moreover, many apoptotic genes show altered expression between these two cell lines. Gene fusion products were also identified from the RNA-seq analysis. Both the cell lines were found to harbor the well-known Ewing's sarcoma gene fusion EWS-FLI1. In addition to EWS-FLI1, we also discovered some new and unique gene fusion candidates in both cell lines. Furthermore, we used exome-capture sequence analysis to identify sequence variation and mutations in these two cell lines. Known mutations reported in COSMIC database and SNPs that are not included in normal populations from the 1000 genome project were carefully studied. Interestingly, some of the non-synonymous changes identified map to kinases and other known cancer-related genes. Conclusions: Our studies suggest that the difference in drug sensitivity of two Ewing's sarcoma cell-lines is probably due to the expression of ATP-dependent efflux pumps as well as alterations in the apoptotic machinery. Further studies are warranted to understand the exact molecular mechanism of drug sensitivity. Citation Format: TABREZ A. MOHAMMAD, Aparna Gorthi, Andrew Robles, Xavier Bernard, Tzu-hung Hsiao, Dawn Garcia, Zhao Lai, Susan L. Mooberry, Gail E. Tomlinson, Alexander JR Bishop, Yidong Chen. Integrative genomic analysis of two Ewing's sarcoma cell lines that show differential drug sensitivity. [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 A30.