Targeting the DNA repair pathway in Ewing sarcoma.
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ABSTRACT:
SUBMITTER: Stewart E
PROVIDER: S-EPMC4386669 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
Publications
Targeting the DNA repair pathway in Ewing sarcoma.
Stewart Elizabeth E Goshorn Ross R Bradley Cori C Griffiths Lyra M LM Benavente Claudia C Twarog Nathaniel R NR Miller Gregory M GM Caufield William W Freeman Burgess B BB Bahrami Armita A Pappo Alberto A Wu Jianrong J Loh Amos A Karlström Åsa Å Calabrese Chris C Gordon Brittney B Tsurkan Lyudmila L Hatfield M Jason MJ Potter Philip M PM Snyder Scott E SE Thiagarajan Suresh S Shirinifard Abbas A Sablauer Andras A Shelat Anang A AA Dyer Michael A MA
Cell reports 20141023 3
Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temo ...[more]
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Project description:Ewing’s Sarcoma is a bone and soft tissue tumor that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive but survival for metastatic and recurrent disease is poor. Whole genome sequencing of 19 Ewing’s Sarcoma tumors showed that STAG2 was mutated in 10% (2/19) of the tumors and STAG2 protein was absent in 14% (13/106) tumors by immunohistochemical staining. Previous studies have shown that glioblastoma cells lacking STAG2 are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors. We found that Ewing’s Sarcoma cell lines are sensitive to PARP inhibitors irrespective of STAG2 protein expression. Ewing Sarcoma cell lines are defective in double strand DNA break repair. PARP inhibitor cytotoxicity in Ewing’s Sarcoma cells was potentiated 10-1,000 fold by DNA damaging agents (irinotecan and temozolomide). To extend these studies in vivo, we developed an orthotopic Ewing’s Sarcoma mouse model and performed pharmacokinetic and pharmacodynamic studies with three different PARP inhibitors (BMN-673, olaparib and veliparib) in clinical development for pediatric cancer. Those data were used to design preclinical phase I studies to identify tolerable drug combinations for pilot efficacy testing (preclinical phase II). Based on the results of the preclinical phase I/II data, we performed a double blind, randomized, placebo controlled preclinical phase III trial with 274 mice in 15 treatment groups. Irinotecan administered in a low-dose protracted schedule optimized for pediatric patients was an effective DNA damaging agent to combine with olaparib and BMN-673 and was better tolerated than combinations with temozolomide. Combining olaparib or BMN-673 with irinotecan and temozolomide gave complete and durable responses in over 80% of the mice.
| EGAS00001000839 | EGA
Project description:We previously demonstrated that RNA helicase DDX3X (DDX3) can be a therapeutic target in Ewing sarcoma (EWS), but its role in EWS biology remains unclear. The present work demonstrates that DDX3 plays a unique role in DNA damage repair (DDR). We show that DDX3 interacts with several proteins involved in homologous recombination, including RAD51, RECQL1, RPA32, and XRCC2. In particular, DDX3 colocalizes with RAD51 and RNA:DNA hybrid structures in the cytoplasm of EWS cells. Inhibition of DDX3 RNA helicase activity increases cytoplasmic RNA:DNA hybrids, sequestering RAD51 in the cytoplasm, which impairs nuclear translocation of RAD51 to sites of double-stranded DNA breaks, thus increasing sensitivity of EWS to radiation treatment, both in vitro and in vivo. This discovery lays the foundation for exploring new therapeutic approaches directed at manipulating DDR protein localization in solid tumors.
| S-EPMC10844834 | biostudies-literature
Project description:We previously demonstrated that RNA helicase DDX3X (DDX3) can be a therapeutic target in Ewing sarcoma (EWS), but its role in EWS biology remains unclear. The present work demonstrates that DDX3 plays a unique role in DNA damage repair (DDR). We show that DDX3 interacts with several proteins involved in homologous recombination, including RAD51, RECQL1, RPA32, and XRCC2. In particular, DDX3 colocalizes with RAD51 and RNA:DNA hybrid structures in the cytoplasm of EWS cells. Inhibition of DDX3 RNA helicase activity increases cytoplasmic RNA:DNA hybrids, sequestering RAD51 in the cytoplasm, which impairs nuclear translocation of RAD51 to sites of double-stranded DNA breaks thus increasing sensitivity of EWS to radiation treatment, both in vitro and in vivo. This discovery lays the foundation for exploring new therapeutic approaches directed at manipulating DDR protein localization in solid tumors.
| S-EPMC10274875 | biostudies-literature
Project description:PurposeEwing sarcoma is a small round blue cell tumor that is highly malignant and predominantly affects the adolescent and young adult population. It has long been suspected that a genetic predisposition exists for this cancer, but the germ-line genetic underpinnings of this disease have not been well established.MethodsWe performed germline variant analysis of whole-genome or whole-exome sequencing of samples from 175 patients affected by Ewing sarcoma.ResultsWe discovered pathogenic or likely pathogenic germline mutations in 13.1% of our cohort. Pathogenic mutations were highly enriched for genes involved with DNA damage repair and for genes associated with cancer predisposition syndromes.ConclusionOur findings reported here have important clinical implications for patients and families affected by Ewing sarcoma. Genetic counseling should be considered for patients and families affected by this disease to take advantage of existing risk management strategies. Our study also highlights the importance of germline sequencing for patients enrolled in precision-medicine protocols.Genet Med advance online publication 26 January 2017.
| S-EPMC5529247 | biostudies-literature
Project description:Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Although the 5-year survival rate of localized disease approaches 75%, the prognosis of metastatic and/or therapy-resistant disease remains dismal despite the wide use of aggressive therapeutic strategies. We previously reported that high expression of glutathione S-transferase M4 (GSTM4) in primary tumors correlates with poor patient outcomes. GSTM4 is required for oncogenic transformation and mediates resistance to chemotherapeutic drugs in Ewing sarcoma cells. Here, we performed RNA-sequencing analyses of Ewing sarcoma cells and combined our results with publicly available datasets to demonstrate that GSTM4 is a major GST specifically expressed in Ewing sarcoma. Pharmacological inhibition of GSTM4 activity using a pan GST inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), significantly limited cellular proliferation and oncogenic transformation of Ewing sarcoma cells. Moreover, combined use of NBDHEX and etoposide synergistically increased cytotoxicity, suggesting a role for GSTM4 as an inhibitor of apoptosis. Mechanistic studies revealed that GSTM4 limits apoptosis owing to its ability to interact with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibit signaling via the c-Jun N-terminal Kinase axis. To exploit our observation that GSTM4 expression is specifically up-regulated in Ewing sarcoma, we tested the effect of a GSTM4-activated anti-cancer agent, O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K, on tumor growth and survival. We found that JS-K robustly decreased Ewing sarcoma cell viability and xenograft tumor growth and improved overall survival of xenograft mice. Our data suggest that GSTM4 is a novel therapeutic target for the treatment of high GSTM4-expressing Ewing sarcoma. Strategies that combine standard chemotherapy with agents that inhibit GSTM4, that are activated by GSTM4, or that block GSTM4/ASK1 interactions, can potentially be more specific and/or efficacious than standard therapeutic approaches.
| S-EPMC4123608 | biostudies-other
Project description:Inhibitors of the bromodomain and extra-terminal domain (BET) family proteins modulate EWS-FLI1 activities in Ewing sarcoma. However, the efficacy of BET inhibitors as a monotherapy was moderate and transient in preclinical models. The objective of this study was to identify the mechanisms mediating intrinsic resistance to BET inhibitors and develop more effective combination treatments for Ewing sarcoma. Using a panel of Ewing sarcoma cell lines and patient-derived xenograft lines (PDX), we demonstrated that IGF1R inhibitors synergistically increased sensitivities to BET inhibitors and induced potent apoptosis when combined with BET inhibitors. Constitutively activated AKT significantly protected Ewing sarcoma cells against BET inhibitors, suggesting that IGF1R regulates responsiveness to BET inhibitors mainly through the PI3K/AKT pathway. Although two Ewing sarcoma cell lines were resistant to IGF1R inhibitors, they retained synergistic response to a combination of BET inhibitors and mTOR inhibitors, suggesting that BET proteins, when IGF1R is not functional, cross-talk with its downstream molecules. Furthermore, the combination of a BET inhibitor and an IGF1R inhibitor induced potent and durable response in xenograft tumors, whereas either agent alone was less effective. Taken together, our results suggest that IGF1R and the downstream PI3K/AKT/mTOR kinase cascade mediate intrinsic resistance to BET inhibitors in Ewing sarcoma. These results provide the proof-of-concept for combining BET inhibitors with agents targeting the IGF1R pathway for treating advanced Ewing sarcoma.
| S-EPMC6497538 | biostudies-literature
Project description:PURPOSE:Recent studies revealed divergent gene expression patterns in Ewing sarcoma (EwS) with canonical EWSR1-ETS gene fusions and undifferentiated round cell sarcomas (URCS) with EWSR1 rearrangements fused to the non-ETS gene NFATc2. Thus, the question arises whether the latter tumors really belong to EwS. METHODS:We collected five cases matching the group of URCS with EWSR1-NFATc2 fusion and performed DNA methylation and copy number profiling. Results were compared to methylation data of 30 EwS with various EWSR1-ETS fusions and one EwS with FUS-ERG fusion, 16 URCS with CIC rearrangement and 10 URCS with BCOR alteration and a total of 81 EWSR1-associated soft tissue sarcomas including 7 angiomatoid fibrous histiocytomas, 7 clear cell sarcomas of the soft tissue, 28 desmoplastic small round cell tumors, 10 extraskeletal myxoid chondrosarcomas and 29 myxoid liposarcomas. RESULTS:Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis of DNA methylation data revealed a homogeneous methylation cluster for URCS with EWSR1-NFATc2 fusion, which clearly segregated from EwS and the other subtypes. Copy number profiles of EWSR1-NFATc2 cases showed recurrent losses on chromosome 9q and segmental gains on 20q13 and 22q12 involving the EWSR1 and NFATc2 loci, respectively. CONCLUSION:In summary, URCS with EWSR1-NFATc2 fusion share a distinct DNA methylation signature and carry characteristic copy number alterations, which emphasizes that these sarcomas should be considered separately from EwS.
| S-EPMC7607515 | biostudies-literature
Project description:MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.
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Project description:More knowledge is needed regarding germline predisposition to Ewing sarcoma to inform biological investigation and clinical practice. Here, we evaluated the enrichment of pathogenic germline variants in Ewing sarcoma relative to other pediatric sarcoma subtypes, as well as patterns of inheritance of these variants. We carried out European-focused and pan-ancestry case-control analyses to screen for enrichment of pathogenic germline variants in 141 established cancer predisposition genes in 1,147 individuals with pediatric sarcoma diagnoses (226 Ewing sarcoma, 438 osteosarcoma, 180 rhabdomyosarcoma, and 303 other sarcoma) relative to identically processed cancer-free control individuals. Findings in Ewing sarcoma were validated with an additional cohort of 430 individuals, and a subset of 301 Ewing sarcoma parent-proband trios was analyzed for inheritance patterns of identified pathogenic variants. A distinct pattern of pathogenic germline variants was seen in Ewing sarcoma relative to other sarcoma subtypes. FANCC was the only gene with an enrichment signal for heterozygous pathogenic variants in the European Ewing sarcoma discovery cohort (three individuals, OR 12.6, 95% CI 3.0-43.2, p = 0.003, FDR = 0.40). This enrichment in FANCC heterozygous pathogenic variants was again observed in the European Ewing sarcoma validation cohort (three individuals, OR 7.0, 95% CI 1.7-23.6, p = 0.014), representing a broader importance of genes involved in DNA damage repair, which were also nominally enriched in individuals with Ewing sarcoma. Pathogenic variants in DNA damage repair genes were acquired through autosomal inheritance. Our study provides new insight into germline risk factors contributing to Ewing sarcoma pathogenesis.
| S-EPMC9247831 | biostudies-literature
Project description:Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
| S-EPMC6388767 | biostudies-literature