Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by Redistributing It within the Nucleus

Harlow, Matt L.; Maloney, Nichole; Roland, Joseph T.; Navarro, Maria Jose Guillen; Easton, Matthew K.; Kitchen-Goosen, Susan M.; Boguslawski, Elissa A.; Madaj, Zachary; Johnson, Ben K.; Bowman, Megan J.; D’Incalci, Maurizio; Winn, Mary E.; Turner, Lisa; Hostetter, Galen; Galmarini, Carlos M.; Avilés, Pablo; Grohar, Patrick J.

doi:10.1158/0008-5472.can-16-0568

Cited by 61 publications

(50 citation statements)

References 50 publications

(48 reference statements)

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“…IC 50 s were determined by nonlinear regression (GraphPad Prism) as the average of three independent experiments using standard MTS assay CellTiter 96 (Promega). The results were confirmed with real-time proliferation assays on the Incucyte Zoom as described previously (26).…”

Section: Cell Proliferation Assayssupporting

confidence: 62%

“…We have previously shown that treatment of Ewing sarcoma cells with trabectedin and a second-generation analogue redistributes EWS-FLI1 within the nucleus to the nucleolus (26). Therefore, we investigated whether the C max exposure was required for nucleolar redistribution and if it would be sustained following drug removal.…”

Section: Ews-fli1 Redistributes In the Nucleus To The Nucleolus Only mentioning

confidence: 99%

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Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

Harlow

Chassé

Boguslawski

et al. 2019

Clinical Cancer Research

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Purpose: The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18 F-FLT imaging.Results: Trabectedin evicts the SWI/SNF chromatinremodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue.Conclusions: These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18 F-FLT PET imaging in patients with Ewing sarcoma.

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Section: Cell Proliferation Assayssupporting

confidence: 62%

Section: Ews-fli1 Redistributes In the Nucleus To The Nucleolus Only mentioning

confidence: 99%

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

Harlow

Chassé

Boguslawski

et al. 2019

Clinical Cancer Research

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“…RNA was extracted and submitted for 1x75bp sequencing. Libraries were prepared from 500ng of total RNA as described (17). Reads were aligned to hg19 using STAR (v2.7.0f) (18).…”

Section: Rna-sequencingmentioning

confidence: 99%

Direct therapeutic targeting of SWI/SNF induces epigenetic reprogramming and durable tumor regression in rhabdoid tumor

Chassé

et al. 2019

Preprint

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Conflict of Interest:The authors disclose no potential conflicts of interest. STATEMENT OF TRANSLATIONAL RELEVANCEThere is a tremendous need for novel therapeutic approaches for rhabdoid tumor and the more than 20% of human cancers characterized by dysregulation of the SWI/SNF chromatin remodeling complex. While approaches to target associated complexes, such as PRC2, known to be influenced by dysregulated SWI/SNF are currently being evaluated in the clinic, the direct therapeutic targeting of SWI/SNF has not been explored. Here we identify an inhibitor of SWI/SNF and thoroughly explore the therapeutic development of this compound from a mechanistic and translational perspective thus providing insight into the targeting of this complex as well as a dose, schedule, and biomarker of target inhibition that is immediately clinically translatable. ABSTRACTPurpose: Rhabdoid tumor is a pediatric cancer characterized by the biallelic inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex.SMARCB1 inactivation leads to SWI/SNF redistribution to favor a proliferative dedifferentiated cellular state. Although this deletion is the known oncogenic driver, SWI/SNF therapeutic targeting remains a challenge. Experimental Design:We define a novel epigenetic mechanism for mithramycin using biochemical fractionation, chromatin immunoprecipitation sequencing (ChIP-seq), and a dual spike-in assay for transposase accessible chromatin sequencing (ATAC-seq). We correlate epigenetic reprogramming with changes with chromatin A/B compartments and promoter accessibility with chromHMM models and RNA-seq. Finally, we demonstrate durable, marked tumor response in an intramuscular rhabdoid tumor xenograft model. Results:Here we show mithramycin and a second-generation analogue EC8042 evict mutated SWI/SNF from chromatin and are effective in rhabdoid tumor. SWI/SNF blockade triggers chromatin compartment remodeling and promoter reprogramming leading to differentiation and amplification of H3K27me3, the catalytic mark of PRC2.Treatment of rhabdoid rumor xenografts with EC8042 leads to marked, durable tumor regression and differentiation of the tumor tissue into benign mesenchymal tissue, including de novo bone formation. Conclusion:Overall, this study identifies a novel therapeutic candidate for rhabdoid tumor and an approach that may be applicable to the 20% of cancers characterized by mutated SWI/SNF.

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“…39 More recently, it was shown that the drug redistributes EWSR1-FLI1 within the nucleus to the nucleolus, leading to a marked change in the nuclear distribution of the fusion protein. 43 Interestingly, this effect requires high concentrations of drug perhaps offering a clue why a patient with ES responded in the phase I trial, but the phase II trial in with trabectedin was given over 24 hours did not demonstrate activity in ES. 44 The effect of trabectedin is potentiated by combination with other agents, including insulin-like growth factor 1 receptor (IGF1R)–directed therapies, which mitigates drug resistance or irinotecan via suppression of the WRN helicase.…”

Section: Using Ewing Sarcoma Biology To Advance Carementioning

confidence: 99%

“…47,48 Finally, the second-generation compound lurbinectedin, which has been found to have an improved toxicity profile compared to trabectedin, also suppresses EWSR1-FLI1, causes the same nuclear redistribution of the protein, maintains synergy with irinotecan, and induces the transdifferentiation of ES cells in xenografts. 43 There is clinical interest in further developing lurbinectedin in combination with irinotecan as an EWSR1-FLI1–directed therapy.…”

Section: Using Ewing Sarcoma Biology To Advance Carementioning

confidence: 99%

Advances in the Treatment of Pediatric Bone Sarcomas

Grohar

Janeway

Mase

et al. 2017

American Society of Clinical Oncology Educational Book

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OVERVIEW Bone tumors make up a significant portion of noncentral nervous system solid tumor diagnoses in pediatric oncology patients. Ewing sarcoma and osteosarcoma, both with distinct clinical and pathologic features, are the two most commonly encountered bone cancers in pediatrics. Although mutations in the germline have classically been more associated with osteosarcoma, there is recent evidence germline alterations in patients with Ewing sarcoma also play a significant role in pathogenesis. Treatment advances in this patient population have lagged behind that of other pediatric malignancies, particularly targeted interventions directed at the biologic underpinnings of disease. Recent advances in biologic and genomic understanding of these two cancers has expanded the potential for therapeutic advancement and prevention. In Ewing sarcoma, directed focus on inhibition of EWSR1-FLI1 and its effectors has produced promising results. In osteosarcoma, instead of a concentrated focus on one particular change, largely due to tumor heterogeneity, a more diversified approach has been adopted including investigations of growth factors inhibitors, signaling pathway inhibitors, and immune modulation. Continuing recently made treatment advances relies on clinical trial design and enrollment. Clinical trials should include incorporation of biological findings; specifically, for Ewing sarcoma, assessment of alternative fusions and, for osteosarcoma, stratification utilizing biomarkers. Expanded cancer genomics knowledge, particularly with solid tumors, as it relates to heritability and incorporation of family history has led to early identification of patients with cancer predisposition. In these patients through application of cost-effective evidence-based screening techniques the ultimate goal of cancer prevention is becoming a realization.

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Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by Redistributing It within the Nucleus

Cited by 61 publications

References 50 publications

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

Direct therapeutic targeting of SWI/SNF induces epigenetic reprogramming and durable tumor regression in rhabdoid tumor

Advances in the Treatment of Pediatric Bone Sarcomas

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