The Experts below are selected from a list of 11433 Experts worldwide ranked by ideXlab platform
Patrick J Grohar - One of the best experts on this subject based on the ideXlab platform.
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emerging novel agents for patients with advanced Ewing Sarcoma a report from the children s oncology group cog new agents for Ewing Sarcoma task force
F1000Research, 2019Co-Authors: Kelly M Bailey, Carrye Cost, Michael Isakoff, Elizabeth Stewart, Nadia Laack, Peter J. Houghton, Patrick J Grohar, Ian J Davis, Julia Gladebender, Jason YusteinAbstract:Ewing Sarcoma is a small round blue cell malignancy arising from bone or soft tissue and most commonly affects adolescents and young adults. Metastatic and relapsed Ewing Sarcoma have poor outcomes and recurrences remain common. Owing to the poor outcomes associated with advanced disease and the need for a clear research strategy, the Children’s Oncology Group Bone Tumor Committee formed the New Agents for Ewing Sarcoma Task Force to bring together experts in the field to evaluate and prioritize new agents for incorporation into clinical trials. This group’s mission was to evaluate scientific and clinical challenges in moving new agents forward and to recommend agents and trial designs to the Bone Tumor Committee. The task force generated a framework for vetting prospective agents that included critical evaluation of each drug by using both clinical and non-clinical parameters. Representative appraisal of agents of highest priority, including eribulin, dinutuximab, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, anti-angiogenic tyrosine kinase inhibitors, and poly-ADP-ribose polymerase (PARP) inhibitors, is described. The task force continues to analyze new compounds by using the paradigm established.
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functional genomic screening reveals splicing of the ews fli1 fusion transcript as a vulnerability in Ewing Sarcoma
Cell Reports, 2016Co-Authors: Patrick J Grohar, Suntae Kim, Guillermo Rangel Rivera, Nirmalya Sen, Sara Haddock, Matt Harlow, Nichole Maloney, Jack Zhu, Maura Oneill, Tamara L JonesAbstract:Ewing Sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing Sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing Sarcoma cells.
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the second european interdisciplinary Ewing Sarcoma research summit a joint effort to deconstructing the multiple layers of a complex disease
Oncotarget, 2016Co-Authors: Heinrich Kovar, Enrique De ,Álava, Uta Dirksen, James F Amatruda, Erika Brunet, Stefan Burdach, Florencia Cidrearanaz, Wietske Van Der Ent, Patrick J GroharAbstract:Despite multimodal treatment, long term outcome for patients with Ewing Sarcoma is still poor. The second "European interdisciplinary Ewing Sarcoma research summit" assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing Sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA-sequencing of recently described Ewing-like Sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intra-tumor heterogeneity of Ewing Sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing Sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing Sarcoma summits.
Brian D Crompton - One of the best experts on this subject based on the ideXlab platform.
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abstract ct195 a phase 2 clinical trial of palbociclib and ganitumab for relapsed refractory Ewing Sarcoma
Cancer Research, 2020Co-Authors: David S Shulman, Kimberly Stegmaier, Katherine Thornton, Edwin Choy, Lillian M Guenther, Kerri Cavanaugh, Megan Forsyth, Kylene Desmith, Catherine Clinton, Brian D CromptonAbstract:Background: Ewing Sarcoma is a classic translocation-associated malignancy, with nearly all cases carrying an identifiable EWSR1 or FUS translocation. The translocation leads to an aberrant fusion oncoprotein that is thought to function as a pathogenic transcription factor. Despite our understanding of the fundamental biology of this disease, targeting this transcription factor has been elusive. The insulin-like growth factor receptor (IGF-1R) pathway has been long implicated in the pathogenesis of Ewing Sarcoma based on early findings that IGF-1 and IGF-1R are overexpressed in Ewing Sarcoma. A range of preclinical studies have demonstrated the activity of IGF-1R targeting antibodies in multiple Ewing Sarcoma cell lines. Multiple early phase studies of IGF-1R inhibitors have demonstrated that approximately 10% of patients with relapsed disease respond to these agents as monotherapy. Recent work has identified CDK4 as a genomic vulnerability in Ewing Sarcoma. Given that CDK4/6 inhibitor monotherapy is prone to resistance, a series of investigations were undertaken to identify potential combination approaches. In an open reading frame experiment designed to determine whether over expression of specific genes would confer resistance, IGF-1R scored highly. Subsequent in vitro and in vivo experiments demonstrated synergy between CDK4/6 inhibition and IGF-1R inhibition. We have translated these findings into an open phase 2 clinical trial of ganitumab, a monoclonal antibody inhibitor of IGF-1R, and palbociclib, a small molecule inhibitor of CDK4/6. Methods: This is a Phase 2, single-arm, single-stage, investigator-initiated clinical trial of palbociclib and ganitumab in patients 12-50 years of age with relapsed/refractory Ewing Sarcoma (NCT04129151). The primary objectives are to estimate the objective radiographic response rate to the combination of palbociclib and ganitumab and to describe the toxicity of this drug combination in patients with relapsed/refractory Ewing Sarcoma. Palbociclib is given at a dose of 100 mg PO on days 1-21 of a 28-day cycle. Ganitumab is given via IV at a dose of 18 mg/kg on days 1 and 15. The primary endpoint is objective response by RECIST, with disease evaluated after every 2 cycles. Up to 18 patients will be enrolled to yield 15 response evaluable patients. With 15 evaluable patients the study will have 91% power and a type-1 error rate of 0.056 to determine whether the response rate in this population is greater than 40% and significantly different from the 10% response rate seen with IGF-1R monotherapy. Pharmacodynamic testing for confirmation of IGF-1R inhibition (serum IGF-related proteins) is required for all patients. Circulating tumor DNA (ctDNA) is measured at study entry, on cycle 1 day 15, at start of cycle 2 and at each disease evaluation. ctDNA levels will be measured using next generation sequencing for quantification of ctDNA and association with response, and for identification of genomic and epigenetic markers of resistance. Enrollment began in December 2019. Citation Format: David S. Shulman, Katherine Thornton, Edwin Choy, Lillian M. Guenther, Kerri Cavanaugh, Megan Forsyth, Kylene DeSmith, Catherine Clinton, Kimberly Stegmaier, Brian Crompton, Wendy B. London, Steven G. DuBois. A phase 2 clinical trial of palbociclib and ganitumab for relapsed/refractory Ewing Sarcoma [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 CT195.
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therapeutic targeting of kdm1a lsd1 in Ewing Sarcoma with sp 2509 engages the endoplasmic reticulum stress response
Molecular Cancer Therapeutics, 2018Co-Authors: Kathleen I. Pishas, Christina D Drenberg, Cenny Taslim, Emily R Theisen, Kirsten M Johnson, Ranajeet S Saund, Ioana L Pop, Brian D CromptonAbstract:Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing Sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing Sarcoma oncogenesis. As KDM1A/LSD1 (Lysine Specific Demethylase 1) is highly expressed in Ewing Sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P=0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing Sarcoma cell line cohort (n=17)( IC50 range 81nM-1593nM), in contrast resistance to the next generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50g300μM). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hyper-sensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum (ER) stress pathway. In addition, ETS1/ HIST1H2BM were specifically induced/repressed respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing Sarcoma.
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functional chemical genomic and super enhancer screening identify sensitivity to cyclin d1 cdk4 pathway inhibition in Ewing Sarcoma
Oncotarget, 2015Co-Authors: Alyssa L Kennedy, Brian D Crompton, Mounica Vallurupalli, Liying Chen, Glenn S Cowley, Francisca Vazquez, Barbara A Weir, Aviad Tsherniak, Sudha Parasuraman, Sunkyu KimAbstract:// Alyssa L. Kennedy 1, 2, * , Mounica Vallurupalli 1, 3, * , Liying Chen 1, * , Brian Crompton 1 , Glenn Cowley 4 , Francisca Vazquez 4 , Barbara A. Weir 4 , Aviad Tsherniak 4 , Sudha Parasuraman 5 , Sunkyu Kim 5 , Gabriela Alexe 1, 4, 6 , Kimberly Stegmaier 1, 4 1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children’s Hospital, Boston, Massachusetts, USA 2 Boston Combined Residency Program in Pediatrics, Boston, Massachusetts, USA 3 Department of Internal Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA 4 Broad Institute, Cambridge, Massachusetts, USA 5 Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA 6 Bioinformatics Graduate Program, Boston University, Boston, Massachusetts, USA * These authors have contributed equally to this work Correspondence to: Kimberly Stegmaier, e-mail: kimberly_stegmaier@dfci.harvard.edu Keywords: CDK4/6 inhibitor, cyclin D1, epigenetics, Ewing Sarcoma, Sarcoma/soft-tissue malignancies Received: June 29, 2015 Accepted: August 07, 2015 Published: August 18, 2015 ABSTRACT Ewing Sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1 . While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing Sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing Sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing Sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing Sarcoma. We demonstrated that Ewing Sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing Sarcoma cell lines in vitro and growth delay in an in vivo Ewing Sarcoma xenograft model. These results demonstrated a dependency in Ewing Sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease.
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long noncoding rna ewsat1 mediated gene repression facilitates Ewing Sarcoma oncogenesis
Journal of Clinical Investigation, 2014Co-Authors: Michelle Marques Howarth, Brian D Crompton, David Simpson, Siu P Ngok, Bethsaida Nieves, Ron Chen, Zurab Siprashvili, Dedeepya Vaka, Marcus R Breese, Gabriela AlexeAbstract:Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing Sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing Sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing Sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing Sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing Sarcoma via the repression of target genes.
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abstract 999 the genomic landscape of pediatric Ewing Sarcoma
Cancer Research, 2014Co-Authors: Brian D Crompton, Monica L Calicchio, Chip Stewart, Amaro Taylorweiner, Kyle C Kurek, Adam Kiezun, Scott L Carter, Sachet A Shukla, Gabriela Alexa, Swapnil MehtaAbstract:The sequencing of aggressive pediatric solid tumors is revealing remarkably stable genomes. In the cases of malignant rhabdoid and retinoblastoma, there is a paucity of recurrently mutated genes, and oncogenesis appears to be driven, at least in part, by epigenetic deregulation. It has been suggested that pediatric tumors characterized by oncogenic fusions will exhibit relatively few additional somatic driver aberrancies. Ewing Sarcoma, the second most common pediatric bone tumor, is characterized by rearrangements of the EWS gene and ETS-family transcription factor genes, most commonly FLI and ERG. In experimental models, Ewing Sarcoma demonstrates dependency on the expression of the resulting chimeric fusion products. As such, Ewing Sarcoma represents a paradigm for studying the genomic landscape of fusion-driven cancers. To this end, we performed whole-exome sequencing of 96 Ewing Sarcoma tumors and 11 Ewing Sarcoma cell lines, as well as whole-genome sequencing, transcriptome sequencing, and copy-number analysis of a subset of these samples. We found that Ewing Sarcoma is one of the most genetically normal cancers sequenced to date, but that treatment, which generally employs genotoxic chemotherapy and radiation, is associated with an increase in mutation rate and single nucleotide substitutions associated with DNA damage. There was a marked absence of recurrent mutations in immediately druggable targets, such as tyrosine kinases, calling into question the feasibility of utilizing tumor sequencing to nominate targeted therapies for patients with Ewing Sarcoma. Rather, these results highlight the importance of directly targeting the EWS/ETS fusion events or identifying synthetic lethal dependencies. To this end, we clarified a number of outstanding questions regarding the EWS/ETS fusions. We found that reciprocal ETS/EWS fusions are not expressed in Ewing Sarcoma and therefore unlikely to play a role in Ewing pathogenesis as is seen with reciprocal fusions of PML-RARα in acute promyelocytic leukemia. We also found that wild-type FLI and wild-type ERG are not expressed in Ewing Sarcoma tumors. However, there appears to be a role for ETS gene deregulation in this disease beyond the expression of EWS/ETS fusion proteins because we found recurrent somatic events in ERF and ETS1. We also identified a small number of other recurrently mutated genes that likely collaborate with EWS/ETS fusions in a minority of cases and confirmed that loss of STAG2 occurs in approximately 15% of Ewing Sarcoma tumors. Thus, massively parallel sequencing of a large collection of Ewing Sarcoma tumors supports the notion that fusion-driven pediatric malignancies bear quiet genomes, underscores the importance of identifying new treatment approaches targeting EWS/ETS fusions, and also identifies new genetic abnormalities that warrant further biological validation. Citation Format: Brian Crompton, Chip Stewart, Amaro Taylor-Weiner, Gabriela Alexa, Kyle Kurek, Monica Calicchio, Adam Kiezun, Scott Carter, Sachet Shukla, Swapnil Mehta, Aaron Thorner, Carmen de Torres, Cinzia Lavarino, Mariona Sunol, Aaron McKenna, Andrey Sivachenko, Kristian Cibulskis, Michael Lawrence, Lauren Ambrogio, Daniel Auclair, Ivan Rosshandler, Angela Schwarz-Cruz y Celis, Miguel Rivera, Carlos Rodriguez-Galindo, Mark Fleming, Todd Golub, Gad Getz, Jaume Mora, Kimberly Stegmaier. The genomic landscape of pediatric Ewing Sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2014-999
David J. Gordon - One of the best experts on this subject based on the ideXlab platform.
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abstract 2956 inhibition of chk1 sensitizes Ewing Sarcoma cells to cdk1 dependent cell death in s phase
Cancer Research, 2019Co-Authors: David J. Gordon, Kelli L. Goss, Stacia L. KoppenhaferAbstract:Ewing Sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The treatment of Ewing Sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing Sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing Sarcoma cells to inhibitors of RNR, such as gemcitabine. The ATR-CHK1 pathway, from a mechanistic standpoint, regulates multiple stages of the cell cycle, including S-phase, the G2/M transition, and M phase. In the current work, we used cell synchronization to identify that the inhibition of CHK1 in S-phase cells results in premature entry into mitosis and cell death. Similarly, the inhibition of ATR serine/threonine kinase (ATR), the upstream activator of CHK1, also causes Ewing Sarcoma cells to inappropriately enter mitosis. Moreover, we have found in Ewing Sarcoma cells that this aberrant entry into mitosis is mediated, in part, by cyclin dependent kinase 1 (CDK1). In addition, activation of CDK1 by inhibiting the WEE1 kinase with AZD1775 also results in entry into mitosis and cell death in Ewing Sarcoma cells. Currently, ongoing work is focused on the in vivo testing of gemcitabine in combination with CHK1 and WEE1 inhibitors as a novel therapeutic approach for the treatment of Ewing Sarcoma. Citation Format: David Gordon, Kelli Goss, Stacia Koppenhafer. Inhibition of CHK1 sensitizes Ewing Sarcoma cells to CDK1-dependent cell death in S-phase [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 2956.
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Inhibition of CHK1 sensitizes Ewing Sarcoma cells to the ribonucleotide reductase inhibitor gemcitabine
Oncotarget, 2017Co-Authors: Kelli L. Goss, Stacia L. Koppenhafer, Kathryn M. Harmoney, William W. Terry, David J. GordonAbstract:// Kelli L. Goss 1 , Stacia L. Koppenhafer 1 , Kathryn M. Harmoney 1 , William W. Terry 1 and David J. Gordon 1 1 Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Iowa, Iowa City, Iowa 52242, USA Correspondence to: David J. Gordon, email: david-j-gordon@uiowa.edu Keywords: Ewing Sarcoma, ribonucleotide reductase, gemcitabine, CHK1, replication stress Received: March 06, 2017 Accepted: May 22, 2017 Published: June 28, 2017 ABSTRACT Ewing Sarcoma is a bone and soft tissue Sarcoma that occurs in children and young adults. The EWS-FLI1 gene fusion is the driver mutation in most Ewing Sarcoma tumors and functions, in part, as an aberrant transcription factor. We recently identified that Ewing Sarcoma cells are sensitive to inhibition of ribonucleotide reductase (RNR), which catalyzes the formation of deoxyribonucleotides from ribonucleotides. In this report, we show that Ewing Sarcoma cells are sensitive to treatment with clofarabine, which is a nucleoside analogue and allosteric inhibitor of RNR. However, clofarabine is a reversible inhibitor of RNR and we found that the effect of clofarabine is limited when using a short (6-hour) drug treatment. Gemcitabine, on the other hand, is an irreversible inhibitor of the RRM1 subunit of RNR and this drug induces apoptosis in Ewing Sarcoma cells when used in both 6-hour and longer drug treatments. Treatment of Ewing Sarcoma cells with gemcitabine also results in activation of checkpoint kinase 1 (CHK1), which is a critical mediator of cell survival in the setting of impaired DNA replication. Notably, inhibition of CHK1 function in Ewing Sarcoma cells using a small-molecule CHK1 inhibitor, or siRNA knockdown, in combination with gemcitabine results in increased toxicity both in vitro and in vivo in a mouse xenograft experiment. Overall, our results provide insight into Ewing Sarcoma biology and identify a candidate therapeutic target, and drug combination, in Ewing Sarcoma.
Stacia L. Koppenhafer - One of the best experts on this subject based on the ideXlab platform.
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abstract 2956 inhibition of chk1 sensitizes Ewing Sarcoma cells to cdk1 dependent cell death in s phase
Cancer Research, 2019Co-Authors: David J. Gordon, Kelli L. Goss, Stacia L. KoppenhaferAbstract:Ewing Sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The treatment of Ewing Sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing Sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing Sarcoma cells to inhibitors of RNR, such as gemcitabine. The ATR-CHK1 pathway, from a mechanistic standpoint, regulates multiple stages of the cell cycle, including S-phase, the G2/M transition, and M phase. In the current work, we used cell synchronization to identify that the inhibition of CHK1 in S-phase cells results in premature entry into mitosis and cell death. Similarly, the inhibition of ATR serine/threonine kinase (ATR), the upstream activator of CHK1, also causes Ewing Sarcoma cells to inappropriately enter mitosis. Moreover, we have found in Ewing Sarcoma cells that this aberrant entry into mitosis is mediated, in part, by cyclin dependent kinase 1 (CDK1). In addition, activation of CDK1 by inhibiting the WEE1 kinase with AZD1775 also results in entry into mitosis and cell death in Ewing Sarcoma cells. Currently, ongoing work is focused on the in vivo testing of gemcitabine in combination with CHK1 and WEE1 inhibitors as a novel therapeutic approach for the treatment of Ewing Sarcoma. Citation Format: David Gordon, Kelli Goss, Stacia Koppenhafer. Inhibition of CHK1 sensitizes Ewing Sarcoma cells to CDK1-dependent cell death in S-phase [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 2956.
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Inhibition of CHK1 sensitizes Ewing Sarcoma cells to the ribonucleotide reductase inhibitor gemcitabine
Oncotarget, 2017Co-Authors: Kelli L. Goss, Stacia L. Koppenhafer, Kathryn M. Harmoney, William W. Terry, David J. GordonAbstract:// Kelli L. Goss 1 , Stacia L. Koppenhafer 1 , Kathryn M. Harmoney 1 , William W. Terry 1 and David J. Gordon 1 1 Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Iowa, Iowa City, Iowa 52242, USA Correspondence to: David J. Gordon, email: david-j-gordon@uiowa.edu Keywords: Ewing Sarcoma, ribonucleotide reductase, gemcitabine, CHK1, replication stress Received: March 06, 2017 Accepted: May 22, 2017 Published: June 28, 2017 ABSTRACT Ewing Sarcoma is a bone and soft tissue Sarcoma that occurs in children and young adults. The EWS-FLI1 gene fusion is the driver mutation in most Ewing Sarcoma tumors and functions, in part, as an aberrant transcription factor. We recently identified that Ewing Sarcoma cells are sensitive to inhibition of ribonucleotide reductase (RNR), which catalyzes the formation of deoxyribonucleotides from ribonucleotides. In this report, we show that Ewing Sarcoma cells are sensitive to treatment with clofarabine, which is a nucleoside analogue and allosteric inhibitor of RNR. However, clofarabine is a reversible inhibitor of RNR and we found that the effect of clofarabine is limited when using a short (6-hour) drug treatment. Gemcitabine, on the other hand, is an irreversible inhibitor of the RRM1 subunit of RNR and this drug induces apoptosis in Ewing Sarcoma cells when used in both 6-hour and longer drug treatments. Treatment of Ewing Sarcoma cells with gemcitabine also results in activation of checkpoint kinase 1 (CHK1), which is a critical mediator of cell survival in the setting of impaired DNA replication. Notably, inhibition of CHK1 function in Ewing Sarcoma cells using a small-molecule CHK1 inhibitor, or siRNA knockdown, in combination with gemcitabine results in increased toxicity both in vitro and in vivo in a mouse xenograft experiment. Overall, our results provide insight into Ewing Sarcoma biology and identify a candidate therapeutic target, and drug combination, in Ewing Sarcoma.
Neekesh V Dharia - One of the best experts on this subject based on the ideXlab platform.
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trim8 modulates the ews fli oncoprotein to promote survival in Ewing Sarcoma
Cancer Cell, 2021Co-Authors: Bo Kyung A Seong, Neekesh V Dharia, Shan Lin, Katherine A Donovan, Shasha Chong, Amanda L RobichaudAbstract:Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing Sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing Sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing Sarcoma and potentially other fusion-TF-driven cancers.
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abstract b50 targeting ews fli fusion oncoprotein stability degradation in Ewing Sarcoma
Cancer Research, 2020Co-Authors: Bo Kyung Alex Seong, Neekesh V Dharia, Shan Lin, Katherine A Donovan, Amanda L Robichaud, Bjorn Stolte, Emily Jue Wang, Behnam Nabet, Federica Piccioni, Nathanael S GrayAbstract:Fusion-transcription factors (fusion-TFs) represent a class of oncoproteins that drive tumorigenesis by activating an aberrant transcriptional program to promote the development and survival of cancer cells. Unlike fusion oncoproteins involving readily druggable proteins such as kinases, fusion-TFs are difficult to therapeutically target. Recent studies have suggested protein degradation as a novel strategy in targeting oncogenic transcription factors. However, the molecular and functional mechanisms that regulate fusion-TFs’ degradation/stability are unknown. Ewing Sarcoma is the second most common bone cancer in children and is driven by EWS-ETS fusion-TFs, most commonly EWS/FLI (~85%). EWS/FLI acts as a pioneer transcription factor to activate oncogenic gene expression by chromatin remodeling that drives the tumorigenesis and survival of Ewing Sarcoma tumors. Despite this, there are no clinically validated targeted therapies against EWS/FLI. Ewing Sarcoma has a relatively quiet genome and lacks recurrent mutations, which hinders target identification for therapeutic intervention. Thus, we examined whether a protein degradation strategy can be exploited to target EWS/FLI by investigating the molecular and functional mechanisms by which EWS/FLI protein stability/degradation is regulated. In a genome-scale, flow cytometry-based CRISPR-Cas9 screen, we discovered that tripartite motif containing 8 (TRIM8) is a novel E3 ubiquitin for EWS/FLI. Biochemical studies showed that TRIM directly binds and ubiquitinates EWS/FLI for proteasome-dependent degradation. Moreover, we identified TRIM8 as a top enriched and selective dependency in Ewing Sarcoma in a genome-scale CRISPR-Cas9 depletion screen. We determined that TRIM8 knockout increased EWS/FLI protein levels and induced apoptosis of Ewing Sarcoma cells in vitro and reduced tumor growth in vivo. Consistent with TRIM8 as an E3 ligase for EWS/FLI, overexpression of TRIM8 reduced EWS/FLI protein levels and decreased growth. TRIM8 lacking the E3 ligase domain (TRIM8E3) functioned as a dominant negative, leading to increased EWS/FLI and decreased growth of Ewing Sarcoma cells. To investigate whether upregulated EWS/FLI protein expression is mediating the TRIM8 knockout phenotype, we engineered Ewing Sarcoma cell line by concurrently knocking out endogenous EWS/FLI using CRISPR and expressing degradable EWS/FLI (FKBP12F36V-EWS/FLI) using the dTAG system. The dTAG molecule is a heterobifunctional molecule that specifically binds to FKBP12F36V and an E3 ligase complex for targeted protein degradation. Using this degradable EWS/FLI cell model, we showed that TRIM8 suppression phenotype can be rescued by degrading the upregulated EWS/FLI protein expression to control levels. Our results demonstrate that the protein stability of fusion-TF oncoproteins is highly regulated to maintain the expression at a precise level and highlight the critical importance of oncogene dosage in cancer cell survival. Citation Format: Bo Kyung Alex Seong, Shan Lin, Katherine Donovan, Amanda Robichaud, Bjorn Stolte, Emily Wang, Neekesh Dharia, Behnam Nabet, Federica Piccioni, Nathanael Gray, Eric Fischer, Kimberly Stegmaier. Targeting EWS/FLI fusion oncoprotein stability/degradation 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 B50.
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genome scale crispr cas9 screen identifies druggable dependencies in tp53 wild type Ewing Sarcoma
Journal of Experimental Medicine, 2018Co-Authors: Neekesh V Dharia, Amanda L Robichaud, Bjorn Stolte, Amanda Balboni Iniguez, Amy Saur Conway, Ann M MorganAbstract:Ewing Sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type TP53, and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for TP53 wild-type Ewing Sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/PPM1D inhibitor, GSK2830371, decreased the viability of Ewing Sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent TP53 knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.
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ews fli confers tumor cell synthetic lethality to cdk12 inhibition in Ewing Sarcoma
Cancer Cell, 2018Co-Authors: Amanda Balboni Iniguez, Neekesh V Dharia, Gabriela Alexe, Bjorn Stolte, Nicholas Kwiatkowski, Emily Jue Wang, Amy Saur Conway, Tinghu ZhangAbstract:Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing Sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing Sarcoma, including a PDX, in vivo without hematopoietic toxicity.
