19 results on '"Saravana P. Selvanathan"'
Search Results
2. Data from The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
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Francesco Bertoni, Jeffrey A. Toretsky, Brian Lannutti, Anastasios Stathis, Emanuele Zucca, B. Hilda Ye, Davide Genini, Monica Testoni, Ivo Kwee, Andrea Rinaldi, Andrea Cavalli, Saravana P. Selvanathan, Garrett Graham, Giulio Sartori, Valdemar Priebe, Laura Carrassa, Katti Jessen, Sara Napoli, Luciano Cascione, Chiara Tarantelli, Eugenio Gaudio, Elaine Yee Lin Chung, and Filippo Spriano
- Abstract
Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity.Experimental Design:The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments.Results:YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell–like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell–type diffuse large B-cell lymphomas.Conclusions:The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.
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- 2023
3. Table S6 from The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
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Francesco Bertoni, Jeffrey A. Toretsky, Brian Lannutti, Anastasios Stathis, Emanuele Zucca, B. Hilda Ye, Davide Genini, Monica Testoni, Ivo Kwee, Andrea Rinaldi, Andrea Cavalli, Saravana P. Selvanathan, Garrett Graham, Giulio Sartori, Valdemar Priebe, Laura Carrassa, Katti Jessen, Sara Napoli, Luciano Cascione, Chiara Tarantelli, Eugenio Gaudio, Elaine Yee Lin Chung, and Filippo Spriano
- Abstract
Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptome after treatment B) Gene-sets significantly enriched after treatment. C) IRF4/SPIB and lenalidomide related gene-sets significantly enriched after treatment.
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- 2023
4. Table S2 from The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
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Francesco Bertoni, Jeffrey A. Toretsky, Brian Lannutti, Anastasios Stathis, Emanuele Zucca, B. Hilda Ye, Davide Genini, Monica Testoni, Ivo Kwee, Andrea Rinaldi, Andrea Cavalli, Saravana P. Selvanathan, Garrett Graham, Giulio Sartori, Valdemar Priebe, Laura Carrassa, Katti Jessen, Sara Napoli, Luciano Cascione, Chiara Tarantelli, Eugenio Gaudio, Elaine Yee Lin Chung, and Filippo Spriano
- Abstract
Anti-tumor activity of YK-4-279 and TK-216 in lymphomas after 72 h of exposure. Genetic features for DLBCL cells (1,2) are also shown.
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- 2023
5. Table S1 from The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
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Francesco Bertoni, Jeffrey A. Toretsky, Brian Lannutti, Anastasios Stathis, Emanuele Zucca, B. Hilda Ye, Davide Genini, Monica Testoni, Ivo Kwee, Andrea Rinaldi, Andrea Cavalli, Saravana P. Selvanathan, Garrett Graham, Giulio Sartori, Valdemar Priebe, Laura Carrassa, Katti Jessen, Sara Napoli, Luciano Cascione, Chiara Tarantelli, Eugenio Gaudio, Elaine Yee Lin Chung, and Filippo Spriano
- Abstract
Cell lines, growth medium and source. All media were supplemented with fetal bovine serum (20%), Penicillin-Streptomycin 5% (Sigma) and L-glutamine (1%).
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- 2023
6. Table S5 from The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
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Francesco Bertoni, Jeffrey A. Toretsky, Brian Lannutti, Anastasios Stathis, Emanuele Zucca, B. Hilda Ye, Davide Genini, Monica Testoni, Ivo Kwee, Andrea Rinaldi, Andrea Cavalli, Saravana P. Selvanathan, Garrett Graham, Giulio Sartori, Valdemar Priebe, Laura Carrassa, Katti Jessen, Sara Napoli, Luciano Cascione, Chiara Tarantelli, Eugenio Gaudio, Elaine Yee Lin Chung, and Filippo Spriano
- Abstract
Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2. A) Supervised analysis of transcriptome after treatment. B) Gene-sets significantly enriched after treatment.
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- 2023
7. Widespread hypertranscription in aggressive human cancers
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Matthew Zatzman, Fabio Fuligni, Ryan Ripsman, Tannu Suwal, Federico Comitani, Lisa-Monique Edward, Rob Denroche, Gun Ho Jang, Faiyaz Notta, Steven Gallinger, Saravana P. Selvanathan, Jeffrey A. Toretsky, Matthew D. Hellmann, Uri Tabori, Annie Huang, and Adam Shlien
- Subjects
Multidisciplinary ,Neoplasms ,Humans ,RNA ,Prognosis - Abstract
Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers, and prognostic significance are undefined in primary human cancer. This is due, in part, to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Last, patients with hypertranscribed mutations have improved response to immune checkpoint therapy. Our results provide fundamental insights into gene dysregulation across human cancers and may prove useful in identifying patients who would benefit from novel therapies.
- Published
- 2022
8. The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents
- Author
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Ivo Kwee, Laura Carrassa, Monica Testoni, Katti Jessen, B. Hilda Ye, Emanuele Zucca, Filippo Spriano, Davide Genini, Valdemar Priebe, Chiara Tarantelli, Giulio Sartori, Brian J. Lannutti, Saravana P. Selvanathan, Jeffrey A. Toretsky, Sara Napoli, Andrea Cavalli, Elaine Yee Lin Chung, Andrea Rinaldi, Anastasios Stathis, Francesco Bertoni, Eugenio Gaudio, Garrett T. Graham, and Luciano Cascione
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0301 basic medicine ,Cancer Research ,Indoles ,Lymphoma ,Antineoplastic Agents ,Apoptosis ,Biology ,Transcriptome ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,Protein Kinase Inhibitors ,Transcription factor ,Cell Proliferation ,Proto-Oncogene Proteins c-ets ,Venetoclax ,Gene Expression Profiling ,Germinal center ,Cancer ,Drug Synergism ,Prognosis ,medicine.disease ,Immunohistochemistry ,Xenograft Model Antitumor Assays ,Gene expression profiling ,Disease Models, Animal ,030104 developmental biology ,Oncology ,Mechanism of action ,chemistry ,030220 oncology & carcinogenesis ,Cancer research ,medicine.symptom ,Protein Binding - Abstract
Purpose: Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity. Experimental Design: The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments. Results: YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell–like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell–type diffuse large B-cell lymphomas. Conclusions: The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.
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- 2019
9. Abstract LB177: Widespread hypertranscription in aggressive human cancer
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Matthew Zatzman, Fabio Fuligni, Ryan Ripsman, Tannu Suwal, Lisa-Monique Edward, Rob Denroche, Gun Ho Jang, Faiyaz Notta, Steven Gallinger, Saravana P. Selvanathan, Jeffrey Toretsky, Matthew D. Hellmann, Uri Tabori, Annie Huang, and Adam Shlien
- Subjects
Cancer Research ,Oncology - Abstract
Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers and prognostic significance are undefined in primary human cancer. This is due in part to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7,494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Finally, patients with hypertranscribed mutations have improved response to immune checkpoint therapy. Our results provide fundamental insights into gene dysregulation across human cancers and may prove useful in identifying patients that would benefit from novel therapies. Citation Format: Matthew Zatzman, Fabio Fuligni, Ryan Ripsman, Tannu Suwal, Lisa-Monique Edward, Rob Denroche, Gun Ho Jang, Faiyaz Notta, Steven Gallinger, Saravana P. Selvanathan, Jeffrey Toretsky, Matthew D. Hellmann, Uri Tabori, Annie Huang, Adam Shlien. Widespread hypertranscription in aggressive human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB177.
- Published
- 2022
10. EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex
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Eleni M. Tomazou, Tabari M Baker, Saravana P. Selvanathan, Mona Batish, J. Robert Hogg, Garrett T. Graham, Jeffrey A. Toretsky, Alexander R Grego, Kimberly Stegmaier, Mark Simpson, Aykut Üren, Brian D. Crompton, and Heinrich Kovar
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ARID1A ,Oncogene Proteins, Fusion ,Carcinogenesis ,Sarcoma, Ewing ,Biology ,medicine.disease_cause ,Chromatin remodeling ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,Protein Isoforms ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Protein Stability ,Proto-Oncogene Protein c-fli-1 ,Alternative splicing ,fungi ,Gene regulation, Chromatin and Epigenetics ,Nuclear Proteins ,Chromatin Assembly and Disassembly ,Chromatin ,Cell biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Alternative Splicing ,030220 oncology & carcinogenesis ,RNA splicing ,RNA-Binding Protein EWS ,Reprogramming ,Transcription Factors - Abstract
Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS–FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS–FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS–FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS–FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS–FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS–FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.
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- 2019
11. Development of an Ewing sarcoma cell line with resistance to EWS‑FLI1 inhibitor YK‑4‑279
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Aykut Üren, Haydar Çelik, Erin J. Conn, Jeffrey A. Toretsky, Sung Hyeok Hong, Sarah Hour, Garrett T. Graham, David V. Allegakoen, Saravana P. Selvanathan, Jeff R. Petro, and Marilyn Kouassi-Brou
- Subjects
Cancer Research ,Indoles ,Oncogene Proteins, Fusion ,medicine.medical_treatment ,Cell ,CD99 ,Gene Expression ,Antineoplastic Agents ,Sarcoma, Ewing ,Biology ,12E7 Antigen ,Biochemistry ,Targeted therapy ,Cell Line, Tumor ,Genetics ,medicine ,Biomarkers, Tumor ,Humans ,Molecular Biology ,Protein Kinase Inhibitors ,Etoposide ,Dose-Response Relationship, Drug ,Proto-Oncogene Protein c-fli-1 ,Articles ,Cell cycle ,Fusion protein ,medicine.anatomical_structure ,Oncology ,Cell culture ,Drug Resistance, Neoplasm ,FLI1 ,Cancer research ,Molecular Medicine ,RNA-Binding Protein EWS ,medicine.drug - Abstract
Despite Ewing sarcoma (ES) being the second most common pediatric malignancy of bone and soft tissue, few novel therapeutic approaches have been introduced over the past few decades. ES contains a pathognomonic chromosomal translocation that leads to a fusion protein between EWSR1 and an ets family member, most often FLI1. EWS-FLI1 is the most common type of fusion protein and is a well-vetted therapeutic target. A small molecule inhibitor of EWS-FLI1, YK-4-279 (YK) was developed with the intention to serve as a targeted therapy option for patients with ES. The present study investigated resistance mechanisms by developing an ES cell line specifically resistant to YK. The ES cell line A4573 was treated with YK to create resistant cells by long term continuous exposure. The results revealed that resistance in A4573 was robust and sustainable, with a >27-fold increase in IC(50) lasting up to 16 weeks in the absence of the compound. Resistant ES cells were still sensitive to standard of care drugs, including doxorubicin, vincristine and etoposide, which may be valuable in future combination treatments in the clinic. Resistant ES cells revealed an increased expression of CD99. RNA sequencing and qPCR validation of resistant ES cells confirmed an increased expression of ANO1, BRSK2 and IGSF21, and a reduced expression of COL24A1, PRSS23 and RAB38 genes. A functional association between these genes and mechanism of resistance remains to be investigated. The present study created a cell line to investigate YK resistance.
- Published
- 2019
12. Inhibition of the oncogenic fusion protein EWS-FLI1 causes G(2)-M cell cycle arrest and enhanced vincristine sensitivity in Ewing’s sarcoma
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Sydney Parks, Aykut Üren, Jessica N. Haladyna, Ryan M. T. Commins, Eric Moseley, Stefan K. Zöllner, Saravana P. Selvanathan, Sung Hyeok Hong, Michael D. Hogarty, Jeffrey A. Toretsky, Hayriye V. Erkizan, Garrett T. Graham, and Uta Dirksen
- Subjects
0301 basic medicine ,Vincristine ,Vinca ,Indoles ,Oncogene Proteins, Fusion ,Apoptosis ,Sarcoma, Ewing ,Biochemistry ,Article ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Humans ,Cyclin B1 ,Molecular Biology ,Transcription factor ,biology ,Proto-Oncogene Protein c-fli-1 ,Ewing's sarcoma ,Cell Biology ,biology.organism_classification ,medicine.disease ,Fusion protein ,Virology ,Ubiquitin ligase ,G2 Phase Cell Cycle Checkpoints ,030104 developmental biology ,Proto-Oncogene Proteins c-bcl-2 ,Drug Resistance, Neoplasm ,FLI1 ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Cancer research ,M Phase Cell Cycle Checkpoints ,Myeloid Cell Leukemia Sequence 1 Protein ,RNA-Binding Protein EWS ,medicine.drug - Abstract
Ewing’s sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G(2)-M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1-mediated generation of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding the ubiquitin ligase UBE2C, which, in part, contributed to the increase in cyclin B1. YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus, a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients.
- Published
- 2017
13. Single Enantiomer of YK-4-279 Demonstrates Specificity in Targeting the Oncogene EWS-FLI1
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Veselin S Dobrev, Lauren J. Scher, Yali Kong, Maksymilian Chruszcz, Christina L. Kobs, Hayriye V. Erkizan, Julie S. Barber-Rotenberg, Jeffrey A. Toretsky, Tara M. Snyder, Steven Summer, Aykut Üren, Philip J. Monroe, Steven J. Metallo, Milton L. Brown, Wladek Minor, Saravana P. Selvanathan, Perrer N. Tosso, S. Peter Hong, and Natalie L. South
- Subjects
Transcriptional Activation ,Proto-Oncogene Protein c-fli-1 ,Indoles ,Oncogene Proteins, Fusion ,Immunoprecipitation ,RHA ,Transplantation, Heterologous ,Sarcoma, Ewing ,Biology ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Cell Line, Tumor ,Animals ,Humans ,Transcription factor ,EWS-FLI1 ,030304 developmental biology ,Cell Proliferation ,YK-4-279 ,0303 health sciences ,Caspase 3 ,Stereoisomerism ,Molecular biology ,Small molecule ,RNA Helicase A ,Fusion protein ,Research Papers ,3. Good health ,Rats ,Oncology ,030220 oncology & carcinogenesis ,Enantiomer ,RNA-Binding Protein EWS ,Transcription Factors - Abstract
Julie S. Barber-Rotenberg 1 , Saravana P. Selvanathan 1 , Yali Kong 1 , Hayriye V. Erkizan 1 , Tara M. Snyder 2 , S. Peter Hong 3 , Christina L. Kobs 3 , Natalie L. South 3 , Steven Summer 3 , Philip J. Monroe 3 , Maksymilian Chruszcz 4 , Veselin Dobrev 5 , Perrer N. Tosso 1 , Lauren J. Scher 1 , Wladek Minor 4 , Milton L. Brown 1 , Steven J. Metallo 5 , Aykut Uren 1 , and Jeffrey A. Toretsky 1 1 Department of Oncology, Georgetown University Lombardi Comprehensive Cancer Center, Washington, DC USA 2 AMRI, Pharmaceutical and Quality Services, Albany, NY USA 3 Battelle Memorial Institute, Health and Life Sciences, Columbus, OH USA 4 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA USA 5 Department of Chemistry, Georgetown University, Washington, DC USA Received: February 17, 2012; Accepted: February 26, 2012; Published: February 29, 2012; Keywords: PRL-3 monoclonal antibody, PRL-3 mouse/human chimeric antibody, antibody therapy, intracellular oncoprotein Correspondence: Jeffrey A. Toretsky, // // Abstract Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be ‘undruggable.’ A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.
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- 2012
14. The Nuclear Export Signal of Splicing Factor Uap56p Interacts with Nuclear Pore-associated Protein Rae1p for mRNA Export in Schizosaccharomyces pombe
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Ganesh Gopal, Andrew D. Patterson, Anjan G. Thakurta, Saravana P. Selvanathan, and Ravi Dhar
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Nucleocytoplasmic Transport Proteins ,RNA Splicing ,Recombinant Fusion Proteins ,Active Transport, Cell Nucleus ,Biochemistry ,DEAD-box RNA Helicases ,Splicing factor ,Nuclear Matrix-Associated Proteins ,Schizosaccharomyces ,Humans ,RNA, Messenger ,Nuclear pore ,Nuclear export signal ,Molecular Biology ,Nuclear Export Signals ,biology ,RNA ,Cell Biology ,biology.organism_classification ,RNA Helicase A ,Molecular biology ,Cell biology ,Schizosaccharomyces pombe ,RNA splicing ,Nuclear Pore ,Schizosaccharomyces pombe Proteins ,Nuclear transport ,HeLa Cells - Abstract
Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.
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- 2007
15. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level
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Said Rahim, Aykut Üren, Haydar Çelik, Kamal P. Sajwan, Jenny Han, Hayriye V. Erkizan, Saravana P. Selvanathan, Tsion Z. Minas, Yasemin Saygideger Kont, Benjamin J. Marsh, Jeffrey A. Toretsky, and Amrita V. Pai
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Proteomics ,Adamantane ,Bone Neoplasms ,macromolecular substances ,Biology ,Cell morphology ,environment and public health ,DEAD-box RNA Helicases ,Mice ,Ezrin ,Stress granule ,RNA interference ,Tandem Mass Spectrometry ,Cell Line, Tumor ,Animals ,Humans ,RNA, Messenger ,RNA, Small Interfering ,Molecular Biology ,Messenger RNA ,Osteosarcoma ,RNA ,Cell Biology ,Articles ,Surface Plasmon Resonance ,Actin cytoskeleton ,RNA Helicase A ,Molecular biology ,Cell biology ,Cytoskeletal Proteins ,Protein Biosynthesis ,Quinolines ,RNA Interference ,Chromatography, Liquid ,Protein Binding - Abstract
Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5' untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.
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- 2015
16. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing
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Thanemozhi G. Natarajan, Aleksandra Dakic, Xuefeng Liu, Cathy H. Wu, Mats Ljungman, Songtao Yu, Hayriye V. Erkizan, Garrett T. Graham, Uta Dirksen, Jeffrey A. Toretsky, Elizabeth R. Lawlor, Saravana P. Selvanathan, Michelle T. Paulsen, and Aykut Üren
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Indoles ,Oncogene Proteins, Fusion ,Exonic splicing enhancer ,Sarcoma, Ewing ,Biology ,Splicing factor ,Exon ,Cell Line, Tumor ,Humans ,Protein Isoforms ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Telomerase ,Genetics ,Multidisciplinary ,Splice site mutation ,Base Sequence ,Proto-Oncogene Protein c-fli-1 ,fungi ,Alternative splicing ,Intron ,Exons ,Introns ,Cell biology ,Alternative Splicing ,PNAS Plus ,RNA splicing ,Spliceosomes ,RNA-Binding Protein EWS ,Minigene ,Protein Binding ,Signal Transduction - Abstract
The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.
- Published
- 2015
17. Abstract 694: TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma
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Saravana P. Selvanathan, Eric Moseley, Katti Jessen, Brian Lannutti, Jeffrey A. Toretsky, Aykut Üren, and Garrett T. Graham
- Subjects
Cancer Research ,Gene knockdown ,Oncogene ,business.industry ,05 social sciences ,Alternative splicing ,02 engineering and technology ,021001 nanoscience & nanotechnology ,medicine.disease ,In vitro ,Oncology ,Apoptosis ,Cell culture ,0502 economics and business ,RNA splicing ,medicine ,Cancer research ,050211 marketing ,Sarcoma ,0210 nano-technology ,business - Abstract
One of the most significant challenges in creating more potent, less toxic treatments for patients is to identify new cancer therapeutic targets that distinguish the malignant from normal cells. EWS-FLI1 is a well-established Ewing sarcoma (ES) oncogene that has the potential to be an ideal therapeutic target by directly impacting malignant cells. We have previously reported the discovery and characterization of YK-4-279, an enantiomer-specific inhibitor of EWS-FLI1, which has been demonstrated to induce apoptosis, inhibit EWS-FLI1 transcription, block RNA helicase A co-immunoprecipitation with EWS-FLI1, and result in alternative splicing to mimic EWS-FLI1 knockdown. Continuous efforts in structure-guided medicinal chemistry has yielded TK-216, an analog of YK-4-279 inhibitor of EWS-FLI1, which is 3-4 fold more potent with excellent drug-like properties. TK-216 potently inhibits the proliferation of ES cells. Induces apoptosis in a dose -dependent manner as measured by caspase-3 activity in multiple ES cell lines with distinct translocation variants. The effects of TK-216 on alternative splicing (AS) were further validated using genes including ARID1A, CLK1, CASP3, PPFIBP1 and RUNX2. The splicing pattern was similar between TK-216 and YK-4-279. In addition to the in vitro activity of TK-216 , we show that TK-216 displays anti-tumor activity in a number of ES xenograft models. In summary, TK-216, a novel, first-in-class therapeutic which directly inhibits EWS-FLI1, offers a promising approach for the treatment of Ewing Sarcoma and is currently in Phase 1 clinical trials in patients with relapsed or refractory Ewing Sarcoma (clinicaltrials.gov - NCT02657005). Citation Format: Saravana P. Selvanathan, Eric Moseley, Garrett T. Graham, Katti Jessen, Brian Lannutti, Aykut Üren, Jeffrey A. Toretsky. TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of 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 694. doi:10.1158/1538-7445.AM2017-694
- Published
- 2017
18. Abstract A39: Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins
- Author
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Elizabeth R. Lawlor, Aykut Üren, Jeffrey A. Toretsky, Saravana P. Selvanathan, Uta Dirksen, Hayriye V. Erkizan, and Garrett T. Graham
- Subjects
Genetics ,Cancer Research ,Spliceosome ,fungi ,Alternative splicing ,Intron ,Exonic splicing enhancer ,Biology ,Cell biology ,Exon ,SR protein ,Oncology ,RNA splicing ,Minigene - Abstract
Alternative splicing has been implicated as an oncogenic process and provides both a categorization of cancer as well as an opportunity for more effective targeted treatments. Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing, leading to oncogenic protein isoforms. Many proteins that participate in altering splicing have regions of low complexity amino acids, such as serine-arginine (SR) repeats. These SR repeats cause these proteins to have unique biophysical properties leading to phase separation and putatively formation of speckles in the nucleoplasm. EWS-FLI1 has low complexity regions and multiple validated direct interactions with spliceosome components. In addition, our work shows that EWS-FLI1 has novel direct binding to mRNA. We hypothesize that EWS-FLI1, the Ewing sarcoma (ES) oncoprotein, modulates post-transcriptional gene regulation through both novel protein interactions (trans-acting factors) and by directly binding to transcribed mRNA (cis-elements). Our initial results led us to build a novel EWS-FLI1 protein interaction model that identifies both direct and indirect binding throughout the spliceosome. Specific protein interactions of EWS-FLI1 include a newly discovered direct binding partner, DDX5, which is part of the U1 subunit. In addition, direct interactions with both U2 and U5 were identified. We have validated a small molecule probe, YK-4-279, as an enantio-specific inhibitor of EWS-FLI1 that directly blocks its binding to both RHA and p68 (DDX5). Reduction of EWS-FLI1 and YK-4-279 treatment alters exon skipping and intron inclusion events identified from RNA-seq. We then identified splicing events that were similarly reverted by both EWS-FLI1 reduction and YK-4-279, including CLK1, PPFIBP1, and CASP3. In addition, YK-4-279 reverts alternative splicing changes seen in the presence of EWS-FLI1, which was not an effect of altering RNA pol II activity. Our recent unpublished work explores the biophysical interactions of EWS-FLI1 in nucleoplasm. Inhibition of EWS-FLI1 protein interactions by YK-4-279 appear to alter nuclear speckle formation and may, through this mechanism, lead to altered spliceosomal activity. To establish that EWS-FLI1 alternative splicing changes the function of a protein through expression of a different isoform, we selected TERT as an enzyme with an isoform shift in the presence of EWS-FLI1 . When EWS-FLI1 is reduced, a γ-isoform of hTERT is synthesized that lacks exon 11. The γ-isoform of hTERT is reported by others to have increased hTERT activity and we confirm this in cell-based assays following EWS-FLI1 reduction. The cellular and oncogenic implications of this enzymatic activity change is under investigation. To confirm the relevance of splicing modulated by EWS-FLI1 occurring in human tumors, we determined splicing patterns from 75 ES patients and matched splicing patterns for 10 genes in cell line models. Exon-specific expression loci in this comparison were statistically significant for their overlap. In addition, overall gene expression levels did not stratify for overall survival, however, principal component isoform specific analyses did segregate survivors. In metastatic patients, this was significant (p=0.05) while in localized patients it approached significance (p=0.1). This work illuminates potentially critical oncogenic splicing shifts as well as provide a potential stratification mechanism for ES patients. A splicing map specific to ES will define and inform potential functional aspects of alternative isoforms. The biophysical role of EWS-FLI1 in creating or maintaining speckles is an ongoing investigation. Assessment of aberrant splicing driven by EWS-FLI1 may inform oncogenesis and reciprocally, EWS-FLI1 activities may inform splicing mechanisms. Citation Format: Jeffrey Toretsky, Garrett Graham, Uta Dirksen, Hayriye Verda Erkizan, Elizabeth Lawlor, Aykut Uren, Saravana Selvanathan. Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A39.
- Published
- 2016
19. Schizosaccharomyces pombe Dss1p is a DNA damage checkpoint protein that recruits Rad24p, Cdc25p, and Rae1p to DNA double-strand breaks
- Author
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Ming Zhou, Timothy D. Veenstra, Anjan G. Thakurta, Jothy Dhakshnamoorthy, Saravana P. Selvanathan, and Ravi Dhar
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Chromatin Immunoprecipitation ,Nucleocytoplasmic Transport Proteins ,DNA Repair ,DNA repair ,DNA damage ,Ultraviolet Rays ,Recombinant Fusion Proteins ,Blotting, Western ,Cell Cycle Proteins ,Biology ,DNA and Chromosomes ,Biochemistry ,chemistry.chemical_compound ,Nuclear Matrix-Associated Proteins ,Schizosaccharomyces ,DNA Breaks, Double-Stranded ,CHEK1 ,Molecular Biology ,ras-GRF1 ,Cell Cycle ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,G2-M DNA damage checkpoint ,biology.organism_classification ,Molecular biology ,Chromatin ,Cell biology ,Genes, cdc ,chemistry ,Schizosaccharomyces pombe ,Schizosaccharomyces pombe Proteins ,DNA ,DNA Damage - Abstract
Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light Deltadss1 cells entered mitosis prematurely with indistinguishable timing from Deltarad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G(2)/M boundary in response to DNA damage.
- Published
- 2010
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