68 results on '"Regina, Cencic"'
Search Results
2. C8ORF88: A Novel eIF4E-Binding Protein
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Lauren Pugsley, Sai Kiran Naineni, Mehdi Amiri, Akiko Yanagiya, Regina Cencic, Nahum Sonenberg, and Jerry Pelletier
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protein synthesis ,translation initiation ,gene expression ,mRNA ,cap-dependent translation ,C8ORF88 ,Genetics ,QH426-470 - Abstract
Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.
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- 2023
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3. A forward genetic screen identifies modifiers of rocaglate responsiveness
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Leo Shen, Lauren Pugsley, Regina Cencic, HanChen Wang, Francis Robert, Sai Kiran Naineni, Ananya Sahni, Geneviève Morin, Wenhan Zhang, Anastasia Nijnik, John A. Porco, David Langlais, Sidong Huang, and Jerry Pelletier
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Medicine ,Science - Abstract
Abstract Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. Rocaglates perturb eIF4A activity by imparting a gain-of-function activity to eIF4A and mediating clamping to RNA. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as identify responsive tumor types. Here, we report on the results of a positive selection, ORFeome screen aimed at identifying cDNAs capable of conferring resistance to rocaglates. Two of the most potent modifiers of rocaglate response identified were the transcription factors FOXP3 and NR1I3, both of which have been implicated in ABCB1 regulation—the gene encoding P-glycoprotein (Pgp). Pgp has previously been implicated in conferring resistance to silvestrol, a naturally occurring rocaglate, and we show here that this extends to additional synthetic rocaglate derivatives. In addition, FOXP3 and NR1I3 impart a multi-drug resistant phenotype that is reversed upon inhibition of Pgp, suggesting a potential therapeutic combination strategy.
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- 2021
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4. CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary
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Yibo Xue, Brian Meehan, Elizabeth Macdonald, Sriram Venneti, Xue Qing D. Wang, Leora Witkowski, Petar Jelinic, Tim Kong, Daniel Martinez, Geneviève Morin, Michelle Firlit, Atefeh Abedini, Radia M. Johnson, Regina Cencic, Jay Patibandla, Hongbo Chen, Andreas I. Papadakis, Aurelie Auguste, Iris de Rink, Ron M. Kerkhoven, Nicholas Bertos, Walter H. Gotlieb, Blaise A. Clarke, Alexandra Leary, Michael Witcher, Marie-Christine Guiot, Jerry Pelletier, Josée Dostie, Morag Park, Alexander R. Judkins, Ralf Hass, Douglas A. Levine, Janusz Rak, Barbara Vanderhyden, William D. Foulkes, and Sidong Huang
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Science - Abstract
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is driven by SMARCA4 loss. Here the authors demonstrate that SCCOHT cells are highly sensitive to CDK4/6 inhibition and provide mechanistic insights, whereby this druggable vulnerability is driven by cyclin D1 deficiency induced by SMARCA4 loss.
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- 2019
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5. Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F
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Jennifer Chu, Wenhan Zhang, Regina Cencic, Patrick B.F. O’Connor, Francis Robert, William G. Devine, Asher Selznick, Thomas Henkel, William C. Merrick, Lauren E. Brown, Pavel V. Baranov, John A. Porco, Jr., and Jerry Pelletier
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Biology (General) ,QH301-705.5 - Abstract
Summary: Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5′ leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA. : Rocaglates are a diverse family of small molecules that inhibit eIF4A. Chu et al. undertake a comparative analysis of the bioactivity of >200 rocaglates and uncover nuances in their mechanisms of action. Rocaglates interfere with eIF4F release from the cap and exert a bystander effect to inhibit translation. Keywords: rocaglates, eIF4A, eIF4F, translation initiation, interfacial inhibitor
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- 2020
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6. Table S3 from eIF4A Inhibitors Suppress Cell-Cycle Feedback Response and Acquired Resistance to CDK4/6 Inhibition in Cancer
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Sidong Huang, Jerry Pelletier, Morag Park, John A. Porco, William D. Foulkes, Marie-Christine Guiot, Veena Sangwan, Virginie Pilon, Zheng Fu, Anie Monast, Xianbing Zhu, Regina Cencic, Yibo Xue, and Tim Kong
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Supplemental Table 3.Overlap of genes whose mRNA translation efficiency is suppressed by silvestrol
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- 2023
7. Data from eIF4A Inhibitors Suppress Cell-Cycle Feedback Response and Acquired Resistance to CDK4/6 Inhibition in Cancer
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Sidong Huang, Jerry Pelletier, Morag Park, John A. Porco, William D. Foulkes, Marie-Christine Guiot, Veena Sangwan, Virginie Pilon, Zheng Fu, Anie Monast, Xianbing Zhu, Regina Cencic, Yibo Xue, and Tim Kong
- Abstract
CDK4/6 inhibitors are FDA-approved drugs for estrogen receptor–positive (ER+) breast cancer and are being evaluated to treat other tumor types, including KRAS-mutant non–small cell lung cancer (NSCLC). However, their clinical utility is often limited by drug resistance. Here, we sought to better understand the resistant mechanisms and help devise potential strategies to overcome this challenge. We show that treatment with CDK4/6 inhibitors in both ER+ breast cancer and KRAS-mutant NSCLC cells induces feedback upregulation of cyclin D1, CDK4, and cyclin E1, mediating drug resistance. We demonstrate that rocaglates, which preferentially target translation of key cell-cycle regulators, effectively suppress this feedback upregulation induced by CDK4/6 inhibition. Consequently, combination treatment of CDK4/6 inhibitor palbociclib with the eukaryotic initiation factor (eIF) 4A inhibitor, CR-1-31-B, is synergistic in suppressing the growth of these cancer cells in vitro and in vivo. Furthermore, ER+ breast cancer and KRAS-mutant NSCLC cells that acquired resistance to palbociclib after chronic drug exposure are also highly sensitive to this combination treatment strategy. Our findings reveal a novel strategy using eIF4A inhibitors to suppress cell-cycle feedback response and to overcome resistance to CDK4/6 inhibition in cancer.
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- 2023
8. Supplemental Figures 1 - 10 from eIF4A Inhibitors Suppress Cell-Cycle Feedback Response and Acquired Resistance to CDK4/6 Inhibition in Cancer
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Sidong Huang, Jerry Pelletier, Morag Park, John A. Porco, William D. Foulkes, Marie-Christine Guiot, Veena Sangwan, Virginie Pilon, Zheng Fu, Anie Monast, Xianbing Zhu, Regina Cencic, Yibo Xue, and Tim Kong
- Abstract
Supplemental Figure 1 shows validation of cyclin E and CDK4 signals with RNAi. Supplemental Figure 2 shows that cyclin D1 dictates response to palbociclib and is limiting in the formation of CDK4/6-Cyclin D1 complexes in ER-positive breast cancer. Supplemental Figure 3 shows that rocaglates preferentially suppresses protein expression of cyclin D1, CDK4, and cyclin E1. Supplemental Figure 4 shows synergism between a second CDK4/6 inhibitor, Abemaciclib, and CR-1-31-B. Supplemental Figure 5 shows functional cell cycle effects of palbociclib and/or CR-1-31-B treatment in ER-positive breast cancer. Supplemental Figure 6 shows sensitivity of KRAS-mutant non-small cell lung cancers to palbociclib and CR-1-31-B. Supplemental Figure 7 shows functional cell cycle effects of palbociclib and/or CR-1-31-B treatment in NSCLC. Supplemental Figure 8 shows weights of subcutaneous xenografted NSG mice treated with palbociclib, CR-1-31-B, or their combination. Supplemental Figure 9 shows colony formation panel of palbociclib-resistant MCF-7 clones. Supplemental Figure 10 shows cell viability assay of CR-1-31-B response.
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- 2023
9. Supplementary Figure 5 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 775K
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- 2023
10. Supplementary Table 1 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 61K
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- 2023
11. Supplementary Figure 2 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 2 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
12. Supplementary Figure 5 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 5 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
13. Supplementary Figure 7 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 7 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
14. Supplementary Figure 4 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 4 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
15. Supplementary Figure 8 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 8 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
16. Supplementary Figure 4 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 476K
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- 2023
17. Supplementary Figure 8 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 453K
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- 2023
18. Supplementary Figure 10 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 274K
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- 2023
19. Data from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (eIF4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. eIF4F is involved in the recruitment of ribosomes to mRNAs and is thought to generally be the rate-limiting phase of translation. Here, we show that c-Myc directly activates transcription of the three subunits of eIF4F (eIF4E, eIF4AI, and eIF4GI). These transcriptional effects are mediated through canonical E-boxes (5′CACGTG3′) present in the promoters of these genes. In addition, the c-Myc antagonist Mad1 down-regulates the expression of eIF4F subunits. We also show that MycER activation stimulates protein synthesis at the level of translation initiation. Increased eIF4F levels result in stimulation of c-Myc mRNA translation specifically, as assessed by quantitative reverse transcription–PCR. We use a murine model of lymphomagenesis to show the expression of eIF4F subunits is also up-regulated by c-Myc in vivo. Our results suggest the presence of a feedforward loop involving c-Myc and eIF4F that serves to link transcription and translation and that could contribute to the effects of c-Myc on cell proliferation and neoplastic growth. [Cancer Res 2008;68(13):5326–34]
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- 2023
20. sSupplementary Figure Legends 1-10 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 98K
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- 2023
21. Supplementary Figure 3 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 3 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
22. Supplementary Figure 9 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 247K
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- 2023
23. Supplementary Figure 7 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
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PDF file - 662K
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- 2023
24. Supplementary Methods, Figure Legends 1-8 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Methods, Figure Legends 1-8 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
25. Supplementary Figure 3 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
- Abstract
PDF file - 280K
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- 2023
26. Supplementary Figure 1 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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Jerry Pelletier, Francis Robert, John R. Mills, Regina Cencic, and Chen-Ju Lin
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Supplementary Figure 1 from c-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation
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- 2023
27. Supplementary Figure 2 from Targeting Protein Synthesis in a Myc/mTOR-Driven Model of Anorexia-Cachexia Syndrome Delays Its Onset and Prolongs Survival
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Jerry Pelletier, Simon S. Wing, Siegfried Hekimi, Imed Eddine Gallouzi, Michel L. Tremblay, Regina Cencic, Sergio DiMarco, Dantong Wang, Aouod Agenor, John R. Mills, and Francis Robert
- Abstract
PDF file - 309K
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- 2023
28. Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma
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Christina Skofler, Florian Kleinegger, Stefanie Krassnig, Anna Maria Birkl-Toeglhofer, Georg Singer, Holger Till, Martin Benesch, Regina Cencic, John A. Porco, Jerry Pelletier, Christoph Castellani, Andrea Raicht, Ewa Izycka-Swieszewska, Piotr Czapiewski, and Johannes Haybaeck
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neuroblastoma ,eukaryotic initiation factor 4AI (eIF4AI) ,rocaglates ,CR-1-31-B ,SH-SY5Y ,Kelly ,Cytology ,QH573-671 - Abstract
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.
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- 2021
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29. CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A
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Jennifer Chu, Gabriela Galicia-Vázquez, Regina Cencic, John R. Mills, Alexandra Katigbak, John A. Porco Jr., and Jerry Pelletier
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rocaglates ,eIF4A ,translational control ,chemical biology ,CRISPR/Cas9 ,Biology (General) ,QH301-705.5 - Abstract
Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.
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- 2016
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30. Haploinsufficiency of the ESCRT Component HD-PTP Predisposes to Cancer
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Sanaz Manteghi, Marie-Claude Gingras, Dmitri Kharitidi, Luc Galarneau, Maud Marques, Ming Yan, Regina Cencic, Francis Robert, Marilène Paquet, Michael Witcher, Jerry Pelletier, and Arnim Pause
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Biology (General) ,QH301-705.5 - Abstract
Endosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis in vivo. Indeed, Ptpn23+/− loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23+/−-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23+/− loss increases integrin β1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking.
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- 2016
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31. Assessing eukaryotic initiation factor 4F subunit essentiality by CRISPR-induced gene ablation in the mouse
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Marilène Paquet, Regina Cencic, Jennifer Chu, Jerry Pelletier, Nahum Sonenberg, Akiko Yanagiya, Francis Robert, and Patrick Senechal
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Pharmacology ,Regulation of gene expression ,0303 health sciences ,EIF4G ,EIF4E ,Cell Biology ,EIF4A1 ,Biology ,EIF4G3 ,Cell biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Eukaryotic initiation factor 4F ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Eukaryotic initiation factor ,eIF4A ,Molecular Medicine ,Molecular Biology ,030304 developmental biology - Abstract
Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e∆38 fs/+/Eμ-Myc or Eif4a1∆5 fs/+/Eμ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.
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- 2021
32. Exploring the Targeting Spectrum of Rocaglates Among eIF4A Homologs
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Sai Kiran Naineni, Regina Cencic, Francis Robert, Lauren Brown, Minza Haque, Jordan Scott-Talib, Patrick Senechal, T. Martin Schmeing, John Porco, and Jerry Pelletier
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Molecular Biology - Abstract
Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (Pat A) and rocaglates – two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3 – a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream of premature termination codons (PTCs), participate in nonsense mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA transcripts. We find that rocaglates can also interact with eIF4A3 to induce RNA clamping. Rocaglates also inhibit EJC-dependent nonsense-mediated decay (NMD) in mammalian cells, but this does not appear to be due to induced eIF4A3-RNA clamping, but rather a secondary consequence of translation inhibition incurred by clamping eIF4A1 and eIF4A2 to mRNA.
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- 2023
33. Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5′ leader regions
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Brahm J. Yachnin, Sai Kiran Naineni, Mehdi Amiri, Jutta Steinberger, Jerry Pelletier, Sarah A.E. Aboushawareb, Stephen J Kiniry, Francis Robert, Regina Cencic, Pavel V. Baranov, Nahum Sonenberg, Jennifer Chu, Leo Shen, and Rayelle Itoua Maïga
- Subjects
AcademicSubjects/SCI00010 ,Hippuristanol ,Biology ,Ribosome ,Helicase ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Eukaryotic initiation factor ,Small-molecule inhibition ,Genetics ,Protein biosynthesis ,Humans ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Gene regulation, Chromatin and Epigenetics ,RNA ,Binding ,RNA Helicase A ,Eukaryotic translation initiation ,Cell biology ,Sterols ,Therapeutic suppression ,chemistry ,Drug Resistance, Neoplasm ,eIF4A ,Eukaryotic Initiation Factor-4A ,Mutation ,CRISPR-Cas Systems ,5' Untranslated Regions ,Ribosomes ,030217 neurology & neurosurgery - Abstract
Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.
- Published
- 2020
34. RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes
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T. Martin Schmeing, Jerry Pelletier, Regina Cencic, Renying Cai, and Francis Robert
- Subjects
RNA Caps ,AcademicSubjects/SCI00010 ,Biology ,RNA Cap Analogs ,Ribosome ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Eukaryotic translation ,Eukaryotic initiation factor ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,EIF4G ,fungi ,030302 biochemistry & molecular biology ,EIF4E ,food and beverages ,EIF4A1 ,Cell biology ,Eukaryotic Initiation Factor-4E ,HEK293 Cells ,Eukaryotic Initiation Factor-4F ,chemistry ,Protein Biosynthesis ,eIF4A ,Eukaryotic Initiation Factor-4G ,Ribosomes ,Protein Binding - Abstract
Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.
- Published
- 2020
35. CRISPR-Based Screen Links an Inhibitor of Nonsense-Mediated Decay to eIF4A3 Target Engagement
- Author
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Sai Kiran Naineni, Jerry Pelletier, Regina Cencic, Patrick Senechal, Ananya Sahni, and Lauren Pugsley
- Subjects
0301 basic medicine ,Nonsense-mediated decay ,Biology ,01 natural sciences ,Biochemistry ,DEAD-box RNA Helicases ,03 medical and health sciences ,Exon ,Eukaryotic initiation factor ,Humans ,CRISPR ,Alleles ,010405 organic chemistry ,Cas9 ,General Medicine ,RNA Helicase A ,mRNA surveillance ,Nonsense Mediated mRNA Decay ,0104 chemical sciences ,Cell biology ,030104 developmental biology ,Eukaryotic Initiation Factor-4A ,Mutation ,RNA splicing ,Molecular Medicine ,CRISPR-Cas Systems - Abstract
Eukaryotic initiation factor (eIF) 4A3 is a DEAD-box RNA helicase and a core component of the exon-junction complex (EJC). The EJC marks the location of exon:exon junctions following the removal of introns by splicing and plays a critical role in an mRNA surveillance program known as nonsense-mediated decay (NMD). NMD is often triggered by the presence of a premature termination codon (PTC) upstream of the EJC, leading to degradation of the variant mRNA which prevents synthesis of a potentially harmful, truncated polypeptide. One approach by which to treat rare diseases where the underlying cause is a PTC is thus to prevent NMD, while stimulating readthrough of the PTC. Hence, there is much interest in inhibiting NMD, and recently a set of small molecules, 1,4-diacylpiperazine derivatives, targeting eIF4A3 has been developed and shown to harbor such activity. Herein, we undertake a CRISPR/Cas9-based variomics screen to identify eIF4A3 alleles resistant to said compounds. Our results provide genetic evidence linking compound bioactivity to eIF4A3 engagement.
- Published
- 2020
36. Effect of 2′-5′/3′-5′ phosphodiester linkage heterogeneity on RNA interference
- Author
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S. Harikrishna, Keith T. Gagnon, Masad J. Damha, Regina Cencic, Pushpangadan Indira Pradeepkumar, Adam Katolik, Hassan H. Fakih, Maria Barton, Maryam Habibian, John J. Rossi, Mayumi Takahashi, Jerry Pelletier, Eman A Ageely, and Johans Fakhoury
- Subjects
Small interfering RNA ,AcademicSubjects/SCI00010 ,Molecular Dynamics Simulation ,Biology ,01 natural sciences ,03 medical and health sciences ,Chemical Biology and Nucleic Acid Chemistry ,Luciferases, Firefly ,RNA interference ,0103 physical sciences ,Carbohydrate Conformation ,Genetics ,Humans ,RNA, Small Interfering ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,010304 chemical physics ,Kinase ,RNA ,Cell biology ,Sense strand ,Argonaute Proteins ,Phosphodiester bond ,Nucleic Acid Conformation ,Phosphorylation ,RNA Interference ,Tumor Suppressor Protein p53 ,HeLa Cells - Abstract
We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2′-5′/3′-5′-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.
- Published
- 2020
37. A comparative study of small molecules targeting eIF4A
- Author
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Abimael D. Rodríguez, Regina Cencic, Andrea Putnam, Luis A. Amador, Rayelle Itoua Maïga, Sai Kiran Naineni, Jerry Pelletier, and Eckhard Jankowsky
- Subjects
Cell Survival ,Hippuristanol ,Antineoplastic Agents ,Biology ,Small Molecule Libraries ,Phosphatidylinositol 3-Kinases ,03 medical and health sciences ,chemistry.chemical_compound ,Eukaryotic translation ,Neoplasms ,Report ,Eukaryotic initiation factor ,Humans ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Benzofurans ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,TOR Serine-Threonine Kinases ,030302 biochemistry & molecular biology ,RNA Helicase A ,Small molecule ,3. Good health ,Cell biology ,Sterols ,Thiazoles ,Eukaryotic Initiation Factor-4F ,chemistry ,Protein Biosynthesis ,eIF4A ,Eukaryotic Initiation Factor-4A ,Epoxy Compounds ,Macrolides ,Proto-Oncogene Proteins c-akt - Abstract
The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity—generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.
- Published
- 2020
38. A forward genetic screen identifies modifiers of rocaglate responsiveness
- Author
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Sidong Huang, David Langlais, Regina Cencic, Wenhan Zhang, Sai Kiran Naineni, Geneviève Morin, Jerry Pelletier, Lauren Pugsley, Francis Robert, Anastasia Nijnik, Leo Shen, Ananya Sahni, John A. Porco, and HanChen Wang
- Subjects
Translation ,Cancer therapy ,Molecular biology ,Science ,Receptors, Cytoplasmic and Nuclear ,Biology ,Article ,Cell Line ,Target validation ,03 medical and health sciences ,0302 clinical medicine ,Eukaryotic translation ,Target identification ,Eukaryotic initiation factor ,Humans ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Genetic Testing ,ORFeome ,Gene ,Transcription factor ,Constitutive Androstane Receptor ,Benzofurans ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Drug discovery ,Forkhead Transcription Factors ,Chemical biology ,RNA Helicase A ,Cell biology ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,eIF4A ,Eukaryotic Initiation Factor-4A ,Medicine ,Genetic screen - Abstract
Rocaglates are a class of eukaryotic translation initiation inhibitors that are being explored as chemotherapeutic agents. They function by targeting eukaryotic initiation factor (eIF) 4A, an RNA helicase critical for recruitment of the 40S ribosome (and associated factors) to mRNA templates. Rocaglates perturb eIF4A activity by imparting a gain-of-function activity to eIF4A and mediating clamping to RNA. To appreciate how rocaglates could best be enabled in the clinic, an understanding of resistance mechanisms is important, as this could inform on strategies to bypass such events as well as identify responsive tumor types. Here, we report on the results of a positive selection, ORFeome screen aimed at identifying cDNAs capable of conferring resistance to rocaglates. Two of the most potent modifiers of rocaglate response identified were the transcription factors FOXP3 and NR1I3, both of which have been implicated in ABCB1 regulation—the gene encoding P-glycoprotein (Pgp). Pgp has previously been implicated in conferring resistance to silvestrol, a naturally occurring rocaglate, and we show here that this extends to additional synthetic rocaglate derivatives. In addition, FOXP3 and NR1I3 impart a multi-drug resistant phenotype that is reversed upon inhibition of Pgp, suggesting a potential therapeutic combination strategy.
- Published
- 2021
39. Assessing eukaryotic initiation factor 4F subunit essentiality by CRISPR-induced gene ablation in the mouse
- Author
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Patrick, Sénéchal, Francis, Robert, Regina, Cencic, Akiko, Yanagiya, Jennifer, Chu, Nahum, Sonenberg, Marilène, Paquet, and Jerry, Pelletier
- Subjects
Male ,Mice, Inbred C57BL ,Heterozygote ,Mice ,Protein Subunits ,Eukaryotic Initiation Factor-4F ,Gene Expression Regulation ,Animals ,Clustered Regularly Interspaced Short Palindromic Repeats ,Female ,Mice, Transgenic ,Spermatogenesis - Abstract
Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e
- Published
- 2021
40. Oxo-aglaiastatin-Mediated Inhibition of Translation Initiation
- Author
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Jennifer Chu, Rayelle Itoua Maïga, Daniel D Waller, Lauren E. Brown, William G. Devine, Jerry Pelletier, John A. Porco, Wenhan Zhang, Michael Sebag, and Regina Cencic
- Subjects
0301 basic medicine ,Lymphoma ,lcsh:Medicine ,Antineoplastic Agents ,Article ,03 medical and health sciences ,0302 clinical medicine ,Eukaryotic translation ,Cell Line, Tumor ,Neoplasms ,Eukaryotic initiation factor ,Animals ,Humans ,Peptide Chain Initiation, Translational ,lcsh:Science ,PI3K/AKT/mTOR pathway ,Sulfonamides ,Multidisciplinary ,Chemistry ,lcsh:R ,Drug Synergism ,Translation (biology) ,Biological activity ,EIF4A1 ,Bridged Bicyclo Compounds, Heterocyclic ,Antineoplastic Agents, Phytogenic ,RNA Helicase A ,3. Good health ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,Doxorubicin ,eIF4A ,Eukaryotic Initiation Factor-4A ,Female ,lcsh:Q ,Aglaia ,030217 neurology & neurosurgery - Abstract
Translation is a highly regulated process that is perturbed in human cancers, often through activation of the PI3K/mTOR pathway which impacts directly on the ribosome recruitment phase of translation initiation. While significant research has focused on “drugging” components of the PI3K/mTOR network, efforts have also been directed towards inhibiting eukaryotic initiation factor (eIF) 4F-dependent translation. Small molecule inhibitors of this complex have been identified, characterized, and used to validate the rationale of targeting this step to curtail tumor cell growth and modulate chemotherapy response. One such class of compounds are the rocaglates, secondary metabolites from the plant genus Aglaia, which target the RNA helicase subunit of eIF4F, eIF4A. Here we explore the ability of synthetic derivatives of aglaiastatins and an aglaroxin derivative to target the translation process in vitro and in vivo and find the synthetic derivative oxo-aglaiastatin to possess such activity. Oxo-aglaiastatin inhibited translation in vitro and in vivo and synergized with doxorubicin, ABT-199 (a Bcl-2 antagonist), and dexamethasone when tested on hematological cancer cells. The biological activity of oxo-aglaiastatin was shown to be a consequence of inhibiting eIF4A1 activity.
- Published
- 2019
41. A cautionary note on the use of cap analogue affinity resins
- Author
-
Jerry Pelletier and Regina Cencic
- Subjects
RNA Caps ,0301 basic medicine ,Biophysics ,Plasma protein binding ,Biochemistry ,Ribosome ,Chromatography, Affinity ,03 medical and health sciences ,Eukaryotic translation ,Affinity chromatography ,Eukaryotic initiation factor ,Escherichia coli ,Humans ,RNA, Messenger ,Molecular Biology ,Chemistry ,Sepharose ,RNA ,Translation (biology) ,Cell Biology ,Recombinant Proteins ,Cell biology ,Eukaryotic Initiation Factor-4E ,030104 developmental biology ,Scientific Experimental Error ,Function (biology) ,HeLa Cells ,Protein Binding - Abstract
All cellular cytoplasmic mRNAs are capped at their 5' ends with an m7GpppN group. Several proteins that mediate cap function have been identified by cap affinity purification, enabling their characterization in a number of biological processes. Among these, eukaryotic initiation factor (eIF) 4E is the best characterized and plays a critical role in regulating ribosome recruitment to mRNAs during translation initiation. Cap affinity chromatography is often used to identify eIF4E-interacting proteins, which could play critical roles in molding the eIF4E-interactome and impacting on eIF4E-directed translation. Here we address how improper implementation of this technology can lead to false conclusions and provide recommendations to ensure correct interpretation of data obtained by this approach.
- Published
- 2018
42. Functional mimicry revealed by the crystal structure of an eIF4A:RNA complex bound to the interfacial inhibitor, desmethyl pateamine A
- Author
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Jerry Pelletier, Mingzhao Zhu, Jason Liang, Peter T. Northcote, Paul H. Teesdale-Spittle, Sai Kiran Naineni, Kenneth G. Hull, Daniel Romo, Regina Cencic, and Bhushan Nagar
- Subjects
Models, Molecular ,Stereochemistry ,Clinical Biochemistry ,Molecular Conformation ,Biology ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Eukaryotic translation ,Rocaglamide ,Eukaryotic initiation factor ,Drug Discovery ,Humans ,Molecular Biology ,Pharmacology ,010405 organic chemistry ,RNA ,RNA Helicase A ,Small molecule ,0104 chemical sciences ,Thiazoles ,chemistry ,eIF4A ,Eukaryotic Initiation Factor-4A ,Epoxy Compounds ,Molecular Medicine ,Macrolides ,Macromolecule - Abstract
Interfacial inhibitors exert their biological effects through co-association with two macromolecules. The pateamine A (PatA) class of molecules function by stabilizing eukaryotic initiation factor (eIF) 4A RNA helicase onto RNA, resulting in translation initiation inhibition. Here, we present the crystal structure of an eIF4A1:RNA complex bound to an analog of the marine sponge-derived natural product PatA, C5-desmethyl PatA (DMPatA). One end of this small molecule wedges itself between two RNA bases while the other end is cradled by several protein residues. Strikingly, DMPatA interacts with the eIF4A1:RNA complex in an almost identical fashion as rocaglamide A (RocA), despite being completely unrelated from a structural standpoint. The structural data rationalize the ability of PatA analogs to target a wider range of RNA substrates compared to RocA. We define the molecular basis of how DMPatA is able to clamp eIF4A1 onto RNA, imparting potent inhibitory properties to this molecule.
- Published
- 2021
43. Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response
- Author
-
Ming Yang, Hsiao Chang Chan, Antonio Iavarone, Wenming Xu, Jerry Pelletier, Anna Lasorella, Ching-Wen Chang, Simon S. Wing, Mira Katz-Egorov, Kin Lam Fok, Regina Cencic, Rohini Bose, Ye Chun Ruan, Makoto Nagano, Martine Culty, Kai Sheng, and Sicheng Su
- Subjects
Male ,0301 basic medicine ,Senescence ,endocrine system ,medicine.medical_specialty ,DNA damage ,Ubiquitin-Protein Ligases ,Down-Regulation ,Biology ,Mice ,03 medical and health sciences ,Endocrinology ,Gonocyte ,Internal medicine ,medicine ,Animals ,Spermatogenesis ,Mitosis ,Mitotic catastrophe ,Cells, Cultured ,Mice, Knockout ,urogenital system ,Tumor Suppressor Proteins ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Embryonic stem cell ,Spermatogonia ,Cell biology ,030104 developmental biology ,Cell culture ,Immunology ,DNA Damage - Abstract
Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.
- Published
- 2017
44. Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma
- Author
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Jerry Pelletier, Stefanie Krassnig, Andrea Raicht, Regina Cencic, Ewa Izycka-Swieszewska, Anna Maria Birkl-Toeglhofer, Georg Singer, John A. Porco, Piotr Czapiewski, Christina Skofler, Christoph Castellani, Florian Kleinegger, Johannes Haybaeck, Holger Till, and Martin Benesch
- Subjects
Male ,CR-1-31-B ,0301 basic medicine ,SH-SY5Y ,Article ,neuroblastoma ,eukaryotic initiation factor 4AI (eIF4AI) ,03 medical and health sciences ,0302 clinical medicine ,Eukaryotic translation ,Eukaryotic initiation factor ,Neuroblastoma ,medicine ,Humans ,Initiation factor ,Eukaryotic Initiation Factors ,lcsh:QH301-705.5 ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Chemistry ,rocaglates ,Kelly ,General Medicine ,Middle Aged ,medicine.disease ,030104 developmental biology ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,eIF4A ,Cancer research ,Female - Abstract
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.
- Published
- 2021
45. CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A
- Author
-
John R. Mills, Alexandra Katigbak, Jennifer Chu, Gabriela Galicia-Vázquez, John A. Porco, Regina Cencic, and Jerry Pelletier
- Subjects
0301 basic medicine ,Drug Resistance ,Mice, Nude ,chemical biology ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Drug Delivery Systems ,0302 clinical medicine ,Eukaryotic translation ,Eukaryotic initiation factor ,Animals ,CRISPR ,CRISPR/Cas9 ,lcsh:QH301-705.5 ,Alleles ,Benzofurans ,Genetics ,Mice, Inbred BALB C ,Base Sequence ,EIF4G ,rocaglates ,EIF4E ,Reproducibility of Results ,translational control ,Translation (biology) ,RNA Helicase A ,Triterpenes ,3. Good health ,Cell biology ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,Genetic Loci ,030220 oncology & carcinogenesis ,eIF4A ,Eukaryotic Initiation Factor-4A ,Mutation ,NIH 3T3 Cells ,Female ,CRISPR-Cas Systems - Abstract
SUMMARY Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition., Graphical abstract
- Published
- 2016
46. Haploinsufficiency of the ESCRT Component HD-PTP Predisposes to Cancer
- Author
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Jerry Pelletier, Maud Marques, Sanaz Manteghi, Regina Cencic, Marilène Paquet, Ming Yan, Luc Galarneau, Arnim Pause, Michael Witcher, Francis Robert, Dmitri Kharitidi, and Marie-Claude Gingras
- Subjects
0301 basic medicine ,Integrins ,Tumor suppressor gene ,Carcinogenesis ,Cell Survival ,Integrin ,Haploinsufficiency ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,ESCRT ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,Downregulation and upregulation ,Cell Movement ,hemic and lymphatic diseases ,Neoplasms ,medicine ,Animals ,Humans ,Genetic Predisposition to Disease ,Neoplasm Invasiveness ,B-cell lymphoma ,lcsh:QH301-705.5 ,Hemizygote ,Endosomal Sorting Complexes Required for Transport ,Protein Tyrosine Phosphatases, Non-Receptor ,medicine.disease ,Mice, Inbred C57BL ,030104 developmental biology ,lcsh:Biology (General) ,Tumor progression ,Cancer research ,biology.protein - Abstract
SummaryEndosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis in vivo. Indeed, Ptpn23+/− loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23+/−-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23+/− loss increases integrin β1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking.
- Published
- 2016
47. Translation Inhibition by Rocaglates Is Independent of eIF4E Phosphorylation Status
- Author
-
John A. Porco, Jennifer Chu, Wenyu Wang, Jerry Pelletier, and Regina Cencic
- Subjects
0301 basic medicine ,Cancer Research ,Eukaryotic Initiation Factor-4E ,Antineoplastic Agents ,Cyclopentanes ,Biology ,Article ,Cell Line ,03 medical and health sciences ,Eukaryotic initiation factor ,Protein biosynthesis ,Animals ,Humans ,Phosphorylation ,Benzofurans ,Genetics ,Biological Products ,EIF4E ,Translation (biology) ,Cell biology ,030104 developmental biology ,Oncology ,Protein Biosynthesis ,eIF4A ,Signal transduction ,Signal Transduction - Abstract
Rocaglates are natural products that inhibit protein synthesis in eukaryotes and exhibit antineoplastic activity. In vitro biochemical assays, affinity chromatography experiments coupled with mass spectrometry analysis, and in vivo genetic screens have identified eukaryotic initiation factor (eIF) 4A as a direct molecular target of rocaglates. eIF4A is the RNA helicase subunit of eIF4F, a complex that mediates cap-dependent ribosome recruitment to mRNA templates. The eIF4F complex has been implicated in tumor initiation and maintenance through elevated levels or increased phosphorylation status of its cap-binding subunit, eIF4E, thus furthering the interest toward developing rocaglates as antineoplastic agents. Recent experiments have indicated that rocaglates also interact with prohibitins 1 and 2, proteins implicated in c-Raf-MEK-ERK signaling. Because increased ERK signaling stimulates eIF4E phosphorylation status, rocaglates are also expected to inhibit eIF4E phosphorylation status, a point that has not been thoroughly investigated. It is currently unknown whether the effects on translation observed with rocaglates are solely through eIF4A inhibition or also a feature of blocking eIF4E phosphorylation. Here, we show that rocaglates inhibit translation through an eIF4E phosphorylation–independent mechanism. Mol Cancer Ther; 15(1); 136–41. ©2015 AACR.
- Published
- 2016
48. Amidino-Rocaglates: A Potent Class of eIF4A Inhibitors
- Author
-
Dmitri Beglov, William G. Devine, Sandor Vajda, Jerry Pelletier, Jennifer Chu, Wenhan Zhang, Lauren E. Brown, Thomas Henkel, John A. Porco, and Regina Cencic
- Subjects
Lymphoma ,Cell Survival ,Clinical Biochemistry ,Amidines ,Antineoplastic Agents ,Biology ,01 natural sciences ,Biochemistry ,Ribosome ,Article ,DEAD-box RNA Helicases ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Eukaryotic translation ,Drug Discovery ,Protein biosynthesis ,Animals ,Humans ,Structure–activity relationship ,Benzofuran ,Molecular Biology ,Benzofurans ,Pharmacology ,010405 organic chemistry ,RNA ,RNA Helicase A ,Recombinant Proteins ,3. Good health ,0104 chemical sciences ,Mice, Inbred C57BL ,chemistry ,Drug Design ,Protein Biosynthesis ,eIF4A ,Eukaryotic Initiation Factor-4A ,Molecular Medicine ,Female ,Ribosomes - Abstract
Summary Rocaglates share a common cyclopenta[b]benzofuran core that inhibits eukaryotic translation initiation by modifying the behavior of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA, which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives, as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date and, taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties.
- Published
- 2019
49. CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary
- Author
-
Alexander R. Judkins, Jerry Pelletier, Sidong Huang, Regina Cencic, Ron M. Kerkhoven, Douglas A. Levine, Yibo Xue, William D. Foulkes, Morag Park, Barbara C. Vanderhyden, Radia M. Johnson, Nicholas Bertos, Janusz Rak, Alexandra Leary, Aurélie Auguste, Jay R. Patibandla, Leora Witkowski, Marie-Christine Guiot, Hongbo Chen, Michelle Firlit, Brian Meehan, Daniel Martinez, Xue Qing D. Wang, Tim Kong, Petar Jelinic, Blaise A. Clarke, Michael Witcher, Geneviève Morin, Josée Dostie, Andreas I. Papadakis, Sriram Venneti, Elizabeth Macdonald, Walter H. Gotlieb, Ralf Hass, Iris de Rink, and Atefeh Abedini
- Subjects
0301 basic medicine ,Chromatin Immunoprecipitation ,Cell Survival ,Pyridines ,Science ,General Physics and Astronomy ,Aminopyridines ,02 engineering and technology ,Mice, SCID ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Piperazines ,Article ,03 medical and health sciences ,Mice ,Cyclin D1 ,Downregulation and upregulation ,RNA interference ,Cell Line, Tumor ,Animals ,Humans ,Kinase activity ,Carcinoma, Small Cell ,RNA, Small Interfering ,lcsh:Science ,Protein Kinase Inhibitors ,Ovarian Neoplasms ,Multidisciplinary ,Oncogene ,Kinase ,DNA Helicases ,Nuclear Proteins ,General Chemistry ,021001 nanoscience & nanotechnology ,3. Good health ,030104 developmental biology ,Purines ,SMARCA4 ,Cancer research ,Hypercalcemia ,lcsh:Q ,Benzimidazoles ,Female ,biological phenomena, cell phenomena, and immunity ,0210 nano-technology ,Chromatin immunoprecipitation ,Transcription Factors - Abstract
Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically., Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is driven by SMARCA4 loss. Here the authors demonstrate that SCCOHT cells are highly sensitive to CDK4/6 inhibition and provide mechanistic insights, whereby this druggable vulnerability is driven by cyclin D1 deficiency induced by SMARCA4 loss.
- Published
- 2018
50. Rocaglates as dual-targeting agents for experimental cerebral malaria
- Author
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Martin Schmeing, Ian Crandall, Kodjo Ayi, Michael J. Tarry, Kevin C. Kain, Lauren E. Brown, Jerry Pelletier, J. Kennedy, David Langlais, Philippe Gros, John A. Porco, Regina Cencic, and Neda Moradin
- Subjects
0301 basic medicine ,Erythrocytes ,Plasmodium berghei ,Plasmodium falciparum ,030106 microbiology ,Malaria, Cerebral ,Protozoan Proteins ,Pharmacology ,Plasmodium chabaudi ,Antimalarials ,Mice ,03 medical and health sciences ,In vivo ,parasitic diseases ,medicine ,Animals ,Humans ,Artemisinin ,Multidisciplinary ,biology ,Plant Extracts ,medicine.disease ,biology.organism_classification ,3. Good health ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Eukaryotic Initiation Factor-4F ,PNAS Plus ,Cerebral Malaria ,Female ,Aglaia ,Malaria ,Ex vivo ,medicine.drug - Abstract
Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.
- Published
- 2018
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