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BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells

Oncogene volume 35, pages 4689–4697 (2016)Cite this article

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Abstract

Inhibiting the bromodomain and extra-terminal (BET) domain family of epigenetic reader proteins has been shown to have potent anti-tumoral activity, which is commonly attributed to suppression of transcription. In this study, we show that two structurally distinct BET inhibitors (BETi) interfere with replication and cell cycle progression of murine Myc-induced lymphoma cells at sub-lethal concentrations when the transcriptome remains largely unaltered. This inhibition of replication coincides with a DNA-damage response and enhanced sensitivity to inhibitors of the upstream replication stress sensor ATR in vitro and in mouse models of B-cell lymphoma. Mechanistically, ATR and BETi combination therapy cause robust transcriptional changes of genes involved in cell death, senescence-associated secretory pathway, NFkB signaling and ER stress. Our data reveal that BETi can potentiate the cell stress and death caused by ATR inhibitors. This suggests that ATRi can be used in combination therapies of lymphomas without the use of genotoxic drugs.

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References

  1. Belkina AC, Denis GV . BET domain co-regulators in obesity, inflammation and cancer. Nat Rev Cancer 2012; 12: 465–477.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sinha A, Faller DV, Denis GV . Bromodomain analysis of Brd2-dependent transcriptional activation of cyclin A. Biochem J 2005; 387: 257–269.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Denis GV, Vaziri C, Guo N, Faller DV . RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F. Cell Growth Differ 2000; 11: 417–424.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K . The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 2005; 19: 523–534.

    Article  CAS  PubMed  Google Scholar 

  5. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 2005; 19: 535–545.

    Article  CAS  PubMed  Google Scholar 

  6. Marshall NF, Peng J, Xie Z, Price DH . Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J Biol Chem 1996; 271: 27176–27183.

    Article  CAS  PubMed  Google Scholar 

  7. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153: 320–334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Venkataraman S, Alimova I, Balakrishnan I, Harris P, Birks DK, Griesinger A et al. Inhibition of BRD4 attenuates tumor cell self-renewal and suppresses stem cell signaling in MYC driven medulloblastoma. Oncotarget 2014; 5: 2355–2371.

    PubMed  PubMed Central  Google Scholar 

  9. Pastori C, Daniel M, Penas C, Volmar CH, Johnstone AL, Brothers SP et al. BET bromodomain proteins are required for glioblastoma cell proliferation. Epigenetics 2014; 9: 611–620.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Loosveld M, Castellano R, Gon S, Goubard A, Crouzet T, Pouyet L et al. Therapeutic targeting of c-Myc in T-cell acute lymphoblastic leukemia, T-ALL. Oncotarget 2014; 5: 3168–3172.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Bandopadhayay P, Bergthold G, Nguyen B, Schubert S, Gholamin S, Tang Y et al. BET bromodomain inhibition of MYC-amplified medulloblastoma. Clin Cancer Res 2014; 20: 912–925.

    Article  CAS  PubMed  Google Scholar 

  12. Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 2014; 510: 278–282.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tolani B, Gopalakrishnan R, Punj V, Matta H, Chaudhary PM . Targeting Myc in KSHV-associated primary effusion lymphoma with BET bromodomain inhibitors. Oncogene 2013; 33: 2928–2937.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Puissant A, Frumm SM, Alexe G, Bassil CF, Qi J, Chanthery YH et al. Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discov 2013; 3: 308–323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gao L, Schwartzman J, Gibbs A, Lisac R, Kleinschmidt R, Wilmot B et al. Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation. PLoS One 2013; 8: e63563.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Cheng Z, Gong Y, Ma Y, Lu K, Lu X, Pierce LA et al. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clin Cancer Res 2013; 19: 1748–1759.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA et al. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proc Natl Acad Sci USA 2011; 108: 16669–16674.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 2011; 146: 904–917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Keene RG, Mueller A, Landick R, London L . Transcriptional pause, arrest and termination sites for RNA polymerase II in mammalian N- and c-myc genes. Nucleic Acids Res 1999; 27: 3173–3182.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kerppola TK, Kane CM . Intrinsic sites of transcription termination and pausing in the c-myc gene. Mol Cell Biol 1988; 8: 4389–4394.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bhadury J, Nilsson LM, Muralidharan SV, Green LC, Li Z, Gesner EM et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc Natl Acad Sci USA 2014; 111: E2721–E2730.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fiskus W, Sharma S, Qi J, Valenta JA, Schaub LJ, Shah B et al. Highly active combination of BRD4 antagonist and histone deacetylase inhibitor against human acute myelogenous leukemia cells. Mol Cancer Ther 2014; 13: 1142–1154.

    Article  CAS  PubMed  Google Scholar 

  23. Pajic A, Spitkovsky D, Christoph B, Kempkes B, Schuhmacher M, Staege MS et al. Cell cycle activation by c-myc in a burkitt lymphoma model cell line. Int J Cancer 2000; 87: 787–793.

    Article  CAS  PubMed  Google Scholar 

  24. Bhadury J, Lopez MD, Muralidharan SV, Nilsson LM, Nilsson JA . Identification of tumorigenic and therapeutically actionable mutations in transplantable mouse tumor cells by exome sequencing. Oncogenesis 2013; 2: e44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ember SW, Zhu JY, Olesen SH, Martin MP, Becker A, Berndt N et al. Acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors. ACS Chem Biol 2014; 9: 1160–1171.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ciceri P, Muller S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt JP et al. Dual kinase-bromodomain inhibitors for rationally designed polypharmacology. Nat Chem Biol 2014; 10: 305–312.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dey A, Chitsaz F, Abbasi A, Misteli T, Ozato K . The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci USA 2003; 100: 8758–8763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Boi M, Gaudio E, Bonetti P, Kwee I, Bernasconi E, Tarantelli C et al. The BET bromodomain inhibitor OTX015 affects pathogenetic pathways in preclinical B-cell tumor models and synergizes with targeted drugs. Clin Cancer Res 2015; 21: 1628–1638.

    Article  CAS  PubMed  Google Scholar 

  29. Stratikopoulos EE, Dendy M, Szabolcs M, Khaykin AJ, Lefebvre C, Zhou MM et al. Kinase and BET inhibitors together clamp inhibition of PI3K signaling and overcome resistance to therapy. Cancer Cell 2015; 27: 837–851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lecona E, Fernandez-Capetillo O . Replication stress and cancer: it takes two to tango. Exp Cell Res 2014; 329: 26–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ferrao PT, Bukczynska EP, Johnstone RW, McArthur GA . Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells. Oncogene 2012; 31: 1661–1672.

    CAS  PubMed  Google Scholar 

  32. Murga M, Campaner S, Lopez-Contreras AJ, Toledo LI, Soria R, Montana MF et al. Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nat Struct Mol Biol 2011; 18: 1331–1335.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Höglund A, Strömvall K, Li Y, Forshell LP, Nilsson JA . Chk2 deficiency in Myc overexpressing lymphoma cells elicits a synergistic lethal response in combination with PARP inhibition. Cell Cycle 2011; 10: 3598–3607.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Höglund A, Nilsson L, Muralidharan SV, Hasvold LA, Merta P, Rudelius M et al. Therapeutic implications for the induced levels of Chk1 in Myc-expressing cancer cells. Clin Cancer Res 2011; 17: 7067–7079.

    Article  PubMed  Google Scholar 

  35. Shortt J, Martin BP, Newbold A, Hannan KM, Devlin JR, Baker AJ et al. Combined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC-driven B-cell lymphomas. Blood 2013; 121: 2964–2974.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Toledo LI, Murga M, Zur R, Soria R, Rodriguez A, Martinez S et al. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nat Struct Mol Biol 2011; 18: 721–727.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol 2011; 7: 428–430.

    Article  CAS  PubMed  Google Scholar 

  38. Floyd SR, Pacold ME, Huang Q, Clarke SM, Lam FC, Cannell IG et al. The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature 2013; 498: 246–250.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Josse R, Martin SE, Guha R, Ormanoglu P, Pfister TD, Reaper PM et al. ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses. Cancer Res 2014; 74: 6968–6979.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Foote KM, Blades K, Cronin A, Fillery S, Guichard SS, Hassall L et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-y l}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem 2013; 56: 2125–2138.

    Article  CAS  PubMed  Google Scholar 

  41. Keller U, Huber J, Nilsson JA, Fallahi M, Hall MA, Peschel C et al. Myc suppression of Nfkb2 accelerates lymphomagenesis. BMC Cancer 2010; 10: 348.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Klapproth K, Sander S, Marinkovic D, Baumann B, Wirth T . The IKK2/NF-{kappa}B pathway suppresses MYC-induced lymphomagenesis. Blood 2009; 114: 2448–2458.

    Article  CAS  PubMed  Google Scholar 

  43. Keller U, Nilsson JA, Maclean KH, Old JB, Cleveland JL . Nfkb 1 is dispensable for Myc-induced lymphomagenesis. Oncogene 2005; 24: 6231–6240.

    Article  CAS  PubMed  Google Scholar 

  44. Dorr JR, Yu Y, Milanovic M, Beuster G, Zasada C, Dabritz JH et al. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. Nature 2013; 501: 421–425.

    Article  PubMed  Google Scholar 

  45. Jing H, Kase J, Dorr JR, Milanovic M, Lenze D, Grau M et al. Opposing roles of NF-kappaB in anti-cancer treatment outcome unveiled by cross-species investigations. Genes Dev 2011; 25: 2137–2146.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chien Y, Scuoppo C, Wang X, Fang X, Balgley B, Bolden JE et al. Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity. Genes Dev 2011; 25: 2125–2136.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Moscat J, Diaz-Meco MT . p62 at the crossroads of autophagy, apoptosis, and cancer. Cell 2009; 137: 1001–1004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Fang J, Barker B, Bolanos L, Liu X, Jerez A, Makishima H et al. Myeloid malignancies with chromosome 5q deletions acquire a dependency on an intrachromosomal NF-kappaB gene network. Cell Rep 2014; 8: 1328–1338.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sanz L, Diaz-Meco MT, Nakano H, Moscat J . The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO J 2000; 19: 1576–1586.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Stanlie A, Yousif AS, Akiyama H, Honjo T, Begum NA . Chromatin reader Brd4 functions in Ig class switching as a repair complex adaptor of nonhomologous end-joining. Mol Cell 2014; 55: 97–110.

    Article  CAS  PubMed  Google Scholar 

  51. Bolden JE, Tasdemir N, Dow LE, van Es JH, Wilkinson JE, Zhao Z et al. Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition. Cell Rep 2014; 8: 1919–1929.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Tang C, Wang X, Soh H, Seyedin S, Cortez MA, Krishnan S et al. Combining radiation and immunotherapy: a new systemic therapy for solid tumors? Cancer Immunol Res 2014; 2: 831–838.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Denis GV, McComb ME, Faller DV, Sinha A, Romesser PB, Costello CE . Identification of transcription complexes that contain the double bromodomain protein Brd2 and chromatin remodeling machines. J Proteome Res 2006; 5: 502–511.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Maruyama T, Farina A, Dey A, Cheong J, Bermudez VP, Tamura T et al. A mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase. Mol Cell Biol 2002; 22: 6509–6520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Herold S, Wanzel M, Beuger V, Frohme C, Beul D, Hillukkala T et al. Negative regulation of the mammalian UV response by Myc through association with Miz-1. Mol Cell 2002; 10: 509–521.

    Article  CAS  PubMed  Google Scholar 

  56. Zou Z, Huang B, Wu X, Zhang H, Qi J, Bradner J et al. Brd4 maintains constitutively active NF-kappaB in cancer cells by binding to acetylated RelA. Oncogene 2013; 33: 2395–2404.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Huang B, Yang XD, Zhou MM, Ozato K, Chen LF . Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA. Mol Cell Biol 2009; 29: 1375–1387.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Sofia Nordstrand for animal care, and Eric Campeau and Zenith Epigenetics for RVX2135 and helpful discussions. This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council, the Region Västra Götaland (Sahlgrenska University Hospital, Gothenburg), the Knut and Alice Wallenberg Foundation, the Sahlgrenska Academy and BioCARE—a National Strategic Cancer Research Program at University of Gothenburg (to JAN), and from the Assar Gabrielsson Foundation and the W&M Lundgren Foundation (to SVM, JB and LCG).

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  1. K G McLure

    Present address: 3Present address: Ermaris Bio Corp, Calgary, Alberta, Canada.,

Authors and Affiliations

  1. Department of Surgery, Sahlgrenska Cancer Center, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

    S V Muralidharan, J Bhadury, L M Nilsson, L C Green & J A Nilsson

  2. Zenith Epigenetics Corp, Calgary, Alberta, Canada

    K G McLure

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Correspondence to J A Nilsson.

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KGM was an employee of Zenith Epigenetics Corp at the begining of this project. The remaining authors declare no conflict of interest.

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Muralidharan, S., Bhadury, J., Nilsson, L. et al. BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells. Oncogene 35, 4689–4697 (2016). https://doi.org/10.1038/onc.2015.521

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