Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Co-inhibition of BET proteins and PI3Kα triggers mitochondrial apoptosis in rhabdomyosarcoma cells

Oncogene volume 39, pages 3837–3852 (2020)Cite this article

Subjects

Abstract

Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-xL). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-xL, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: JQ1/BYL719 co-treatment synergistically induces cell death in RMS cells.
Fig. 2: JQ1/BYL719 co-treatment causes G1 arrest prior to inducing caspase-dependent apoptosis.
Fig. 3: JQ1/BYL719 co-treatment shifts the ratio of pro- and antiapoptotic BCL-2 family proteins towards apoptosis.
Fig. 4: JQ1/BYL719 co-treatment modulates TFs regulating BCL-2 protein family members.
Fig. 5: BIM, NOXA, and BMF contribute to JQ1/BYL719-induced apoptosis.
Fig. 6: JQ1/BYL719 co-treatment stimulates activation of BAK and BAX, thereby promoting apoptosis.
Fig. 7: Overexpression of BCL-2 or MCL-1 rescues cells from JQ1/BYL719-induced apoptosis.

Similar content being viewed by others

Data availability

Sequencing data generated during the current study will be available in the publicly accessible database Gene Expression Omnibus (GEO) under accession number GSE145469 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE145469).

References

  1. Dagher R, Helman L. Rhabdomyosarcoma: an overview. Oncologist. 1999;4:34–44.

    Article  CAS  PubMed  Google Scholar 

  2. Hayes-Jordan A, Andrassy R. Rhabdomyosarcoma in children. Curr Opin Pediatr. 2009;21:373–8.

    Article  PubMed  Google Scholar 

  3. Dantonello TM, Leuschner I, Vokuhl C, Gfroerer S, Schuck A, Kube S, et al. Malignant ectomesenchymoma in children and adolescents: report from the Cooperative Weichteilsarkom Studiengruppe (CWS). Pediatr Blood Cancer. 2013;60:224–9.

    Article  PubMed  Google Scholar 

  4. Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006;25:4798–811.

    Article  CAS  PubMed  Google Scholar 

  5. Adams J, Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol. 2007;19:488–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. Nat Rev Drug Discov. 2010;9:447–64.

    Article  CAS  PubMed  Google Scholar 

  7. Wu SY, Chiang CM. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem. 2007;282:13141–5.

    Article  CAS  PubMed  Google Scholar 

  8. Zuber V, Bettella F, Witoelar A, Consortium P, Cruk G, Consortium B, et al. Bromodomain protein 4 discriminates tissue-specific super-enhancers containing disease-specific susceptibility loci in prostate and breast cancer. BMC Genomics. 2017;18:270.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Mujtaba S, Zeng L, Zhou MM. Structure and acetyl-lysine recognition of the bromodomain. Oncogene. 2007;26:5521–7.

    Article  CAS  PubMed  Google Scholar 

  10. Gryder BE, Yohe ME, Chou HC, Zhang X, Marques J, Wachtel M, et al. PAX3-FOXO1 establishes myogenic super enhancers and confers BET bromodomain vulnerability. Cancer Discov. 2017;7:884–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tang Y, Gholamin S, Schubert S, Willardson MI, Lee A, Bandopadhayay P, et al. Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition. Nat Med. 2014;20:732–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Seal J, Lamotte Y, Donche F, Bouillot A, Mirguet O, Gellibert F, et al. Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A). Bioorg Med Chem Lett. 2012;22:2968–72.

    Article  CAS  PubMed  Google Scholar 

  13. 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;153:320–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee DH, Qi J, Bradner JE, Said JW, Doan NB, Forscher C, et al. Synergistic effect of JQ1 and rapamycin for treatment of human osteosarcoma. Int J Cancer. 2015;136:2055–64.

    Article  CAS  PubMed  Google Scholar 

  15. Da Costa D, Agathanggelou A, Perry T, Weston V, Petermann E, Zlatanou A, et al. BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia. Blood Cancer J. 2013;3:e126.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Fiskus W, Sharma S, Qi J, Shah B, Devaraj SG, Leveque C, et al. BET protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD. Mol Cancer Ther. 2014;13:2315–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Schaffer M, Chaturvedi S, Davis C, Aquino R, Stepanchick E, Versele M, et al. Identification of potential ibrutinib combinations in hematological malignancies using a combination high-throughput screen. Leuk Lymphoma. 2018;59:931–40.

  18. Yohe ME, Gryder BE, Shern JF, Song YK, Chou HC, Sindiri S, et al. MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma. Sci Transl Med. 2018;10:448.

    Article  CAS  Google Scholar 

  19. Bid HK, Phelps DA, Xaio L, Guttridge DC, Lin J, London C, et al. The bromodomain BET Inhibitor JQ1 suppresses tumor angiogenesis in models of childhood sarcoma. Mol Cancer Ther. 2016;15:1018–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Satheesha S, Manzella G, Bovay A, Casanova EA, Bode PK, Belle R, et al. Targeting hedgehog signaling reduces self-renewal in embryonal rhabdomyosarcoma. Oncogene. 2016;35:2020–30.

    Article  CAS  PubMed  Google Scholar 

  21. Graab U, Hahn H, Fulda S. Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma. Oncotarget. 2015;6:8722–35.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Hugle M, Belz K, Fulda S. Identification of synthetic lethality of PLK1 inhibition and microtubule-destabilizing drugs. Cell Death Differ. 2015;22:1946–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Haydn T, Metzger E, Schuele R, Fulda S. Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells. Cell Death Dis. 2017;8:e2879.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Heinicke U, Kupka J, Fulda S. JNJ-26481585 primes rhabdomyosarcoma cells for chemotherapeutics by engaging the mitochondrial pathway of apoptosis. Oncotarget. 2015;6:37836–51.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Czymai T, Viemann D, Sticht C, Molema G, Goebeler M, Schmidt M. FOXO3 modulates endothelial gene expression and function by classical and alternative mechanisms. J Biol Chem. 2010;285:10163–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hornsveld M, Tenhagen M, van de Ven RA, Smits AM, van Triest MH, van Amersfoort M, et al. Restraining FOXO3-dependent transcriptional BMF activation underpins tumour growth and metastasis of E-cadherin-negative breast cancer. Cell Death Differ. 2016;23:1483–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kuang WH, Dong ZQ, Tian LT, Li J. IGF-1 defends against chronic-stress induced depression in rat models of chronic unpredictable mild stress through the PI3K/Akt/FoxO3a pathway. Kaohsiung J Med Sci. 2018;34:370–6.

    Article  PubMed  Google Scholar 

  28. Filippakopoulos P, Knapp S. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov. 2014;13:337–56.

    Article  CAS  PubMed  Google Scholar 

  29. Ma Y, Wang L, Neitzel LR, Loganathan SN, Tang N, Qin L, et al. The MAPK pathway regulates intrinsic resistance to BET inhibitors in colorectal cancer. Clin Cancer Res. 2017;23:2027–37.

    Article  CAS  PubMed  Google Scholar 

  30. Gilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol. 2003;162:613–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li GQ, Guo WZ, Zhang Y, Seng JJ, Zhang HP, Ma XX, et al. Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression. Oncotarget. 2016;7:2462–74.

    PubMed  Google Scholar 

  32. Eijkelenboom A, Mokry M, de Wit E, Smits LM, Polderman PE, van Triest MH, et al. Genome-wide analysis of FOXO3 mediated transcription regulation through RNA polymerase II profiling. Mol Syst Biol. 2013;9:638.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Zibat A, Missiaglia E, Rosenberger A, Pritchard-Jones K, Shipley J, Hahn H, et al. Activation of the hedgehog pathway confers a poor prognosis in embryonal and fusion gene-negative alveolar rhabdomyosarcoma. Oncogene. 2010;29:6323–30.

    Article  CAS  PubMed  Google Scholar 

  34. Tinsley S, Meja K, Shepherd C, Khwaja A. Synergistic induction of cell death in haematological malignancies by combined phosphoinositide-3-kinase and BET bromodomain inhibition. Br J Haematol. 2015;170:275–8.

    Article  PubMed  Google Scholar 

  35. Hensel T, Giorgi C, Schmidt O, Calzada-Wack J, Neff F, Buch T, et al. Targeting the EWS-ETS transcriptional program by BET bromodomain inhibition in Ewing sarcoma. Oncotarget. 2016;7:1451–63.

    Article  PubMed  Google Scholar 

  36. Toren P, Kim S, Cordonnier T, Crafter C, Davies BR, Fazli L, et al. Combination AZD5363 with enzalutamide significantly delays enzalutamide-resistant prostate cancer in preclinical models. Eur Urol. 2015;67:986–90.

    Article  CAS  PubMed  Google Scholar 

  37. Packer LM, Geng X, Bonazzi VF, Ju RJ, Mahon CE, Cummings MC, et al. PI3K inhibitors synergize with FGFR inhibitors to enhance antitumor responses in FGFR2mutant endometrial cancers. Mol Cancer Ther. 2017;16:637–48.

    Article  CAS  PubMed  Google Scholar 

  38. Costa C, Ebi H, Martini M, Beausoleil SA, Faber AC, Jakubik CT, et al. Measurement of PIP3 levels reveals an unexpected role for p110beta in early adaptive responses to p110alpha-specific inhibitors in luminal breast cancer. Cancer Cell. 2015;27:97–108.

    Article  CAS  PubMed  Google Scholar 

  39. 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;27:837–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 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;10:305–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Dittmann A, Werner T, Chung CW, Savitski MM, Falth Savitski M, Grandi P, et al. The commonly used PI3-kinase probe LY294002 is an inhibitor of BET bromodomains. ACS Chem Biol. 2014;9:495–502.

    Article  CAS  PubMed  Google Scholar 

  42. Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM. The CD95 (APO-1/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res. 1997;57:3823–9.

    CAS  PubMed  Google Scholar 

  43. Heinicke U, Fulda S. Chemosensitization of rhabdomyosarcoma cells by the histone deacetylase inhibitor SAHA. Cancer Lett. 2014;351:50–8.

    Article  CAS  PubMed  Google Scholar 

  44. Chou T. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou T, editors. Synergism and antagonism in chemotherapy. San Diego, USA: Academic Press; 1991. p. 61–102.

    Google Scholar 

  45. Ianevski A, He L, Aittokallio T, Tang J. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinformatics. 2017;33:2413–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We thank Genentech Inc. for providing WT and phospho-mutant MCL-1 plasmids and C. Hugenberg for expert secretarial assistance.

Author information

Authors and Affiliations

  1. Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany

    Cathinka Boedicker, Nadezda Dolgikh, Michael T. Meister, Julius C. Enßle & Simone Fulda

  2. German Cancer Consortium (DKTK), Partner Site, Frankfurt, Germany

    Cathinka Boedicker, Michael T. Meister, Stefan Knapp & Simone Fulda

  3. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Cathinka Boedicker, Michael T. Meister & Simone Fulda

  4. Laboratory for Epigenomics and Tumor genetics, University Hospital Cologne, Cologne, Germany

    Michelle Hussong, Christina Grimm & Michal R. Schweiger

  5. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Michelle Hussong & Michal R. Schweiger

  6. Department of Pediatric Hematology and Oncology, University Hospital Frankfurt, Frankfurt, Germany

    Michael T. Meister

  7. Institute for Pharmaceutical Chemistry and BMLS (SGC), Frankfurt, Germany

    Marek Wanior & Stefan Knapp

Authors
  1. Cathinka Boedicker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle Hussong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christina Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nadezda Dolgikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael T. Meister
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julius C. Enßle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marek Wanior
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan Knapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michal R. Schweiger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Simone Fulda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Simone Fulda.

Ethics declarations

Conflict of interest

The authors declare that there are no competing financial interests in relation to the work described. This work has been partially supported by grants from the BMBF (to SF and SK), German Cancer Aid (to SF), SGC (to SK) and the Else Kröner-Fresenius-Stiftung (to SF, MTM, and MW), the Volkswagenstiftung (Lichtenberg program to MRS) and the Center for Molecular Medicine Cologne (CMMC to MRS and MH).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Boedicker, C., Hussong, M., Grimm, C. et al. Co-inhibition of BET proteins and PI3Kα triggers mitochondrial apoptosis in rhabdomyosarcoma cells. Oncogene 39, 3837–3852 (2020). https://doi.org/10.1038/s41388-020-1229-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-020-1229-0

Search

Advanced search

Quick links