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Simulating and predicting cellular and in vivo responses of colon cancer to combined treatment with chemotherapy and IAP antagonist Birinapant/TL32711

Cell Death & Differentiationvolume 25pages19521966 (2018) | Download Citation


Apoptosis resistance contributes to treatment failure in colorectal cancer (CRC). New treatments that reinstate apoptosis competency have potential to improve patient outcome but require predictive biomarkers to target them to responsive patient populations. Inhibitor of apoptosis proteins (IAPs) suppress apoptosis, contributing to drug resistance; IAP antagonists such as TL32711 have therefore been developed. We developed a systems biology approach for predicting response of CRC cells to chemotherapy and TL32711 combinations in vitro and in vivo. CRC cells responded poorly to TL32711 monotherapy in vitro; however, co-treatment with 5-fluorouracil (5-FU) and oxaliplatin enhanced TL32711-induced apoptosis. Notably, cells from genetically identical populations responded highly heterogeneously, with caspases being activated both upstream and downstream of mitochondrial outer membrane permeabilisation (MOMP). These data, combined with quantities of key apoptosis regulators were sufficient to replicate in vitro cell death profiles by mathematical modelling. In vivo, apoptosis protein expression was significantly altered, and mathematical modelling for these conditions predicted higher apoptosis resistance that could nevertheless be overcome by combination of chemotherapy and TL32711. Subsequent experimental observations agreed with these predictions, and the observed effects on tumour growth inhibition correlated robustly with apoptosis competency. We therefore obtained insights into intracellular signal transduction kinetics and their population-based heterogeneities for chemotherapy/TL32711 combinations and provide proof-of-concept that mathematical modelling of apoptosis competency can simulate and predict responsiveness in vivo. Being able to predict response to IAP antagonist-based treatments on the background of cell-to-cell heterogeneities in the future might assist in improving treatment stratification approaches for these emerging apoptosis-targeting agents.

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Shared senior authorship: Daniel B Longley and Markus Rehm.


  1. 1.

    de Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol. 2000;18:2938–47.

  2. 2.

    De Roock W, De Vriendt V, Normanno N, Ciardiello F, Tejpar S. KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. Lancet Oncol. 2011;12:594–603.

  3. 3.

    Hurwitz HI, Tebbutt NC, Kabbinavar F, Giantonio BJ, Guan ZZ, Mitchell L, et al. Efficacy and safety of bevacizumab in metastatic colorectal cancer: pooled analysis from seven randomized controlled trials. Oncologist. 2013;18:1004–12.

  4. 4.

    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

  5. 5.

    Delbridge AR, Grabow S, Strasser A, Vaux DL. Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nat Rev Cancer. 2016;16:99–109.

  6. 6.

    Fulda S. Promises and challenges of smac mimetics as cancer therapeutics. Clin Cancer Res. 2015;21:5030–6.

  7. 7.

    Senzer NN, LoRusso P, Martin LP, Schilder RJ, Amaravadi RK, Papadopoulos KP, et al. Phase II clinical activity and tolerability of the SMAC-mimetic birinapant (TL32711) plus irinotecan in irinotecan-relapsed/refractory metastatic colorectal cancer. J Clin Oncol. 2013;31 Suppl 15:3621.

  8. 8.

    Fulda S. Molecular pathways: targeting inhibitor of apoptosis proteins in cancer--from molecular mechanism to therapeutic application. Clin Cancer Res. 2014;20:289–95.

  9. 9.

    Gyrd-Hansen M, Meier P. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer. 2010;10:561–74.

  10. 10.

    Hector S, Prehn JH. Apoptosis signaling proteins as prognostic biomarkers in colorectal cancer: a review. Biochim Biophys Acta. 2009;1795:117–29.

  11. 11.

    Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res. 2000;6:1796–803.

  12. 12.

    Obexer P, Ausserlechner MJ. X-linked inhibitor of apoptosis protein - a critical death resistance regulator and therapeutic target for personalized cancer therapy. Front Oncol. 2014;4:197.

  13. 13.

    Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P, et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell. 2007;131:669–81.

  14. 14.

    Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell. 2007;131:682–93.

  15. 15.

    Allensworth JL, Sauer SJ, Lyerly HK, Morse MA, Devi GR. Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-alpha-independent mechanism. Breast Cancer Res Treat. 2013;137:359–71.

  16. 16.

    Tenev T, Bianchi K, Darding M, Broemer M, Langlais C, Wallberg F, et al. The Ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs. Mol Cell. 2011;43:432–48.

  17. 17.

    Hellwig CT, Kohler BF, Lehtivarjo AK, Dussmann H, Courtney MJ, Prehn JH, et al. Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation. J Biol Chem. 2008;283:21676–85.

  18. 18.

    Laussmann MA, Passante E, Dussmann H, Rauen JA, Wurstle ML, Delgado ME, et al. Proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8. Cell Death Differ. 2011;18:1584–97.

  19. 19.

    Wang C, Youle RJ. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak. Oncogene. 2012;31:3177–89.

  20. 20.

    Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn JH. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3. J Biol Chem. 2002;277:24506–14.

  21. 21.

    Rehm M, Huber HJ, Dussmann H, Prehn JH. Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein. EMBO J. 2006;25:4338–49.

  22. 22.

    Schmid J, Dussmann H, Boukes GJ, Flanagan L, Lindner AU, O’Connor CL, et al. Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization. J Biol Chem. 2012;287:41546–59.

  23. 23.

    Dussmann H, Rehm M, Kogel D, Prehn JH. Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis. J Cell Sci. 2003;116(Pt 3):525–36.

  24. 24.

    Albeck JG, Burke JM, Aldridge BB, Zhang M, Lauffenburger DA, Sorger PK. Quantitative analysis of pathways controlling extrinsic apoptosis in single cells. Mol Cell. 2008;30:11–25.

  25. 25.

    Benzekry S, Lamont C, Beheshti A, Tracz A, Ebos JM, Hlatky L, et al. Classical mathematical models for description and prediction of experimental tumor growth. PLoS Comput Biol. 2014;10:e1003800.

  26. 26.

    Simeoni M, Magni P, Cammia C, De Nicolao G, Croci V, Pesenti E, et al. Predictive pharmacokinetic-pharmacodynamic modeling of tumor growth kinetics in xenograft models after administration of anticancer agents. Cancer Res. 2004;64:1094–101.

  27. 27.

    Van Schaeybroeck S, Allen WL, Turkington RC, Johnston PG. Implementing prognostic and predictive biomarkers in CRC clinical trials. Nat Rev Clin Oncol. 2011;8:222–32.

  28. 28.

    Feoktistova M, Geserick P, Kellert B, Dimitrova DP, Langlais C, Hupe M, et al. cIAPs Block Ripoptosome Formation, a RIP1/Caspase-8 Containing Intracellular Cell Death Complex Differentially Regulated by cFLIP Isoforms. Mol Cell. 2011;43:449–63.

  29. 29.

    Tenev T, Bianchi K, Darding M, Broemer M, Langlais C, Wallberg F, et al. The ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs. Mol Cell. 2011;43:432–48.

  30. 30.

    Clarke PR, Allan LA. Cell-cycle control in the face of damage--a matter of life or death. Trends Cell Biol. 2009;19:89–98.

  31. 31.

    Vetma V, Rozanc J, Charles EM, Hellwig CT, Alexopoulos LG, Rehm M. Examining the in-vitro efficacy of the IAP antagonist birinapant as a single-agent or in combination with dacarbazine to induce melanoma cell death. Oncol Res. 2017;25:1489–94.

  32. 32.

    Krepler C, Chunduru SK, Halloran MB, He X, Xiao M, Vultur A, et al. The novel SMAC mimetic birinapant exhibits potent activity against human melanoma cells. Clin Cancer Res. 2013;19:1784–94.

  33. 33.

    Salvucci M, Wurstle ML, Morgan C, Curry S, Cremona M, Lindner AU, et al. A stepwise integrated approach to personalized risk predictions in stage III colorectal cancer. Clin Cancer Res. 2017;23:1200–12.

  34. 34.

    Eissing T, Kuepfer L, Becker C, Block M, Coboeken K, Gaub T, et al. A computational systems biology software platform for multiscale modeling and simulation: integrating whole-body physiology, disease biology, and molecular reaction networks. Front Physiol. 2011;2:4.

  35. 35.

    Altrock PM, Liu LL, Michor F. The mathematics of cancer: integrating quantitative models. Nat Rev Cancer. 2015;15:730–45.

  36. 36.

    Rehm M, Parsons MJ, Bouchier-Hayes L. Measuring caspase activity by Forster resonance energy transfer. Cold Spring Harb Protoc. 2015;2015:pdbprot082560.

  37. 37.

    Wilson TR, McLaughlin KM, McEwan M, Sakai H, Rogers KM, Redmond KM, et al. c-FLIP: a key regulator of colorectal cancer cell death. Cancer Res. 2007;67:5754–62.

  38. 38.

    Huber HJ, Rehm M, Plchut M, Dussmann H, Prehn JH. APOPTO-CELL--a simulation tool and interactive database for analyzing cellular susceptibility to apoptosis. Bioinformatics. 2007;23:648–50.

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The authors kindly acknowledge support for their work by grants from the European Union (FP7 APO-DECIDE) and Science Foundation Ireland/Department for Employment and Learning Northern Ireland Investigator Programme (14/IA/2582; 13/IA/1881). CTH and FAL received support from the Irish Research Council (GOIPD/2013/102; GOIPG/2014/1299). MR receives further support from the German Research Foundation (FOR2036, MO 3226/1-1).

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Author notes

  1. Edited by S. Fulda


  1. Cell Death & Drug Resistance Group, Centre for Cancer Research & Cell Biology, Queen’s University Belfast, Belfast, UK

    • Nyree Crawford
    •  & Daniel B Longley
  2. Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland

    • Manuela Salvucci
    • , Frank A Lincoln
    • , Ruth E Mooney
    • , Carla L O’Connor
    • , Jochen HM Prehn
    •  & Markus Rehm
  3. Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland

    • Manuela Salvucci
    • , Frank A Lincoln
    • , Ruth E Mooney
    • , Jochen HM Prehn
    •  & Markus Rehm
  4. Institute of Cell Biology and Immunology, University of Stuttgart, D-70569, Stuttgart, Germany

    • Christian T Hellwig
    •  & Markus Rehm
  5. Stuttgart Research Center Systems Biology, University of Stuttgart, D-70569, Stuttgart, Germany

    • Christian T Hellwig
    •  & Markus Rehm


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The authors declare that they have no conflict of interest.

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Correspondence to Daniel B Longley or Markus Rehm.

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