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Identifying and Validating MYC:Protein Interactors in Pursuit of Novel Anti-MYC Therapies

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The Myc Gene

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2318))

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Abstract

By identifying MYC protein–protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein–protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo–in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.

Brian Raught and Linda Z. Penn are Lead Contacts

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References

  1. Chacon Simon S, Wang F, Thomas LR et al (2020) Discovery of WD repeat-containing protein 5 (WDR5)-MYC inhibitors using fragment-based methods and structure-based design. J Med Chem 63(8):4315–4333. https://doi.org/10.1021/acs.jmedchem.0c00224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Beaulieu ME, Jauset T, Massó-Vallés D et al (2019) Intrinsic cell-penetrating activity propels omomyc from proof of concept to viable anti-myc therapy. Sci Transl Med 11(484):eaar5012. https://doi.org/10.1126/scitranslmed.aar5012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Han H, Jain AD, Truica MI et al (2019) Small-molecule MYC inhibitors suppress tumor growth and enhance immunotherapy. Cancer Cell 36(5):483–497.e15. https://doi.org/10.1016/j.ccell.2019.10.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kalkat M, Resetca D, Lourenco C et al (2018) MYC protein interactome profiling reveals functionally distinct regions that cooperate to drive tumorigenesis. Mol Cell 72(5):836–848.e7. https://doi.org/10.1016/j.molcel.2018.09.031

    Article  CAS  PubMed  Google Scholar 

  5. Martinato F, Cesaroni M, Amati B, Guccione E (2008) Analysis of myc-induced histone modifications on target chromatin. PLoS One 3(11):e3650. https://doi.org/10.1371/journal.pone.0003650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dang CV (2013) MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harb Perspect Biol 3(8):a014217. https://doi.org/10.1101/cshperspect.a014217

    Article  CAS  Google Scholar 

  7. Kalkat M, De Melo J, Hickman KA et al (2017) MYC deregulation in primary human cancers. Genes (Basel) 8(6):151. https://doi.org/10.3390/genes8060151

    Article  CAS  Google Scholar 

  8. Salghetti SE, Kim SY, Tansey WP (1999) Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. EMBO J 18(3):717–726. https://doi.org/10.1093/emboj/18.3.717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Farrell AS, Sears RC (2014) MYC degradation. Cold Spring Harb Perspect Med 4(3):a014365. https://doi.org/10.1101/cshperspect.a014365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Blackwood EM, Eisenman RN (1991) Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251(4998):1211–1217. https://doi.org/10.1126/science.2006410

    Article  CAS  PubMed  Google Scholar 

  11. Nair SK, Burley SK (2003) X-ray structures of Myc-Max and Mad-Max recognizing DNA: molecular bases of regulation by proto-oncogenic transcription factors. Cell 112(2):193–205. https://doi.org/10.1016/s0092-8674(02)01284-9

    Article  CAS  PubMed  Google Scholar 

  12. Lavigne P, Crump MP, Gagné SM et al (1998) Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper. J Mol Biol 281(1):165–181. https://doi.org/10.1006/jmbi.1998.1914

    Article  CAS  PubMed  Google Scholar 

  13. McMahon SB, Van Buskirk HA, Dugan KA et al (1998) The novel ATM-related protein TRRAP is an essential cofactor for the c- Myc and E2F oncoproteins. Cell 94(3):363–374. https://doi.org/10.1016/s0092-8674(00)81479-8

    Article  CAS  PubMed  Google Scholar 

  14. Frank SR, Parisi T, Taubert S et al (2003) MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Rep 4(6):575–580. https://doi.org/10.1038/sj.embor.embor861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Thomas LR, Wang Q, Fesik SW et al (2015) Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC. Mol Cell 58(3):440–452. https://doi.org/10.1016/j.molcel.2015.02.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang A, Ho CSW, Ponzielli R et al (2005) Identification of a novel c-Myc protein interactor, JPO2, with transforming activity in medulloblastoma cells. Cancer Res 65(13):5607–5619. https://doi.org/10.1158/0008-5472.CAN-05-0500

    Article  CAS  PubMed  Google Scholar 

  17. Hirst M, Ho C, Sabourin L et al (2001) A two-hybrid system for transactivator bait proteins. Proc Natl Acad Sci U S A 98(15):8726–8731. https://doi.org/10.1073/pnas.141413598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Peukert K, Staller P, Schneider A et al (1997) An alternative pathway for gene regulation by Myc. EMBO J 16(18):5672–5686. https://doi.org/10.1093/emboj/16.18.5672

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sakamuro D, Elliott KJ, Wechsler-Reya R, Prendergast GC (1996) BIN1 is a novel MYC-interacting protein with features of a tumour suppressor. Nat Genet 14(1):69–77. https://doi.org/10.1038/ng0996-69

    Article  CAS  PubMed  Google Scholar 

  20. Fields S, Song OK (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230):245–246. https://doi.org/10.1038/340245a0

    Article  CAS  PubMed  Google Scholar 

  21. Baluapuri A, Hofstetter J, Dudvarski Stankovic N et al (2019) MYC recruits SPT5 to RNA polymerase II to promote Processive transcription elongation. Mol Cell 74(4):674–687.e11. https://doi.org/10.1016/j.molcel.2019.02.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kalkat M, Wasylishen AR, Kim SS, Penn LZ (2011) More than MAX: discovering the Myc interactome. Cell Cycle 10(3):374–375. https://doi.org/10.4161/cc.10.3.14645

    Article  CAS  PubMed  Google Scholar 

  23. Taipale M, Tucker G, Peng J et al (2014) A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell 158(2):434–448. https://doi.org/10.1016/j.cell.2014.05.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Barrios-Rodiles M, Ellis JD, Blencowe BJ, Wrana JL (2017) Lumier: a discovery tool for mammalian protein interaction networks. Methods Mol Biol 1550:137–148. https://doi.org/10.1007/978-1-4939-6747-6_11

    Article  CAS  PubMed  Google Scholar 

  25. Dingar D, Kalkat M, Chan PK et al (2015) BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J Proteome 118:95–111. https://doi.org/10.1016/j.jprot.2014.09.029

    Article  CAS  Google Scholar 

  26. Roux KJ, Kim DI, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196(6):801–810. https://doi.org/10.1083/jcb.201112098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Samavarchi-Tehrani P, Samson R, Gingras A-C (2020) Proximity dependent biotinylation: key enzymes and adaptation to proteomics approaches. Mol Cell Proteomics 19(5):757–773. https://doi.org/10.1074/mcp.R120.001941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim DI, Kc B, Zhu W et al (2014) Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A 111(24):E2453–E2461. https://doi.org/10.1073/pnas.1406459111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Choi H, Larsen B, Lin Z et al (2011) SAINT : probabilistic scoring of affinity purification – mass spectrometry data. Nat Methods 8(1):70–73. https://doi.org/10.1038/nmeth.1541

    Article  CAS  PubMed  Google Scholar 

  30. Wenger CD, Phanstiel DH, Lee MV et al (2011) COMPASS: a suite of pre- and post-search proteomics software tools for OMSSA. Proteomics 11(6):1064–1074. https://doi.org/10.1002/pmic.201000616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jäger S, Cimermancic P, Gulbahce N et al (2012) Global landscape of HIV-human protein complexes. Nature 481(7381):365–370. https://doi.org/10.1038/nature10719

    Article  CAS  Google Scholar 

  32. Teo G, Liu G, Zhang J et al (2013) ScienceDirect SAINTexpress : improvements and additional features in significance analysis of INTeractome software. J Proteome 100:37–43. https://doi.org/10.1016/j.jprot.2013.10.023

    Article  CAS  Google Scholar 

  33. Wei Y, Resetca D, Li Z et al (2019) Multiple direct interactions of TBP with the MYC oncoprotein. Nat Struct Mol Biol 26(11):1035–1043. https://doi.org/10.1038/s41594-019-0321-z

    Article  CAS  PubMed  Google Scholar 

  34. Sturm M, Leitner A, Lindner W (2011) Development of an indole-based chemically cleavable linker concept for immobilizing bait compounds for protein pull-down experiments. Bioconjug Chem 22(2):211–217. https://doi.org/10.1021/bc100330a

    Article  CAS  PubMed  Google Scholar 

  35. Söderberg O, Leuchowius KJ, Gullberg M et al (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45(3):227–232. https://doi.org/10.1016/j.ymeth.2008.06.014

    Article  CAS  PubMed  Google Scholar 

  36. Weibrecht I, Lundin E, Kiflemariam S et al (2013) In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay. Nat Protoc 8(2):355–372. https://doi.org/10.1038/nprot.2013.006

    Article  CAS  PubMed  Google Scholar 

  37. Dingar D, Tu WB, Resetca D et al (2018) MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability. Nat Commun 9(1):3502. https://doi.org/10.1038/s41467-018-05660-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Debaize L, Jakobczyk H, Rio AG et al (2017) Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes. Mol Cytogenet 10:27. https://doi.org/10.1186/s13039-017-0328-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Concepcion J, Witte K, Wartchow C et al (2009) Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb Chem High Throughput Screen 12(8):791–800. https://doi.org/10.2174/138620709789104915

    Article  CAS  PubMed  Google Scholar 

  40. Sultana A, Lee JE (2015) Measuring protein-protein and protein-nucleic acid interactions by biolayer interferometry. Curr Protoc Protein Sci 79:19251–192526. https://doi.org/10.1002/0471140864.ps1925s79

    Article  Google Scholar 

  41. Cooper MA (2006) Optical biosensors: where next and how soon? Drug Discov Today 11(23–24):1061–1067. https://doi.org/10.1016/j.drudis.2006.10.003

    Article  CAS  PubMed  Google Scholar 

  42. De Munter S, Gornemann J, Derua R, Lesage B, Qian J, Heroes E, Waelkens E, Van Eynde A, Beullens M, Bollen M (2017) Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett 591(2):415–424. https://doi.org/10.1002/1873-3468.12548

    Article  CAS  PubMed  Google Scholar 

  43. Branon TC, Bosch JA, Sanchez AD et al (2018) Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 38(1):108. https://doi.org/10.1038/s41587-019-0355-0

    Article  CAS  Google Scholar 

  44. Wallner B (2020) Estimating local protein model quality: prospects for molecular replacement. Acta Crystallogr Sect D Struct Biol 76(Pt 3):285–290. https://doi.org/10.1107/S2059798320000972

    Article  CAS  Google Scholar 

  45. Bertolin G, Sizaire F, Herbomel G et al (2016) A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells. Nat Commun 7:12674. https://doi.org/10.1038/ncomms12674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Carabet LA, Rennie PS, Cherkasov A (2019) Therapeutic inhibition of myc in cancer. Structural bases and computer-aided drug discovery approaches. Int J Mol Sci 20(1):120. https://doi.org/10.3390/ijms20010120

    Article  CAS  Google Scholar 

  47. Helander S, Montecchio M, Pilstål R et al (2015) Pre-anchoring of Pin1 to unphosphorylated c-Myc in a fuzzy complex regulates c-Myc activity. Structure 23(12):2267–2279. https://doi.org/10.1016/j.str.2015.10.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We would like to thank the members of the Penn laboratory for helpful discussions and critical review of this chapter. This work was supported by the Canada Research Chairs Program (FRN# 87152, L.Z.P.), Canadian Institutes of Health Research (FRN# 156167, L.Z.P), The Princess Margaret Foundation (B.R., Structure Genomics Consortium (SGC, C.A.), Ontario Graduate Scholarship to D.R. and A.M., and the Peterborough K. M. Hunter Graduate Award (from Peterborough K. M. Hunter Charitable Foundation) to A.M. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome.

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Authors and Affiliations

  1. Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

    Diana Resetca, Alannah S. MacDonald, Tristan M. G. Kenney, Cheryl H. Arrowsmith, Brian Raught & Linda Z. Penn

  2. Structural Genomics Consortium, Toronto, ON, Canada

    Tristan M. G. Kenney, Yong Wei & Cheryl H. Arrowsmith

  3. Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada

    Brian Raught & Linda Z. Penn

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  1. Diana Resetca
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  2. Alannah S. MacDonald
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  3. Tristan M. G. Kenney
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  4. Yong Wei
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  5. Cheryl H. Arrowsmith
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  6. Brian Raught
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  7. Linda Z. Penn
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Correspondence to Linda Z. Penn .

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Editors and Affiliations

  1. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Laura Soucek

  2. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Jonathan Whitfield

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Resetca, D. et al. (2021). Identifying and Validating MYC:Protein Interactors in Pursuit of Novel Anti-MYC Therapies. In: Soucek, L., Whitfield, J. (eds) The Myc Gene. Methods in Molecular Biology, vol 2318. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1476-1_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1476-1_4

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