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:

CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers

Oncogene volume 40, pages 3287–3302 (2021)Cite this article

Subjects

Abstract

Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using β-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the β-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.

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: Genome-wide CRISPR/Cas9 knockout screens for ABT-263 sensitivity in colorectal cancer cells with KRAS mutations.
Fig. 2: Correlation between the WNT signaling pathway signature and BCL-2 family genes in colorectal cancer patients from The Cancer Genome Atlas database.
Fig. 3: Effect of β-catenin knockdown on ABT-263 sensitivity in KRAS-mutated colorectal cancer cells.
Fig. 4: Synergistic effect of ABT-263 and NCB-0846 in KRAS-mutated colorectal cancer cells.
Fig. 5: Molecular mechanisms of MCL1 downregulation via combination of ABT-263 and NCB-0846.
Fig. 6: Transcriptomic analysis after ABT-263 and NCB-0846 treatment in KRAS-mutated colorectal cancer cells.
Fig. 7: In vivo efficacy of ABT-263 and NCB-0846 treatment in KRAS-mutated patient-derived xenograft (PDX) models.

Similar content being viewed by others

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    PubMed  Google Scholar 

  2. Martini G, Troiani T, Cardone C, Vitiello P, Sforza V, Ciardiello D, et al. Present and future of metastatic colorectal cancer treatment: a review of new candidate targets. World J Gastroenterol. 2017;23:4675–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Punt CJA, Koopman M, Vermeulen L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat Rev Clin Oncol. 2017;14:235–46.

    Article  CAS  PubMed  Google Scholar 

  4. DeStefanis RA, Kratz JD, Emmerich PB, Deming DA. Targeted Therapy in Metastatic Colorectal Cancer: current Standards and Novel Agents in Review. Curr Colorectal Cancer Rep. 2019;15:61–9.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Phipps AI, Limburg PJ, Baron JA, Burnett-Hartman AN, Weisenberger DJ, Laird PW, et al. Association Between Molecular Subtypes of Colorectal Cancer and Patient Survival. Gastroenterology. 2015;148:77–87.

    Article  CAS  PubMed  Google Scholar 

  6. Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, et al. Wild-Type BRAF Is Required for Response to Panitumumab or Cetuximab in Metastatic Colorectal Cancer. J Clin Oncol. 2008;26:5705–12.

    Article  PubMed  CAS  Google Scholar 

  7. Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006;66:3992–5.

    Article  CAS  PubMed  Google Scholar 

  8. Cho SY, Han JY, Na D, Kang W, Lee A, Kim J, et al. A Novel Combination Treatment Targeting BCL-XL and MCL1 for KRAS/BRAF-mutated and BCL2L1-amplified Colorectal Cancers. Mol Cancer Ther. 2017;16:2178–90.

    Article  CAS  PubMed  Google Scholar 

  9. Corcoran RB, Cheng KA, Hata AN, Faber AC, Ebi H, Coffee EM, et al. Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models. Cancer Cell. 2013;23:121–8.

    Article  CAS  PubMed  Google Scholar 

  10. Faber AC, Coffee EM, Costa C, Dastur A, Ebi H, Hata AN, et al. mTOR Inhibition Specifically Sensitizes Colorectal Cancers with KRAS or BRAF Mutations to BCL-2/BCL-XL Inhibition by Suppressing MCL-1. Cancer Disco. 2014;4:42–52.

    Article  CAS  Google Scholar 

  11. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–73.

    Article  CAS  PubMed  Google Scholar 

  12. Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–7.

    Article  CAS  Google Scholar 

  13. Yoshida N, Kinugasa T, Ohshima K, Yuge K, Ohchi T, Fujino S, et al. Analysis of Wnt and beta-catenin Expression in Advanced Colorectal Cancer. Anticancer Res. 2015;35:4403–10.

    CAS  PubMed  Google Scholar 

  14. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44–57.

    Article  CAS  Google Scholar 

  15. Wang BB, Wang M, Zhang WB, Xiao TF, Chen CH, Wu A, et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat Protoc. 2019;14:756–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019;47:D607–D13.

    Article  CAS  PubMed  Google Scholar 

  17. Gao JJ, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal. Sci Signal. 2013;6:pl1.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Cerami E, Gao JJ, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio Cancer Genomics Portal: an Open Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Disco. 2012;2:401–4.

    Article  Google Scholar 

  19. Herbst A, Jurinovic V, Krebs S, Thieme SE, Blum H, Goke B, et al. Comprehensive analysis of beta-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/beta-catenin signaling. BMC Genom. 2014;15:74.

    Article  Google Scholar 

  20. Masuda M, Uno Y, Ohbayashi N, Ohata H, Mimata A, Kukimoto-Niino M, et al. TNIK inhibition abrogates colorectal cancer stemness. Nat Commun. 2016;7:12586.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang L, Doherty GA, Judd AS, Tao ZF, Hansen TM, Frey RR, et al. Discovery of A-1331852, a first-in-class, potent, and orally-bioavailable BCL-X-L inhibitor. ACS Med Chem Lett. 2020;11:1829–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schuijers J, Junker JP, Mokry M, Hatzis P, Koo BK, Sasselli V, et al. Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts. Cell Stem Cell. 2015;16:158–70.

    Article  CAS  PubMed  Google Scholar 

  23. Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009;25:1091–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hoeflich KP, Merchant M, Orr C, Chan J, Den Otter D, Berry L, et al. Intermittent administration of MEK inhibitor GDC-0973 plus PI3K inhibitor GDC-0941 triggers robust apoptosis and tumor growth inhibition. Cancer Res. 2012;72:210–9.

    Article  CAS  PubMed  Google Scholar 

  25. Holt SV, Logie A, Davies BR, Alferez D, Runswick S, Fenton S, et al. Enhanced apoptosis and tumor growth suppression elicited by combination of MEK (selumetinib) and mTOR kinase inhibitors (AZD8055). Cancer Res. 2012;72:1804–13.

    Article  CAS  PubMed  Google Scholar 

  26. Halilovic E, She QB, Ye Q, Pagliarini R, Sellers WR, Solit DB, et al. PIK3CA mutation uncouples tumor growth and cyclin D1 regulation from MEK/ERK and mutant KRAS signaling. Cancer Res. 2010;70:6804–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bendell J, Ciardiello F, Tabernero J, Tebbutt N, Eng C, Di Bartolomeo M, et al. Efficacy and safety results from IMblaze370, a randomised Phase III study comparing atezolizumab plus cobimetinib and atezolizumab monotherapy vs regorafenib in chemotherapy-refractory metastatic colorectal cancer. Ann Oncol. 2018;29:123.

    Article  Google Scholar 

  28. Billard C. BH3 mimetics: status of the field and new developments. Mol Cancer Ther. 2013;12:1691–700.

    Article  CAS  PubMed  Google Scholar 

  29. Croce CM, Reed JC. Finally, An Apoptosis-Targeting Therapeutic for Cancer. Cancer Res. 2016;76:5914–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. McBride A, Houtmann S, Wilde L, Vigil C, Eischen CM, Kasner M, et al. The Role of Inhibition of Apoptosis in Acute Leukemias and Myelodysplastic Syndrome. Front Oncol. 2019;9:192.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68:3421–8.

    Article  CAS  PubMed  Google Scholar 

  32. Ho SY, Keller TH. The use of porcupine inhibitors to target Wnt-driven cancers. Bioorg Med Chem Lett. 2015;25:5472–6.

    Article  CAS  PubMed  Google Scholar 

  33. Yeung P, Beviglia L, Cancilla B, Dee-Hoskins C, Evans JW, Fischer MM, et al. Wnt pathway antagonist OMP-54F28 (FZD8-Fc) inhibits tumor growth and reduces tumor-initiating cell frequency in patient-derived hepatocellular carcinoma and ovarian cancer xenograft models. Cancer Res. 2014;74:S1907.

    Google Scholar 

  34. Gang EJ, Hsieh YT, Pham J, Zhao Y, Nguyen C, Huantes S, et al. Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia. Oncogene. 2014;33:2169–78.

    Article  CAS  PubMed  Google Scholar 

  35. Eguchi M, Nguyen C, Lee SC, Kahn M. ICG-001, A Novel Small Molecule Regulator of TCF/beta-Catenin Transcription. Med Chem. 2005;1:467–72.

    Article  CAS  PubMed  Google Scholar 

  36. Shitashige M, Satow R, Jigami T, Aoki K, Honda K, Shibata T, et al. Traf2-and Nck-Interacting Kinase Is Essential for Wnt Signaling and Colorectal Cancer Growth. Cancer Res. 2010;70:5024–33.

    Article  CAS  PubMed  Google Scholar 

  37. Shkoda A, Town JA, Griese J, Romio M, Sarioglu H, Knofel T, et al. The Germinal Center Kinase TNIK Is Required for Canonical NF-kappa B and JNK Signaling in B-Cells by the EBV Oncoprotein LMP1 and the CD40 Receptor. PloS Biol. 2012;10:e1001376.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gui JH, Yang BT, Wu JY, Zhou XM. The enormous influence of TNIK knockdown on intracellular signals and cell survival. Hum Cell. 2011;24:121–6.

    Article  CAS  PubMed  Google Scholar 

  39. Krishnamurthy N, Kurzrock R. Targeting the Wnt/beta-catenin pathway in cancer: update on effectors and inhibitors. Cancer Treat Rev. 2018;62:50–60.

    Article  CAS  PubMed  Google Scholar 

  40. Jung YS, Park JI. Wnt signaling in cancer: therapeutic targeting of Wnt signaling beyond beta-catenin and the destruction complex. Exp Mol Med. 2020;52:183–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006;10:375–88.

    Article  CAS  PubMed  Google Scholar 

  42. van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE, et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell. 2006;10:389–99.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Senichkin VV, Streletskaia AY, Zhivotovsky B, Kopeina GS. Molecular Comprehension of Mcl-1: from Gene Structure to Cancer Therapy. Trends Cell Biol. 2019;29:549–62.

    Article  CAS  PubMed  Google Scholar 

  44. Mills JR, Hippo Y, Robert F, Chen SMH, Malina A, Lin CJ, et al. mTORC1 promotes survival through translational control of Mcl-1. Proc Natl Acad Sci USA. 2008;105:10853–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343:84–7.

    Article  CAS  PubMed  Google Scholar 

  46. Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017;12:828–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharm Rev. 2006;58:621–81.

    Article  CAS  PubMed  Google Scholar 

  49. Booy EP, Henson ES, Gibson SB. Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer. Oncogene. 2011;30:2367–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (grant No. 2017R1C1B2002183); the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science & ICT (grant No. 2018M3A9F3056902 and 2019M3E5D4066900); Creative-Pioneering Researchers Program through Seoul National University (grant No. 800-20200510); and the Collaborative Research Program of SNU Boramae Medical Center and Basic Medical Science from Seoul National University College of Medicine (grant No. 800-20200005).

Author information

Author notes

  1. These authors contributed equally: Hae Rim Jung, Yumi Oh

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea

    Hae Rim Jung & Yumi Oh

  2. Medical Research Center, Genomic Medicine Institute, Seoul National University College of Medicine, Seoul, Korea

    Hae Rim Jung, Yumi Oh & Sung-Yup Cho

  3. Ewha Institute of Convergence Medicine, Ewha Womans University Mokdong Hospital, Seoul, Korea

    Deukchae Na

  4. Department of Life Science, Ewha Womans University, Seoul, Korea

    Seoyeon Min, Jinjoo Kang & Charles Lee

  5. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea

    Dongjun Jang, Seungjae Shin, Jiwon Kim, Sang Eun Lee & Sung-Yup Cho

  6. Department of Pharmacy, College of Pharmacy, Jeju National University, Jeju Special Self-Governing Province, Korea

    Eui Man Jeong

  7. Interdisciplinary Graduate Program in Advanced Convergence Technology and Science, Jeju National University, Jeju Special Self-Governing Province, Korea

    Eui Man Jeong

  8. Bio-Health Materials Core-Facility Center, Jeju National University, Jeju Special Self-Governing Province, Korea

    Eui Man Jeong

  9. Practical Translational Research Center, Jeju National University, Jeju Special Self-Governing Province, Korea

    Eui Man Jeong

  10. School of Biosystem and Biomedical Science, College of Health Science, Korea University, Seoul, Korea

    Joon Yong An

  11. Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

    Chang Ohk Sung

  12. Department of Surgery, Gil Medical Center, Gachon University, Incheon, Korea

    Won-Suk Lee

  13. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA

    Charles Lee

  14. Cancer Research Institute, Seoul National University, Seoul, Korea

    Sung-Yup Cho

Authors
  1. Hae Rim Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yumi Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Deukchae Na
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Seoyeon Min
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jinjoo Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dongjun Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Seungjae Shin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jiwon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sang Eun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Eui Man Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Joon Yong An
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Chang Ohk Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Won-Suk Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Charles Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sung-Yup Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CL and SYC designed research. HRJ, YO, SM, JK, DJ, SS, JK, and SEL performed the experiments. WSL provided colorectal tumor specimens. DN, EMJ, JYA, CL, and SYC analyzed data. CL and SYC wrote the paper and all authors reviewed the paper.

Corresponding author

Correspondence to Sung-Yup Cho.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

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

Jung, H.R., Oh, Y., Na, D. et al. CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers. Oncogene 40, 3287–3302 (2021). https://doi.org/10.1038/s41388-021-01777-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-021-01777-7

This article is cited by

Search

Advanced search

Quick links