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-Cas9 screens identify regulators of antibody–drug conjugate toxicity

Nature Chemical Biology volume 15, pages 949–958 (2019)Cite this article

Subjects

Abstract

Antibody–drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine–citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.

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 screen uncovers diverse endolysosomal regulators of ADC toxicity.
Fig. 2: A subset of endolysosomal trafficking regulators are critical only for toxicity of the noncleavable linker ADC.
Fig. 3: Lysosomal delivery is not required for processing of VC cleavable linkers.
Fig. 4: C18ORF8/RMC1 is required for endosomal trafficking and lysosomal delivery of ADCs.
Fig. 5: Inhibition of sialic acid synthesis sensitizes cells to CD22 ADC toxicity.
Fig. 6: Depletion of sialic acid enhances rate of ADC lysosomal delivery.

Similar content being viewed by others

Data availability

The complete results of genome-wide screens and secondary screens are in Supplementary Datasets 1–4. All data are available from the corresponding author upon reasonable request.

Code availability

casTLE v.1.0 is available at https://bitbucket.org/dmorgens/castle.

References

  1. Beck, A., Goetsch, L., Dumontet, C. & Corvaïa, N. Strategies and challenges for the next generation of antibody–drug conjugates. Nat. Rev. Drug Discov. 16, 315–337 (2017).

    Article  CAS  Google Scholar 

  2. Lyon, R. Drawing lessons from the clinical development of antibody-drug conjugates. Drug Discov. Today Technol. 30, 105–109 (2018).

    Article  Google Scholar 

  3. St Pierre, C. A., Leonard, D., Corvera, S., Kurt-Jones, E. A. & Finberg, R. W. Antibodies to cell surface proteins redirect intracellular trafficking pathways. Exp. Mol. Pathol. 91, 723–732 (2011).

    Article  Google Scholar 

  4. Ritchie, M., Tchistiakova, L. & Scott, N. Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates. MAbs 5, 13–21 (2013).

    Article  Google Scholar 

  5. Loganzo, F. et al. Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments. Mol. Cancer Ther. 14, 952–963 (2015).

    Article  CAS  Google Scholar 

  6. Donaghy, H. Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates. MAbs 8, 659–671 (2016).

    Article  CAS  Google Scholar 

  7. Lewis Phillips, G. D. et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 68, 9280–9290 (2008).

    Article  CAS  Google Scholar 

  8. Polson, A. G. et al. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res. 69, 2358–2364 (2009).

    Article  CAS  Google Scholar 

  9. Rabuka, D., Rush, J. S., deHart, G. W., Wu, P. & Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).

    Article  CAS  Google Scholar 

  10. Agarwal, P. et al. Hydrazino–Pictet–Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug. Chem. 24, 846–851 (2013).

    Article  CAS  Google Scholar 

  11. Lopus, M. et al. Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules. Mol. Cancer Ther. 9, 2689–2699 (2010).

    Article  CAS  Google Scholar 

  12. Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).

    Article  CAS  Google Scholar 

  13. Morgens, D. W., Deans, R. M., Li, A. & Bassik, M. C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016).

    Article  CAS  Google Scholar 

  14. Hamblett, K. J. et al. SLC46A3 is required to transport catabolites of noncleavable antibody maytansine conjugates from the lysosome to the cytoplasm. Cancer Res. 75, 5329–5340 (2015).

    Article  CAS  Google Scholar 

  15. Hyttinen, J. M. T., Niittykoski, M., Salminen, A. & Kaarniranta, K. Maturation of autophagosomes and endosomes: a key role for Rab7. Biochim. Biophys. Acta 1833, 503–510 (2013).

    Article  CAS  Google Scholar 

  16. Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010).

    Article  CAS  Google Scholar 

  17. Liu, K. et al. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion. J. Cell Biol. 212, 181–198 (2016).

    Article  CAS  Google Scholar 

  18. Rapiteanu, R. et al. A genetic screen identifies a critical role for the WDR81–WDR91 complex in the trafficking and degradation of tetherin. Traffic 17, 940–958 (2016).

    Article  CAS  Google Scholar 

  19. Burger, J. A. & Wiestner, A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat. Rev. Cancer 18, 148–167 (2018).

    Article  CAS  Google Scholar 

  20. Mould, A. W. et al. Global expression profiling of murine MEN1-associated tumors reveals a regulatory role for menin in transcription, cell cycle and chromatin remodelling. Int. J. Cancer 121, 776–783 (2007).

    Article  CAS  Google Scholar 

  21. Wood, K., Tellier, M. & Murphy, S. DOT1L and H3K79 methylation in transcription and genomic stability. Biomolecules 8, 11 (2018).

    Article  Google Scholar 

  22. Schröder, B. et al. Integral and associated lysosomal membrane proteins. Traffic 8, 1676–1686 (2007).

    Article  Google Scholar 

  23. Dubowchik, G. M. et al. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. 13, 855–869 (2002).

    Article  CAS  Google Scholar 

  24. Caculitan, N. G. et al. Caculitan, N. G. et al. Cathepsin B is dispensable for cellular processing of cathepsin B-cleavable antibody-drug conjugates. Cancer Res. 77, 7027–7037 (2017).

    Article  CAS  Google Scholar 

  25. Zmolek, W., Bañas, S., Barfield, R. M., Rabuka, D. & Drake, P. M. A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations. J. Chromatogr. B 1032, 144–148 (2016).

    Article  CAS  Google Scholar 

  26. Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M. & Tashiro, Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 266, 17707–17712 (1991).

    CAS  PubMed  Google Scholar 

  27. Pontano Vaites, L., Paulo, J. A., Huttlin, E. L. & Harper, J. W. Systematic analysis of human cells lacking ATG8 proteins uncovers roles for GABARAPs and the CCZ1/MON1 regulator C18orf8/RMC1 in macro and selective autophagic flux. Mol. Cell. Biol. 38, e00392-17 (2017).

    Article  Google Scholar 

  28. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  29. Rillahan, C. D. et al. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat. Chem. Biol. 8, 661–668 (2012).

    Article  CAS  Google Scholar 

  30. Xiao, H., Woods, E. C., Vukojicic, P. & Bertozzi, C. R. Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc. Natl Acad. Sci. USA 113, 10304–10309 (2016).

    Article  CAS  Google Scholar 

  31. Wu, A. M. et al. Differential affinities of erythrina cristagalli lectin (ECL) toward monosaccharides and polyvalent mammalian structural units. Glycoconj. J. 24, 591–604 (2007).

    Article  CAS  Google Scholar 

  32. Polson, A. G. et al. Antibody-drug conjugates targeted to CD79 for the treatment of non-Hodgkin lymphoma. Blood 110, 616–623 (2007).

    Article  CAS  Google Scholar 

  33. Li, L. et al. The effect of the size of fluorescent dextran on its endocytic pathway. Cell Biol. Int. 39, 531–539 (2015).

    Article  CAS  Google Scholar 

  34. Liu, Y. et al. LC-MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1). J. Pharm. Biomed. Anal. 137, 170–177 (2017).

    Article  CAS  Google Scholar 

  35. Lu, J., Jiang, F., Lu, A. & Zhang, G. Linkers having a crucial role in antibody-drug conjugates. Int. J. Mol. Sci. 17, 561 (2016).

    Article  Google Scholar 

  36. Abdollahpour-Alitappeh, M. et al. Antibody-drug conjugates (ADCs) for cancer therapy: strategies, challenges, and successes. J. Cell Physiol. 234, 5628–5642 (2018).

    Article  Google Scholar 

  37. Lee, B.-C. et al. FRET reagent reveals the intracellular processing of peptide-linked antibody-drug conjugates. Bioconjug. Chem. 29, 2468–2477 (2018).

    Article  CAS  Google Scholar 

  38. Wang, H. et al. Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells. Cancer Sci. 108, 1458–1468 (2017).

    Article  CAS  Google Scholar 

  39. Li, G. et al. Mechanisms of acquired resistance to trastuzumab emtansine in breast cancer cells. Mol. Cancer Ther. 17, 1441–1453 (2018).

    Article  CAS  Google Scholar 

  40. Liu, K. et al. WDR91 is a Rab7 effector required for neuronal development. J. Cell Biol. 216, 3307–3321 (2017).

    Article  CAS  Google Scholar 

  41. Kinneer, K. et al. SLC46A3 as a potential predictive biomarker for antibody-drug conjugates bearing non-cleavable linked maytansinoid and pyrrolobenzodiazepine warheads. Clin. Cancer Res. 24, 6570–6582 (2018).

    Article  Google Scholar 

  42. Ríos-Luci, C. et al. Resistance to the antibody-drug conjugate T-DM1 Is based in a reduction in lysosomal proteolytic activity. Cancer Res. 77, 4639–4651 (2017).

    Article  Google Scholar 

  43. Sung, M. et al. Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1). Mol. Cancer Ther. 17, 243–253 (2018).

    Article  CAS  Google Scholar 

  44. Zhang, M. & Varki, A. Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis. Glycobiology 14, 939–949 (2004).

    Article  CAS  Google Scholar 

  45. Mathew, M. P. et al. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells. Oncotarget 7, 66491–66511 (2016).

    PubMed  PubMed Central  Google Scholar 

  46. Nabi, I. R., Shankar, J. & Dennis, J. W. The galectin lattice at a glance. J. Cell Sci. 128, 2213–2219 (2015).

    Article  CAS  Google Scholar 

  47. Pearce, O. M. T. & Läubli, H. Sialic acids in cancer biology and immunity. Glycobiology 26, 111–128 (2016).

    Article  CAS  Google Scholar 

  48. Wang, L., Liu, Y., Wu, L. & Sun, X.-L. Sialyltransferase inhibition and recent advances. Biochim. Biophys. Acta 1864, 143–153 (2016).

    Article  CAS  Google Scholar 

  49. Macauley, M. S. et al. Systemic blockade of sialylation in mice with a global inhibitor of sialyltransferases. J. Biol. Chem. 289, 35149–35158 (2014).

    Article  CAS  Google Scholar 

  50. Büll, C. et al. Sialic acid blockade suppresses tumor growth by enhancing T-cell-mediated tumor immunity. Cancer Res. 78, 3574–3588 (2018).

    PubMed  Google Scholar 

  51. Deans, R. M. et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nat. Chem. Biol. 12, 361–366 (2016).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Hess, P. Drake, A. Lu and S. Pfeffer for assistance and discussions, A. Gupta for help with LC–MS/MS and M. Dubreuil and E. Jeng for comments on the manuscript. We also thank M. Pegram and W. Liang for generously providing the T-DM1 used in our experiments. This work was funded by grants from NIH (grant nos. F31 GM126688-01A1 to C.K.T., NIH 1DP2HD084069-01 to M.C.B. and NIH R01 CA227942 to C.R.B.).

Author information

Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    C. Kimberly Tsui, David W. Morgens, Amy Li & Michael C. Bassik

  2. Catalent Biologics, Emeryville, CA, USA

    Robyn M. Barfield & David Rabuka

  3. Stanford ChEM-H, Stanford University, Stanford, CA, USA

    Curt R. Fischer, Benjamin A. H. Smith, Melissa Anne Gray, Carolyn R. Bertozzi & Michael C. Bassik

  4. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA

    Benjamin A. H. Smith

  5. Department of Chemistry, Stanford University, Stanford, CA, USA

    Melissa Anne Gray & Carolyn R. Bertozzi

  6. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Carolyn R. Bertozzi

Authors
  1. C. Kimberly Tsui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robyn M. Barfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Curt R. Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David W. Morgens
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amy Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Benjamin A. H. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Melissa Anne Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carolyn R. Bertozzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David Rabuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michael C. Bassik
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.K.T. designed and performed experiments, analyzed data and wrote the manuscript. R.M.B. generated ADC reagents. C.R.F. helped to design and assisted with the experiments performed with LC–MS/MS. D.W.M. assisted in screen data analysis. A.L. assisted in library cloning and screens. B.A.H.S., M.A.G. and C.R.B. helped design sialic acid inhibition experiments. M.C.B. and D.R. supervised the study.

Corresponding author

Correspondence to Michael C. Bassik.

Ethics declarations

Competing interests

R.M.B. and D.R. are employees of Catalent Biologics. C.R.B. is a cofounder and Scientific Advisory Board member of Redwood Bioscience, which generated antibody–drug conjugates used in this work. Stanford University has filed a patent application based on the findings in this article.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Table 1, Supplementary Figures 1–8.

Reporting Summary

Supplementary Dataset 1

Genome-wide CRISPR screen results with anti-CD22-maytansine ADC in Ramos cells.

Supplementary Dataset 2

ADC/endolysosomal sublibrary screen results with anti-CD22-maytansine (noncleavable ADC), anti-CD22-VC–maytansine (cleavable ADC) and free maytansine in Ramos cells.

Supplementary Dataset 3

Drug target, kinases and phosphatases sublibrary screen results with anti-CD22-maytansine (noncleavable ADC) and free maytansine in Ramos cells.

Supplementary Dataset 4

Count files for all screens in Ramos cells.

Supplementary Dataset 5

ADC/endolysosomal sublibrary sgRNA composition.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tsui, C.K., Barfield, R.M., Fischer, C.R. et al. CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity. Nat Chem Biol 15, 949–958 (2019). https://doi.org/10.1038/s41589-019-0342-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0342-2

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research