Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC

Abstract

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014198. CRISPR-Cas9 screen data were summarized in Supplementary File 1 and Supplementary File 2.

The following data sets were generated

Article and author information

Author details

  1. Hao Zeng

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    For correspondence
    hao-1.zeng@novartis.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4967-9555
  2. Johnny Castillo-Cabrera

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Mika Manser

    Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Bo Lu

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Zinger Yang

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8543-4841
  6. Vaik Strande

    Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Damien Begue

    Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Raffaella Zamponi

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Shumei Qiu

    Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Frederic Sigoillot

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Qiong Wang

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Alicia Lindeman

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. John S Reece-Hoyes

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Carsten Russ

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Debora Bonenfant

    Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  16. Xiaomo Jiang

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Youzhen Wang

    Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Feng Cong

    Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, United States
    For correspondence
    feng.cong@novartis.com
    Competing interests
    The authors declare that no competing interests exist.

Funding

No external funding was received for this work.

Ethics

Animal experimentation: All animal work was performed in accordance with Novartis Animal Care and Use Committee (ACUC) regulations and guidelines (reference number 120137). All animals were allowed to acclimate in the Novartis animal facility with access to food and water ad libitum for 3 days prior to manipulation. All cell lines were confirmed as mycoplasma- and rodent pathogens-negative (IMPACT VIII PCR Profile, IDEXX) before implantation.

Copyright

© 2019, Zeng et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 9,506
    views
  • 1,244
    downloads
  • 39
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Hao Zeng
  2. Johnny Castillo-Cabrera
  3. Mika Manser
  4. Bo Lu
  5. Zinger Yang
  6. Vaik Strande
  7. Damien Begue
  8. Raffaella Zamponi
  9. Shumei Qiu
  10. Frederic Sigoillot
  11. Qiong Wang
  12. Alicia Lindeman
  13. John S Reece-Hoyes
  14. Carsten Russ
  15. Debora Bonenfant
  16. Xiaomo Jiang
  17. Youzhen Wang
  18. Feng Cong
(2019)
Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC
eLife 8:e50223.
https://doi.org/10.7554/eLife.50223

Share this article

https://doi.org/10.7554/eLife.50223

Further reading

    1. Cancer Biology
    Han V Han, Richard Efem ... Richard Z Lin
    Research Article

    Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca−/− KPC (named αKO) cancer cells induces clonal enrichment of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression, and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally enriched CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally enriched T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

    1. Cancer Biology
    2. Immunology and Inflammation
    Almudena Mendez-Perez, Andres M Acosta-Moreno ... Esteban Veiga
    Short Report

    In this study, we present a proof-of-concept classical vaccination experiment that validates the in silico identification of tumor neoantigens (TNAs) using a machine learning-based platform called NAP-CNB. Unlike other TNA predictors, NAP-CNB leverages RNA-seq data to consider the relative expression of neoantigens in tumors. Our experiments show the efficacy of NAP-CNB. Predicted TNAs elicited potent antitumor responses in mice following classical vaccination protocols. Notably, optimal antitumor activity was observed when targeting the antigen with higher expression in the tumor, which was not the most immunogenic. Additionally, the vaccination combining different neoantigens resulted in vastly improved responses compared to each one individually, showing the worth of multiantigen-based approaches. These findings validate NAP-CNB as an innovative TNA identification platform and make a substantial contribution to advancing the next generation of personalized immunotherapies.