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Background <p>Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate that is an effective therapy for HER2-positive breast cancer; however, its efficacy is limited by drug resistance. While multiple mechanisms of resistance have been proposed, these are not yet well understood. Greater understanding of T-DM1 sensitivity and resistance could provide new combination strategies to overcome resistance or predictive biomarkers to guide therapy.</p> Methods <p>We have conducted CRISPR/Cas9 functional genomics modifier screens in HER2-positive breast cancer cell lines to allow for unbiased discovery of T-DM1 sensitivity and resistance genes. Whole-genome knockout screens were carried out in MDA-MB-361 and MDA-MB-453 cells treated with T-DM1 and its payload cytotoxin DM1. Hits were validated in secondary T-DM1 screens using a focused single-guide RNA (sgRNA) library and subsequently by individual gene knockout.</p> Results <p>The whole-genome CRISPR screens with T-DM1 and DM1 identified 599 genes as potential modifiers of T-DM1 sensitivity and resistance. Of these, 17 genes were significantly enriched and 3 genes depleted at <i>P</i> &lt; 0.001 in either or both MDA-MB-361 and MDA-MB-453 libraries in the secondary screens. Among the top hits, were known T-DM1 sensitivity genes <i>ERBB2</i> and <i>SLC46A3</i>, in addition to negative regulators of mTOR complex 1: <i>TSC1</i> and <i>TSC2</i>. MDA-MB-453 clones with knockout of <i>TSC1</i> or partial knockout of <i>TSC2</i> were more resistant to T-DM1 than wild type cells in competition growth assays and to T-DM1 and other HER2 targeting therapies (T-DXd, lapatinib and neratinib) in growth inhibition assays, and had increased internalisation of T-DM1 at 6&#xa0;h. T-DM1 and the mTOR inhibitor everolimus demonstrated synergistic activity at inhibiting cell proliferation at multiple T-DM1 concentrations across four HER2-positive breast cancer cell lines.</p> Conclusions <p>Our CRISPR screening approach with T-DM1 in HER2-positive breast cancer cell lines identified genes not previously implicated in T-DM1 sensitivity or resistance, including <i>TSC1</i> and <i>TSC2</i>. These genes may inform new strategies to enhance T-DM1 therapy in the clinic.</p>

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CRISPR screens with trastuzumab emtansine in HER2-positive breast cancer cell lines reveal new insights into drug resistance

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月31日

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  • Barbara A. Lipert,
  • Kyla N. Siemens,
  • Aziza Khan,
  • Rebecca Airey,
  • Gech Heng Dam,
  • Man Lu,
  • Marcella Flinterman,
  • Queenie Yong,
  • Tet Woo Lee,
  • Francis W. Hunter,
  • Stephen M. F. Jamieson

摘要

Background

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate that is an effective therapy for HER2-positive breast cancer; however, its efficacy is limited by drug resistance. While multiple mechanisms of resistance have been proposed, these are not yet well understood. Greater understanding of T-DM1 sensitivity and resistance could provide new combination strategies to overcome resistance or predictive biomarkers to guide therapy.

Methods

We have conducted CRISPR/Cas9 functional genomics modifier screens in HER2-positive breast cancer cell lines to allow for unbiased discovery of T-DM1 sensitivity and resistance genes. Whole-genome knockout screens were carried out in MDA-MB-361 and MDA-MB-453 cells treated with T-DM1 and its payload cytotoxin DM1. Hits were validated in secondary T-DM1 screens using a focused single-guide RNA (sgRNA) library and subsequently by individual gene knockout.

Results

The whole-genome CRISPR screens with T-DM1 and DM1 identified 599 genes as potential modifiers of T-DM1 sensitivity and resistance. Of these, 17 genes were significantly enriched and 3 genes depleted at P < 0.001 in either or both MDA-MB-361 and MDA-MB-453 libraries in the secondary screens. Among the top hits, were known T-DM1 sensitivity genes ERBB2 and SLC46A3, in addition to negative regulators of mTOR complex 1: TSC1 and TSC2. MDA-MB-453 clones with knockout of TSC1 or partial knockout of TSC2 were more resistant to T-DM1 than wild type cells in competition growth assays and to T-DM1 and other HER2 targeting therapies (T-DXd, lapatinib and neratinib) in growth inhibition assays, and had increased internalisation of T-DM1 at 6 h. T-DM1 and the mTOR inhibitor everolimus demonstrated synergistic activity at inhibiting cell proliferation at multiple T-DM1 concentrations across four HER2-positive breast cancer cell lines.

Conclusions

Our CRISPR screening approach with T-DM1 in HER2-positive breast cancer cell lines identified genes not previously implicated in T-DM1 sensitivity or resistance, including TSC1 and TSC2. These genes may inform new strategies to enhance T-DM1 therapy in the clinic.