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Figure 3 from ESR1 F404 Mutations and Acquired Resistance to Fulvestrant in ESR1-Mutant Breast Cancer

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posted on 2024-02-08, 08:20 authored by Belinda Kingston, Alex Pearson, Maria Teresa Herrera-Abreu, Li-Xuan Sim, Rosalind J. Cutts, Heena Shah, Laura Moretti, Lucy S. Kilburn, Hannah Johnson, Iain R. Macpherson, Alistair Ring, Judith M. Bliss, Yingwei Hou, Weiyi Toy, John A. Katzenellenbogen, Sarat Chandarlapaty, Nicholas C. Turner

F404 does not activate estrogen signaling. A, CRISPR clones of MCF7 cells expressing ESR1 F404L (1210T>C, CRISPR edit indicated by red arrows) or D538G (1613A>G; CRISPR edit indicated by black arrows) were identified by RT-PCR followed by Sanger sequencing (left-hand panels). Similarly, a second round of CRISPR was used to introduce ESR1 F404L (1210T>C) into a clone (D6C) that expressed D538G (1613A>G; right-hand panels). B, Estrogen-dependent growth was assessed in colony formation assay. Parental MCF7 cells and indicated ESR1-mutant models were grown in either the absence or presence of estradiol (1 nmol/L) for 14 days. C, Quantification of colony formation assays of ESR1-mutant models treated with and without estradiol (1 nmol/L). Sulforhodamine B (SRB)-stained colonies were dissolved, and absorbance at 565 nm was measured. Mean with SEM, n = 3 independent experiments, nonparametric one-way ANOVA with Dunn multiple comparisons test; **, P < 0.01. D, Expression of estrogen target genes, progesterone receptor (PgR), and trefoil factor-1 (TFF1), assessed by western blot in parental MCF7 cells and indicated ESR1-mutant models grown in either the absence or presence of estradiol (1 nmol/L) for 24 hours. E, MCF7 cells were transfected with ESR1 expression constructs with indicated ESR1 variants. Expression of ERα was determined by western blot. F, MCF7 cells were cotransfected with the indicated ESR1 expression constructs ERE-luciferase reporter and control construct. Cells were treated in either the absence or presence of estradiol (1 nmol/L) for 24 hours, and ERE-luciferase activity was assessed. Two-way repeated-measures ANOVA with Dunnett multiple comparisons test, n = 4 mean with SD; *, P < 0.05. WT, wild-type.

Funding

Cancer Research UK (CRUK)

NIHR Biomedical Research Centre, Royal Marsden NHS Foundation Trust/Institute of Cancer Research (BRC)

Breast Cancer Now (BCN)

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ARTICLE ABSTRACT

Fulvestrant is used to treat patients with hormone receptor–positive advanced breast cancer, but acquired resistance is poorly understood. PlasmaMATCH Cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Baseline ESR1 mutations Y537S are associated with poor outcomes and Y537C with good outcomes. Sequencing of baseline and EOT ctDNA samples (n = 69) revealed 3/69 (4%) patients acquired novel ESR1 F404 mutations (F404L, F404I, and F404V), in cis with activating mutations. In silico modeling revealed that ESR1 F404 contributes to fulvestrant binding to estrogen receptor–alpha (ERα) through a pi-stacking bond, with mutations disrupting this bond. In vitro analysis demonstrated that single F404L, E380Q, and D538G models were less sensitive to fulvestrant, whereas compound mutations D538G + F404L and E380Q + F404L were resistant. Several oral ERα degraders were active against compound mutant models. We have identified a resistance mechanism specific to fulvestrant that can be targeted by treatments in clinical development. Novel F404 ESR1 mutations may be acquired to cause overt resistance to fulvestrant when combined with preexisting activating ESR1 mutations. Novel combinations of mutations in the ER ligand binding domain may cause drug-specific resistance, emphasizing the potential of similar drug-specific mutations to impact the efficacy of oral ER degraders in development.This article is featured in Selected Articles from This Issue, p. 201

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