TRIM24 as a therapeutic target in endocrine treatment-resistant breast cancer

Abstract

While Estrogen receptor alpha (ERα)+ breast cancer treatment is considered effective, resistance to endocrine therapy is common. Since ERα is still the main driver in most therapy-resistant tumors, alternative therapeutic strategies are needed to disrupt ERα transcriptional activity. In this work, we position TRIM24 as a therapeutic target in endocrine resistance, given its role as a key component of the ERα transcriptional complex. TRIM24 interacts with ERα and other well-known ERα cofactors to facilitate ERα chromatin interactions and allows for maintenance of active histone marks including H3K23ac and H3K27ac. Consequently, genetic perturbation of TRIM24 abrogates ERα-driven transcriptional programs and reduces tumor cell proliferation capacity. Using a recently developed degrader targeting TRIM24, ERα-driven transcriptional output and growth were blocked, effectively treating not only endocrine-responsive cell lines but also drug-resistant derivatives thereof as well as cell line models bearing activating ESR1 point mutations. Finally, using human tumor-derived organoid models, we could show the efficacy of TRIM24 degrader in the endocrine-responsive and -resistant setting. Overall, our study positions TRIM24 as a central component for the integrity and activity of the ERα transcriptional complex, with degradation-mediated perturbation of TRIM24 as a promising therapeutic avenue in the treatment of primary and endocrine resistance breast cancer.

Keywords: TRIM24; breast cancer; estrogen receptor alpha; heterobifunctional protein degrader; therapy resistance breast cancer.

Fig. 1.

TRIM24 is a member of the ERα transcriptional complex, and its expression is correlated with poor BC prognosis. (A) Volcano plot summarizing the quantitative results of ERα RIME in MCF7. TRIM24 is depicted in red. ERα (ESR1 gene) and several known ERα interactors are labeled. (B) Coimmunoprecipitation of TRIM24, followed by western blot for TRIM24 and ERα in MCF7 cells. (C) Volcano plot summarizing the quantitative results of TRIM24 RIME. ERα (ESR1 gene), several known ERα interactors, and members of the TIF1 family (TRIM28 and TRIM33) are labeled. (D) Box plot showing TRIM24 expression in different ERα+ BC grade tumors. Gene expression data originated from the METABRIC dataset (26), filtered for ERα+/HER2- BC patients (n = 997) that have received hormone therapy. Gene expression category per patient was defined by the median gene expression for the group (above = HIGH, below = LOW). The t test was performed for association with Grade (1,2,3) and gene expression categories. (E) Correlation plot of normalized TPM expression (RNA-seq) between TRIM24 and Ki-67 in 879 ERα+ female BC patient samples from TCGA-BRCA cohort (www.cancer.gov/tcga). Pearson’s correlation coefficient is displayed. (F) Violin plot showing the RNA-seq TRIM24 gene expression from Breast Invasive Carcinoma TNMplot data including tumor, metastatic, and normal samples as control. Average of 113 healthy, 1,097 tumors, and 7 metastatic samples is shown. Global P-value of the Kruskal–Wallis test, as well as post hoc Wilcoxon’s rank-sum test, is indicated.

Fig. 2.

TRIM24 binds together with ERα at the same genomic locations, and its binding is hormone dependent. (A) Snapshots of ERα and TRIM24 ChIP-seq signal at GREB1, TFF1, and IGFBP4 loci. The genomic coordinates are annotated. (B) Heatmap disclosing ChIP-seq signal for Input, ERα, and TRIM24 in full medium. Regions were sorted according to decreasing ERα signal. Data are centered at each factor peak, depicting a ±1 kb window around the peak center. (C) Correlation plot between ERα and TRIM24 ChIP-seq signal at ERα peaks. Pearson’s correlation coefficient (R) is indicated. Red line depicts the linear regression (glm, y ~ x) ± SE. Color scale indicates the dot density. (D) Motif enrichment analysis at TRIM24 binding sites. Font size represents log₁₀(P-value) × 10³. (E) GIGGLE enrichment analysis for the top transcription factors binding at the enriched TRIM24 sites. (F) Snapshots of TRIM24 ChIP-seq signal at GREB1, TFF1, and IGFBP4 loci upon treatment with DMSO or E2 for 3 h. The genomic coordinates are annotated.

Fig. 3.

Depletion of TRIM24 leads to reduced ERα activity. (A) Western blot for TRIM24-KO in MCF7 cells. NT: nontarget. Actin is used as a loading control. (B) Cell viability assay on TRIM24-KO MCF7 cells and NT MCF7 cells, by CTG (n = 7). P-value represents the two-tailed paired t test. (C) Snapshots of ERα, H3K23ac, and H3K27ac ChIP-seq peaks at GREB1, TFF1, and IGFBP4 loci. The genomic coordinates are annotated. (D) Heatmap disclosing ChIP-seq signal for ERα, H3K23ac, and H3K27ac in NT (clone 1) or TRIM24-KO (Clone A) MCF7 cells at ERα binding regions in NT MCF7 cells. Regions were sorted according to decreasing ERα average signal. Data are centered at each factor peak, depicting a ±2.5 kb window around the peak center. (E) Mean signal intensity for p-Ser RNA Polymerase 2 at genes found near an ERα binding site in NT1. Below is a zoom shot of the starting site ±2.5 kb distance. (F) Enrichment plot of the hallmark estrogen response late pathway performed on differential transcriptomic data between TRIM24-KO A and NT1. Genes are ranked by differential expression upon TRIM24-KO. (G) Heatmap depicting the differential expression of genes included in the Hallmark Estrogen Response Late gene set between both TRIM24-KO MCF7 clones and both NT. Color scale represents the gene expression (z-score).

Fig. 4.

TRIM24 degrader impairs BC cell proliferation through inhibition of ERα transcriptional activity. (A) Representative western blot for TRIM24 and ERα protein levels upon dTRIM24 treatment for 24 h in MCF7 (TRIM24+ and ERα+ BC cell line) and MDA-MB-231 (TRIM24− and ERα− BC cell line). Actin is used as a loading control. (B) Relative growth of MCF7 or MDA-MB-231 cells treated with 5 μM of dTRIM24 compared to DMSO control (n = 7). P-value is determined by two-way ANOVA with Bonferroni’s post hoc test ****P < 0.0001; n.s.; nonsignificant. (C) Cell viability analysis of MCF7 and MDA-MB-231 cells treated with 5 μM of dTRIM24. CTG was performed after 8 d of treatment with dTRIM24 (n = 4). P-value is determined by the two-tailed paired t test. **P < 0.01, n.s.; nonsignificant. (D) Cell viability assay of MCF7 treated with 5 μM of dTRIM24_2. CTG was performed after 8 d of treatment (n = 6). P-value is determined by the two-tailed paired t test: ***P < 0.001. (E) Cell viability assay by CTG for MCF7 cells deprived for 3 d of hormones and incubated for 8 d with the depicted treatments (n = 3). P-value is determined by two-way ANOVA with Sidak’s correction: **P < 0.01, ***P < 0.001. (F) GSEA for hallmark gene sets. Shown are the top differentially enriched pathways upon 24 h treatment with dTRIM24 (5 µM) or vehicle (DMSO). The X-axis indicates the normalized enrichment score. Only significantly enriched pathways are indicated (P < 0.05). (G) Volcano plot summarizing the quantitative results of ERα RIME in MCF7 treated for 24 h with 5 μM dTRIM24 or vehicle (DMSO).

Fig. 5.

TRIM24 degrader blocks therapy-resistant BC cell growth and impedes the formation of ERα+ patient-derived BC organoids. (A) TRIM24 protein levels upon treatment with 5 μM of dTRIM24 in several endocrine-resistant cell lines. Actin is used as a loading control. (B) Cell proliferation percentage relative to DMSO of MCF7 TAMR and LTED treated with 5 μM of dTRIM24 for 12 d (n = 3). Results shown are normalized to the vehicle control. P-values are determined by two-way ANOVA with Bonferroni post hoc test: ****P < 0.0001. (C) Cell viability assay by CTG in MCF7 TAMR (n = 5) and LTED (n = 6) treated with 5 μM of dTRIM24 for 12 d. P-value is determined by the two-tailed paired t test: *P < 0.05, ***P < 0.001. (D) Colony Assay quantification of MCF7 TAMR treated with increased concentrations of dTRIM24_2 (n = 3). P-values represent two-way ANOVA with Tukey’s multiple test correction. ***P < 0.001, ****P < 0.0001. (E) Cell proliferation analysis of MCF7 ESR1-mutant Y537S C1, C2, and MCF7 ESR1-mutant D538G C3, treated with 5 μM of dTRIM24 for 8 d (n = 5). Results shown are normalized to the vehicle control. P-values are determined by two-way ANOVA with Bonferroni post hoc test: ***P < 0.001; ****P < 0.0001. (F) TRIM24 and ERα protein levels in the depicted organoid models after treatment with 10 μM of dTRIM24. Actin is used as a loading control. (G) 3D patient-derived organoid culture cell viability assay. Each patient-derived organoid was incubated either with dTRIM24 (5 or 10 μM) and vehicle control for 7 d before cell-viability was measured (n = 6). TNBC: triple negative breast cancer. Two-way ANOVA test: *P < 0.05; **P < 0.01; ***P < 0.001. (H) GREB1 protein level after treatment with 10 μM of dTRIM24 in the two depicted organoid models. Actin is used as a loading control. (I) mRNA level of GREB1, XBP1, and RARα upon treatment with 10 μM of dTRIM24 in T347 (Left) and T4-1 (Right) organoid models. Results are representative of n = 3 biological replicates. Two-tailed t test: *P < 0.05; **P < 0.01; ***P < 0.001.