RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

Abstract

Background: Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improve the outcome of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, resistance to this treatment and disease progression remains a major clinical challenge. High expression of the receptor tyrosine kinase REarranged during Transfection (RET) has been associated with resistance to endocrine therapy in breast cancer, but the role of RET in CDK4/6i treatment response/resistance remains unexplored.

Methods: To identify gene expression alterations associated with resistance to combined endocrine therapy and CDK4/6i, we performed RNA sequencing of two ER+ breast cancer cell models resistant to this combined therapy. The functional role of RET was assessed by siRNA-mediated RET silencing and targeted inhibition with the FDA/EMA-approved RET-selective inhibitor selpercatinib in resistant breast cancer cells and patient-derived organoids (PDOs). RET silencing was evaluated mechanistically using global gene expression and pathway analysis. The clinical relevance of RET expression in ER+ breast cancer was investigated by gene array analysis of primary tumors treated with endocrine therapy and by immunohistochemical scoring of metastatic lesions from patients who received combined CDK4/6i and endocrine therapy.

Results: We show that RET is upregulated in ER+ breast cancer cell lines resistant to combined CDK4/6i and fulvestrant compared to isogenic cells resistant to fulvestrant alone. siRNA-mediated silence of RET in high RET-expressing, combined CDK4/6i- and fulvestrant-resistant cells reduced their growth partially by affecting cell cycle regulators of the G2-M phase and E2F targets. Notably, targeting RET with selpercatinib in combination with CDK4/6i inhibited the growth of CDK4/6i-resistant cell lines and resensitized ER+ breast cancer patient-derived organoids resistant to CDK4/6i. Finally, analysis of RET expression in ER+ breast cancer patients treated with endocrine therapy showed that high RET expression correlated with poor clinical outcomes. We further observed a shorter median survival to combined CDK4/6i and endocrine therapy in patients with RET-positive compared to RET-negative tumors, but this difference did not reach statistical significance.

Conclusions: Our findings show that RET is overexpressed in ER+ metastatic breast cancer resistant to combined CDK4/6i and endocrine therapy, rendering RET inhibition a promising therapeutic approach for patients who experience disease progression on combined CDK4/6i and endocrine therapy.

Keywords: CDK4/6 inhibitor; RET; drug resistance; estrogen receptor-positive breast cancer; selpercatinib.

Figure 1

RET is overexpressed in ER+ breast cancer cell lines resistant to combined CDK4/6i and endocrine therapy. Dot plots of Hallmark gene set significantly enriched in (A) MPF-R vs. MF-R and (B) TPF-R vs. TF-R. (C) List of genes enriched in gene set “Hallmark estrogen response early”. (D) Evaluation of RET expression in ER+ breast cancer cell lines resistant to combined palbociclib and fulvestrant (MPF-R and TPF-R), resistant to fulvestrant only (MF-R and TF-R), and parental sensitive cells (M-S and T-S) using RNA sequencing. Statistical comparison is shown relative to double-resistant cells. TPM = transcripts per million. The data represent independent experiments in triplicates ± SEM. (E) Quantitative RT-PCR verifying the gene expression alterations of RET. The expression was normalized using the PUM1 gene and shown as a relative expression in MPF-R vs. M-S and TPF-R vs T-S cells. Data represent three independent experiments ± SEM (*0.01 < p < 0.05). (F) Western blotting analysis of lysates from M-S, MF-R, MPF-R, T-S, TF-R and TPF-R cells. 10 µg and 50 µg of total protein of MCF-7- and T47D-derived cells, respectively, were loaded. β-actin was used as a loading control. A representative for three biological replicates is shown. Asterisk indicate significant differences in students t-test (*0.01 < p < 0.05, **0.001 < p < 0.01 and ***0.0001< p <0.001).

Figure 2

RET-specific siRNA-mediated knockdown inhibits the growth of MPF-R breast cancer cells. The efficiency of RET silencing in combined CDK4/6i- and fulvestrant-resistant cell lines (MPF-R and TPF-R) and their parental sensitive cell lines (M-S and T-S), respectively, transfected with two different RET-specific siRNAs (RET15 and RET17) or scrambled siRNA (control). (A) RT-qPCR verifying reduction of RET mRNA level 48 h post-transfection with RET-specific siRNA. The expression was normalized using the PUM1 gene. The knockdown efficiency is represented as the average percentage compared to the control (scr) of triplicates (mean ± SEM). (B) Western blot validation of protein levels 96 h post-transfection with RET-specific siRNAs. GAPDH was used as a protein loading control. (C) Cell growth at different time points following RET-specific siRNA transfection as assessed by crystal violet assay. Graph columns show cell growth at days 6 and 10 for MCF7-derived cell lines and T47D-derived cell lines, respectively. Scrambled siRNA: control siRNA; RET15, and RET17: two different RET-specific siRNAs. RET15 + 17: combination of both RET-specific siRNAs. Asterisks indicate significant differences in the one-way ANOVA test (****p < 0.0001).

Figure 3

RET gene knockdown impairs cell growth of combined CDK4/6i- and fulvestrant-resistant breast cancer cells by blocking the G2-M phase progression of the cell cycle. Bar graphs and enrichment plots of Hallmark gene sets significantly enriched in (A) MPF-R RET-siRNA vs. control-siRNA and (B) TPF-R RET-siRNA vs. control-siRNA. RET-siRNA 15 and RET-siRNA 17 pools were used. Statistical significance (nominal P-value) of the enrichment score (ES) is calculated using an empirical phenotype-based permutation test. (C) Western blotting of cell cycle regulators in three biological replicates (BR) of MPF-R RET-siRNA versus control-siRNA. GAPDH was used as a loading control.

Figure 4

RETi resensitizes combined CDK4/6i- and fulvestrant-resistant ER+ breast cancer cells. Cell growth of (A) MPF-R and M-S cells and (B) TPF-R and T-S cells over six days in the presence of fulvestrant (100nM), CDK4/6i (200 nM) and RETi (5µM) alone or different combinations analyzed by crystal violet assay. Growth at day six is represented by columns. The data represents the mean of three biological replicates ± SEM. Asterisks indicate significant differences in one-way ANOVA tests at day six. Means are compared to the mean of the standard combined CDK4/6i and fulvestrant (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and ****p < 0.0001). (C) Western blotting of cell cycle regulators in MPF-R and TPF-R cells treated with RETi alone or combined with CDK4/6i and/or fulvestrant. GAPDH was used as a loading control.

Figure 5

Combined CDK4/6i and RETi efficiently reduces viability of breast cancer patient-derived organoids resistant to CDK4/6i. (A) RT-qPCR verifying CDK6, CDK4, and CCND1 gene expression alterations upon palbociclib treatment. The expression was normalized using the gene PUM1 and shown as relative expression in control vs. treated with CDK4/6i. (B) Effect of increasing concentrations of CDK4/6i palbociclib and RETi selpercatinib, alone or RETi combined with a fixed concentration of CDK4/6i (1 µM), on the viability of patient-derived breast cancer organoid PDO-P48 for seven days. The results represent the mean ± SEMs of three replicates relative to the control (untreated). (C) Dose-effect curves of each single drug, CDK4/6i and RETi, or RETi combined with a fixed concentration of CDK4/6i (1 µM), during treatment of PDO-P48 for seven days. IC50 were calculated by normalizing the transformed data and using the non-linear curve fitting method “log(inhibitor) vs. normalized response – Variable slope”. (D) Viability of PDO-P48 treated with 10 µM of CDK4/6i and RETi single agents or their combination for seven days. (E) Brightfield images depicting PDO-P48 control (untreated) and treated with combined CDK4/6i and RETi. Scale bars: black 400 µm; blue 100 µm; orange 200 µm.

Figure 6

High RET expression correlates with shorter overall and relapse-free survival in patients with ER+ breast cancer receiving endocrine therapy. Kaplan-Meier survival curves for (A) OS and (B) RFS for RET expression by KM plotter analysis. (C, D) Kaplan-Meier survival curves evaluating PFS according to RET intensity score in ER+ metastatic lesions from patients treated with combined CDK4/6i and endocrine therapy. (D) Cut-off values: negative RET: intensity = 0; positive RET: intensity ≥ 1. A two-sided p-value calculated using Log-rank testing is shown.