Radiotherapy Delivered before CDK4/6 Inhibitors Mediates Superior Therapeutic Effects in ER+ Breast Cancer

Abstract

Purpose: Recent preclinical data suggest that cyclin-dependent kinase 4/6 (CDK4/6) inhibition may be harnessed to sensitize estrogen receptor-positive (ER⁺) breast cancer to radiotherapy. However, these findings were obtained in human ER⁺ breast cancer cell lines exposed to subclinical doses of CDK4/6 inhibitors with limited attention to treatment schedule. We investigated the activity of radiotherapy combined with the prototypic CDK4/6 inhibitor palbociclib placing emphasis on therapeutic schedule.

Experimental design: We combined radiotherapy and palbociclib in various doses and therapeutic schedules in human and mouse models of ER⁺ and ER-negative (ER^-) breast cancer, including an immunocompetent mouse model that recapitulates key features of human luminal B breast cancer in women. We assessed proliferation, cell death, cell-cycle control, and clonogenic survival in vitro, as well as tumor growth, overall survival, and metastatic dissemination in vivo.

Results: Radiotherapy and palbociclib employed as standalone agents had partial cytostatic effects in vitro, correlating with suboptimal tumor control in vivo. However, while palbociclib delivered before focal radiotherapy provided minimal benefits as compared with either treatment alone, delivering focal radiotherapy before palbociclib mediated superior therapeutic effects, even in the absence of p53. Such superiority manifested in vitro with enhanced cytostasis and loss of clonogenic potential, as well as in vivo with improved local and systemic tumor control.

Conclusions: Our preclinical findings demonstrate that radiotherapy delivered before CDK4/6 inhibitors mediates superior antineoplastic effects compared with alternative treatment schedules, calling into question the design of clinical trials administering CDK4/6 inhibitors before radiotherapy in women with ER⁺ breast cancer.

Figure 1.

Radiotherapy followed by palbociclib mediates robust cytostatic effects against human ER⁺ breast cancer cells. A, Experimental setting. P, palbociclib; RT, radiotherapy. B and C, MCF7 cells were cultured in control conditions or exposed to P or a single radiotherapy fraction in the indicated doses and combinations, as schematized in A, followed by the flow cytometry-assisted quantification of residual cell number, mitochondrial depolarization, and plasma membrane rupture. Representative dot plots (B) and quantitative data (C) are reported. In B, total number of cells and percentage of cells in each quadrant are indicated. Results are means ± SEM plus individual data points from n = 8–10 independent biological samples from 4–5 independent experiments. Statistical significance was assessed with paired one-way ANOVA plus Fisher LSD test. ***, P < 0.001, as compared with MCF7 cells maintained in control conditions; ^#n.s., not significant; ^##, P < 0.01; ^###, P < 0.001, as compared with MCF7 cells treated with the same P dose; ^†n.s., not significant; ^†, P < 0.05; ^††, P < 0.01; ^†††, P < 0.001, as compared with MCF7 cells treated the same radiotherapy dose; ^‡, P < 0.05, as compared with MCF7 cells treated with the same doses of P→RT.

Figure 2.

Superior short- and long-term cell-cycle control by radiotherapy followed palbociclib in human ER⁺ breast cancer cells. A-D, MCF7 cells were cultured in control conditions or exposed to palbociclib (P) or a single radiotherapy (RT) fraction in the indicated doses and sequential combinations, as depicted in Fig. 1A, followed by the flow cytometry-assisted quantification of cell-cycle distribution. Representative dot plots (A and C) and quantitative data (B and D) are reported. In A and C, the percentage of cells in each quadrant is indicated. Results are mean cumulative cell-cycle distribution as well as means ± SEM plus individual data points for each cell-cycle phase from n = 6 independent biological samples collected over three independent experiments. Statistical significance was assessed with paired one-way ANOVA plus Fisher LSD test. *n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with MCF7 cells maintained in control conditions; ^#n.s., not significant; ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001, as compared with MCF7 cells treated with P alone; ^†n.s., not significant; ^††, P < 0.01; ^†††, P < 0.001, as compared with MCF7 cells treated with radiotherapy alone; ^‡n.s., not significant; ^‡‡, P < 0.01; ^‡‡‡, P < 0.001, as compared with MCF7 cells treated with the same doses of P→RT. Please note that control samples in B and D are the same. E-G, Residual clonogenic potential of MCF7 cells optionally treated with the indicated radiotherapy dose alone or in sequential combination with 100 nmol/L palbociclib and then allowed to form colonies for 14 days. Representative images (E) and quantitative data (F and G) are reported. In E and F, number of colonies and SERs for 50% and 80% efficacy are indicated, respectively. Results are means ± SEM normalized to untreated MCF7 cells (F, radiotherapy; G) or MCF7 cells treated with P alone (F, P→RT, RT→P) optionally fitted to the linear-quadratic model (F) from n = 12–18 independent biological samples collected over 2–3 independent experiments. Statistical significance was assessed with unpaired one-way ANOVA plus Fisher LSD test. ***, P < 0.001, as compared with MCF7 cells maintained in control conditions; ^###, P < 0.001, as compared with MCF7 cells treated with P alone;^‡‡, P < 0.01; ^‡‡, P < 0.001, as compared with MCF7 cells treated with radiotherapy alone; ^‡, P < 0.05, as compared with MCF7 cells treated with the same doses of P→RT.

Figure 3.

Radiotherapy followed by palbociclib enables superior local control of endogenous ER⁺ mammary tumors developing in immunocompetent mice. A, Experimental setting. P, palbociclib; RT, radiotherapy. ¹Only for TS/A and M/D-driven tumors; ²replaced by palbociclib for 4T1 tumors. B, Female BALB/c mice bearing palpable TS/Amammary carcinomas were randomly allocated to no treatment or to palbociclib (150 mg/kg per dose), radiotherapy (6Gy per fraction), or their combination as illustrated in A, and monitored over time for tumor growth and survival. Results are mean tumor areas ± SEM from the indicated number of mice per group and OS, as obtained in two independent experiments. Statistical assessments for tumor growth were performed with RM one-way ANOVA plus Geisser–Greenhouse and Bonferroni’s multiple comparisons test. Statistical assessments forOSwere performed with the Gehan–Breslow–Wilcoxon test. *n.s., not significant; ***, P < 0.001, as compared with untreated mice; ^###, P < 0.001, as compared with P-treated mice; ^†n.s., not significant, as compared with radiotherapy-treated mice; ^‡, P < 0.05; ^‡‡‡, P < 0.001, as compared with mice treated with P→RT. C and D, Female BALB/c mice bearing palpable 4T1 mammary carcinomas were randomly allocated to no treatment or to palbociclib (150 mg/kg per dose), radiotherapy (12 Gy in a single fraction), or their combination as illustrated in A, and monitored over time for tumor growth. Results are mean tumor areas ± SEM from the indicated number of mice per group (C), or mean number of surface lung metastases on d21 ± SEM and individual data points (inclusive of representative images; D). Statistical assessments for tumor growth were performed with RM one-way ANOVA plus Geisser–Greenhouse and Bonferroni’s multiple comparisons test. Statistical assessments for metastatic dissemination were performed with paired one-way ANOVA plus Fisher LSD test, respectively. *n.s., not significant; *, P < 0.05; ***, P < 0.001, as compared with untreated mice; ^#n.s., not significant; ^###, P < 0.001, as compared with P-treated mice; ^†n.s., not significant; ^†, P < 0.05; ^††, P < 0.01, as compared with radiotherapy-treated mice; ^‡, P < 0.05; ^‡‡‡, P < 0.001, as compared with mice treated with P→RT. E, Female C57BL/6 mice bearing palpableM/D-driven carcinomas were randomly allocated to no treatment or to P (150 mg/kg per dose), radiotherapy (10 Gy per fraction), or their combination as illustrated in A, and monitored over time for the growth of the target lesion. Results are individual growth curves and mean tumor areas ± SEM from the indicated number of mice per group. Statistical assessments for tumor growth were performed with RM one-way ANOVA plus Geisser–Greenhouse and Bonferroni’s multiple comparisons test. ***, P < 0.001, as compared with untreated mice; ^#n.s., not significant; ^###, P < 0.001, as compared with P-treated mice; ^†n.s., not significant; ^†††, P < 0.001, as compared with radiotherapy-treated mice; ^‡‡‡, P < 0.001, as compared with mice treated with P→RT.