Efficacy of SERD/SERM Hybrid-CDK4/6 Inhibitor Combinations in Models of Endocrine Therapy-Resistant Breast Cancer

Abstract

Purpose: Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)-positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy-resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant ESR1(+) breast cancer.

Experimental design: SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy-resistant ESR1(+) breast cancer.

Results: The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone.

Conclusions: A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. See related commentary by DeMichele and Chodosh, p. 4999.

Figure 1. SERDs and SERM/SERD hybrids inhibit ESR1 action with similar efficacy despite differences in potency and efficiency of estrogen receptor turnover

A) Chemical structures of SERM/SERD hybrids (SSHs) and SERDs evaluated. B) MCF7 cells were incubated with increasing concentrations (10⁻¹¹–10⁻⁵ M) SERD ICI 182,780 (ICI), or SSHs bazedoxifene (BZA), pipendoxifene (PIP), BPN1, or BPL2 for 18 hrs. ESR1 protein levels (left) were assessed using an In Cell Western. Data was normalized to DNA content using DRAQ5 (center) and quantitated (average of duplicate wells) using GraphPad Prism 6 (right). C) MCF7 breast cancer cells were treated for 24 hours with 1 nM E2 in the presence of increasing concentrations (10⁻¹¹ – 10⁻⁶ M) of antagonist. mRNA levels of ESR1 target genes progesterone receptor (PGR) and trefoil factor 1 (TFF1) were assessed using real time quantitative PCR (RT qPCR) following RNA isolation. mRNA expression was normalized to the 36B4 housekeeping gene, and expression levels are presented as fold change as compared to an untreated control. D) MCF7 cells were plated in phenol red free media supplemented with charcoal stripped FBS 24 hours prior to treatment, and were treated with 1nM E2 as well as with the indicated ligands (10⁻¹¹ – 10⁻⁶ M) on days 1, 4, and 6 of an 8 day proliferation assay. DNA content, as assessed by fluorescence (FluoReporter assay), was measured as a surrogate for cell proliferation. The relative increase in DNA fluorescence was calculated by normalizing to baseline values detected in a duplicate plate of cells that was harvested on day 1 prior to the initial treatment. Data are representative of at least 3 independent experiments.

Figure 2. Fulvestrant, bazedoxifene, and pipendoxifene inhibit ESR1 activity in relevant models of endocrine therapy resistant breast cancer

A) LTED MCF7 cells were plated in phenol red free media supplemented with FBS that was stripped of growth factors twice using charcoal. Cells were treated with ICI, BZA, and PIP (10⁻¹² – 10⁻⁷ M) on days 1, 4, and 6 of an 8 day proliferation assay and analyzed as in Figure 1. B) TamR cells were plated in media supplemented with CFS 24 hours prior to treatment, and were treated with 1nM E2 as well as with ICI, BZA, and PIP (10⁻¹¹ – 10⁻⁶ M) on days 1, 4, and 6 of an 8 day proliferation assay. Cell proliferation was quantitated as in Figure 1. C) TamR cells were plated in phenol red free media supplemented with charcoal stripped FBS 48 hours prior to treatment with ICI, BZA, or PIP (10⁻¹³ – 10⁻⁶ M) for 24 hours. Expression of ESR1 (C) and loading control cytokeratin 18 (CK18 – Suppl. Figure 2A) in whole cell extracts were detected by immunoblot (left). ESR1 levels relative to CK18 were quantitated by densitometry using Adobe Photoshop (right). D) MCF7 cells engineered to express wt ESR1 or Y537S, Y537N, or D538G mutations of ESR1 were treated for 7 days with increasing (10⁻¹¹ – 10⁻⁶ M) concentrations of ICI, BZA, or PIP. DNA content, as assessed by luminescence (Cell Titer Glo assay), serves as an indicator of cell number in each condition. Data represent average detection +/− SD of triplicate wells. Data are representative of at least 3 independent experiments. IC₅₀ values listed in the figure legend were calculated by non-linear curve regression using GraphPad Prism 6.

Figure 3. SERDs or SERM/SERD hybrids and CDK4/6 inhibitors impact breast cancer growth by distinct mechanisms

A) MCF7 cells were plated in media supplemented with FBS prior to 24 hours treatment with increasing concentrations of palbociclib (10^−8.6 – 10^−6.6 M). Levels of pRb, Rb, ESR1, and CK18 were detected by immunoblot of whole cell extracts (left) followed by densitometry analysis and normalization (right) as in Figure 2C. Red arrow (→) indicates protein band corresponding to pRb. Protein levels were normalized to the control (no palbociclib treatment) present in the first lane. B) MCF7 breast cancer cells were treated for 24 hours with increasing concentrations (10⁻¹³ – 10⁻⁹ M) E2 in the presence of palbociclib (0, 25, 50 or 100 nM). mRNA expression of TFF1 was analyzed as in Figure 1. C) Proliferation of MCF7 cells was analyzed as in Figure 1 after 8 days treatment with 1nM E2 as well as increasing concentrations of SSH/SERD (ICI, BZA, or PIP) and palbociclib (0 or 25 nM). Data are representative of at least 3 independent experiments.

Figure 4. Palbociclib increases the efficacy with which SERDs or SERM/SERD hybrids inhibit the growth of tamoxifen-resistant breast tumor xenografts

TamR tumors were implanted into tamoxifen-treated mice. When Tam-stimulated tumors attained ~0.1cm³ tumor volume, animals were randomized (7-9 mice per group) to receive continued tamoxifen treatment as well as vehicle or SSH/SERD (BZA, 5 or 10 mg/kg/day s.c.; PIP, 5 or 10 mg/kg/day s.c.; or ICI, 5 mg/mouse 1× weekly i.m.), and also vehicle or palbociclib (100 mg/kg/day, p.o.). Tumor growth for each group (separated by SERD treatment for legibility) is presented as average tumor volume +/− SEM per study arm at each day of treatment, with the initial day of treatment at randomization considered to be day 0. Tam control and palbociclib only treatments presented on each graph are identical. Tumor growth for animals treated with 5 mg/kg BZA or PIP are shown above, while measurements for animals treated with 10 mg/kg BZA or PIP are depicted in Suppl. Figure 4C. By day 14, responses to all treatments were significant as compared to the tam treatment only control (p < 0.01). Significant differences (p < 0.05) between combination treatments and SERD only (*), palbociclib only ($), or both single treatments (#) are indicated at appropriate time points.

Figure 5. Palbociclib and BZA inhibit the growth of xenograft tumors derived from patients resistant to endocrine interventions

A-C) WHIM11 (A), WHIM16 (B), or WHIM43 (C) PDX tumors were implanted into intact NSG mice. Animals bearing tumors of equivalent size (200-500 mm³ depending on the tumor model) were randomized (7-10 mice per group) to treatment with vehicle or palbociclib (125 mg/kg/day, p.o.). Upper panels: Tumor growth for each group is presented as average tumor volume +/− SEM per study arm at each day of treatment, with the initial day of treatment at randomization considered to be day 0. Lower panels: pRb and Ki67 were detected by IHC analysis of representative tumors for each group. A-D) Asterisk (*) denotes significance (p < 0.0001) as compared to vehicle control. D) WHIM20 PDX tumors were implanted as above. When tumors reached ~200 mm³ volume, animals (4 mice per group) were randomized to palbociclib (125 mg/kg/day, p.o) or BZA (10 mg/kg/day sc) alone or in combination. Left: Tumor growth for each group is presented as average tumor volume +/− SEM per study arm at each day of treatment, with the initial day of treatment at randomization considered to be day 0. Center: Ki67 expression in one representative tumor per treatment group as detected by IHC analysis. Right: 38 sectors per tumor were quantitated by duplicate manual scoring of 3 tumors per group. Mean % positive cells/tumor of individual tumors, as well as the average (+/− SEM) per treatment group, are depicted in the right panel. Significant differences between treatments were detected by ANOVA followed by Holm-Sidak multiple comparison test and are indicated: a - p < 0.0001, b - p < 0.001, c - p < 0.01, d - p < 0.05, n.s. - comparison did not detect significant difference.