Cyclin-dependent Kinase 4/6 Inhibitor Palbociclib in Combination with Ralaniten Analogs for the Treatment of Androgen Receptor–positive Prostate and Breast Cancers

The cell cycle is precisely controlled in benign cells by cyclin-dependent kinases (CDKs), regulatory cyclins, and checkpoint regulators. However, in cancer, the cell cycle is frequently altered because of somatic mutations in genes encoding cell-cycle regulators and defects in checkpoint proteins (1, 2). CDK4/6 inhibitors used clinically include palbociclib, abemaciclib, and ribociclib, and these are administered in combination with hormone therapy for the treatment of breast cancer. When the RB pathway is intact, these CDK 4/6 inhibitors block the transition of cells from the G₁- to the S-phase, which leads to accumulation of cells in G₁ (3).

Androgen receptor (AR) is a transcription factor that regulates the expression of thousands of genes including those involved in cell growth and survival. Altered expression and transcriptional activity of AR contribute to the development and progression of castration-resistant prostate cancer (CRPC) and some triple-negative breast cancers (TNBCs). AR associates with regulators of the cell cycle (4). Overexpression of CDK6 occurs in approximately 50% of prostate cancer and enhances AR transcriptional activity to promote disease progression (5). Increased AR transcriptional activity is associated with disruption of cyclin D1 repression in prostate cancer (6–8). In LNCaP human prostate cancer cells, AR interacts with the pre-replication complex during mid-G₁-phase and associates with DNA synthesis, indicating its essential role during the G₁–S transition (9). Luminal AR is a subtype of TNBC that is particularly sensitive to CDK4/6 inhibitors, and this sensitivity is associated with AR expression and low cyclin E1 levels (10).

Full-length AR consists of N-terminal domain (NTD), DNA-binding domain, a hinge region, and a ligand-binding domain (LBD) to which androgens and antiandrogens bind. Deletion of LBD results in constitutively active AR variants (AR-Vs), of which AR-V7 is most clinically relevant. AR-V7 is associated with the failure of CRPC to therapies targeting the full-length AR such as antiandrogens and androgen deprivation therapies (11, 12). AR-V7 is also detected in human breast cancer cell lines as well as in clinical samples of breast cancer (13–16). AR-V7 mediates a growth response and regulates expression of a subset of genes that is unique from those regulated by the full-length AR (15, 17).

Ralaniten (aka EPI-002) and its analogs specifically bind to transactivation unit-5 (Tau-5) within activation function 1 (AF-1) of the intrinsically disordered AR NTD (18–22). Uniquely, ralaniten compounds inhibit the transcriptional activities of full-length AR, AR-Vs, as well as gain-of-function mutations in AR LBD that are found in patients with CRPC who are resistant to antiandrogens and abiraterone (18, 20, 22). Antiandrogens and AR-NTD inhibitors cause prostate cancer cells that express full-length AR to arrest in the G₁ phase and not progress to the S-phase of the cell cycle (9, 20, 23). Thus, a functional AR is required for G₁–S progression. Cyclin D1-CDK4/6 and late G₁ cyclin E-CDK2 kinases phosphorylate the RB protein, which leads to E2F regulation of expression of cyclins that are required for the transition to the S-phase (24). Based on the importance of CDK4/6 in controlling G₁–S progression, we propose a therapy of inhibiting CDK4/6 combined with an AR-NTD antagonist that blocks transcription activities of all AR species (full-length AR and AR-Vs).

Monotherapies for metastatic cancers rarely lead to durable responses or cures primarily due to tumor plasticity and heterogeneity with the survival and outgrowth of resistant clones. The goals of combination therapies include deeper and more durable responses and can include approaches such as targeting pathway reactivation and/or targeting multiple components of a signaling pathway. In this study, we examined the effects of targeting both AR-NTD and CDK4/6 in CRPC and AR-expressing breast cancer with a combination of a second-generation ralaniten analog and palbociclib. EPI-7170 is a more potent analog of ralaniten that specifically blocks AR transcriptional activity (23, 25, 26). Here, we show that EPI-7170 disrupted the cell cycle in CRPC and AR-expressing breast cancer cell lines, and when combined with palbociclib had improved antitumor activity. As expected, palbociclib caused G₁ accumulation, whereas EPI-7170 caused both G₁ accumulation and also delayed the cell cycle. Sequential versus concomitant combinations resulted in differential effects on the cell cycle.

LNCaP cells were obtained from Leland Chung (Cedars-Sinai Medical Center, Los Angeles, CA) and maintained in RPMI1640 supplemented with 5% [volume for volume (v/v)] FBS. LNCaP95 cells were obtained from Stephen Plymate (University of Washington, Seattle, WA) and maintained in RPMI1640 supplemented with 10% (v/v) charcoal-stripped serum (CSS). SUM159PT cells were obtained from Sharon Gorski (Canada's Michael Smith Genome Sciences Centre at BC Cancer, Vancouver, British Columbia, Canada) and maintained in Ham's F-12 Nutrient Mix supplemented with 5% (v/v) FBS, l-Glutamine, HEPES, 1 μg/mL hydrocortisone, and 0.2 U/mL bovine insulin. MFM-223 cells were purchased from DSMZ (DSMZ No.ACC422) and maintained in MEM supplemented with 10% (v/v) FBS, 1% (v/v) L-Gln and insulin-transferrin-selenium. MDA-MB-453 cells were purchased from ATCC (ATCC HTB-131) and maintained in RPMI1640 supplemented with 10% (v/v) FBS. MDA-MB-468 cells were purchased from ATCC (ATCC HTB-132) and maintained in MEM supplemented with 10% (v/v) FBS and 1% (v/v) L-Gln. All cell lines were incubated at 37°C with 5.0% CO₂. All cell lines have been authenticated using short tandem repeat profiling and were routinely tested every 6 months to ensure Mycoplasma free using the Venor GeM Mycoplasma Detection kit (Sigma-Aldrich). The last test for Mycoplasma was done July 23, 2021. All treatments with inhibitors or vehicle controls were carried out in culture maintaining media supplemented with FBS for each of the cell lines except LNCaP95 cells which were in 5% CSS. All repeated experiments were conducted using cells within five to 10 passages. EPI-7170 was synthesized by Raymond Andersen (University of British Columbia, Vancouver, British Columbia, Canada; refs. 23, 25). Enzalutamide (ENZA) was purchased from Omega Chem. Palbociclib (PD-0332991) HCl was purchased from Selleckchem (catalog No. S1116). These three inhibitors were dissolved in DMSO. Vehicle controls were DMSO (final = 0.02–0.04% v/v). For combination treatments, concomitant regimens (two inhibitors were mixed in media and added to the cultures at the same time) were performed unless a sequential regimen was otherwise stated.

LNCaP (3,000 cells/well), LNCaP95 (3,000 cells/well), and SUM159PT (2,000 cells/well) were plated in 96-well culture plate (Corning Primaria). The next day (as day 0), cells were treated with inhibitors at the indicated concentrations or with vehicle control. On days 2 and 3 of treatment, alamarBlue was added to the treated cells and incubated at 37°C for 2 to 4 hours before fluorescence measurement (560-nm excitation and 590-nm emission) by Infinite M1000 (Tecan). EPI or enzalutamide in medium without cells did not change alamarBlue fluorescence measurement as compared to medium only. GraphPad Prism 8 was used for plotting data and statistical analyses.

LNCaP (400–600 cells/well), LNCaP95 (500 cells/well), and SUM159PT (400 cells/well) were plated in 24-well culture plates. The next day (as day 0), cells were treated with inhibitors at indicated concentrations or with vehicle control without medium change. For consistency, the same cell numbers were plated before treatments, instead of plating the same cell number after treatments which would require adjusting cell numbers after collecting cells from each of the treatments. For experiments with a one-time medium change: on day 3 (LNCaP95) or day 4 (SUM159PT) of treatment, the media from one set of treated cells was changed to fresh media without inhibitors. On day 15 (LNCaP and LNCaP95) or day 7 (SUM159PT) of treatments, cells were fixed with 4% paraformaldehyde, washed with PBS, and stained with 0.01% (w/v) crystal violet. Images of colonies were captured by ChemiDoc XRS+ Imaging System with a trans-white conversion screen and Image Lab 5.1 software (Bio-Rad).

For cell cycle with bromodeoxyuridine (BrdU) incorporation, non-synchronized cells were plated (200,000 or 300,000 cells per dish for LNCaP; 300,000 cells per dish for LNCaP95 and MDA-MB-453; 150,000 cells per dish for SUM159PT; 500,000 for MFM-223; 250,000 for MDA-MB-468) in 60-mm culture dishes. The next day (as day 0), cells were treated with inhibitors at the indicated concentrations or with vehicle control. At the indicated timepoints, cells were labeled with 10 μmol/L BrdU for 2 hours. After BrdU incorporation, culture media containing both nonattached cells and attached cells that were trypsinized by TrypLE were collected. Cells were then passed through a 50-μm filter to obtain single cells and fixed by cold 70% (v/v) ethanol. Fixed cells were stained with anti–BrdU-FITC antibody (clone B44, BD Biosciences, catalog No. 347583) and 7-aminoactinomycin D (7-AAD from Sigma, catalog No. A9400), analyzed by flow cytometry using FACSCalibur and data were acquired by CellQuest Pro software (BD Biosciences). For cell-cycle tracing with BrdU incorporation, experimental procedures were the same as described above except cells were labeled with BrdU for 2 hours prior to inhibitor treatments. Cells were harvested and collected at the indicated timepoints. All cell-cycle data were analyzed with FlowJo V.10. Single-cell gating was performed on FSC-H versus FL3-W profiles. Data calculations for bar or line graphs used GraphPad Prism 8.

For analysis of LNCaP95 cell cycle with EdU incorporation and marker expression, Click-iT EdU Alexa Fluor 647 Flow Cytometry assay kit (Invitrogen) was used, and manufacturers' protocol was followed with modifications on fixation and intracellular marker staining. Cells (1,200,000 per dish) were plated on a 100-mm culture dish. The next day (as day 0), cells were treated with inhibitors or with vehicle control. On day 3 of treatments, cells were labeled with 10 μmol/L EdU for 2 hours prior to collecting nonattached and attached cells. Collected cells were fixed with 70% cold ethanol. Incorporated EdU was detected using Click chemistry. Antibody against phospho-histone H2A.X (Ser 139; clone: 20E3; Cell Signaling Technology No. 9718) was used for intracellular marker staining with staining carried out using TBS/FBS (4% v/v). Secondary antibody conjugated with Alexa Fluor 488 was used (Invitrogen A11008 for anti-rabbit IgG). After intracellular marker staining, cells were stained with 7-AAD, analyzed by LSRFortessa, and data were acquired by FACSDiva software (BD Biosciences). All cell-cycle data were analyzed using FlowJo V.10. Single-cell gating was performed on SSC-A versus SSC-W profiles. Data calculations for bar graphs were done using GraphPad Prism 8.

Cell number counting was carried out using an automatic cell counter (EVE Automated Cell Counter, NanoEnTek Inc.). The same number of cells from different treatment conditions at different timepoints were used for analyses of the cell cycle (BrdU) and EdU/γH2AX except for cell-cycle tracing experiments where all collected cells were used for BrdU staining to show cell number changes using 7AAD staining.

Animal studies were approved by the University of British Columbia Animal Care Committee. Mice were maintained in Animal Resource Centre at BC Cancer Research. Six-week-old male NSG mice were castrated. Two weeks after castration, 2 × 10⁶ cells of LNCaP95-D3 cells prepared in 1:1 ratio of RPMI and Matrigel (Corning, Product 354234) were inoculated subcutaneously on the back of mice. When tumor volume reached 50 to 90 mm³, mice were randomly separated into four treatment groups: vehicle control, EPI-7170 (30 mg/kg body weight), palbociclib (75 mg/kg body weight), and combination (EPI-7170 at 30 mg/kg body weight and palbociclib at 75 mg/kg body weight). Treatments were given daily by oral gavage for 21 days. Treatment solutions were freshly prepared each day. A working solution of palbociclib (isethionate salt purchased from LC Laboratories P-7766) was prepared daily from powder by dissolving in sterile Milli-Q water. To be completely soluble, each treatment solution was prepared in the following order of DMSO or EPI-7170 (dissolved in DMSO), Tween-80, methyl cellulose solution (Sigma M0512; 0.5% w/v prepared in sterile Milli-Q water), and sterile Milli-Q water or palbociclib solution to have final treatment concentrations in 5% DMSO/1.5% Tween-80/0.5% methyl cellulose. Body weight was measured every day. Tumor volume was measured every 3 days and calculated using formula: length × width × height × 0.5236.

Whole-cell lysates were extracted from LNCaP95 cells treated with inhibitors or vehicle control after 3 days by lysing cells in lysis buffer RIPA buffer containing protease inhibitors (cOmplete EDAT-free protease inhibitor cocktail and PhosStop from Roche). Xenograft tumors were homogenized in lysis buffer in Lysing Matrix M with FastPrep-24 homogenizer (MP Biomedicals). Protein concentrations in cell lysates were quantified by the bicinchoninic acid assay (Pierce BCA Protein assay kit). An equal amount of protein (40 μg) of each sample was loaded and resolved on 10% or 12.5% SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting analyses. Antibodies were diluted in TBS (pH 8.0)/Tween20 (0.05%) containing 5% milk or 5% BSA (w/v). Washes were done using TBS (pH8.0)/Tween20 (0.05%). Antibodies included AR [clone: ER179(2); abcam ab108341], RB (clone: 4H1; Cell Signaling Technology No. 9309), phospho-RB at Ser780 (clone: D59B7; Cell Signaling Technology No. 8180), CDK4 (clone: D9G3E; Cell Signaling Technology No. 12790), CDK6 (clone: DCS83; Cell Signaling Technology No. 3136), CDK2 (clone: D-12; Santa Cruz Biotechnology sc-6248), cyclin D1 (clone: 92G2; Cell Signaling Technology No. 2978), cyclin A2 (clone: BF683; Cell Signaling Technology No. 4656), p27 Kip1 (clone: D69C12; Cell Signaling Technology No. 3686), Skp2 (clone: D3G5; Cell Signaling Technology No. 2652), RAD51 (clone: D4B10; Cell Signaling Technology No. 8875), and β-actin (clone: AC-15; Sigma-Aldrich A5441). Secondary antibodies conjugated with horseradish peroxidase were used (Cell Signaling Technology No. 7074 for anti-rabbit IgG and Cell Signaling Technology No. 7076 for anti-mouse IgG) and enhanced ECL (GE Healthcare Lifescience Amersham ECL Prime Western Blotting Detection Reagent) was applied for chemiluminescence detection by ChemiDoc XRS+ Imaging System and Image Lab 5.1 software (Bio-Rad). Relative protein levels were quantified by Image Lab 5.1 software. Data and bar graphs were calculated and generated by GraphPad Prism 8.

LNCaP95 cells were plated on an 8-well chamberslide (15,000 cells/well). On the day, cells were treated with vehicle control, EPI-7170, palbociclib, or EPI-7170 combined with palbociclib as indicated. After 3 days of treatments, cells were fixed with 4% paraformaldehyde (v/v) and washed with TBS, pH8.0. Fixed cells were penetrated with TBS/0.1% TritonX-100 (v/v), blocked with TBS/0.05% Tween20 (v/v)/5% BSA (w/v) and stained in the same blocking buffer with antibodies against phospho-histone H2AX (Ser 139; clone: D7T2V; Cell Signaling Technology No.80312) and 53BP1 (Cell Signaling Technology No.4937S). Secondary antibody conjugated with Alexa Fluor 488 (Invitrogen A11008 for anti-rabbit IgG) and Alexa Fluor 568 (Invitrogen A11031 for anti-mouse IgG) was used. Secondary antibodies only were used as negative controls. After staining, cells were washed and mounted with mounting media containing DAPI. Stained cells were imaged by Axio Imager.M2 (ZEISS) and images of two-dimensional planes were acquired and processed by ZEN blue edition 2.6.

Foci numbers were quantified by ImageJ 1.45s and cell nuclei identified by ImageJ were manually confirmed. Cell clusters without clear separation or boundary were excluded. Thresholds (or noise tolerance in ImageJ) to identify foci of both 53BP1 and γH2AX were set to have a 1:1 ratio of both proteins in control within each independent experiment to allow comparison among experiments. At least three different fields from each condition and total number of cells (control: 1753, EPI-7170: 854, palbociclib: 1207, and E+P: 617) were analyzed from independent experiments (n = 4).

Statistics analyses were facilitated using GraphPad Prism 8. Two-way ANOVA with Tukey, Dunnett, or Sidak multiple comparisons test and One-way ANOVA with Tukey multiple comparisons test were used to calculate statistical significance among treatments. t test was used to determine statistical significance for tumor weight, Ki67, and TUNEL analyses in xenograft study. For estimation of cell doubling times, the exponential growth model (Malthusian model) was used to estimate the best-fit values.

All data are available within the article and its Supplementary Data.

A combination of palbociclib with EPI-7170 was tested in CRPC and AR-expressing breast cancer cells to determine their effective concentrations to reduce tumor growth, colony formation, and cell viability. Enzalutamide was included as a control because it targets the full-length AR by binding to the AR-LBD and has no effect on AR-Vs. Antiandrogens including more potent second-generation compounds such as enzalutamide are routinely used in cell culture at a concentration of 10 μmol/L (27) presumably to saturate the AR (28).

LNCaP human prostate cancer cells express full-length AR, are negative for AR-Vs, and are sensitive to androgen and enzalutamide. These cells exhibited a dose-dependent decrease in cell viability in response to palbociclib when used as a monotherapy and when combined with EPI-7170 or enzalutamide (Fig. 1A). As expected, enzalutamide caused an approximately 25% to 40% decrease in the viability of LNCaP cells that plateaued at 2.5 μmol/L (Fig. 1B, orange line). The addition of 0.25 μmol/L palbociclib resulted in a synergistic effect with enzalutamide (Fig. 1B, light orange line; Supplementary Fig. S1) in LNCaP cells.

LNCaP95 cells express full-length AR and AR-Vs, but their proliferation is driven by AR-Vs, and hence they are resistant to enzalutamide (22). The addition of 5 μmol/L enzalutamide had no effect on its own nor did it improve the response achieved with palbociclib in LNCaP95 cells, whereas 5 μmol/L EPI-7170 had a profound inhibitory effect on its own in the absence of palbociclib, which could not be improved with the addition of higher doses of palbociclib (plateaued; Fig 1A). Maintaining the concentration of palbociclib at 0.25 μmol/L and adding increasing amounts of EPI-7170 yielded dose-dependent decreases in the viability of LNCaP and LNCaP95 cells (Fig. 1B, light blue line) that were synergistic at lower concentrations of EPI-7170 in LNCaP cells but at higher concentrations of EPI in LNCaP95 cells (Supplementary Fig. S1). No increase in cell density or in cell number was detected with increased concentrations of EPI-7170 with or without palbociclib (Supplementary Figs. S2 and S3).

The TNBC cell line, SUM159PT, exhibited a dose-dependent decrease in cell viability in response to EPI-7170 monotherapy (Fig. 1C, dark blue line), and its combination with palbociclib resulted in synergistic decreases at higher concentrations of EPI-7170 (Fig. 1C, green and light blue lines; Supplementary Fig. S1).

Interestingly, LNCaP cells were unable to form colonies in the presence of 0.25 μmol/L palbociclib (Fig. 1D) even though cell viability and cell number were not reduced to zero on day 3 (Fig. 1A; Supplementary Fig. S2). On the contrary, LNCaP95 cells were able to form colonies that were reduced in a dose-dependent manner by EPI-7170 and by palbociclib, whereas enzalutamide had no effect (Fig. 1E, left: no medium change; Supplementary Fig. S4A). Holding the concentrations of EPI-7170 and enzalutamide constant at 5 μmol/L, palbociclib inhibited colony formation in a dose-dependent manner regardless of inhibitor in LNCaP95 cells. A combination of EPI-7170 with palbociclib resulted in a synergistic effect at higher concentrations of EPI-7170 (Supplementary Fig. S4B and S4C). In addition, the inhibitory effect was reversible as indicated by increased colonies after removal of both inhibitors after 3 days of treatments compared with conditions without removal of the inhibitors (Fig. 1E, right: medium change on day 3). The reversible effect was not 100% complete, as colony numbers were less at the higher concentrations tested (10 or 15 μmol/L EPI-7170; 500 nmol/L palbociclib) than vehicle control, which may suggest that some of the cells were either dead or permanently affected in their ability to form colonies or proliferate. Similar results were observed in SUM159PT cells (Fig. 1F).

Attenuation of AR transcriptional activity and inhibition of CDK4/6 activity both independently block cell-cycle progression from G₁- to the S-phase (4, 9, 29–31). Hence, we next examined whether EPI-7170 that targets AR NTD would affect the cell cycle in both prostate cancer and breast cancer cells and whether it would deepen any response mediated by palbociclib. Non-synchronized cells were used to reflect what would be encountered in tumor tissues with all phases of the cell cycle present. In addition, one particular method of synchronizing cells may result in different phases in differing types of cells. Single cells were selected for cell-cycle analyses as shown in Supplementary Fig. S5. On the basis of the above results from colony formation assays and cell viability, concentrations of 5 and 10 μmol/L of EPI-7170 were chosen with 10 μmol/L being approximately 10-fold its IC₅₀ for blocking AR transcriptional activity in LNCaP cells (23). Consistent with previous reports (23, 25), EPI-7170 significantly inhibited the transcriptional activities of androgen-induced full-length AR as well as androgen-independent AR-V7 transcriptional activity as demonstrated by luciferase reporter gene assays (Supplementary Fig. S6A and S6B) and measurement of expression of endogenous genes regulated by AR and AR-V7 with quantitative RT-PCR (Supplementary Fig. S6C and S6D). Palbociclib had no effect on the transcriptional activities of androgen-induced full-length AR or AR-V7 as measured using two reporter gene constructs (Supplementary Fig. S6A and S6B). As expected palbociclib had no effect on androgen-regulated genes (KLK3 for PSA and KLK2) while impacting the cell-cycle genes UBE2C, CDC20, and CCNA2 that are also known AR-V7 target genes (ref. 17; Supplementary Fig. S6C and S6D). These data support that the synergistic effects observed on cell viability and colony formation measured with the combination therapy of palbociclib with EPI-7170 are not due to palbociclib impacting AR transcriptional activity.

As expected in a cell line that depends on a transcriptionally active full-length AR for proliferation, the percentages of cells in the S-phase were reduced when LNCaP cells were treated with enzalutamide and EPI-7170 (Fig. 2A; Supplementary Fig. S7A). At 10 μmol/L EPI-7170, LNCaP cells in the S-phase decreased to 1.71 ± 0.52% (mean ± SEM) on day 2 and 3.75 ± 0.59% on day 3, and importantly decreased to almost 0% when EPI-7170 was combined with palbociclib. Unique to treatment with 10 μmol/L EPI-7170, either alone or with palbociclib, was the increase in the percentage of cells in G₂–M (∼twofold compared with controls) that was not observed with enzalutamide, thereby differentiating these two inhibitors of AR in a cell line that expresses solely full-length AR. A combination of 5 μmol/L EPI-7170 and palbociclib resulted in a similar effect on the S-phase, but not on G₁ or G₂–M, as measured with 10 μmol/L EPI-7170 monotherapy (i.e., reduced S percentages compared with controls). Palbociclib monotherapy increased the percentage of LNCaP cells in G₁ on day 2 as expected, with little to no effect on day 3 compared with controls. However, the combination on day 3 with either enzalutamide or EPI-7170 had a significant effect compared with the individual treatments. Similar cell distribution patterns (i.e., ratios among different phases of cell cycle) were detected between day 2 and day 3 of treatments when comparing among the different treatment conditions. However, S percentage increased on day 3 (compared with day 2) under EPI-7170 treatment with or without palbociclib, whereas S percentage decreased on day 3 (compared with day 2) for enzalutamide treatment with or without palbociclib.

Another important finding was the unique increase of percentages of cells in the S-phase without BrdU incorporation [S BrdU-negative (S BrdU−)] seen in cells treated with 10 μmol/L EPI-7170 and with a combination of EPI-7170 (5 and 10 μmol/L) with palbociclib that was not observed with enzalutamide monotherapy or combination. This S BrdU− population was more profoundly increased in cells treated with a combination of 10 μmol/L EPI-7170 plus palbociclib (day 2: 3.72 ± 0.69% in combination and 0.24 ± 0.03% in control; day 3: 3.38 ± 0.61% in combination and 0.34 ± 0.06% in control).

Proliferation of LNCaP95 cells is driven by AR-Vs and in these cells EPI-7170 had similar impact on the cell-cycle profile as seen with LNCaP cells, but this was not the case for enzalutamide that had no effect when compared with controls (Fig. 2B; Supplementary Fig. S7B). Interestingly, even though higher S percentages were detected in LNCaP95 controls (day 2: 38.95 ± 0.68%; day 3: 29.70 ± 1.10%) than in LNCaP controls (day 2: 18.66 ± 0.62%; day 3: 14.52 ± 0.59%), the combination treatment of EPI-7170 and palbociclib caused reduction in the S population of LNCaP95 cells to almost 0% as observed in LNCaP cells. The G₁ accumulation induced by palbociclib in LNCaP95 was consistent with a previous report using other AR-V–driven CRPC cells treated with this compound (29). In addition, the percentages of S BrdU− cells increased to approximately 13% with 10 μmol/L EPI-7170 plus palbociclib compared with controls.

The impact of these inhibitors on the cell cycle of several breast cancer cell lines was next examined. SUM159PT TNBC cells exhibited two distinct the S-phase populations (early S and late S) on days 3 and 4 (Supplementary Fig. S8A). EPI-7170 treatment with or without palbociclib in these cells did not result in substantial changes in total percentages of cells in the S-phase (Fig. 2C) as compared with that measured in LNCaP or LNCaP95. However, the percentages of SUM159PT cells in early the S-phase were reduced by EPI-7170 on day 4 (Supplementary Fig. S8A and red bars in Supplementary Fig. S8B). Enzalutamide (10 μmol/L) reduced the percentages of cells in early the S-phase but to a lesser extent than EPI-7170. As expected, palbociclib increased the percentage of cells in the G₁-phase (Fig. 2C; Supplementary Fig. S8A). However, palbociclib reduced the S BrdU− population (gray bars in Fig. 2C; Supplementary Fig. S8A) and reduced the number of tetraploid cells which are the cells with doubled DNA content (i.e., non-gated cells, higher 7AAD intensity, and excluded from calculation in bar graphs).

In MFM-223 TNBC cells that express both full-length AR and AR-V7 (15), the percentages of cells in the S-phase were reduced by EPI-7170, but there were no distinct the S-phase populations (Fig. 2D; Supplementary Fig. S8C). The combination of 10 μmol/L EPI-7170 with palbociclib reduced the percentage of MFM-223 cells in the S-phase to almost zero, which was consistent to that observed in LNCaP and LNCaP95 cells as shown above.

MDA-MB-453 breast cancer cells are negative for both estrogen and progesterone receptors but importantly express AR-V7 (15). These cells responded to EPI-7170 more robustly compared to enzalutamide especially with respect to accumulation in G₁ and reduction of cells in the S-phase (Fig. 2E; Supplementary Fig. S8D).

MDA-MB-468 TNBC cells were included as a negative control because they do not express either AR or RB (Supplementary Fig. S9A). As predicted, there was no impact on the cell-cycle phases of MDA-MB-468 cells in response to EPI-7170, enzalutamide, or palbociclib (Fig. 2F; Supplementary Fig. S9B), thereby supporting the specificity of these inhibitors.

Taken together, these results indicate EPI-7170 disrupted the cell cycle in prostate cancer and breast cancer cells that express AR and that its effects were greater in combination with palbociclib. Enzalutamide did not disrupt cell cycle as profoundly as EPI-7170 for cell lines that express AR-Vs such as in LNCaP95, MFM-223, and MDA-MB-453.

To investigate the effectiveness of combination treatments in AR-V7–driven tumors, the LNCaP95-D3 human CRPC xenograft model was employed. This enzalutamide-resistant cell line expresses AR-Vs and is less heterogeneous than parental LNCaP95 cells (32). Reduced tumor growth was observed in animals receiving EPI-7170 monotherapy and palbociclib monotherapy (Fig. 3A–C). The combination treatment significantly reduced the tumor growth compared with the control group and EPI-7170 monotherapy (Fig. 3A and B). Body weight was not significantly altered among the different treatment groups (Fig. 3D). The xenograft tumors from three individual best responders under combination treatment exhibited decreased percentage of proliferating cells (i.e., Ki67-positive cells) and increased percentage of apoptotic cell death (TUNEL; Supplementary Fig. S10).

The essential role of AR on cell-cycle progression has been shown by experiments examining androgen depletion, antiandrogens, and knockdown of AR (9, 33–36). Knockdown of AR or inhibition of AR transcriptional activity by antiandrogens or androgen depletion blocks progression from G₁ to S by mechanisms involving decreased levels of CDK2, and cyclin A and increased levels of p27^Kip1 (34–36). Here we examined in vivo and in vitro levels of AR and cell cycle–associated proteins in response to individual and combination treatments. Levels of these proteins detected from the harvested xenografts were not statistically significantly different between treatment groups and control group due to a wide range of variability (Fig. 3E; Supplementary Table S1). However, individual tumors from the best responders in each of the treatment groups appeared to be distinct from the average values as shown for AR, AR-V, pS780-RB, CDK6, cyclin A2, p27^Kip1, and Skp2 (labeled orange in Fig. 3E). The original immunoblot images are provided in Supplementary Figs. S11 and S12.

Levels of these cell cycle–associated proteins were also measured from in vitro studies using LNCaP95 cells that were treated with EPI-7170 and palbociclib. Consistent with previous studies in enzalutamide-resistant prostate cancer cell lines (25), EPI-7170 treatment was associated with decreased protein levels of AR-V7, whereas not altering levels of full-length AR (Fig. 3F). EPI-7170 altered the protein levels of pS780-RB, CDK2, cyclin A2, p27^Kip1, Skp2, and RAD51, which was consistent with its on-target effects on AR and mechanism of action in blocking cell-cycle progression from G₁ to S (34–36). Palbociclib significantly altered the protein levels of CDK4, cyclin D1, and CDK2 (Fig. 3F; original immunoblot images are provided in Supplementary Fig. S13). The increased levels of CDK4 and cyclin D1 proteins induced by palbociclib are consistent with stabilization of CDK4–cyclin D1 complex as demonstrated by others with this compound (37). Interestingly, when compared with the control, the combination treatment significantly decreased the protein levels of total RB, pS780-RB, CDK2, cyclin A2, Skp2, and RAD51, but significantly increased the levels of p27^Kip1 protein (Fig. 3F). This increased level of p27^Kip1 protein was also detected in LNCaP95-D3 xenografts from the best responders treated with EPI-7170 monotherapy and the combination (Fig. 3E); whereas palbociclib did not alter the level of p27^Kip1. The differential effects of EPI-7170 and palbociclib on the levels of these cell cycle–associated molecules supports their distinct inhibitory mechanisms on the cell cycle. Importantly, EPI-7170, palbociclib and the combination reduced the levels of CDK2 and cyclin A2 proteins, which have critical roles in DNA replication (38). These reduced protein levels were consistent with decreased percentages of cells in the S-phase with EPI-7170, palbociclib and combination treatments. EPI-7170, palbociclib, and the combination significantly reduced the protein levels of RAD51, which is important for DNA damage repair.

Antiandrogens and abiraterone have been reported to increase DNA damage in LNCaP, LNCaP-AR, and VCaP cells (39, 40). Decreased levels of RAD51 protein as measured here with EPI-7170 are also consistent with studies using antiandrogens and androgen ablation (41, 42). Hence, we examined whether EPI-7170 behaved similarly to other AR antagonists and would increase DNA damage. Phosphorylated H2AX (γH2AX) was detected in LNCaP95 cells treated with EPI-7170, and these levels could be further increased by combination treatment (Fig. 4A and B). Interestingly, with EPI-7170 or the combination treatment, the majority of the S-phase EdU− (same as S BrdU−) cells expressed γH2AX (∼60%–70% in Fig. 4A, bottom and 4C). On the contrary, only a small portion (∼20%) of the S-phase EdU− cells expressed γH2AX in the control or palbociclib-treated cells. Cells in G₂–M also expressed γH2AX at a significantly higher level with the combination than cells treated with control or solely palbociclib (Fig. 4C). The expression trend of γH2AX in cells in G₁ was similar to the trend in G₂–M cells but to a lesser extent (Fig. 4C). Immunofluorescence staining demonstrated that the foci of γH2AX and 53BP1 were increased in cells treated with EPI-7170 and the combination as compared with control and indicated by the number of foci per cell and the percentage of cells with more than five foci in the nucleus (Fig. 4D–G). Even though the number of foci for both proteins was correlated in a linear manner in each cell regardless of treatment (Fig. 4G), they did not always colocalize (Fig. 4D). The increase of DNA double-strand breaks was also observed in LNCaP cells treated with EPI-7170 or combination (Supplementary Fig. S14). Taken together, these data suggest that EPI-7170 may induce DNA double-strand breaks or impair the DNA-damage response similar to other inhibitors of the androgen axis.

Cell-cycle tracing experiments were conducted in LNCaP95 cells to reveal a more detailed mechanism of the inhibition mediated by EPI-7170 and palbociclib on the cell cycle. Each phase of the cell cycle from either BrdU-positive or BrdU-negative populations was measured after selecting single cells (Supplementary Fig. S15A). Compared with control cells, the cells treated with the combination had increased granularity in SSC and slightly decreased cell size in FSC over a 4-day treatment (Fig. 5A, left). Cells treated with the combination exhibited distinct BrdU-positive and BrdU-negative populations with decreased BrdU intensities to a much lesser extent over time compared with controls (Fig. 5A, right). This suggests a slower rate of division/proliferation for the combination-treated cells compared with the control cells. Surprisingly, a portion of cells in the S-phase from the BrdU-positive population were sustained in the S-phase with a 4-day combination treatment, as shown in the diagonally distributed population in Fig. 5B (red gates). However, another portion of cells in the S-phase continued to progress through the cell cycle and had already entered a new G₁ phase on day 1 of treatment albeit at a slower rate; that is, it took slightly longer to reach regular cell-cycle profiles as control as demonstrated in BrdU-positive cells (Fig. 5B and C; Supplementary Fig. S15B, top). For BrdU-negative cells, the combination treatment stopped the cells from continuing the cell cycle over 4 days which was unique from control cells (Fig. 5B and C; Supplementary Fig. S15B, bottom). Measurement of the number of live cells indicated that the control cells were proliferating more rapidly than the cells treated with the combination, which appeared to stop proliferating over the 4 days (Supplementary Fig. S15C).

To further understand how each inhibitor affected different phases of the cell cycle, we examined shorter intervals of treatment durations. BrdU-labeled cells were collected and analyzed at several timepoints during a course of 36-hour treatments. Consistent with the observation under a 4-day treatment, the combination treatment caused a portion of cells in the S-phase to be sustained in the S-phase over 36 hours (Supplementary Fig. S16). This sustained population was only observed with the combination and was not observed in the control or either monotherapy. Next, we examined BrdU-positive and BrdU-negative populations separately. Interestingly, for BrdU-positive cells, at 6 hours of treatment with inhibitors, there was approximately 8% of new G₁ population with the control or palbociclib while there was no new G₁ population with EPI-7170 or the combination (Fig. 5D; Supplementary Fig. S17; note: cells at t = 0 were all in the S-phase and gates were drawn as reference only). Approximately 2% of new G₁ cells started to form at 9 hours with EPI-7170 or the combination. After 18 hours, control cells reached a plateau with approximately 60% of new G₁ population as cells were trying to reach a "disrupted" tumor cell-cycle distribution. However, palbociclib-treated cells resulted in a higher percentage of new G₁ population compared with the control. This suggests that these cells were accumulating in the G₁-phase. Cells treated with EPI-7170 or the combination also displayed a higher percentage of new G₁ cells than the control, although this accumulation was delayed until 30 hours of treatment. These data indicate that for the S-phase cells, palbociclib resulted in accumulation of cells in a new G₁ population, whereas EPI-7170 delayed entry into a new G₁-phase as well as caused cells to accumulate in a new G₁ population. Moreover, G₂–M cells were likely to be affected by each of the inhibitors because the percentage changes were different from controls. Possibly the small populations with lower BrdU intensity within G₂–M gates were new G₂–M cells, which were detected in early timepoints (within 12 hours) and detected in combination treatments during 36 hours as well as 4-day time courses (Supplementary Figs. S17 and S15B, top). This new population of G₂–M cells were neither detected in later timepoints over a 36-hour course nor any other timepoint over 4 days in control cells. The BrdU intensities in the new G₂–M populations were similar to those in the new G₁ populations in these control cells, which was more obvious after 18 hours. Note that the gates for the new G₂–M population of cells could not be drawn precisely to calculate the percentages due to the fact that late S and G₂–M cells were not shown as distinct populations as clearly as the new G₁ cells (Supplementary Fig. S17). Nevertheless, it is possible that with the combination treatment, there were sustained populations of cells in late-S and new-G₂–M phases of the cell cycle.

For the BrdU-negative cells at t = 0, the majoritiy of the cells were in G₁ and G₂–M with approximately 1% of the cells in the S-phase (Fig. 5E; Supplementary Fig. S18). After 6 hours of treatment with inhibitors or in the control, G₂–M cells had already started moving to the G₁-phase as indicated by increased percentages of cells in G₁ and decreased percentages of cells in G₂–M compared with t = 0 (Fig. 5E, left and right; Supplementary Fig. S18). In control cells, G₁ cells started to enter the S-phase after 9 hours (Fig. 5E, left and right). This S-phase entry slowed down after 18 hours as the percentage of cells in G₁ started increasing, whereas those in the S-phase started decreasing. At the same time, new cells in the G₂–M phase, from the new cells in the S-phase, started to increase until 24 hours after which new G₂–M cells started entering into the G₁-phase. These data indicate that over 36 hours, there were two events of G₂–M cells entering the G₁-phase, and the G₁ cells entered the S-phase once. Both monotherapy with EPI-7170 or palbociclib altered these cell-cycle distributions but with slightly different patterns. EPI-7170 altered G₂–M cells at the beginning of treatment, that is, different from the control prior to 6 hours, whereas palbociclib altered G₂–M cells after 12 hours of treatment, that is, different from the control after 12 hours. Both EPI-7170 and palbociclib caused different G₁ and S percentages after 9 hours when compared with controls. However, EPI-7170 caused different G₁ and S patterns after 9 hours when compared with palbociclib. Combination treatment disrupted the cell cycle to a greater degree than the monotherapies as determined by the percentages of cells in G₁ and G₂–M compared with the single treatments over time (Fig. 5E). These results suggest that the cell cycle did not progress, but rather arrested with combination treatment and this cell-cycle arrest was observed in nearly constant cell number counts as shown in the cell-cycle histograms (BrdU-negative cells in Fig. 5F). BrdU-positive cells slowed down in the cell cycle with EPI-7170 alone or in combination, whereas with palbociclib monotherapy, the cells accumulated in the G₁-phase. Of note, 7-AAD intensities decreased over time in the control cells as shown in the shift to the left in the histograms from 6 to 36 hours (Fig. 5F) and from 1 to 4 days (Fig. 5C). This suggests that the control cells were proliferating. As noted in the Materials and Methods, all collected cells were used for DNA staining without cell number adjustment for cell-cycle tracing experiments, thereby the DNA dye intensity may decrease as the cell number increases. The decreases in 7-AAD intensities were also detected with both monotherapies and the combination albeit to lesser extent. Consistent with these observations, the numbers of total live cells from the cells treated with either monotherapy or the combination were significantly lower compared with control cells (Fig. 5G). This was consistent with the greater than twofold increase in cell doubling time determined for the combination therapy at approximately 63 hours compared with the control at approximately 25 hours. Collectively, these cell-cycle tracing studies revealed that EPI-7170 delayed the cell cycle for the S-phase cells although EPI-7170–treated cells eventually accumulated in the G₁-phase. With the combination treatment, BrdU-negative cells were stopped in the cell cycle; however, BrdU-positive cells were able to continue in the cycle but with delayed entry into the G₁-phase, and with only a portion of cells sustained in the S- and G₂–M-phase, thereby suggesting a partial cell-cycle arrest.

The unique inhibitory effects on each phase of the cell cycle mediated by EPI-7170 and palbociclib suggest that sequential treatments may yield different responses from concomitant treatments. To investigate this, LNCaP95 cells were used with two treatments that were 24 hours apart so that the first inhibitor would have shown its effect on the cell cycle or have altered the cell-cycle pattern before the second inhibitor was applied. Of note, day 1 and day 2 after the second treatments are day 2 and day 3, respectively, after the first treatments. Single treatments and concomitant combination were the same treatment durations as in Fig. 2B. Although the percentages of cells in each phase of the cell cycle were not identical in both sets of data (Fig. 2B; Fig. 6A), the trends were the same. As shown in Fig. 6A and B, the distribution patterns of cell-cycle phases were similar between day 1 and day 2 after the second treatment. When 10 μmol/L EPI-7170 was applied first (i.e., E10+D, E10/P+D and E10+P; symbol “+” is used to denote sequential and symbol “/” is used to denote concomitant), the S BrdU− population increased substantially (range, 12%–17%) as compared with the control and other treatment conditions (range, 0.7%–5.1%). Interestingly, the G₂–M population was also increased by approximately twofold with these three treatment conditions that used 10 μmol/L EPI-7170. As expected, the percentages of cells in the S-phase decreased to almost 0 regardless of the sequence of addition of 10 μmol/L EPI-7170. Compared with E5+P treatment (day 1: 4.25 ± 0.72%; day 2: 9.64 ± 1.47%), P+E5 caused a greater reduction of cells in the S-phase (day 1: 3.12 ± 0.95%; day 2: 6.35 ± 2.17%). The treatment of P+E5 also caused a greater increase of cells in the G₁-phase compared with E5+P, although all three conditions (E5/P, E5+P, and P+E5) caused a substantial increase of cells in the G₁-phase compared with the control and to the single treatments (D+D, E5+D, E10+D, and P+D). The percentages of cells in the G₁-phase were higher when cells were first treated with palbociclib before the addition of EPI-7170. Overall, the cell-cycle distribution patterns for P+E10 and E10+P were very different, whereas the patterns for P+E5 and E5+P were quite similar. The total number of live cells significantly increased from day 1 to day 2 in the control, E5+D, and P+D, but not with any other treatments (Fig. 6C). Monotherapy with 10 μmol/L EPI-7170 stopped the cells from proliferating. Similarly, for the combination treatments, both concentrations of EPI-7170 blocked the cell numbers from increasing regardless of the order of addition. Taken together, these data revealed that a lower concentration of 5 μmol/L EPI-7170 can be used when it was administered in combination with palbociclib to prevent cells from proliferating (i.e., reducing cells in the S-phase) and to induce G₁ arrest.

Most benign/nonmalignant cells are able to proliferate in the absence of CDK4/6 activities. Thus, inhibitors of these kinases were predicted to have selectivity against cancer cells to cause cell-cycle arrest in G₁, senescence, and induce apoptosis while sparing benign cells. There are three CDK4/6 inhibitors that are FDA approved for use in combination with hormonal therapies for the treatment of patients with advanced and metastatic breast cancer that have estrogen receptor–positive and HER2-negative disease. Palbociclib is highly specific for inhibiting CDK4 and CDK6 (43, 44). Dose-limiting toxicity to palbociclib is neutropenia. Ribociclib has a similar structure and toxicity profile to palbociclib, whereas abemaciclib has preference to inhibit CDK4 but also inhibits CDK6 and 9 (45) with toxicities being diarrhea and fatigue. The success of CDK4/6 inhibitors for some breast cancers has spurred interest for their potential application for other types of cancers including its combination with androgen-deprivation therapy for prostate cancer that expresses full-length AR (NCT02059213). However, to date, there are no studies that have examined the feasibility of a combination of a CDK4/6 inhibitor with an AR-NTD inhibitor that targets both full-length AR and AR-Vs for the treatment of CRPC and TNBC. Expression of AR-V7 is considered a major mechanism of resistance to clinically approved therapies that target the full-length AR (11, 12).

Here, we investigated the combination of palbociclib with EPI-7170 in breast cancer and prostate cancer and reveal that the combination: (i) blocked the in vivo growth of CRPC xenografts; (ii) maximized the reduction of cells in the S-phase with accumulation in the G₁-phase compared with monotherapies; (iii) caused a portion of cells in the S-phase to not continue in the cell cycle; and (iv) caused cells in G₁ and G₂–M to arrest. Palbociclib and EPI-7170 have differential effects on the progression of the cell cycle, thereby supporting their unique mechanisms of action. EPI-7170 monotherapy delayed cells in the S-phase to continue the cell-cycle thereby resulting in accumulation of cells in G₁. EPI-7170 also induced DNA damage and/or impaired the DNA repair response. The order in which the combination was applied as either sequential or concomitant had a differing impact on the cell cycle.

Combining a CDK4/6 inhibitor with another targeted therapy may overcome the resistance that can develop when using CDK4/6 inhibitors as monotherapies (1, 3). These resistance mechanisms include RB status, amplified cyclin E1, PIK3CA mutations, and CDK2 activation (3, 37, 46). AR regulates the expression and activities of CDK1 and CDK2 in prostate cancer cells in response to androgen (47, 48). Consistent with these reports, here the AR-NTD inhibitor, EPI-7170 attenuated the level of CDK2 protein, which may be beneficial in preventing resistance to palbociclib by a mechanism of upregulation of this kinase. CDK4/6 inhibitors have shown some efficacy against full-length AR-positive prostate cancer cells that are resistant to antiandrogens due to a gain-of-function point mutation in the LBD of AR such as AR F876 L (49). A combination of palbociclib with inhibition of the full-length AR by application of either enzalutamide or short hairpin RNA knockdown of AR have shown in vitro efficacy in breast cancer (50, 51). However, clinical samples of breast cancer can express both full-length AR and AR-V7 (13–16). Recently, it was shown that expression of AR-V7 measured in circulating tumor cells of patients with metastatic breast cancer was associated with bone metastases and also failure of TNBC to therapies (13, 52). Thus, application of AR-NTD inhibitors such as EPI-7170 that inhibit the transcriptional activities of both AR-V7 and full-length AR (23, 25, 26) may be beneficial for the treatment of TNBC as well as CRPC.

This is the first report to examine a CDK4/6 inhibitor in combination with an AR-NTD inhibitor for the treatment of AR-expressing breast cancer and prostate cancer. Palbociclib inhibited the phosphorylation of RB at Ser780 in LNCaP95 cells, and this is consistent with previous studies in LNCaP, LAPC4, and VCaP cell lines (29). Ser780 on RB is specifically phosphorylated by CDK4 and not CDK2 and is a robust marker of mitosis. Palbociclib may stabilize CDK4/6–cyclin D1 complexes as interpreted from the increased levels of these proteins in palbociclib-treated LNCaP95 cells. These results are consistent with reports of palbociclib stabilizing the activated CDK4/6–cyclin D complex (37) to suggest an incomplete cell cycle arrest that may lead to resistance when using a discontinuous treatment regimen (3 weeks on therapy and 1-week off) (37, 53). The moderately decreased levels of CDK4, CDK6, and cyclin D1 proteins shown here in cultured LNCaP95 cells in response to EPI-7170 may be beneficial when using a combination with palbociclib to prevent drug resistance.

AR has an important role in the cell cycle with its transcriptional activity depending on the phase of the cell cycle. Full-length AR is transcriptionally active at G₀- and the S-phases, but its activity is markedly reduced during G₁–S transition when levels of cyclin D1 and CDK4 are at their peak in a mouse fibroblast model that measured endogenous AR transcriptional activity on integrated promoters (54). In human prostate cancer cells, AR transcriptional activity also varies during cell cycle, and it is required for cells to progress from the G₁- to the S-phase and also for DNA synthesis during the S-phase (9).

The p27^Kip1 protein blocks activation of the cyclin D–CDK4 complex to regulate progression of the cell cycle at G₁. Levels of this protein are regulated by proteasomal degradation in prostate cancer cells by AKT and mTOR complex 2 in response to a transcriptionally active AR (55). The loss of expression of p27^Kip1 is associated with increased tumor grade and increased proliferative index in prostate cancer (56), which is consistent with the resumed transactivation of the AR axis in CRPC. Hence, inhibition of AR transcriptional activity as shown here with EPI-7170, either as a monotherapy or in combination with palbociclib, was expected to lead to increased levels of p27^Kip1 protein as shown in both cultured cells and in xenografts harvested from the best responders. The consequence of EPI-7170 enhanced protein levels of p27^Kip1 would be cell-cycle arrest or slowing down the cell division cycle.

Here, we provide a comprehensive analysis of the impact of inhibition of AR transcriptional activity by EPI-7170 and the CDK4/6 inhibitor palbociclib on the cell cycle of prostate cancer and breast cancer cells. These data reveal that EPI-7170 reduced the protein levels of cyclin D1 and CDK4/6, which caused accumulation of cells in G₁, and prolonged the S-phase, which resulted in a subset of cells to delay the cell cycle. Accumulation of DNA damage likely occurred in cells in the prolonged the S-phase with EPI-7170 or with the combination because the majority of γH2AX-positive cells were in the S-phase (S EdU− in Fig. 4A and prolonged S in Fig. 5B). Full-length AR regulates DNA repair in prostate cancer (39–42). AR-Vs also regulate DNA repair (23, 57) and EPI-7170 has been proposed to enhance the sensitivity of prostate cancer cells to radiotherapy by targeting AR-V7 to block the expression of DNA damage repair genes (23). Here, we show that EPI-7170 significantly decreased levels of both cyclin A2 and RAD51 proteins. Cyclin A2 is a regulator of homologous recombination in DNA repair that maintains protein levels of MRE11 and RAD51 (38). Increased expression of cyclin A2 has been found in many types of cancers, and its reduction results in increased double-strand breaks in breast cancer cells and hypersensitivity to DNA-damaging agents (58). Thus, reduced levels of cyclin A2 (both xenograft and cultured LNCaP95) and RAD51 (in both xenograft and cultured LNCaP95 but more obvious in culture LNCaP95) proteins shown here in response to EPI-7170 were consistent with previous studies and support a role for functional AR in the DNA repair response. We noted that the combination therapy of EPI-7170 with palbociclib increased the % of γH2AX-positive cells beyond levels achieved with the individual treatments. Thus, in addition to the AR playing an important role in DNA damage and repair, the cyclinD-CDK4/6-RB pathway is also involved in DNA repair (59) and inhibitors of CDK4/6 are being pursued as a strategy for tumor radiosensitization for numerous malignancies [for a review, see Yang and colleagues (60)]. This is the first report of enhanced DNA damage in prostate cancer cells using a combination of an AR-NTD inhibitor with a CDK4/6 inhibitor, which may have potential application with radiotherapy for high-risk prostate cancer.

The beneficial inhibitory effect of EPI-7170 was optimally enhanced with the addition of palbociclib administered sequentially rather than concomitantly. Compared with control cells, EPI-7170 decreased the cells in the S-phase and increased the cells in G₁ and G₂–M, whereas palbociclib caused arrest in G₁ to decrease the percentage of cells in S and G₂–M. Moreover, sequential treatments, regardless of whether the concentration of EPI-7170 was 5 or 10 μmol/L, caused more G₁ accumulation and less cells in the S-phase than concomitant treatments. These effects were more profound, especially at saturating concentrations of EPI-7170 (10 μmol/L) and when palbociclib was administered before EPI-7170. Enzalutamide saturates AR in prostate cancer between 10.76 to 32 μmol/L (5–15 μg/mL; ref. 28). The addition of EPI-7170 subsequent to palbociclib further enhanced the accumulation of cells in G₁ with a reduction of cells in the S-phase to almost zero. These data are consistent with the inhibitory mechanism of each drug as seen previously with other cell-cycle inhibitors and cells. The unique mechanisms of action of EPI-7170 and palbociclib to block the progression of the cell cycle support a strategy for sequential combination therapies for patients with breast cancer and prostate cancer.

A.H. Tien reports grants from the NCI of the NIH during the conduct of the study; in addition, A.H. Tien has a patent for Pharmaceutical Compositions and Combinations Comprising Inhibitors of the Androgen Receptor and uses thereof pending to ESSA Pharma Inc. M.D. Sadar reports grants from NCI of the NIH during the conduct of the study; personal fees from ESSA Pharma Inc outside the submitted work; in addition, M.D. Sadar has a patent for Pharmaceutical Compositions and Combinations Comprising Inhibitors of the Androgen Receptor and uses thereof pending to ESSA Pharma Inc; and M.D. Sadar has equity in and is a scientific advisor for ESSA Pharma Inc.

A.H. Tien: Conceptualization, formal analysis, methodology, writing–original draft. M.D. Sadar: Conceptualization, formal analysis, supervision, funding acquisition, writing–review and editing.

The authors acknowledge Nasrin R. Mawji and Jun Wang for their technical support. This research was supported by funding from the NCI of the NIH (R01CA105304).

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