Purpose:

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor–positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models.

Experimental Design:

Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy.

Results:

Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21–1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08–1.68) and abemaciclib-treated (rER: 1.19–2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94–1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth.

Conclusions:

These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

Translational Relevance

Although CDK4/6 inhibitors are currently indicated for patients with metastatic, ER+ breast cancer, their utility in the nonmetastatic setting is still being established. Our understanding of the interaction between CDK4/6 inhibitors and the ionizing radiotherapy given as part of the standard of care is lacking, and the utility of this approach in women at high risk for locoregional failure is unknown. In this article, we demonstrate using multiple nonoverlapping in vitro and in vivo models that combination therapy with radiotherapy and each of the three clinically approved CDK4/6 inhibitors is more effective at decreasing cell proliferation and tumor growth when compared with either radiotherapy or CDK4/6 inhibition alone. Furthermore, this sensitization is due, at least in part, through the suppression of homologous recombination (HR)-mediated DNA repair. In contrast, preclinical models with acquired resistance to CDK4/6 inhibition do not demonstrate radiosensitization or suppression of HR. These data suggest that combination CDK4/6 inhibition and radiotherapy represent a novel indication for CDK4/6 inhibitors and a clinically feasible strategy for the radiosensitization of ER+ breast cancers that warrants clinical exploration.

The treatment of breast cancer is guided, in part, by the presence or absence of hormone receptors including the estrogen receptor (ER). Nearly 75% of new breast cancer diagnoses will be classified as estrogen receptor–positive (ER+) disease (1). For these patients, precision medicine strategies that target the ER using selective estrogen receptor modulators, selective estrogen receptor degraders, and aromatase inhibitors that block ER signaling have resulted in significant improvements in recurrence-free and overall survival rates (1). While many women with ER+ metastatic breast cancer initially respond to endocrine therapy, nearly all will become refractory to endocrine therapy (1). Treatment options in the metastatic setting are expanding, and the recent introduction of cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors into the clinic has significantly improved outcomes for these patients (2, 3).

In contrast to antiestrogen therapies, CDK4/6 inhibitors work by targeting the cell cycle (4). When cells are actively proliferating, the levels of cyclin proteins rise and fall in a series of predetermined, cyclic patterns. The activation of specific cyclins at fixed points in the cell cycle is crucial for proper regulation of CDKs. CDKs are serine/threonine kinases that act as master regulators of the cell cycle; they phosphorylate downstream target proteins necessary to proceed through cell-cycle “checkpoints” designed to control abnormal proliferation (4). For example, cyclin D1 forms a complex with CDK4/6 and leads to the phosphorylation of many downstream targets, including the inactivation of the retinoblastoma (RB1) tumor suppressor (4). The ability of cancer cells to evade growth suppressors, one of the hallmarks of cancer, has long been appreciated in many cancer types based on the dysregulation of cyclins and CDKs (4).

With the development of selective CDK4/6 inhibitors such as palbociclib, the ability to selectively target this cell-cycle dysregulation in metastatic, ER+ breast cancer became possible. There are currently three FDA-approved CDK4/6 inhibitors: palbociclib (PD0332991; ref. 5), ribociclib (LEE011; ref. 6), and abemaciclib (LY2835219; ref. 7). All three are orally bioavailable ATP-competitive inhibitors of CDK4 and CDK6. Early preclinical studies suggested that ER+ breast cancer cell lines are more sensitive to the antiproliferative effects of specific CDK4/6 inhibitors compared with other breast cancer subtypes, like triple-negative breast cancer, where alterations in RB1 are more frequent (5). This differential response, later validated by others, provided the rationale to restrict early clinical trials to ER+ breast cancers (3). On the basis of several practice-changing clinical trials (2, 3, 8, 9), CDK4/6 inhibitors are now standard of care for women diagnosed with metastatic ER+ breast cancer in combination with hormone therapies such as letrozole or tamoxifen.

For patients with metastatic ER+ breast cancer, CDK4/6 inhibitors have improved progression-free survival, but acquired resistance to these drugs remains a critical clinical issue (4). While the exact mechanism(s) of therapy resistance remain unclear, recent data suggest changes in phosphorylation of RB1 (5, 10, 11), and changes in cyclin/CDK expression (12–16), may contribute to drug resistance; however, currently, there is no known consensus pathway of resistance. CDK4/6 inhibitors have also demonstrated the ability to slow progression in many types of cancers as well as the potential to synergize with other agents for more durable responses.

Although use of CDK4/6 inhibitors is currently limited to the metastatic setting, there are ongoing efforts to evaluate the efficacy of CDK4/6 inhibitors in the upfront setting for women with locally advanced or high-risk ER+ disease (17). This resistance may become a more critical issue as these inhibitors make their way into the clinical management of patients with locally advanced, nonmetastatic disease where cure remains the therapeutic goal. Thus, there is a critical unmet need to identify strategies to improve the local efficacy of CDK4/6 inhibitor therapy in patients with ER+ breast cancer.

Radiotherapy remains a mainstay in the treatment of women with locally advanced ER+ breast cancer (18). Despite its ubiquitous use, combination studies testing the use of radiotherapy in combination with CDK4/6 inhibition are lacking. It is well established that pharmacologic CDK4/6 inhibition interferes with cell-cycle regulation (4), but recent studies have also shown that single-agent palbociclib can also affect the regulation of DNA damage response pathways (19, 20). However, our understanding of this interaction and the resulting effects of CDK4/6 inhibitor therapy and radiotherapy is incomplete. Therefore, we sought to determine whether combining CDK4/6 inhibitors with radiotherapy would prove to be more effective than either treatment alone in multiple models of ER+ breast cancer, and to evaluate the physiologic significance of this phenomenon in vivo.

Cell culture

All cell lines were obtained from ATCC and cultured at 37°C and 5% CO2 at subconfluent densities. Cell culture media and additives can be found in Supplementary Methods. Parental cell lines (MCF-7, T47D, CAMA-1, ZR-75–1) are ER+ breast cancer cells that are sensitive to both estrogen supplementation and hormone therapies. CDK4/6 inhibitor–resistant cell lines were developed through serial passaging with dose escalation of either palbociclib, ribociclib, or abemaciclib every 2–3 weeks. Cells were selected for approximately 3 months (Fig. 1D) and resistant pools were continuously cultured in 1 μmol/L CDK4/6 inhibitor. Before use in assays, drug was removed for at least 24 hours and cells were plated in drug-free media. The identity of the cell lines was confirmed by short tandem repeat profiling and Mycoplasma testing was done monthly (Lonza, catalog no. LT07-318).

Drugs

All drugs were solubilized in 100% DMSO for a stock concentration of 10 mmol/L for use in all cell culture assays. Palbociclib (Sigma, catalog no. PZ0199), ribociclib (Med Chem Express, catalog no. HY-15777A), abemaciclib (Med Chem Express, catalog no. HY-16297A), staurosporine (Sigma, catalog no. S6942), NU7441 (Selleck, catalog no. S2638), and AZD7762 (Sigma, catalog no. SML0350) were all purchased commercially.

Clonogenic survival assay

Cells were seeded at single-cell density in 6-well plates and allowed to adhere overnight. The following morning, cells were pretreated with drug for 1 hour (except where indicated otherwise) and radiated. Colony counts were used to determine toxicity, the surviving fraction of cells at 2 Gy (SF 2 Gy), and the radiotherapy enhancement ratio (rER) for each treatment condition. Clonogenic data were fit to a linear-quadratic model and enhancement ratios were calculated as the ratio of the AUC from control cells/experimental conditions.

Immunoblotting

Cell lysates were prepared with RIPA buffer (Thermo Fisher Scientific, catalog no. 89901) containing commercially available phosphatase and protease inhibitor tablets (Sigma #PHOSS-RO, #CO-RO). Protein lysates were sonicated and reduced with 2% β-Mercaptoethanol and 4× Nu-Page buffer (Life Technologies, catalog no. NP0007). Immunoreactivity was detected using the following antibodies: anti-RAD51 (Millipore ABE257 1:1,000), pKu80 (Invitrogen, catalog no. 38118, 1:1,000), Ku80 (Cell Signaling Technology, catalog no. 2180S, 1:1,000), γH2AX (Milipore, catalog no. 05-636, 1:1,000), cleaved PARP (Cell Signaling Technology, catalog no. 5625S, 1:1,000), PARP1 (Cell Signaling Technology, catalog no. 9542S, 1:1,000), and anti-β-Actin-HRP (Cell Signaling Technology, catalog no. 12262S, 1:50,000).

Irradiation

Irradiation was carried out as described previously in the University of Michigan Experimental Irradiation Core (21, 22). Briefly, a Philips RT250 (Kimtron Medical), which is calibrated to meet the standards of the National Institute of Standards and Technology (NIST), was used at a dose rate of approximately 2 Gy/minute for both in vitro and in vivo irradiation experiments.

Immunofluorescence

Immunofluorescence was performed as described previously (21, 22). Antibody and dilution information can be found in the Supplementary Methods.

Nonhomologous end joining reporter and qPCR

Nonhomologous end joining (NHEJ) reporter assays were performed as described previously (21, 22). Briefly, a linearized GFP reporter plasmid was transfected into cells and plasmid DNA was isolated to perform comparative qPCR (ΔΔCt) using GFP and internal control primers. All Ct values were normalized to untreated control cells. Additional information can be found in Supplementary Methods.

Xenograft studies

MCF-7 cells (n = 4 × 106) were injected bilaterally into the mammary fat pads of 8–10 weeks old CB17-SCID female mice in 50% Matrigel (Thermo Fisher Scientific, catalog no. CB-40234). Estrogen pellets (Innovative Research of America, #SE-121) were implanted subcutaneously in the nape of the neck on the day of tumor injection and removed after visible tumor formation. When tumors reached approximately 80 mm3, mice were randomized into four groups (14–16 tumors per group): vehicle (sodium L-lactate, 50 mmol/L pH 4.0, Sigma, catalog no. L-7022), palbociclib only, radiotherapy only, or combination treatment. Mice in the palbociclib only or combination groups were treated with 25 mg/kg palbociclib by oral gavage for 6 days. Mice receiving radiotherapy only received fractions of 2 Gy for 5 days. Mice in the combination group started palbociclib treatment one day before radiotherapy, but drug in all groups was discontinued after the last radiotherapy fraction. Tumor growth was measured 1–3 times a week and tumor volume was calculated using the equation V = (L*W2)*π/6. All xenograft experiments and procedures were done with the approval of the Institutional Animal Care & Use Committee at the University of Michigan (Ann Arbor, MI).

Transcriptomic analysis

RNA was isolated using QIAzol and the RNeasy Mini Kit (Qiagen, catalog no. 74104) and sent to the University of Michigan Advanced Genomics Core. For transcriptomic analyses, expression values were calculated using a robust multiarray average (23) to convert probe values into log2 expression values for each gene which were then fit using linear models (24). The SE for each gene was standardized across all arrays used for a median SE of 1. All P values were corrected for an FDR. Analyses were done using the oligo and limma packages of Bioconductor in R at the University of Michigan Bioinformatics Core. Data from this study are publicly available through Gene Expression Omnibus (GSE155570).

Proteomic analysis

Protein for reverse phase protein array (RPPA) was extracted from cells and standardized to 1.5 μg/μL in RIPA buffer. Cell lysate was reduced with β-mercaptoethanol and 4× SDS and sent to the Functional Proteomics RPPA Core Facility at M.D. Anderson Cancer Center for analysis (25). Briefly, serial dilutions of each sample were prepared and used to capture the linear antibody/antigen reaction for accurate data analysis using validated antibodies. In addition, 48 unique cell lysates were printed on each slide and served as controls to develop replicate-based normalization used for quality control for data generation and analysis and RPPA data merging across different slides. Subsequent algorithms of spatial correction, quality control of antibody probing, protein loading correction, replicate-based normalization, and quality of antibody batches were performed with each run and an automated program for RPPA Pipeline processing was used. Each sample was quantitated and run in triplicate for each condition.

Statistical analysis

The SF 2 Gy values and the NHEJ reporter data were compared with control cells using a one-way ANOVA with Dunnett test. A t test was used to compare radiotherapy and combination groups in the immunofluorescence experiments, and P values were corrected for multiple comparisons. All in vitro experiments were completed as the average of at least three independent experiments and pooled for statistical analysis. Xenograft tumor size and mouse weights were compared using a two-way ANOVA, and survival curves were compared using the log-rank (Mantel–Cox) test. Fractional tumor volume (FTV) was calculated in a manner consistent with previous studies (22, 26).

Single-agent CDK4/6 inhibition leads to a suppression of cell cycle and DNA damage response pathways

To determine the effects of CDK4/6 inhibition in breast cancer, we performed proliferation assays and calculated the IC50 values of palbociclib, ribociclib, and abemaciclib in the estrogen-dependent, ER+ breast cancer cell lines MCF-7, T47D (Supplementary Fig. S1), CAMA-1, and ZR-75–1 (Supplementary Fig. S2). To understand the biological changes that are induced by short-term CDK4/6 inhibition, we analyzed transcriptomic changes of T47D cells treated with 40 nmol/L palbociclib for 16 hours (Fig. 1A). Overrepresentation (pORA) and total pathway accumulation (pAcc) were computed using iPathway (Advaita) to find pathways that were significantly differentially expressed.

As expected, the cell-cycle pathway was differentially expressed between vehicle-treated and palbociclib-treated T47D cells (P = 9.283 × 10−5) and expression of RB1, a canonical target of CDK4/6, was significantly decreased compared with control cells (P = 2.014 × 10−4). Surprisingly, pathway analysis identified DNA damage response as the pathway most significantly overrepresented after palbociclib treatment. These significantly altered pathways (with FDR-corrected P values) include DNA replication (P = 6.198 × 10−23), mismatch repair (P = 3.209 × 10−7), base excision repair (P = 1.249 × 10−5), Fanconi anemia (P = 1.585 × 10−5), nucleotide excision repair (P = 4.935 × 10−5), and homologous recombination (HR; P = 8.874 × 10−5). Cell-cycle downregulation also led to the global suppression of cell-cycle genes (Fig. 1D) including Cyclin E2 (CCNE2), the transcription factor E2F (E2F1), and RB1 (RB1). T47D cells treated with either 100 nmol/L ribociclib (Fig. 1B) or 20 nmol/L abemaciclib (Fig. 1C) demonstrated similar pathway changes in cell cycle and DNA damage response pathways. MCF-7 cells treated with low concentrations of CDK4/6 inhibition for 16 hours (Supplementary Fig. S3) showed less robust changes in gene expression overall and no significant changes in DNA damage response pathways, but showed some change in cell-cycle response along with significant changes in IL and chemokine signaling.

To understand how these pathway changes might be altered after the development of CDK4/6 inhibitor resistance, we developed models of acquired resistance (Fig. 1E) to palbociclib (PalAR) ribociclib, (RibAR), and abemaciclib (AbeAR) in MCF-7 and T47D cells. After selection, CDK4/6 inhibitor–resistant MCF-7 and T47D cells demonstrated a 10- to 100-fold greater resistance to CDK4/6 inhibition as evident by a significant shift in the dose-response curves (Supplementary Fig. S1). CDK4/6 inhibitor–resistant cell lines also developed cross resistance to all three CDK4/6 inhibitors, suggesting commonality in resistance mechanisms (Supplementary Fig. S4). In contrast to the changes observed after CDK4/6 inhibition in CDK4/6 inhibitor–sensitive parental cell lines, short-term treatment of palbociclib-resistant T47D PalAR cells with 40 nmol/L palbociclib (Fig. 1F) predominately led to changes in pathways involved in adhesion, cytokine signaling, and immune regulation, which has been demonstrated by others (19). T47D RibAR (Fig. 1G) and T47D AbeAR (Fig. 1H) cells and CDK4/6 inhibitor–resistant MCF-7 cells (Supplementary Fig. S3D–S3F) also showed minimal changes in cell cycle and DNA damage response pathways, suggesting that CDK4/6 inhibitor–resistant cell lines are not as susceptible to manipulations of DNA repair and DNA damage as their wild-type counterparts.

To confirm these observed transcriptomic changes, we used RPPA to quantify changes in protein and phosphoprotein expression in MCF-7 and MCF-7 PalAR cells 16 hours after treatment with 75 nmol/L palbociclib. Along with expected changes in cell-cycle proteins—including decreased pRB1 in MCF-7 parental cells—we saw a decrease in the expression of a significant number of proteins and phosphoproteins involved in the DNA damage response (Fig. 1I). CDK4/6 inhibition with palbociclib did not cause significant suppression of these proteins in MCF-7 PalAR cells treated with 75 nmol/L palbociclib, suggesting that CDK4/6 inhibitor–resistant cells are no longer susceptible to CDK4/6 inhibitor–mediated suppression of DNA repair.

CDK4/6 inhibition radiosensitizes CDK4/6 inhibitor–naïve ER+ breast cancer cell lines

Because CDK4 and CDK6 act at the G1/S checkpoint, it has been hypothesized that the use of CDK4/6 inhibitors would be radioprotective by arresting cells in the G1 phase; cells are typically most sensitive to radiotherapy in G2–M. Our data demonstrating significant changes in DNA damage response proteins and phosphoproteins suggested that CDK4/6 inhibitors may be directly impacting these pathways independent of the cell-cycle effects and may potentiate the effects of DNA damaging agents (including ionizing radiotherapy). To assess the ability of CDK4/6 inhibition to influence the radiosensitivity of ER+ breast cancer cell lines, we performed clonogenic cell survival assays. In these assays, ER+ breast cancer cells were pretreated with a low concentration of CDK4/6 inhibition 1 hour prior to radiotherapy to minimize potential confounding effects of cell-cycle reassortment.

We demonstrate that escalating doses of palbociclib produced a dose-dependent increase in radiosensitization in MCF-7 cells (rER: 1.15–1.67) at concentrations at or below the IC50 value (Fig. 2A). The rER of clinically approved radiosensitizing agents such as cisplatin (27) is approximately 1.2, suggesting that this radiosensitization is clinically meaningful (27, 28). Similar results were observed in T47D (rER: 1.12–1.65; Fig. 2B), CAMA-1 (rER: 1.14–1.42; Supplementary Fig. S2D), and ZR-75–1 cells (rER: 1.12–1.43; Supplementary Fig. S2F) when treated with sub-IC50 concentrations of palbociclib. Radiosensitization occurred in MCF-7 and T47D cells to a similar degree with ribociclib (Fig. 2C and D) and abemaciclib (Fig. 2E and F), suggesting that all three CDK4/6 inhibitors led to comparable levels of radiosensitization in vitro. Furthermore, all three drugs showed modest single-agent toxicity (Supplementary Table S1), predominately at concentrations closer to the IC50 value. However, at these concentrations, CDK4/6 inhibition did not significantly radiosensitize the transformed, mammary epithelial cell line MCF-10A (Supplementary Fig. S5), suggesting that the combination treatment would be unlikely to cause significant toxicity to normal breast tissue when treated with radiotherapy (Supplementary Table S3).

Because CDK4/6 inhibitors change the cell-cycle distribution of exponentially growing ER+ breast cancer cells, we sought to understand how cell-cycle changes may play a role in the radiosensitization. To that end, we performed propidium iodide–based cell-cycle analysis in T47D and MCF-7 cells (Supplementary Fig. S6A and S6C) to determine the time course of G1 arrest in our cell lines. In T47D cells, G1 arrest did not occur at 1 or 6 hours after CDK4/6 inhibitor treatment, but cells were significantly arrested by 16 hours and remained arrested at 24 and 48 hours. MCF-7 cells did not arrest after 1 hour of drug treatment—equivalent to our 1 hour pretreatment in other assays—but did demonstrate cell-cycle arrest at 48 hours even at these low concentrations. In addition, we performed clonogenic assays in MCF-7 cells with varied CDK4/6 inhibitor pretreatment times before radiotherapy. In spite of these differences in cell-cycle accumulation, pretreatment with 50–100 nmol/L palbociclib for either 6 or 24 hours led to nearly equivalent levels of radiosensitization in MCF-7 cells (rER: 1.29–1.58 at 6 hours, 1.13–1.71 at 24 hours; Supplementary Fig. S6E and S6F; Supplementary Table S4), suggesting that radiosensitization is cell-cycle independent and that radiosensitization occurs even in cells arresting at the G1 checkpoint.

Given that CDK4/6 inhibitor–resistant cell lines respond differently to short-term CDK4/6 inhibition compared with their parental cell lines, we were interested in understanding whether CDK4/6 inhibitor resistance would play a role in the response of ER+ breast cancer cells to ionizing radiotherapy. As expected, cell-cycle analysis demonstrated that CDK4/6 inhibitor–resistant MCF-7 and T47D cells did not arrest at G1 after treatment with palbociclib, ribociclib, or abemaciclib (Supplementary Fig. S6B and S6D) even after 48 hours. Much higher concentrations of all three drugs (100–500 nmol/L) also failed to radiosensitize CDK4/6 inhibitor–resistant MCF-7 (Fig. 3A, C, and E) and T47D cells (Fig. 3B, D, and F) in clonogenic cell survival assays. Because these cells were selected for acquired resistance to CDK4/6 inhibitor monotherapy, single-agent toxicity (Supplementary Table S2) was minimal, as expected.

Short-term CDK4/6 inhibition leads to a decrease in HR efficiency

Radiosensitization can occur through a variety of mechanisms, including changes in the efficiency of the DNA damage response, cell-cycle reassortment, changes in oxygenation, and upregulation of other cellular response pathways such as apoptosis or senescence. Given that we observed cellular changes in multiple DNA damage response pathways at the transcriptomic and proteomic/phosphoproteomic level after short-term CDK4/6 inhibition, we wanted to understand whether CDK4/6 inhibition affects specific DNA damage response pathways, including the two major double-strand break repair pathways of HR and NHEJ.

To assess HR-mediated effects, we performed RAD51 foci formation assays. RAD51 is a recombinase responsible for protecting single-stranded DNA at the site of DNA strand breaks and initiating the catalysis of HR-mediated DNA repair. RAD51 foci are indicative of active HR and can be quantified using immunofluorescence microscopy to assess HR competency. In MCF-7 (Fig. 4A) and T47D (Fig. 4B) cells, radiotherapy (4 Gy) led to an increase in RAD51 foci at 6 and 16 hours following radiotherapy. In contrast, a 1-hour pretreatment with either palbociclib, ribociclib, or abemaciclib led to a significant decrease in RAD51 foci at 6 and 16 hours postradiotherapy compared with radiotherapy alone. The inability of ER+ breast cancer cells to respond to and repair double-stranded breaks using HR cannot solely be attributed to an absence of RAD51 protein (Fig. 4C), though there was a slight decrease in total RAD51 protein levels in the palbociclib- and combination-treated groups in T47D cells at these time points. In contrast to CDK4/6 inhibitor–sensitive cell lines, CDK4/6 inhibitor–resistant MCF-7AR (Fig. 4D–F) and T47DAR cells (Fig. 4G–I) did not demonstrate changes in RAD51 foci formation at either 6 or 16 hours. CDK4/6 inhibitor–resistant cell lines still formed RAD51 foci and retained the ability to perform HR, but the addition of a CDK4/6 inhibitor did not further suppress the repair capacity of these cells. Representative foci are shown in both CDK4/6 inhibitor–sensitive (Supplementary Fig. S7A and S7B) and CDK4/6 inhibitor–resistant (Supplementary Fig. S7C and S7D) cells.

CDK4/6 inhibition does not suppress NHEJ repair

To understand how CDK4/6 inhibition may affect NHEJ efficiency, we used a transient GFP reporter system (22) to assess NHEJ proficiency in MCF-7 (Fig. 5A) and CAMA-1 (Fig. 5B) cells. In this system, CDK4/6 inhibitor monotherapy did not affect NHEJ efficiency in either cell line compared with vehicle controls. As a control, treatment with 1 μmol/L NU7441 (a DNAPK inhibitor) significantly decreases NHEJ activity in both MCF-7 and CAMA-1 cells, but the CHK1/2 inhibitor AZD7762 does not affect NHEJ repair efficiency. Furthermore, when combined with radiotherapy, we observed stable or increased expression of the NHEJ protein mediator pKu80 in MCF-7 (Fig. 5C), CAMA-1 (Fig. 5D), and T47D cells (Fig. 5E) suggesting that NHEJ repair was not inhibited and may be activated in response to decreases in HR. In addition, at concentrations that are double the IC50 values of each CDK4/6 inhibitor in MCF-7 cells, pKu80 expression was significantly higher in cells treated with the combination of drug and radiotherapy (Fig. 5F).

If CDK4/6 inhibition suppresses the ability of ER+ breast cancer cells to undergo HR, NHEJ becomes the predominant form of double-stranded DNA (dsDNA) break repair. Thus, we hypothesized that combining CDK4/6 inhibition with pharmacologic or genetic inhibition of proteins in the NHEJ pathway would be synergistic. In both MCF-7 and T47D cells (Fig. 5G and J), palbociclib in combination with 500 nmol/L NU7441 leads to extremely significant levels of radiosensitization (rER: 1.80–3.46) compared with either compound alone. However, in MCF-7 cells, pharmacologic CHK1/2 inhibition is not synergistic with CDK4/6 inhibition, consistent with the hypothesis that CDK4/6 inhibitors act redundantly to suppress HR repair. Similar results were obtained using genetic knockdown of Ku70 (XRCC6) and RAD51 (RAD51) in MCF-7 cells (Fig. 5H and I). Although the ability of single-agent DNAPK inhibition to radiosensitize MCF-7 cells is retained in MCF-7 PalAR cells (Fig. 5K), the addition of palbociclib does not lead to additional or synergistic levels of radiosensitization.

These studies demonstrate that CDK4/6 inhibition impairs the ability of cells to undergo HR and may shunt dsDNA break repair through the NHEJ pathway. In our models, neither immunofluorescent γH2AX foci (Supplementary Fig. S8A, S8B, S8E, and S8F) or γH2AX total protein (Supplementary Fig. S8C and S8D) were significantly different between cells treated with radiotherapy alone (2 Gy) or the combination of radiotherapy and palbociclib, suggesting that combination treatment did not significantly affect the persistence of dsDNA damage in the cell. In our proteomic analysis using RPPA, we did not see any changes in γH2AX (pS139) expression in our MCF-7 cells treated with 75 nmol/L palbociclib (Fig. 1I). To further confirm this, we performed the neutral COMET assay in MCF-7 cells to detect changes in dsDNA breaks (Supplementary Fig. S8G and S8H). Although radiotherapy (4 Gy) caused an increase in dsDNA breaks at both 6 and 16 hours after radiotherapy, CDK4/6 inhibition did not potentiate a delay in dsDNA break repair. Thus, the ability of cells to repair dsDNA breaks in breast cancer cells treated with CDK4/6 inhibition and radiotherapy may be limited to low-fidelity NHEJ repair.

CDK4/6 inhibition radiosensitizes ER+ breast cancer cells in vivo

To understand whether CDK4/6 inhibition leads to clinically relevant levels of radiosensitization in vivo, we generated orthotopic xenograft models with the MCF-7 cells (Fig. 6A). In the combination group, palbociclib treatment was started one day before fractionated radiotherapy and was discontinued after the last fraction to measure the radiosensitizing effects of CDK4/6 inhibition with palbociclib independent from its single-agent efficacy. Treatment with the combination of palbociclib and radiotherapy significantly suppressed tumor growth (P < 0.01; Fig. 6B) and prolonged time to tumor doubling (P < 0.0001; Fig. 6C) compared to mice treated with radiotherapy or palbociclib alone. These treatments did not lead to any visible toxicities or significant changes in body weights of mice (Fig. 6D) throughout the duration of the study, suggesting that the therapy was generally well tolerated. In addition, we calculated the expected and observed FTV (26) for each treatment condition (Supplementary Table S6) and our results suggest that combination treatment with palbociclib and radiotherapy has synergistic (expected/observed ratio > 1) rather than additive effects.

In this study, we demonstrate that short-term treatment of ER+ breast cancer cell lines with the CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib led to alterations in many cellular pathways, including suppression of cell-cycle signaling and changes in the DNA damage response (Fig. 1). In ER+ breast cancer cells that are sensitive to CDK4/6 inhibitor monotherapy, the combination of CDK4/6 inhibition and ionizing radiotherapy led to significant radiosensitization with each of the three clinically approved CDK4/6 inhibitors (Fig. 2). This radiosensitizing ability, however, was lost in ER+ breast cancer cells with acquired resistance to CDK4/6 inhibition and was not observed in normal breast epithelial cells (Fig. 3; Supplementary Fig. S5). Mechanistically, the radiosensitization observed in CDK4/6 inhibitor–sensitive models was mediated by impaired HR that shunted dsDNA break repair toward error-prone NHEJ (Fig. 5). In contrast, both HR and NHEJ repair remained intact in CDK4/6 inhibitor–resistant cell lines (Figs. 4 and 5). In xenograft models of ER+ breast cancer, CDK4/6 inhibition led to tumor radiosensitization (Fig. 6). Taken together, these results demonstrate that the combination of CDK4/6 inhibition and radiotherapy is a potentially effective strategy for the radiosensitization of ER+ breast cancer that is lost in cells that have become resistant to CDK4/6 inhibitor monotherapy. Our data also suggest that concurrent administration of CDK4/6 inhibition with radiotherapy (instead of adjuvant therapy) may be a more effective strategy to decrease the rates of disease recurrence in patients with ER+ breast cancer at high risk of locoregional recurrence and that this strategy warrants clinical investigation.

In contrast to the conventional use of CDK4/6 inhibitors in the metastatic setting, our work challenges the standard treatment paradigm and highlights the therapeutic potential of using CDK4/6 inhibitors in combination with ionizing radiotherapy to radiosensitize CDK4/6 inhibitor–naïve ER+ breast cancers. In contrast to studies that seek to overcome CDK4/6 inhibitor resistance or propose therapeutic alternatives for CDK4/6 inhibitor–resistant tumors, our approach in combining CDK4/6 inhibitors and radiotherapy is a novel therapeutic strategy that can be utilized prior to the development of drug resistance; thus, this approach has the potential to cure women prior to the development of metastatic disease. Finally, in our study, all three clinically approved CDK4/6 inhibitors demonstrated the ability to radiosensitize ER+ breast cancer cell lines at similar concentrations, suggesting that this effect could be achieved in patients regardless of the specific CDK4/6 inhibitor chosen for therapy.

In current practice, radiotherapy is only given in combination with CDK4/6 inhibitors for palliative management in patients with metastatic disease. While there have been a few studies that report additional skin and gastrointestinal toxicities for these patients (29, 30), recent analyses report that combination therapy in the palliative setting has been generally well tolerated (31–34). Our data in normal breast epithelial cells suggested that CDK4/6 inhibition did not potentiate radiotherapy effects and therefore should be well tolerated by the normal breast tissue when translated clinically.

It is important to note that our in vivo murine studies were designed to test the effect of low-dose CDK4/6 inhibition (25 mg/kg) as a radiosensitizing strategy, rather than the efficacy of continued combination therapy at standard, optimal doses (50–100 mg/kg). Because drug was not continued after completion of fractionated radiotherapy, we would expect that long-term CDK4/6 inhibition after completion of radiotherapy would lead to even further reduction in overall tumor burden. Importantly, our data suggest that much lower doses of CDK4/6 inhibition can confer radiosensitivity, and one potential translational strategy would be to use these low doses in combination with radiotherapy which would potentially limit the frequency of systemic toxicities that lead to the discontinuation of therapy. Alternatively, future studies will assess whether monotherapy doses of CDK4/6 inhibition lead to an even greater degree of radiosensitization with an acceptably low toxicity profile. These two competing strategies are currently being considered in the planned phase I/II clinical trials testing this combination treatment.

While this work may be beneficial for patients with ER+ breast cancer, our study may also provide valuable mechanistic insights that could be applied to other cancers where CDK4/6 inhibitors are being studied preclinically. Indeed, whether CDK4/6 inhibitor–mediated radiosensitization is clinically effective in other subtypes of breast cancer (basal-like, HER2 enriched, etc.) remains an unanswered question, as well as if this is effective in other histologies, including invasive lobular or inflammatory breast cancer. Previous studies have, however, demonstrated that CDK4/6 inhibition may radiosensitize head and neck squamous cell carcinomas (35, 36), glioblastomas (37, 38), and colorectal and lung cancer cell lines (39). In line with findings in head and neck squamous cell carcinoma cell lines (35, 36), impaired HR efficiency might be important for CDK4/6 inhibitor–mediated radiosensitization in multiple cancer types.

Along the same lines, other preclinical studies performed in pancreatic cancer (40) and triple-negative breast cancer (20, 41) cell lines have suggested that CDK4/6 inhibition impairs HR efficiency after administration of cytotoxic chemotherapies or radiotherapy (42). This will be an important clinical consideration for future studies with CDK4/6 inhibitors as radiosensitizing agents, as cytotoxic chemotherapies are routinely used to treat patients with ER+ breast cancer in the neoadjuvant setting. However, in contrast to studies in lung and colorectal cancer cell lines that suggest that radiosensitization is p53 dependent (39), our data showed that CDK4/6 inhibitor–mediated radiosensitization occurs in both p53 wild-type (MCF-7, ZR-75–1) and p53 mutant (T47D, CAMA-1) models. In this study, all of our models express the tumor suppressor RB1 which has recently been shown to directly promote HR in breast cancer cell lines (43).

There are some limitations to this study that need to be considered. Although we focused on CDK4/6 inhibition and the DNA damage response, other mechanisms of radiosensitization may play a minor role in this phenotype. CDK4/6 inhibitor monotherapy has been shown to increase apoptosis in triple-negative breast cancer cell lines (44–46), but in our models we did not see an increase in apoptosis with CDK4/6 inhibition or combination treatment (Supplementary Fig. S9). There are conflicting reports about the effect of CDK4/6 inhibition on senescence in breast and other cancer types (47–52), and further studies could address any potential contributions of senescence to the radiosensitization phenotype that we see in ER+ breast cancer models. We also did not explore mechanisms of single-strand break repair, such as mismatch repair, base excision repair, or nucleotide excision repair, but our transcriptomic data (Fig. 1) suggested that these pathways could play a minor role in radiosensitization. Finally, confirmatory animal studies demonstrating radiosensitization in CDK4/6 inhibitor treatment–naïve patient-derived xenograft (PDX) models and lack of radiosensitization in CDK4/6 inhibitor–resistant PDXs (from women whose disease progressed on CDK4/6 inhibitor therapy) are needed. These studies were underway when the COVID-19 pandemic arose and are still planned when circumstances allow.

It is possible that other CDK inhibitors may be able to radiosensitize ER+ breast cancer cells. Flavopiridol, a nonspecific CDK inhibitor, has been shown to radiosensitize cancer cell lines (53, 54) and to potentiate cell death after cytotoxic therapy (55), though it is has an unacceptable safety profile that has prevented its clinical development (56). Furthermore, studies of CDK12/13 in triple-negative breast cancer have demonstrated changes in radiotherapy sensitivity based on direct interaction with transcriptional machinery and changes in polyadenylation (57), and support the idea that radiosensitization in ER+ breast cancers may be achieved with inhibition of other CDKs as well. Finally, there is recent evidence to suggest that hormone therapy and CDK4/6 inhibitor resistance may lead to alterations in genes like AKT1 and AURKA that are involved in DNA repair (58), which may have important clinical implications for patients receiving both types of therapy.

In conclusion, our results suggest that CDK4/6 inhibitor therapy would be effective in decreasing tumor growth in patients with ER+ breast cancer by radiosensitizing tumor cells during fractionated radiotherapy. Our data also suggests that the development of CDK4/6 inhibitor resistance with one drug leads to cross-resistance with the others in its class, consistent with what others have shown, which is an important clinical consideration as clinicians start to use CDK4/6 inhibitors in the adjuvant setting with radiotherapy. We also found that CDK4/6 inhibitor–mediated radiosensitization can be used as a therapeutic strategy in the absence of (or prior to) the initiation of hormone therapies, but future studies will seek to understand the interaction between CDK4/6 inhibition and radiotherapy with concurrent endocrine therapy. A complete understanding of the mechanism of CDK4/6 inhibitor–mediated radiosensitization will provide further insight into future treatment protocols and strategies to more effectively treat patients with ER+ breast cancers.

A.M. Pesch reports grants from NIGMS T32GM007767 and NCI F31CA254138 during the conduct of the study. A.R. Michmerhuizen reports grants from NIGMS T32-GM007315 and NIGMS T32-GM113900 during the conduct of the study. L.J. Pierce reports other from UpToDate (royalties as section reviewer and author) outside the submitted work; in addition, L.J. Pierce has two patents on signatures to identify local control following radiotherapy, one owned by the University of Michigan and one by PFS Genomics, neither licensed and both outside the submitted work. C. Speers reports other from PFS Genomics (co-founder and unpaid consultant) outside the submitted work. No potential conflicts of interest were disclosed by the other authors.

A.M. Pesch: Conceptualization, data curation, formal analysis, investigation, methodology, writing-original draft, writing-review and editing. N.H. Hirsh: Data curation, formal analysis, investigation, writing-review and editing. B.C. Chandler: Data curation, formal analysis, investigation, visualization, methodology, writing-review and editing. A.R. Michmerhuizen: Data curation, formal analysis, investigation, writing-review and editing. C.L. Ritter: Formal analysis, investigation, writing-review and editing. M.P. Androsiglio: Investigation, writing-review and editing. K. Wilder-Romans: Data curation, formal analysis, investigation, writing-review and editing. M. Liu: Formal analysis, investigation, writing-review and editing. C.L. Gersch: Conceptualization, resources, writing-review and editing. J.M. Larios: Conceptualization, resources, writing-review and editing. L.J. Pierce: Supervision, funding acquisition, project administration, writing-review and editing. J.M. Rae: Resources, supervision, funding acquisition, project administration, writing-review and editing. C.W. Speers: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing.

A.M. Pesch is supported by T32-GM007767, F31-CA254138, and the University of Michigan Center for the Education of Women (CEW+) Irma M. Wyman Fund. A.R. Michmerhuizen is supported by T32-GM007315 and T32-GM113900. B.C. Chandler is supported by T32-CA140044. A.M. Pesch, A.R. Michmerhuizen, and B.C. Chandler are all supported by Rackham Graduate School Research Grants.

The authors would also like to thank the Breast Cancer Research Foundation (N02600 to L.J. Pierce, N003173 to J.M. Rae) and the University of Michigan Rogel Cancer Center (P30CA046592 and G023324). Finally, the authors greatly appreciate the contributions of the University of Michigan core facilities (Flow Cytometry, Advanced Genomics, Bioinformatics), and the M.D. Anderson Functional Proteomics RPPA Core Facility (NCI #CA16672) for support and technical assistance with the experiments in this manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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