Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in >2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R 2 = 0.72, p < 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9–3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22–1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors.

tumor volume was calculated using the equation: volume = (length × width 2) × π/6. Additive and synergistic effects were calculated using the FTV method as previously described (59, 60). Study approval. The protocols used in this study were approved by the IACUC of the University of Michigan. Statistics. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). A log-rank (Mantel-Cox) test was used for survival curve analyses. One-way ANOVA was performed for BC subtype analysis. A 2-sided Student's t test and 1-way ANOVA with Dunnett's multiple comparisons test were used for in vitro statistical analyses. A 1-way ANOVA with Dunnett's multiple comparisons test and a log-rank (Mantel-Cox) test were used for in vivo analyses. A P value of 0.05 or less was considered statistically significant.

Michmerhuizen et al. Seviteronel Radiosensitizes AR+ TNBC RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC 50 > 10 mmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12-1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT AE PARPi in women with IBC (SWOG 1706, NCT03598257).

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 &lt; 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 &lt; 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P &gt; 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.