Bcl-xL inhibition radiosensitizes PIK3CA/PTEN wild-type triple negative breast cancers with low Mcl-1 expression.

Patients with radioresistant breast cancers, including a large percentage of women with triple negative breast cancer (TNBC), demonstrate limited response to radiation (RT) and increased locoregional recurrence; thus, strategies to increase the efficacy of RT in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant PIK3CA/PTEN TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of PIK3CA/PTEN status (rER: 0.95 - 1.07). In wild-type PIK3CA/PTEN TNBC, pan Bcl-2 family inhibition or Bcl-xL specific inhibition with RT led to increased levels of apoptosis (p < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated PTEN knockout in wild-type PIK3CA/PTEN MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor-mediated radiosensitization (rER: 0.94 - 1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02 - 1.04) but transient MCL1 knockdown in CAL-51 cells promoted Bcl-xL-inhibitor mediated radiosensitization (rER 2.35 ± 0.05). In vivo, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence.

Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers.

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.

A Signature That May Be Predictive of Early Versus Late Recurrence After Radiation Treatment for Breast Cancer That May Inform the Biology of Early, Aggressive Recurrences.

PURPOSE: Unmet clinical needs in breast cancer (BC) management include the identification of patients at high risk of local failure despite adjuvant radiation and an understanding of the biology of these recurrences. We previously reported a radiation response signature and here extend those studies to identify a signature predictive of recurrence timing (before or after 3 years). METHODS AND MATERIALS: Two independent patient cohorts were used. The training cohort included 119 patients with in-breast tumor recurrence (343 total), and the validation testing cohort had 16 patients with recurrences (112 total). All patients received radiation treatment after breast-conserving surgery. Initial feature selection used Spearman rank correlation, and a linear model was trained and locked before testing and validation. Cox regression was used for univariate and multivariable analyses (UVA and MVA, respectively). Biologically related concepts were identified using gene set enrichment analysis. RESULTS: Spearman correlation identified 485 genes whose expression was significantly associated with recurrence time (early vs late). Feature reduction further refined the list to 41 genes retained within the signature. In training, the correlation of score to recurrence time was 0.85 (P value < 1.3 × 10-31) with an area under the curve (AUC) of 0.91. Application of this early versus late signature to an independent BC testing and validation set accurately identified patients with early versus late recurrences (Spearman correlation = 0.75, P value = .001, AUC = 0.92, sensitivity = 0.75, specificity = 1.0, positive predictive value = 1.0, and negative predictive value = 0.8). Unique associations of breast cancer intrinsic subtype to timing of local recurrence were identified. In UVA and MVA the early versus late recurrence signature remained the most significant factor associated with recurrence. Gene set enrichment analysis identified proliferation and epidermal growth factor receptor concepts associated with early recurrences and luminal and ER-signaling pathways associated with late recurrences. Knockdown of genes associated with the early and late recurrences demonstrated novel effects on proliferation and clonogenic survival, respectively. CONCLUSIONS: We report a breast cancer gene signature that may identify patients unlikely to respond to adjuvant radiation and may be used to predict timing of recurrences with implications for potential treatment intensification and duration of follow-up for women with breast cancer treated with radiation.

Seviteronel, a Novel CYP17 Lyase Inhibitor and Androgen Receptor Antagonist, Radiosensitizes AR-Positive Triple Negative Breast Cancer Cells.

Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15-35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 µM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20-1.89 in models of TNBC with high AR expression. AR-negative (AR-) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 µM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an in vivo AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and 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.

TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination.

Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients' tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown; however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.

PARP1 Inhibition Radiosensitizes Models of Inflammatory Breast Cancer to Ionizing Radiation.

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 (IC50 > 10 µmol/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 ± PARPi in women with IBC (SWOG 1706, NCT03598257).

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer.

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 (R2 = 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.