Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Standard radiation (RT) therapy does not reliably provide locoregional control for women with multi-node positive and triple-negative (TNBC) breast cancers. We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor positive (ER+) cells, but also TNBC that express retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB wild-type TNBC (n=4, rER 1.49 -2.22), but failed to radiosensitize RB-null TNBC (n=3, rER: 0.84 -1.00). RB expression predicted response to CDK4/6i + RT (R 2 =0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88 -1.13) after RB1 knockdown in isogenic and non-isogenic models. CDK4/6i suppressed homologous recombination (HR) in RB wild-type cells, but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB re-expression. Radiosensitization was independent of non-homologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts, but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult to control RBintact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

Background: Clinical management of BC includes radiation therapy (RT), with most women receiving RT as part of their treatment. Although effective, many women develop locoregional recurrence, including a disproportionate number of women with triple-negative or inflammatory BC. Unfortunately, the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. We hypothesized that transcriptomic and proteomic changes that occur after ionizing radiation in intrinsically radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response. Methods: Intrinsic radiosensitivity across all 10 cell lines was measured with clonogenic survival assays as the surviving fraction (SF) after 2 Gy RT. Gene expression changes were assessed by RNA-Seq 24 hours after 4 Gy RT. For long-course RT, cell lines were treated with fractionated RT (2 Gy x 5 fractions). For in vivo mouse xenograft experiments mice received fractionated RT (2 Gy x 6 fractions). Differential gene expression analysis with DeSeq2 was performed on all samples, followed by pathway analysis with Advaita Bioinformatics’ iPathwayGuide. Protein was collected 1, 12, and 24 hours after RT for RPPA analysis evaluating expression changes in 100 proteins and phospho-proteins with SuperCurve. Results: Clonogenic survival identified a wide range of radiation sensitivity in human BC cell lines (SF 83% - 19%) with no significant correlation (r %lt 0.3) to intrinsic BC subtype. The most highly affected pathways in both resistant and sensitive cell lines 24 hours after RT include cell cycle, cellular senescence, and estrogen signaling pathways. For the long-course RT samples, several pathways were significantly altered in fractionated samples only, including MAPK and Hippo signaling and EGFR tyrosine kinase inhibitor resistance. From the in vivo experiments, pathways uniquely affected in the in vivo samples include IL-17 signaling and transcriptional misregulation in cancer. From the proteomic data, we found that proteins including p53, Bcl-2 family proteins, and cell cycle proteins exhibit expression changes after 1 hour. A significant number of pathways (N=69, p %lt 0.01, FDR 0.05) were affected in radioresistant BC models compared to radiosensitive cell lines and these pathways may underlie intrinsic radioresistance. Conclusions: Ionizing radiation induces transcriptomic and proteomic expression changes that differ between intrinsically sensitive and resistant BC models in both single fraction and fractionated studies. Pathways identified in these analyses offer potential insight into the mechanisms underlying intrinsic radioresistance and suggest biologic vulnerabilities that may be targeted to more effectively treat women at a high risk of local BC recurrence. Genome wide CRIPSR-Cas9 screens are currently underway in these breast cancer models to confirm these vulnerability targets. Citation Format: Breanna N. McBean, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Meilan Liu, Alan P. Boyle, Corey W. Speers. Molecular mechanisms of intrinsic radioresistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2401.

Purpose Radiation therapy (RT) and hormone receptor (HR) inhibition are used for the treatment of HR-positive breast cancers; however, little is known about the interaction of the androgen receptor (AR) and estrogen receptor (ER) in response to RT in AR-positive, ER-positive (AR+/ER+) breast cancers. Here we assessed radiosensitisation of AR+/ER+ cell lines using pharmacologic or genetic inhibition/degradation of AR and/or ER. Methods Radiosensitisation was assessed with AR antagonists (enzalutamide, apalutamide, darolutamide, seviteronel, ARD-61), ER antagonists (tamoxifen, fulvestrant) or using knockout of AR. Results Treatment with AR antagonists or ER antagonists in combination with RT did not result in radiosensitisation changes (radiation enhancement ratios [rER]: 0.76–1.21). Fulvestrant treatment provided significant radiosensitisation of CAMA-1 and BT-474 cells (rER: 1.06–2.0) but not ZR-75-1 cells (rER: 0.9–1.11). Combining tamoxifen with enzalutamide did not alter radiosensitivity using a 1 h or 1-week pretreatment (rER: 0.95–1.14). Radiosensitivity was unchanged in AR knockout compared to Cas9 cells (rER: 1.07 ± 0.11), and no additional radiosensitisation was achieved with tamoxifen or fulvestrant compared to Cas9 cells (rER: 0.84–1.19). Conclusion While radiosensitising in AR + TNBC, AR inhibition does not modulate radiation sensitivity in AR+/ER+ breast cancer. The efficacy of ER antagonists in combination with RT may also be dependent on AR expression.

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