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Background: Acquired mutations in oncogenic drivers in response to systemic therapies is a common mechanism of treatment resistance. How such mutations alter response to radiation therapy is poorly understood. The increasing adoption of radiation therapy in the treatment of oligometastatic disease further amplifies the need to define the role of such mutations in modifying response to radiation therapy. Over 70% of breast cancers are Estrogen Receptor (ER)-positive. Endocrine therapies, which block ER signaling, are the mainstay in the treatment of such cancers. Acquired mutations in the gene encoding for ER (ESR1) are a common mechanism of acquired resistance to endocrine therapies in metastatic breast cancer. In this study, we define the role of ESR1 mutations in altering response to radiation therapy in pre-clinical models of ER-positive breast cancer. Methods: To test if ESR1 mutations influence response to radiation therapy, we employed ER-positive MCF7 and T47D cell lines which have been genome edited to knockin two of the most common ESR1 mutations found in breast cancer patients (Y537S and D538G). Clonogenic survival assays were carried out to characterize differences in radiation sensitivity of these cells. γ-H2AX and Comet assays were employed to study the differences in residual unrepaired DNA damage following radiation in these cell lines. MCF7 cell line-derived xenografts harboring the wild type or mutant ESR1 were employed to study differences in radiation resistance of these tumors in vivo. Results: ER-positive cell lines harboring ESR1 mutations exhibited profound resistance to radiation therapy compared to cell lines harboring the wild type ESR1. These radioresistant cell lines also demonstrated significantly less unrepaired DNA damage following radiation therapy. Mice harboring ESR1 mutant xenografts showed remarkable resistance to radiation therapy. Conclusion: ESR1 mutations, which confer resistance to endocrine therapies, also confer profound resistance to radiation therapy. ESR1 mutations have been primarily reported in metastatic ER-positive breast cancer patients with prior history of treatment with endocrine therapies. Our findings suggest that oligometastatic ER-positive breast cancer patients who are being treated with radiation therapy (such as those enrolled in NRG BR002 trial) may show significant differences in response to radiation therapy based on their ESR1 mutation status. Recent studies are also exploring the use of neo-adjuvant endocrine therapy in non-metastatic ER-positive breast cancer patients. It is conceivable that some of these patients may acquire ESR1 mutations under selective pressure of neoadjuvant endocrine therapy and may respond poorly to subsequent adjuvant radiation therapy. Finally, our findings support potential personalization of radiation treatments in breast cancer patients based on ESR1 mutation status, heralding an era of precision radiation oncology. Citation Format: Nashir Udden, Qian Wang, Prasanna G Alluri. ESR1 mutations confer radiation resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-01.

Background: Approximately 15% of breast cancers lack expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER-2), and are referred to as Triple Negative Breast Cancer (TNBC). Clinical outcomes in patients with TNBC continue to be poor due to inherently aggressive biology of the disease and paucity of targeted therapies. In this study, we identified Cell Division Cycle protein 20 (CDC20), a key regulatory protein that mediates sister chromatid separation in mitosis, as a novel therapeutic target in TNBC. Methods: To uncover new drivers of TNBC, we nominated genes that are significantly overexpressed in patients with TNBC, compared to non-TNBCs. After identifying CDC20 as a top hit in this analysis, we used expression microarrays, RNA-seq analyses and Western blotting to assess CDC20 expression levels in breast cancer patients and cell lines. Kaplan-Meier analyses were performed to study the correlation between CDC20 expression levels and clinical outcomes, including recurrence-free survival, metastasis-free survival and overall survival. shRNA-mediated knockdown of CDC20 in TNBC cell lines was employed to study the role of CDC20 in TNBC proliferation, invasion, migration and xenograft growth in mice. Results: CDC20 was significantly overexpressed in TNBCs (compared to non-TNBCs) in The Cancer Genome Atlas (TCGA) breast dataset, and in several other independent breast cancer datasets, including Curtis, Stickeler and Kao. CDC20 expression was also significantly higher in TNBC cell lines compared to non-TNBC cell lines. Furthermore, expression levels of CDC20 were highly significantly correlated with clinical outcomes, including recurrence-free survival, metastasis-free survival and overall survival. shRNA-mediated knockdown of CDC20 expression in TNBC cell lines, MDA-MB-231 and MDA-MB-468, resulted in decreased proliferation, invasion and migration. Our preliminary findings suggest that CDC20 knockdown also results in inhibition of cell line-derived xenograft growth in mice. Conclusion: We identified CDC20 is a novel therapeutic target in TNBC. Our findings support development of pharmacologic strategies to inhibit CDC20 function as a potential targeted therapy against TNBC. Citation Format: Sharma J, Raicu I, Nguyen J, Udden N, Wang Q, Alluri P. CDC20 is a novel therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-20.

Background: Approximately 15% of breast cancers lack expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER-2), and are referred to as Triple-Negative Breast Cancer (TNBC). Due to a lack of targeted therapies, clinical outcomes in patients with TNBC continue to be poor. Likewise, due to the inherently aggressive biology of the disease, patients are forced to deal with the toughest breast cancer while having the least treatment options. In this study, we identified Cell Division Cycle protein 20 (CDC20), a key regulatory protein that mediates sister chromatid separation in mitosis, as a novel therapeutic target in TNBC. Methods: To uncover new drivers of TNBC, we nominated genes that are significantly overexpressed in patients with TNBC, compared to non-TNBCs. After identifying CDC20 as a top hit in this analysis, we used RNAseq analyses, expression microarrays, and Western blotting to assess CDC20 expression levels in breast cancer patients and cell lines. Kaplan-Meier analyses were formed to study the correlation between clinical outcomes and CDC20 expression levels, including recurrence-free survival, metastasis-free survival and overall survival. shRNA and siRNA-mediated knockdown of CDC20 in TNBC cell lines were employed to study the role of CDC20 in TNBC proliferation, invasion, and migration. Results: CDC20 was significantly overexpressed in TNBCs (compared to non-TNBCs) patients and is a predictor of poor clinical outcomes and radiation resistance. CDC20 expression was also significantly higher in TNBC cell lines compared to non-TNBC cell lines, reflecting what is seen in clinical data sets, such as in The Cancer Genome Atlas. Sh and siRNA-mediated knockdown of CDC20 expression resulted in decreased proliferation, invasion and migration in TNBC cell lines. Conclusions and future directions: Our findings support the development of pharmacologic strategies to inhibit CDC20 function as a potential targeted therapy against TNBC. Efforts are underway to validate our findings in vivo in xenograft models of TNBC in mice. Citation Format: Jayesh K. Sharma, Prasanna Alluri, Nashir Udden. CDC20 is a novel therapeutic target in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6378.