Abstract 6270: A radiosensitizer screen identifies a novel role for MDM2 inhibition in the radiosensitization of ER+ breast cancers in a p53 dependent manner
Authors: Ritter Cassandra L. , Chandler Benjamin C. , Pesch Andrea M. , Michmerhuizen Anna R. , Hirsh Nicole , Zhang Amanda , Ward Tanner , Cremona Mattia , Hennessy Bryan , Pierce Lori J. , Speers Corey W. 
Published: September 2020

Abstract
Background: Radiation therapy (RT) is a mainstay of treatment for most women with breast cancer (BC). Despite this treatment, response remains heterogenous for women with estrogen receptor positive (ER+) BC. Thus, approaches that result in radiosensitization of aggressive ER+ disease are critically needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data from ER+ models treated +/-RT to identify potential mediators of RT resistance.
Methods: Clonogenic survival assays were used to determine RT sensitivity of 21 BCC lines as well as radiosensitization with drug treatment. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Microarray and RPPA data was used for differential gene/protein expression and pathway analysis. AlamarBlue was used to determine IC50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP was used to measure apoptosis and Cyclins A and E to measure cell cycle progression.
Results: Our radiosensitizer screen nominated the MDM2 inhibitor (JNJ-26854165) as one of the most effective drugs in treating RT-resistant BC cell lines (R2= 0.43, p-value &lt;0.01). In addition, differential gene expression and pathway analysis in multiple non-overlapping ER+ BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced by RT in ER+ cell lines. Within these pathways MDM2 was significantly overexpressed after RT compared to RT- in ER+ p53 wild-type (WT) cells. However, in p53 mutant (MT) cell lines MDM2 was not differentially expressed suggesting MDM2 may mediate radioresistance in a p53 dependent manner. The MDM2 inhibitor AMG-232 inhibited cell growth in the p53 WT cell lines MCF-7 and ZR-75-1 (IC-50 values of 554nM and 264nM, respectively). In contrast, p53 MT ER+ cell lines were not sensitive to MDM2 inhibition (IC-50&gt; 10uM). Clonogenic survival assays demonstrated that MDM2 inhibition at sub-IC50 doses leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.37-1.66;ZR751 rER: 1.30-1.65). In contrast, p53 MT ER+ cells did not demonstrate significant radiosensitization (T47D rER: 0.94-1.11). Combination of AMG-232 and RT led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Additionally, combination treatment led to differential cylin expression in p53 WT cells but not p53 MT cells. In vivo studies testing MDM2 inhibition with RT in p53 WT and MT orthotopic and PDX models are ongoing.
Conclusions: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC. Additionally, MDM2 inhibition confers radiosensitization in a p53 dependent manner in ER+ BC and may represent a tractable clinical strategy in women with p53 WT BC.
Citation Format: Cassandra L. Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Amanda Zhang, Tanner Ward, Mattia Cremona, Bryan Hennessy, Lori J. Pierce, Corey W. Speers. A radiosensitizer screen identifies a novel role for MDM2 inhibition in the radiosensitization of ER+ breast cancers in a p53 dependent manner [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 6270.