Abstract 1386: The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers
Authors: Ritter Cassandra Lynne , Chandler Benjamin C. , Pesch Andrea M. , Michmerhuizen Anna R. , Hirsh Nicole , Ward Tanner , Zhang Amanda , Cremona Mattia , Pierce Lori J. , Hennessy Bryan , Speers Corey W. 
Published: July 2021

Abstract
Background​: Radiation therapy (RT) is standard in the treatment of many women with breast cancer (BC). Despite this, women with estrogen receptor positive (ER+) BC respond heterogeneously to RT. Radiosensitization methods for aggressive ER+ disease are 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 IC-50 values of the MDM2 inhibitor AMG-232. Western blot analysis of Cleaved PARP and Annexin V staining for FLOW was used to measure apoptosis and Cyclins A, E, B and p-Histone H3 and flow cytometry to measure cell cycle progression. yH2AX immunofluorescence was used to measure dsDNA breaks.
Results​: A MDM2 inhibitor (JNJ-26854165) was nominated as an effective drug in treatment for RT-resistant BC cell lines (R2​ = 0.43, p-value &lt;0.01) in our novel radiosensitizer screen. 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 in ER+ cell lines by RT. Within these MDM2 was significantly overexpressed after RT+ compared to RT- in ER+ p53 wild-type (WT) cells. In p53 mutant (MT) cell lines, however, MDM2 was not differentially expressed. This suggests MDM2 may mediate radioresistance in a p53 dependent manner. Cell growth in the p53​ ​WT cell lines MCF-7 and ZR-75-1 was inhibited by AMG-232, an MDM2 inhibitor (IC-50 values of 554nM and 264nM). p53 MT ER+ cell lines were not sensitive to MDM2 inhibition with this drug (IC-50&gt; 10uM). Clonogenic survival assays demonstrated that at sub-IC50 doses MDM2 inhibition leads to radiosensitization in p53 WT ER+ cell lines (MCF-7 rER: 1.17-2.13; ZR751 rER: 1.30-1.65), however, p53 MT ER+ cells were not radiosensitized (T47D rER: 0.94-1.11; CAMA-1 rER: 0.88-0.95). AMG-232 and RT combined led to an increase in apoptosis compared to RT alone in ER+ p53 WT cells but not p53 MT cells. Combination treatment led to differential cyclin and p-Histone H3 expression in p53 WT cells but not p53 MT cells. ​G1 cell cycle arrest was a secondary effect of MDM2 inhibition and radiation. Experiments investigating the role of dsDNA breaks in radiosensitization are ongoing.
Conclusions​: Our novel radiosensitizer screen identifies MDM2 as a potential mediator of radioresistance in ER+ BC in a p53-dependent manner and suggests that MDM2 targeting concurrent with RT may represent a tractable clinical strategy in women with locally advanced ER+, p53 WT BC.
Citation Format: Cassandra Lynne Ritter, Benjamin C. Chandler, Andrea M. Pesch, Anna R. Michmerhuizen, Nicole Hirsh, Tanner Ward, Amanda Zhang, Mattia Cremona, Lori J. Pierce, Bryan Hennessy, Corey W. Speers. The role of MDM2 inhibition in the radiosensitization of ER+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1386.