Abstract 215: MDM2 inhibition as a non-hormone dependent radiosensitizing strategy in p53 wild-type breast cancer models
Authors: Nino Charles A. , Ritter Cassandra L. , Pesch Andrea M. , Michmerhuizen Anna R. , Chandler Benjamin C. , Kaur Yashmeet , Fields Maria , Lerner Lynn , Pierce Lori J. , Speers Corey W. 
Published: June 2022

Background: Radiation therapy (RT) is standard in the treatment of most women with breast cancer (BC), though efficacy of RT remains inadequate for women with locally advanced BC. Thus, more effective radiosensitization strategies are needed. We performed a radiosensitizer screen paired with transcriptomic and proteomic data to identify potential mediators of RT resistance and identify novel therapeutic targets.

Methods: Microarray and RPPA data were used for gene/protein expression and pathway analysis. Clonogenic survival assays were performed to assess radiosensitization and calculate relative enhancement ratios (rERs) with MDM2 inhibitors navtemadlin (AMG-232) and alrizomadlin (APG-115) in p53 wild-type (WT) and mutant (MT) models of estrogen receptor positive (p53 WT: MCF-7, p53 MT: T47D) and triple-negative BC (p53 WT: CAL-51, p53 MT: MDA-MB-231). AlamarBlue was used to determine IC50 concentrations of MDM2 inhibitors. Clonogenic and IC50 experiments performed in parallel with p53 CRISPR knockout models of p53 WT cell lines. DNA damage response was assessed with γ-H2AX Immunofluorescence microscopy. Flow Cytometry with Annexin V staining was used to evaluate apoptosis and Propidium Iodide staining for cell cycle progression. Light microscopy with β-gal staining was used to evaluate senescence. In vivo efficacy of combination therapy was evaluated with CAL-51 and CAL-51 p53 CRISPR xenograft models.

Results: An 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 non-overlapping p53 WT BC cell lines treated +/-RT identified apoptosis, cell cycle, and p53 signaling as the top pathways induced in p53 WT cell lines by RT. MDM2 was significantly overexpressed after RT compared to no RT in p53 WT cells. Cell growth was decreased by MDM2 inhibition with navtemadlin and alrizomadlin in p53 WT cells (IC50s: 264-592nM) and not in p53 MT cells (IC50s &gt;10μm). MDM2 inhibition radiosensitized p53 WT cells (rERs: 1.81-2.85) but not p53 MT cells (rERs: 1.00-1.03). This phenotype was reversed in p53 CRISPR knockout cells (rERs: 1.06-1.12). MDM2 inhibition in combination with RT demonstrated delayed DNA damage repair compared to RT alone. p53 WT cells demonstrated increased G1 cell cycle arrest with RT and combination therapy. Combination MDM2 inhibition and RT resulted in increased apoptosis and senescence in p53 WT cell lines. In vivo xenograft studies are ongoing.

Conclusions: These results demonstrate the combination of RT and MDM2 inhibition may be an effective therapeutic strategy in patients with p53-wild type breast cancer, regardless of hormone receptor status.

Citation Format: Charles A. Nino, Cassandra L. Ritter, Andrea M. Pesch, Anna R. Michmerhuizen, Benjamin C. Chandler, Yashmeet Kaur, Maria Fields, Lynn Lerner, Lori J. Pierce, Corey W. Speers. MDM2 inhibition as a non-hormone dependent radiosensitizing strategy in p53 wild-type breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 215.