Case Western Reserve School of Medicine Cleveland, OH
R. Abou Zeidane1, S. Lichtman-Mikol1, C. Nino2, C. Ritter3, A. Pesch2, A. R. Michmerhuizen2, B. Chandler2, N. Hirsh3, T. Ward2, Y. Kaur2, B. Hauk1, D. Gurdak1, M. Tao1, V. Mercer1, L. J. Pierce2, J. Lyons1, and C. Speers1; 1Department of Radiation Oncology, University Hospitals Seidman Cancer Center and Case Western Reserve University, Cleveland, OH, 2Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 3University of Michigan, Ann Arbor, MI
Purpose/Objective(s): Most women with breast cancer (BC) receive radiation therapy (RT) as part of the standard of care, however its efficacy remains inadequate for those with locally advanced disease. Thus, more effective radiosensitization strategies are needed. We performed a radiosensitizer screen to identify potential mediators of RT resistance and identify novel therapeutic targets.Materials/
Methods: We screened 130 clinically available drugs for radiosensitization using the Genomics of Drug Sensitivity in Cancer database and analyzed their effectiveness based on clonogenic survival with IC50 values. MDM2 inhibitors navtemadlin (AMG-232) and alrizomadlin (APG-115) were used in p53 wild-type and mutant models of estrogen receptor (p53-WT:MCF-7, p53-MT:T47D) and ER-negative BC (p53-WT: CAL-51, p53-MT: MDA-MB-231). Alterations to DNA damage response were assessed with gamma-H2AX Immunofluorescence. Flow Cytometry with Annexin V staining was used to evaluate apoptosis and with Propidium Iodide staining for cell cycle progression. ß-gal staining was used to evaluate senescence. In vivo efficacy of MDM2 inhibition and RT combination was evaluated with CAL-51 and CAL-51 p53 CRISPR xenograft models. Results: An MDM2 inhibitor was nominated as a potent radiosensitizer (R2 = 0.43, p-value <0.01). MDM2 was significantly overexpressed after RT compared to no RT in p53-WT cells. Cell growth was decreased with navtemadlin and alrizomadlin in p53-WT cells (IC50s:264-592nM) but not in p53-MT cells (IC50s >10µm). MDM2 inhibition with either drug radiosensitized p53-WT cells (rERs: 1.81-2.85) but not p53-MT cells (rERs: 1.00-1.03), regardless of hormone receptor status, or p53 CRISPR 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. Additionally, in vivo experiments showed MDM2 inhibition, using both genetic and pharmacological (navtemadlin) inhibition in combination with RT led to a significantly increased time to tumor tripling in CAL51-WT cells, but not in isogenic CAL-51 p53 CRISPR models (tripling time 31 days in WT vs not reached in RT+MDM2 KO or navtemadlin treated groups, p<0.01; 26 days vs. 30 days in WT vs RT+navtemadlin treated p53 CRISPR CAL-51 models, p>0.4. Mechanistically, combination MDM2 inhibition with navtemadlin and RT resulted in significantly increased apoptosis and senescence in p53-WT cell lines (CAL-51, ZR-75, and CAMA-1 cells % apoptotic: DMSO 6-9%, combination 31-54%, p<0.01; senescent cells: DMSO 3-5%, combination 37-64%, p<0.01) Conclusion: These results demonstrate the combination of RT and MDM2 inhibition may be an effective therapeutic strategy in patients with p53-WT BC which represent the majority of BCs, regardless of hormone receptor status. Clinical trial development is currently underway to test this in women with locally advanced p53-WT BCs at high risk of locoregional recurrence.
