Abstract 1943: Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition
Authors: Pesch Andrea M. , Chandler Benjamin C. , Michmerhuizen Anna R. , Hirsh Nicole , Wilder-Romans Kari , Liu Meilan , Ward Tanner , Messinger Dana , Nino Charles , Ritter Cassandra , Rae James M. , Speers Corey W. 
Published: July 2021

Abstract
Purpose: Compared to other breast cancer subtypes, triple negative breast cancers (TNBC) derive the least benefit from adjuvant radiation (RT) which contributes to higher rates of locoregional recurrence. Thus, there is a critical need to identify clinical strategies to increase the effectiveness of RT therapy in TNBC.
Methods: Alamar blue proliferation assays were used to calculate half maximal inhibitory concentration (IC50) values for each Bcl-2 family inhibitor 72 hours after drug treatment. Clonogenic survival assays were used to evaluate radiosensitivity and to calculate the radiation enhancement ratio (rER) after combination treatment. Apoptosis was assessed through formation of cleaved PARP and annexin V/PI-based flow cytometry. Xenograft models with MDA-MB-231 cells and TNBC patient-derived xenografts (PDX4664) were used to assess radiosensitization in vivo.
Results: A novel radiosensitizer screen identified Bcl-2 family inhibition as a potentially effective treatment strategy in radioresistant breast cancer models. Single-agent response to pan Bcl-2 family inhibition (ABT-263) or Bcl-xL inhibition (WEHI-539, A-1331852) was more effective in PIK3CA wild type (wt) TNBC (IC50 &lt; 1µM) compared to PIK3CA mutant TNBC. Inhibition of apoptosis with ABT-263 led to radiosensitization of PIK3CA/PTEN wild-type TNBC cell lines (rER: 1.09-1.74), but had no effect on PIK3CA/PTEN mutant TNBC (rER: 0.87-1.18). Radiosensitization was observed to be Bcl-xL-dependent, with Bcl-xL inhibitor-specific radiosensitization (rER: 1.12-2.38) but a lack of Bcl-2 inhibitor (ABT-199, rER: 0.94 - 1.21) or MCL-1 inhibitor-mediated radiosensitization (S63845, rER: 0.91 - 1.06). In PIK3CA wt TNBC, combination treatment of Bcl-2 family inhibition and RT significantly increased the percent of apoptotic cells (p &lt; 0.001) and led to increased formation of cleaved PARP 48 hours after RT. Sensitivity to RT was dependent on expression of MCL-1, an anti-apoptotic protein that is overexpressed in PIK3CA/PTEN mutant TNBC. Overexpression of MCL-1 in PIK3CA/PTEN wild type TNBC rescued radioresistance (rER: 0.99-1.09), whereas co-inhibition of MCL-1 and Bcl-xL in PIK3CA/PTEN mutant TNBC was sufficient to overcome radioresistance (rER: 2.32 - 2.35). In vivo, nonspecific Bcl-2 family inhibition or specific Bcl-xL inhibition in combination with RT decreased tumor growth and increased time to tumor tripling (p &lt; 0.0001) in PIK3CA wt models of TNBC.
Conclusions: In this study, we demonstrated that inhibition of Bcl-2 family proteins in combination with RT led to increased levels of apoptosis and cell death in PIK3CA/PTEN wt - but not PIK3CA/PTEN mutant - TNBC and we identified MCL-1 as a critical mediator of this radiosensitIvity. Together, these results indicate that Bcl-xL inhibition may be a feasible clinical strategy for the radiosensitization of PIK3CA/PTEN wild-type TNBC.
Citation Format: Andrea M. Pesch, Benjamin C. Chandler, Anna R. Michmerhuizen, Nicole Hirsh, Kari Wilder-Romans, Meilan Liu, Tanner Ward, Dana Messinger, Charles Nino, Cassandra Ritter, James M. Rae, Corey W. Speers. Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition [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 1943.