Abstract PO-028: RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of estrogen receptor positive (ER+) breast cancers
Authors: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Erin Cobain, Lori J. Pierce, James M. Rae, Corey Speers
Journal: Clinical Cancer Research (2021)

Abstract Purpose: Fractionated radiation (RT) is used in the adjuvant setting for locoregional control and to prevent the development of metastatic lesions in estrogen receptor positive (ER+) breast cancers. We previously demonstrated that inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) along with RT leads to the radiosensitization of ER+ breast cancers, but the exact mechanism by which this occurs is unknown. We hypothesized that the presence of RB is necessary for effective double strand repair of radiation-induced DNA damage mediated through homologous recombination (HR), and that this is prevented using CDK4/6 inhibition. Methods: Pharmacological CDK4/6 inhibition was achieved using three FDA approved CKD4/6 inhibitors: palbociclib, ribociclib, and abemaciclib. Genetic knockdown of RB1 was performed using siRNA and knockout was achieved using a CRISPR-Cas9 system. Parental and RB1 knockdown/CRISPR MCF-7 and T47D cells were treated for 72 hours to assess drug sensitivity. Cells were pretreated with a CDK4/6 inhibitor one hour prior to RT and colony formation was quantified to assess changes in radiosensitivity. MCF-7 cells expressing an HR-specific GFP reporter were used to assess HR competency. RB, yH2AX, and RAD51 cellular localization following RT and CDK4/6 inhibition was assessed using immunofluorescence assays. Flow cytometry with propidium iodide staining was used to assess cell cycle distribution. Protein expression was assessed by immunoblotting. Results: CDK4/6 inhibition with palbociclib, ribociclib, and abemaciclib + RT radiosensitizes ER+ breast cancer cells at sub-IC50 concentrations in vitro (rER: 1.21 – 2.05) through impaired HR, which we confirmed using MCF-7 cells that express a stable HR-GFP reporter system (p &amp;lt; 0.01). RB1 knockdown decreased single-agent efficacy of CDK4/6 inhibition on the proliferation of ER+ breast cancer cell lines, leading to an increase in the IC50 for each CDK4/6 inhibitor. Palbociclib, ribociclib, and abemaciclib failed to induce G1 cell cycle accumulation after RB1 knockdown (p &amp;gt; 0.05). Genetic knockdown of RB1 led to a decrease in the ability of breast cancer cells to perform HR-directed DNA repair (p &amp;lt; 0.01) independent of drug treatment, and further suppression of HR with CDK4/6 inhibition was lost in MCF-7 and T47D cells lacking RB expression (p &amp;gt; 0.05). Furthermore, RB protein is necessary for CDK4/6i mediated radiosensitization as evidenced by the abrogation of radiosensitization in RB null isogenic models of ER+ breast cancer (MCF-7 rER: 0.97 ± 0.13). Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization and HR suppression is dependent on RB expression. Thus, RB might serve as an effective biomarker for patient selection in future clinical trials that seek to combine CDK4/6 inhibition + RT. Citation Format: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Erin Cobain, Lori J. Pierce, James M. Rae, Corey Speers. RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of estrogen receptor positive (ER+) breast cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-028.