Search

Supplemental Figures (8) and Tables

Supplementary Figures 1-9 and Supplementary Tables 1-6

Supplementary Materials and Methods Section

Purpose:Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor–positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models.Experimental Design:Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy.Results:Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21–1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08–1.68) and abemaciclib-treated (rER: 1.19–2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94–1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth.Conclusions:These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

Captions for Supplementary Figures 1-9

Patients with radioresistant breast cancers, including a large percentage of women with triple-negative breast cancer (TNBC), demonstrate limited response to radiation and increased locoregional recurrence; thus, strategies to increase the efficacy of radiation in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition [ABT-263, radiation enhancement ratio (rER): 1.52–1.56] or Bcl-xL–specific inhibition (WEHI-539, A-1331852; rER: 1.31–2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize PIK3CA/PTEN-mutant TNBC (rER: 0.90–1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of PIK3CA/PTEN status (rER: 0.95–1.07). In wild-type PIK3CA/PTEN TNBC, pan Bcl-2 family inhibition or Bcl-xL–specific inhibition with radiation led to increased levels of apoptosis (P < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated PTEN knockout in wild-type PIK3CA/PTEN MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor–mediated radiosensitization (rER: 0.94–1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02–1.04) but transient MCL1 knockdown in CAL-51 cells promoted Bcl-xL inhibitor–mediated radiosensitization (rER: 2.35 ± 0.05). In vivo, ABT-263 or A-1331852 in combination with radiation decreased tumor growth and increased tumor-tripling time (P < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early-phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and radiation in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence. Significance: This study proposes a novel strategy for the treatment of radioresistant TNBCs using FDA-approved compounds that target apoptosis to improve local disease control in this patient population.