164 - Synergistic Potential of CDK4/6 Inhibitors and Radiotherapy with Anti-PD-L1 Immunotherapy in Triple-Negative Breast Cancer

W. C. Yang^1,2, M. F. Wei³, C. S. Huang⁴, Y. C. Shen^2,5, and S. H. Kuo^2,6; ¹Department of Radiation Oncology, National Taiwan University Cancer Center, Taipei, Taiwan, ²Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan, ³Division of Radiation Oncology, Department of Oncology, National Taiwan University, Taipei, Taiwan, ⁴Departments of Surgery, National Taiwan University Hospital, Taipei, Taiwan, ⁵Department of Medical Oncology, National Taiwan University Cancer Center, Taipei city, Taiwan, ⁶Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan

Purpose/Objective(s): Triple-negative breast cancer (TNBC) represents the subtype with the poorest survival outcomes among all breast cancer molecular classifications. While immunotherapy has shown promising anti-tumor effects in TNBC treatment, its efficacy is not universal across all patients. CDK4/6 inhibitors have been recognized for their ability to radiosensitize and modulate the immune system. Additionally, high-dose radiotherapy (RT) has been observed to bolster the effects of immunotherapy. This study investigates the potential of enhancing immunotherapy effectiveness in TNBC by integrating RT with CDK4/6 inhibitors, focusing on modulating the tumor microenvironment. Materials/

Methods: We employed three human TNBC cell lines—MDA-MB-231, MDA-MB-453, and MDA-MB-468—along with the 4T1 mouse TNBC cell line to assess CDK4/6 inhibitor, abemaciclib, in radiosensitizing effects of TNBC using the clonogenic assays. We assessed the anti-tumor efficacy of RT, abemaciclib, and anti-PD-L1 antibody combination through the 4T1 cell line-derived immunocompetent mouse model. Interferon-? (IFN-?) levels in mouse blood were monitored before, during, and after treatment to gauge the immune response. Tumor-infiltrating lymphocytes (TILs) were analyzed in excised tumor samples through flow cytometry assays and immunohistochemical staining.
Results: Clonogenic assays showed that RT combined with abemaciclib pretreatment had synergistic effects across all tested TNBC cell lines. A single 8 Gy fraction of RT increased PD-L1 expression on the surface of tumor cells, while abemaciclib (at 100nm or 200nm concentrations) did not alter PD-L1 expression in all TNBC cell lines. The combination of abemaciclib, RT, and anti-PD-L1 in the 4T1 mouse model significantly decreased the tumor growth (p < 0.05) and elevated circulating IFN-? levels during treatment (p < 0.001) when compared with control, RT alone, abemaciclib with anti-PD-L1, abemaciclib with RT, and anti-PD-L1 with RT. TILs assays showed the combination treatment of abemacilcib, RT, and anti PD-L1 increased CD4 and CD8 positive T cells proportion (p < 0.05 and p <0.01, respectively) as well as tumor associated macrophage (p < 0.001) when compared to the control group. Immunohistochemical staining revealed an increase in CD8 positive T cells and monocyte chemoattractant protein (MCP)-1 positive macrophages in the triple-combination treatment group compared to the other four groups.
Conclusion: Combined CDK4/6 inhibitors with RT enhance anti-tumor effects of anti-PD-L1 immunotherapy in TNBC through increasing secretion of IFN-? and modulation of tumor microenvironments via recruiting CD4 and CD8 positive T-cells, as well as M1 type tumor associated macrophage.