Identification and Characterization of Novel Kinases Regulating Programmed Death Ligand-1 (PD-L1) in Immune Evasion of Triple Negative Breast Cancer Cells
The programmed death ligand-1 (PD-L1) is an immune checkpoint protein expressed on a variety of antigen-presenting cells to normalize immune system. Recently, overexpression of PD-L1 on the surface of cancer cells has been proven to contribute to cancer immune evasion and tumor progression. Specifically, PD-L1 is found highly upregulated in Triple negative breast cancer (TNBC), a subtype of breast cancer that currently has no effective clinical therapies, allowing blockade immune checkpoint therapy a potentially effective treatment. However, the overall response rate for PD-L1 targeted immunotherapy in PD-L1 positive TNBC is less than 20%. The mechanism for this is largely unknown. It has been suggested that the levels of many other genes regulating PD-L1 may contribute to the sensitivity of cancers to immunotherapy. Therefore, there is a critical need to understand how PD-L1 is upregulated and interacts with its upstream regulators in the modulation of anti-PD-L1 immunotherapy response in TNBC. Over the past years, the literature that implicated the regulation of PD-L1 was primarily on transcription levels. For this reason, my project aims to identify post-translational modification of PD-L1 in efforts to better target it in TNBC immunotherapy. To identify novel kinase regulators of PD-L1, we first performed a proteomic analysis of PD-L1 and identified several tyrosine kinases as novel PD-L1 interacting proteins. We provide evidence that some of these kinases (EphA3, FAK, FES, and FER) could phosphorylate PD-L1 in vitro and co-expression of FAK, FES, and FER could stabilize PD-L1 in ectopic co-expression models. Moreover, FAK inhibition, knockdown, or ectopic over-expression resulted in decreased or increased PD-L1 levels, respectively; and that inhibition of EphA2 was associated with reduced endogenous PD-L1 levels. Most significantly, knockout of FAK in BT549 TNBC cells reduces immune evasion by inhibiting cancer cells-induced T-cell apoptosis in coculture experiments. In summary, we have identified several novel kinase regulators of PD-L1 stability and immune evasion. This is a critical first step in unveiling the molecular mechanism underlying PD-L1 upregulation in TNBC immune evasion and tumorigenesis. These findings present exciting opportunities for developing a new potential target and effective combinational therapies for immunotherapy-resistant TNBC.