Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

PurposeCyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also demonstrate antineoplastic activity through triggering T cell-mediated immunity. One of the potential mechanisms responsible for this immunological effect might be qualitative and quantitative changes in human leukocyte antigen (HLA) ligands on the cell surface after treatment with CDK4/6i. These changes may increase the immunogenicity of breast cancer cells offering potential synergies for combinations with cancer immunotherapies. Experimental DesignWe investigated the ability of two CDK4/6 inhibitors (CDK4/6i), Abemaciclib and Palbociclib, to alter the immunopeptidome at subclinical, non-toxic, levels in different breast cancer cell lines. Biochemical isolation of HLA ligands, identification by mass spectrometry and subsequent network analysis after drug treatment were used to characterize the changes in the immunopeptidome. The mechanisms for altered CDK4/6 presentation were explored. ResultsLow-dose treatment with 100nM of Abemaciclib and Palbociclib led to upregulation of cell surface HLA levels and induced hundreds of HLA ligands in breast cancer cell lines. These new ligands were significantly and most strongly enriched for peptides derived from proteins involved in the "G1/S phase transition of cell cycle" pathway and included among others, HLA ligands from CDK4, CDK6, Cyclin D1 and Cyclin E1. An increase in transcript, protein, and subsequent ubiquitination for Cyclin D1, which could lead to enhanced degradation of the target protein, was identified as a potential mechanism for the altered presentation of peptides. ConclusionsCDK4/6i treatment gave rise to drug-induced antigens through cell cycle disruption and increased antigen presentation. Interestingly, these induced HLA ligands are often sourced from the proteins of the CDK4/CDK6/CCND1 complex or more downstream interaction partners, providing evidence that inhibition of a distinct cellular pathway leads to increased presentation of the proteins involved. These findings suggested CDK4/6i provided a tool for highly selective induction of HLA ligands that may be targeted by T cell-based immunotherapeutics. Translational RelevanceThese data demonstrated that low-dose treatment of breast cancer cells with CDK4/6 inhibitors, Abemaciclib and Palbociclib, induced marked changes in presentation of HLA ligands, especially from proteins involved in the G1/S phase transition, the phase in which these drugs arrest the cells. Enhanced ubiquitination and degradation was identified as a mechanism for the altered presentation for one of the relevant proteins. The induced HLA ligands may provide ideal specific targets for combination immunotherapies. The data show for the first time that selective inhibition of a distinct pathway can lead to specific presentation of HLA ligands in breast cancer cells. This work supports the rationale for testing the combination of low-dose CDK4/6i with immunotherapeutic agents, such as immune checkpoint blockade antibodies or T-cell-based approaches specifically directed against one of the induced HLA ligands.