靶向拮抗激活蛋白1转录因子复合物在HNSCC模型中产生强效抗肿瘤活性

The AP-1 transcription factor complex, comprised of Fos and Jun family heterodimers, plays a pivotal role in tumor progression and metastasis of head and neck squamous cell carcinoma (HNSCC), where expression of AP-1 component Fra1 positively correlates with poor prognosis. As dimerization is required for AP-1 complex DNA binding and transcriptional activity, we designed Fra1 antagonizing peptide (FraAP) to disrupt complex formation and prevent associated activity. Low nanomolar FraAP binding affinity to Fra1 and Jun and selectivity towards AP-1 family members was demonstrated by bio-layer interferometry (BLI), fluorescence polarization, and DNA ELISA assays. Immunofluorescence imaging reveals rapid FraAP cell entry into both the cytoplasmic and nuclear compartments of HNSCC cells, while proximity ligation assays (PLA) and co-immunoprecipitation experiments demonstrate FraAP antagonism of cJun and Fra1 protein-protein interactions and reporter assays confirm inhibition of AP-1 transcriptional activity in vitro. To investigate the impact of FraAP on tumor cell transcriptomics, RNAseq and pathway enrichment analysis identified a significant impact of FraAP on AP-1-regulated pathways including tumor cell apoptosis, invasion and proliferation. Corresponding functional in vitro assays support transcriptomic observations, as FraAP induces dose-dependent Annexin V+ apoptosis by flow cytometry; promotes a phenotypic mesenchymal to epithelial transition characterized by decreased mesenchymal marker N-cadherin and increased epithelial marker E-cadherin, leading to an inhibitory effect on invasion in Boyden chamber assays; and induces G1 arrest characterized by upregulation of CDKN1A, reduction of CDK4, CDK6, and CCND1 expression, and hypophosphorylation of Rb. The impact of FraAP on CDK4 and CDK6 expression prompted investigation of the combination activity of FraAP with the CDK4/6 inhibitor abemaciclib. Data identifies synergistic anti-tumor activity in vitro in assays of cytotoxicity, clonogenic formation, orosphere formation, and cell signaling. Further, while FraAP results in significant anti-tumor activity as a monotherapy in HNSCC subcutaneous xenograft models, combination of subpharmacologic FraAP and abemaciclib results in enhanced tumor growth inhibition. In summary, these data support FraAP as a potent peptide antagonist of the AP-1 transcription factor family that warrants further development as a novel therapeutic option for AP-1 driven tumors such as HNSCC.