Integrated single cell and bulk gene expression and ATAC-seq reveals heterogeneity and early changes in pathways associated with resistance to cetuximab in HNSCC sensitive cell lines (scRNA-Seq)

The identification of the potential mechanisms of resistance while tumor cells still respond to therapy is critical to develop combination therapies to delay acquired resistance. Cetuximab, an anti-EGFR therapy, is the only targeted therapy available for head and neck squamous cell carcinoma (HNSCC). We generated the first comprehensive multi-omics, bulk and single cell data in sensitive HNSCC cells to identify relevant transcriptional and epigenetic changes that are an immediate response to cetuximab in sensitive cells. These changes include genes from two pathways potentially associated with resistance: regulation of growth factor receptors through the transcription factor TFAP2A, and epithelial-to-mesenchymal transition (EMT) process. Single cell RNA-sequencing demonstrates inter-cell lines heterogeneity, with cell specific expression profiles of TFAP2A and VIM gene expression in cetuximab treated and untreated clones, and an independent role of each pathway. RNA-seq and ATAC-seq demonstrate that there are global transcriptional and epigenetic changes within the first five days of anti-EGFR therapy. We also experimentally verified that lack of TFAP2A reduces HNSCC growth in vitro and that this effect is enhanced with cetuximab and a stronger effect is observed with JQ1, an inhibitor of alternative receptor tyrosine kinases. Corroborating our scRNA-seq observation, TFAP2A silencing does not affect cell migration, supporting the lack of interplay with the EMT pathway. Overall, our study shows that the immediate adaptive transcriptional and epigenetic changes induced by cetuximab include relevant pathways associated with acquired resistance. Although heterogeneous, these changes can be targeted by a multiple-target drug as JQ1 that in combination with cetuximab in the early stage of treatment present better efficacy in controlling tumor growth.