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). 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. Overall design: 3 head and neck squamous cell carcinoma cell lines treated with cetuximab or PBS (untreated controls) each in duplicate.

在肿瘤细胞仍对治疗产生应答时识别潜在耐药机制，对于开发联合疗法以延缓获得性耐药至关重要。西妥昔单抗（Cetuximab）是一种抗表皮生长因子受体（EGFR）疗法，也是目前头颈部鳞状细胞癌（HNSCC）唯一可用的靶向治疗药物。我们在敏感型头颈部鳞状细胞癌细胞中生成了首个全面的多组学、批量及单细胞数据集，用以识别敏感细胞对西妥昔单抗产生即刻应答的相关转录组与表观遗传学改变。这些改变涉及两条可能与耐药相关的通路：经由转录因子TFAP2A调控生长因子受体的通路，以及上皮间质转化（EMT）过程。单细胞RNA测序（scRNA-seq）结果显示细胞系间存在异质性：西妥昔单抗处理组与未处理克隆中，TFAP2A和VIM基因的表达具有细胞特异性，且两条通路各自发挥独立作用。RNA测序（RNA-seq）与ATAC测序（ATAC-seq）结果表明，在抗EGFR疗法启动后的前5天内，存在全局性的转录组与表观遗传学改变。我们还通过实验证实，敲低TFAP2A可在体外抑制头颈部鳞状细胞癌细胞的增殖，且该抑制效果可通过联合西妥昔单抗得到增强；而使用JQ1——一种替代受体酪氨酸激酶抑制剂——时，抑制效果更为显著。与我们的scRNA-seq观察结果一致，TFAP2A沉默并不会影响细胞迁移，这证实其与EMT通路不存在相互作用。综上，本研究表明，西妥昔单抗诱导的即刻适应性转录组与表观遗传学改变，包含与获得性耐药相关的关键通路。尽管这些改变存在异质性，但可通过JQ1这类多靶点药物进行靶向干预；在治疗早期联合西妥昔单抗时，可更有效地控制肿瘤生长。总体实验设计：3株头颈部鳞状细胞癌细胞系分别经西妥昔单抗或磷酸盐缓冲液（PBS，未处理对照组）处理，每组设置2个生物学重复。