LAUSR

Introduction Phosphoinositide-3-kinase (PI3K) pathway is often hyper activated in Head and Neck and Esopgageal squamous cell carcinoma leading to tumour cell proliferation and survival. Inhibition of the PI3K pathway using the isoform specific inhibitor BYL719, has shown clinical activity in patients bearing mutations or amplification of PIK3CA gene which encods the a-subunit of PI3K. Unfortunately, the efficacy of the drug was limited by the emergence of resistance, that was driven by overexpression of the receptor tyrosine kinase AXL. The molecular mechanisms underlying AXL overexpression in resistance to PI3Ki in HNSCC and ESCC remained elusive. Material and methods RNA-seq was performed to identify transcription factors linked with resistance. Immunohistochemistry (IHC), western blotting and quantitative-PCR(qPCR) were applied demonstrating the expression levels of AXL, c-JUN, c-FOS Biomedical studies ilustrated the effect of drug combination in-vitro and in vivo. Results and discussions Q-PCR analysis of AXL mRNA level indicated a 25-fold increase in BYL719 resistant compared to sensitive cells, indicating the upregulation of AXL on the transcriptional level. By performing an RNA-seq analysis we screened for transcription factor segnitures linked with resistance and correlated them with the potential binding sites for the AXL promotor. We found that the AP-1 trancription complex may play a role in AXL overexpression in resistance to BYL719. In agreement, silencing of c-JUN and c-FOS downregulted AXL mRNA and protein levels and sensitised the cells to BYL719. A positive correlation between AXL and c-JUN expression levels was demonstrated across a panel of HNSCC and ESCC cell lines, and patient tumour samples. More over, resistance to BYL719 was associated with upregulation of the transcription factor c-JUN concomitantly with an increase of AXL expression. Importantly, blocking of c-JUN-N-terminal kinase (JNK) using SP600125, inhibited c-JUN activation and AXL expression. Also, the combination of SP600125 with BYL719 showed a synergistic anti-proliferative activity in-vitro. In agreement, the combination of SP600125 and BYL719 had a superior anti-tumour activity in vivo, in both cell line derived and patient-derived xenograft models. Conclusion The AP-1 transcriptional complex plays an important role in the resistance mechanism of HNSCC and ESCC cancer to inhibition of the PI3k pathway. These results support the rational for combined inhibition of JNK\c-JUN\AXL axis and PI3K in HNSCC and ESCC patients.