The AP-1 transcriptional complex regulates AXL-induced resistance to PI3K pathway inhibition in Head and Neck and Esophageal cancer

Introduction:

The Phosphoinositide-3-kinase (PI3K) pathway is often hyper activated in head and neck and esophagus squamous cell carcinoma (HNSCC and ESCC, respectively), leading to tumor cell proliferation and survival. Inhibition of the PI3K pathway using BYL719 has shown clinical activity in patients bearing mutations or amplification of PIK3CA gene, which encodes the a-subunit of PI3K. Unfortunately, the efficacy of the drug was limited by the emergence of resistance. The resistance was suggested to be 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.

Materials and Methods:

RNA-seq was performed to identify transcription factors linked with resistance to PI3Ki- BYL719. The expression levels of AXL, c-JUN, c-FOS and related signaling pathways were analyzed using Immunohistochemistry (IHC), western blotting and qPCR. In vitro IC50 analysis and synergy tests were used to study the effect of BYL719 and SP600125 combination. In vivo experiments using cell line derived and patient derived xenografts (CDX and PDX, respectively), were conducted to test the efficacy of PI3Ki and JNK inhibitor combinations.

Results:

Here we demonstrate that AXL expression levels determine the sensitivity HNCC and ESCC cells to BYL719, in vitro and in vivo, as silencing of AXL using shRNAs sensitized HNSCC and ESCC cells and tumors to BYL719. RNA-seq data of BYL719 resistant cells showed that the AP-1 transcriptional complex is upregulated in BYL719 resistance. Indeed, the expression of c-JUN (belonging to the AP-1 complex) was elevated in BYL719 resistant HNSCC and ESCC cells concomitantly with AXL over-expression. A positive correlation between AXL and c-JUN expression levels was also shown in a cohort of 17 HNSSC and ESCC cell lines and a tissue array of 48 HNSCC patients. AXL expression was downregulated by the silencing of c-JUN and c-FOS (both associate in the AP-1 complex), which was concomitant with enhanced sensitivity to BYL719 in vitro. The combination of BYL719 and SP60015, an inhibitor of c-Jun N-terminal kinase (JNK), had a synergistic anti-proliferative activity in vitro. In vivo, the efficacy of drug combination showed a potent anti-tumor activity in two HNSCC cell lines and two patients derived xenograft models (PDXs).

Conclusions:

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.