Inhibition of jun N-terminal kinase (JNK) enhances the efficacy of PI3Ka inhibitor in vivo in a murine model of head and neck squamous cell carcinoma

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the common and lethal cancer types worldwide. The phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway is commonly hyper-activated in HNSCC, leading to the activation of the AKT/mTOR signaling pathway that enhance cancer cell proliferation and survival. Blocking PI3K using the isoform specific inhibitor of the PI3K alpha (PI3Ka), BYL719, showed a promising anti-tumor activity in PIK3CA-mutated HNSCC. However, the efficacy of BYL719 was limited by the development of resistance. This resistance is linked with the upregulation of AXL receptor tyrosine kinase that re-activated the mTOR pathway. Our unpublished data suggests that AXL expression in HNSCC is regulated by the transcription factor c-JUN. Therefore, we hypothesized that inhibition of Jun N-terminal kinase (JNK) may down regulate AXL expression and thus enhance the efficacy of BYL719 inhibitors in HNSCC.

Objectives: Investigate the role of JNK inhibition on AXL expression, and its effect on tumor growth in the presence of BYL719.

Methods: HNSCC murine cell lines were developed from mice. Cell proliferation assay was performed to evaluate the response to BYL719 and SP600125 in vitro. Western blot analysis was performed to determine AXL, cJUN and, the downstream signaling p-AKT, p-ERK1/2, pS6 levels following treatments with BYL719 and SP600125. The efficacy of drug combination was tested in vivo. Immunohistochemistry was used to measure AXL, c-JUN, p-AKT, p-ERK1/2, pS6 levels in tumor tissues.

Results: 4NQO, 4-nitroquinoline-1-oxide, was utilized to develop carcinogen-induced murine HNSSC cancer model. C57Bl/6 mice were exposed to 4NQO in the drinking water to develop tumors in the oral cavity. Tumor cell lines were developed from the lips and the tongue for further drug studies. A significant additive anti-tumor effect of BYL719 and SP600125 combination was observed as indicated by a reduction in cell proliferation. Western blot analysis revealed that the combination inhibited pAKT, pERK1/2 and pS6 levels, and the expression of AXL was reduced. In vivo, BYL719 and SP600125 combined treatment had a superior anti-tumor effect.

Conclusions: Blocking c-JUN using a JNK inhibitor the SP600125 sensitized tumor cells to BYL719, in vitro and in vivo. Thus, the combination of SP600125 and BYL719 may be considered as an alternative approach in treating HNSSC patients.