49 Inhibition of AXL along with c-Met potentially halts resistance development in renal cell carcinoma

Abstract Background c-Met, a receptor tyrosine kinase (RTK), is overexpressed in renal cell carcinoma (RCC) and correlates with a decreased survival rate. Upon binding to its specific ligand, Hepatocyte Growth Factor (HGF), c-Met activates pro-tumorigenic signaling pathways. Cabozantinib (Cabo) inhibits c-Met and a few other RTKs, including AXL, and is approved for the treatment of patients with advanced-stage RCC. AXL and its ligand, GAS6, are overexpressed in RCC and are also markers of poor prognosis. The pro-tumorigenic role of AXL and its potential crosstalk with c-Met in renal cancer need to be thoroughly investigated. Methods We generated a Cabo-resistant (CaboR) cell line from wild-type Cabo-sensitive (CaboS) Caki-1 clear-cell RCC cells, and CRISPR/Cas9-mediated AXL knock-out cells (AXL-KO) from 786-O clear-cell RCC cells. Chromatin immunoprecipitation and sequencing (ChIP-seq) was performed to profile H3K27ac, a histone post-translational modification associated with active promoters and enhancers, in CaboR and CaboS cell lines. We also performed RNA sequencing (RNA-seq) on CaboR and CaboS and evaluated differentially expressed genes and enriched pathways using gene set enrichment analysis (GSEA). Transcriptional profiles of control clones and AXL-KO cells were also compared to identify the effect of AXL-KO on pro-tumorigenic signaling. We utilized an AXL-specific small molecule inhibitor, TP-0903, in combination with Cabo to validate our data. Finally, we studied how AXL silencing or inhibition may affect resistance to Cabo. Results Our findings revealed that AXL forms a complex with c-Met and may have a significant crosstalk which can be involved in therapeutic resistance. We found that prolonged treatment with c-Met inhibitors Cabo, Crizotinib, and PF-4217903, induced c-Met and AXL overexpression in RCC cells. Interestingly, c-Met inhibitor(s)-induced overexpressed c-Met can also be increasingly phosphorylated (at low concentrations) in the presence of HGF, which may cause enhanced downstream tumor-promoting signaling. We found that silencing AXL can prevent this c-Met-inhibitors-mediated c-Met overexpression. Moreover, we found marked activation of the promoters and enhancers of several transcription factors, including ETV1, HOXA9, and FOXK1, in CaboR cells compared to CaboS cells. In the CaboR cells, genes involved in oxidative phosphorylation, progression through the division cycle, DNA replication, the NRF2 pathway, and DNA repair were upregulated, whereas genes associated with glycolysis, angiogenesis, hypoxia, and the Met pathway were downregulated (Figure 1). Finally, silencing AXL (using siRNA) or inhibiting AXL (using TP0903) in CaboR cells, induced significant apoptosis. Conclusions Together, our data suggest that CaboR cells have a distinct epigenomic and transcriptomic profile, and that targeting AXL along with c-Met inhibition can be beneficial in preventing acquired therapeutic resistance in RCC.