A majority of patients with BRAF-mutated metastatic melanoma respond to therapy 
with BRAF inhibitors (BRAFi), but relapses are common owing to acquired 
resistance. To unravel BRAFi resistance mechanisms we have performed gene 
expression and mass spectrometry based proteome profiling of the sensitive 
parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell 
lines with induced BRAFi resistance. Increased expression of two novel 
resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription 
factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In 
addition, increased levels of the previously reported resistance mediators, 
receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth 
factor receptor MET were also identified. The expression of these proteins was 
assessed in matched tumor samples from melanoma patients obtained before BRAFi 
and after disease progression. MET was overexpressed in all progression samples 
while the expression of the other candidates varied between the individual 
patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both 
parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells 
with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, 
previously demonstrated to cause inhibition of the migratory capacity. AKT and 
RSK, both reported to induce EPHA2 S897 phosphorylation, were also 
dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused 
decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In 
addition, silencing of FLI1 sensitized the resistant cells to BRAFi. 
Furthermore, we show that BRAFi in combination with the multi kinase inhibitor 
dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 
phosphorylation and total FLI1 protein expression. This is the first report 
presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF 
inhibition with potential as drug targets in BRAFi refractory melanoma.