Abstract
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.
Highlights
Cytotoxic chemotherapy in disseminated cutaneous malignant melanoma (CMM) results in a low proportion of clinical responses and no improved survival.[1]
In the mass spectrometry based protein profiling we identified two novel resistance candidates, aminopeptidaseN (CD13/ANPEP) and ETS transcription factor FLI1, which are associated with resistance to vemurafenib and confirmed a number of recently reported druggable resistance mediators such as MET, EGFR and ephrine receptor A2 (EPHA2)
Among the significantly differentially expressed proteins we focused on two novel potential candidates; CD13/ANPEP and FLI1, both with markedly higher protein expression in the BRAFi-resistant cells, and on the recently reported BRAFi resistance mediator EPHA2 (Table 2).[11]
Summary
Cytotoxic chemotherapy in disseminated cutaneous malignant melanoma (CMM) results in a low proportion of clinical responses and no improved survival.[1]. Treatment of disseminated CMMcarrying activating BRAF mutations (V600E/K) with inhibitors targeting the mitogen-activated protein kinase (MAPK) signaling pathway, either as single agent treatment with BRAF inhibitor ((BRAFi) dabrafenib or vemurafenib) or in combination with MEK inhibitor ((MEKi) trametinib) significantly prolongs overall survival in patients with BRAF-mutated CMM.[2,3,4,5] Still, remissions with these agents are often not durable and research aimed at improving existing therapies by identifying predictive factors for long response and at reversing both intrinsic and acquired resistance to targeted therapies has a high priority. CD13/ANPEP and FLI1 role in BRAF inhibitor resistance A Azimi et al parental BRAFi-sensitive A375 human melanoma cell line and three daughter cell lines with induced BRAFi resistance
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