Oral communication, CS16 / C77

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

cKIT expression level and NRAS/BRAF mutation status predict the response to the tyrosine kinase inhibitor dasatinib in melanoma cell lines

BACKGROUND: Patients with advanced melanoma have limited effective therapy. New targeted drugs have to be evaluated in melanoma lines with regards to mutation status and active signalling pathways. OBJECTIVES: We evaluated the cytotoxic effects of the SRC tyrosine kinase inhibitor dasatinib (BMS-354825) in a panel of melanoma cell lines, we examined the molecular targets of the drug in sensitive cells, and we proposed markers to select melanoma patients who would benefit from dasatinib therapy. METHODS: We assessed the effects of dasatinib on melanoma cell proliferation (crystal violet staining) and apoptosis (annexin-V-PE detection) in relation with cKIT, NRAS and BRAF mutation status (PCR/sequencing) and key proteins involved in signalling pathways (Western blotting). RESULTS: We examined 33 melanoma cell lines and found that 8 lines were highly sensitive to dasatinib (IC50<10−9M), 13 were moderately sensitive (IC50 from 10−8 to 10−6M) and 12 were resistant (IC50>10−5M). All highly sensitive lines expressed high cKIT levels, whereas others had undetectable cKIT expression. Then, we checked for the most common mutation in cKIT (L576P), NRAS (Q61L/R) and BRAF (V600E/K), and showed that the highly sensitive cells did not bear any of these mutations, while 60% of the moderately sensitive and 75% of the resistant lines had NRAS or BRAF mutations. We also found that, in the sensitive cells, dasatinib induced apoptosis in time and concentration-dependent manner. Interestingly, we showed that the combination of a cKIT inhibitor (10-10M sunitinib) and a SRC inhibitor (10-8M PP2) resulted in a synergistic effect, suggesting that dasatinib is most effective at low concentrations by targeting cKIT and SRC simultaneously. To confirm this, we showed that, in the sensitive cells, 10-12M dasatinib completely inhibited the phosphorylation of cKIT, SRC, ERK and AKT, while in the resistant cells, at least 10-4M concentration was needed to inhibit ERK phosphorylation but without any effect on AKT phosphorylation. CONCLUSIONS: Dasatinib appears as a promising agent for the treatment of a selected group of melanoma patients with tumor tissues harbouring cKIT overexpression and no mutations of cKIT, NRAS or BRAF. By targeting wildtype BRAF tumors, dasatinib use might be complementary to the recent unprecedented encouraging therapeutic responses obtained with RG7204 (PLX4032), a specific inhibitor of V600EBRAF activating mutation.

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