Abstract B36: The effects of dasatinib in KIT L579P and NRAS Q61K mutant canine melanoma cells

Abstract

Purpose: Malignant melanoma is a common malignant tumor in dog. It is a fatal disease with local invasiveness and frequently metastatic propensity and the poor response has been observed. The aim of this study was to establish the drug-screening platform and used that platform to evaluate small molecular inhibitor, Dasatinib, for therapy of canine melanoma. Experimental Design: Various phenotypes of eight canine melanoma cell lines, CM01, M1, M3, M4, M5, Pu, C1 and KMeC, were examined in this study. Mutation analysis of kit, Nras and Braf was done by PCR/DNA sequencing. Expression of S100, KIT and KIT downstream signaling cascades NRAS, phospho-ERK and phospho-AKT were evaluated by immunocytochemistry. KMeC, M5 and CM01 cell lines were selected for further evaluation of Dasatinib. Proliferation assay and wound healing assay were performed to assess cell viability and motility in the presence of dasatinib. Cell cycle analysis and Annexin V apoptosis assay were also applied to realize the mechanism of cell death caused by Dasatinib. Results: Expressions of S100 and KIT were observed in all of the cell lines. NRAS and activated ERK were identified in several cell lines with different expression levels. However, no activated AKT was identified in any cell lines. kit T1736C mutation causing KIT L579P change was detected in M3, KMeC and C1 cells, while Nras C181A mutation causing NRAS Q61K change was found in M5 cell. ERK activity in KMeC (KIT L579P), M5 (NRAS Q61K) and CM01 (KIT & NRAS wild type) cells was down-regulated when Dasatinib was administered. Dasatinib reduced cell viability with the IC50 values of 0.64 μM in KMeC, 0.24 μM in M5 and 0.37 μM in CM01 cells. In wound healing assay, Dasatinib inhibited cell motility in all of the three cell lines. Cell cycle analysis and Annexin V apoptosis assay revealed that the treatment of Dasatinib causes a G1 cell cycle arrest and induction of apoptosis. The analysis of Dasatinib caused signaling change in canine melanoma cell is ongoing. Conclusions: Various phenotypes of eight canine melanoma cell lines were identified in DNA and protein levels. The NRAS Q61K mutation was first found in canine melanoma. The results showed that KIT L579P mutant type, NRAS Q61K mutant type and wild type canine melanoma cells are sensitive to the treatment of Dasatinib. It caused the reduction of cell viability, and inhibition of motility, moreover, the promotion of G1 cell cycle arrest and cell apoptosis. Dasatinib may potentially be selected as a candidate for therapy of canine melanoma. Citation Format: Lu-Ping Lu, Chen-Si Lin, Albert Taiching Liao. The effects of dasatinib in KIT L579P and NRAS Q61K mutant canine melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B36.