Abstract P5-05-08: Sensitivity to c-Met inhibition is increased in dasatinib resistant TNBC cells
Authors: Gaule Patricia , Corkery Brendan , Crown John , Duffy Micheal J , O' Donovan Norma 
Published: May 2015

Pre-clinical models TNBC cells have demonstrated sensitivity to the multi-targeted Src kinase inhibitor dasatinib, however clinical trials with single agent dasatinib showed limited efficacy in unselected populations. Trials of dasatinib in combination with chemotherapy are ongoing. In order to study potential mechanisms of resistance to dasatinib in TNBC we established a cell line model of acquired dasatinib resistance (231-DasB).

The dasatinib resistant cell line (231-DasB) was developed by constant exposure to incrementally increasing concentrations of dasatinib, from 200 nM to 500 nM over a period of 13 weeks.Cell proliferation was measured by acid phosphatase assay after 5 day treatment with SRC inhibitors (dasatinib, PD180970), EGFR inhibitors (gefitinib and neratinib), chemotherapy drugs (carboplatin, docetaxel and doxorubicin) and a c-Met inhibitor (CpdA, Amgen). P values were calculated using the Student’s T-Test (2 tailed with unequal variance). Expression and phosphorylation of c-Met and Src was measured by immunoblotting and multiplex magnetic bead assays carried out on a MAGPIX® Instrument.

Following approximately 3 months exposure to dasatinib, 231-DasB cells were resistant to dasatinib with IC50 &gt; 5μM compared to 0.04 ± 0.001 µM in MDA-MB-231. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970 [Table 1]. 231-DasB cells showed small but statistically significantly increases in sensitivity to docetaxel and doxorubicin.

Table 1: Sensitivity to chemotherapy and targeted therapies in the MDA-MB-231 and 231-DasB cells.DrugMDA-MB-231231DasBp valuePD180970 IC50 (µM)0.40 ± 0.040.87 ± 0.070.003CpdA IC50 (µM)&gt;102.1 ± 0.10.0001Docetaxel IC50 (nM)1.9 ± 0.11.2 ± 0.10.003Doxorubicin IC50 (nM)138.6 ±1.097.2 ±6.60.007Carboplatin IC50 (µM)13.2 ± 0.811.5 ± 1.30.147Gefitinib IC50 (µM)23.1 ± 2.220.9 ± 2.10.289Neratinib (% growth @ 10µM)94.5 ± 2.095.9 ± 7.60.834

No significant change in sensitivity to carboplatin or to the EGFR inhibitors gefitinib and neratinib was observed. However, the 231-DasB cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpDA, with an IC50 value of 2.1 ± 0.1 µM compared to an IC50 greater than 10 µM in the parental MDA-MB-231 cells. Treatment of MDA-MB-231 cells with dasatinib (100 nM) blocked phosphorylation of Src kinase. In contrast, dasatinib treatment (100 nM) did not decrease p-Src levels in the 231-DasB cells. p-Met levels were significantly increased in 231-DasB cells relative to MDA-MB-231. Treatment with 2 µM CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Other key receptor tyrosine kinases (EGFR, HER2, HER3, HER4, IGFIR and IR) show no significant changes in phosphorylation in 231-DasB cells compared to MDA-MB-231.

Constitutive activation of p-Src through increased c-Met signalling may be a potential mechanism of resistance and suggests that combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance.

Citation Format: Patricia Gaule, Brendan Corkery, John Crown, Micheal J Duffy, Norma O' Donovan. Sensitivity to c-Met inhibition is increased in dasatinib resistant TNBC cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-08.