Kinome-profiling using patient-derived cell lines to discover novel therapeutic targets for treating dermatofibrosarcoma protuberans with resistance to imatinib

[Introduction] Dermatofibrosarcoma protuberans (DFSP) is a rare sarcoma, characterized by a unique fusion gene, COL1A1-PDGFB. Imatinib, a kinase inhibitor which blocks PDGF receptors, is included in the standard treatment of DFSP, and 50% of patients with DFSP demonstrate resistance. The purpose of this study is to reveal the response to the imatinib treatments at the molecular level, and identify the kinases, which could be predictive biomarkers or therapeutic targets in the resistant patients. We hypothesized that the alternative kinases pathways are activated in the patients exhibiting the resistance. Examining the kinase activity in the DFSP cell lines with the different sensitivity to the treatments with imatinib, we investigated the kinases, which contribute to the resistance. These kinases and the activated pathways will be candidates of predictive biomarkers and therapeutic targets in DFSP. [Materials and Method] Five cell lines established from tumor tissues of five patients with DFSP were used in the study. The tumor tissues were obtained from the surgically resected tumor prior to imatinib treatment. The cell lines were exposed to different concentrations of imatinib, and the half maximal inhibitory concentration (IC50; μM) was calculated. The cell lines were classified into two groups; Group S as sensitive to imatinib treatments with IC50 < 10 μM, and Group R as resistant to imatinib treatments with IC50 > 10 μM. Activity of 98 kinases was examined by in vitro kinase assay (PamStation, PamGene). All cell lines were treated with imatinib with concentration of each IC50 or dimethyl sulfoxide as control. The activity of kinases was subjected to the comparative study, and the effects of imatinib treatments were evaluated by student-t analysis with the statistical significance of 0.05. The kinases exhibiting the fold activity change greater than 2 with statistical significance were considered as those affected by the exposure of imatinib. We determined the kinases whose activity was differently regulated by the imatinib exposure between the cell lines in Group R and Group S. [Result] Two and three DFSP cell lines were classified into Group S (IC50: 2.4-4.6 μM) and Group R (IC50: 13 – 42 μM), respectively. All cell lines underwent the kinome-profiling after exposure to imatinib or dimethyl sulfoxide. We observed the activity from 142 phosphorylated substrate peptides, and compared them between both groups. By the statistical comparison, we found that the kinases that are the targets of Ponatinib, which was approved for the treatments of the patients with chronic myelogenous leukemia, were activated in Group R, but not in Group S. [Discussion] The kinases whose activity contributes to the resistance to imatinib treatments can be potent candidates for novel therapeutic targets. Ponatinib and its target kinases are worth investigating for treating of the patients resistant to imatinib.