Introduction

Canine lymphoma, the most common hematopoietic neoplasia in the dog, is routinely treated with a multi-drug chemotherapy protocol. Despite a high initial response rate, tumor relapse is common and often resistant to chemotherapy, resulting in treatment failure. Alternative treatment options are mandatory and since masitinib showed a mild anti-proliferative effect on lymphoid cells, other protein kinase inhibitors (PKIs) might provide this alternative.

Materials and Methods

GL-1 is a canine B-cell lymphoid cell line and GL-40 its doxorubicin/vincristine resistant subline demonstrating P-gp overexpression. Cell lines were cultured as previously described. Cell viability was assessed using a colorimetric assay (AlamarBlue®). Cells were seeded in 96 well plates at a density of 2 x10⁴ cells per well in cell culture medium containing a concentration range (0, 0,1, 1 and 10 µM) of the PKI tested and incubated for 48 hours at 37°C, 5% CO₂. AlamarBlue® (resazurin) was added three hours prior to analysis and the reduced fluorescent molecule (resorufin) was measured by light absorbance in a fluorescence spectrophotometer (560EX nm/590EM nm). Experiments were performed in triplicate.

Cell survival was calculated by dividing light absorbance in treated cells by that in control cells after correction for background absorbance. Concentration-dependent effects were analyzed by non-linear regression after log transformation of PKI concentration. Median inhibitory concentration (IC50) was calculated as a measure of the PKI's antiproliferative effect. Graphs were fitted according to a sigmoïd dose-response curve.

Results

IC50 for the various PKIs in the GL-1 cells ranged from <0.1 µM (BI-2536, sorafenib, quizartinib, sunitinib, toceranib), 0.1–1 µM (TAE684, SGI-1776, TAE226, tozasertib), 1–10 µM (dasatinib, pazopanib, nilotinib, erlotinib, axitinib) and >10 µM (gefitinib, imatinib, masitinib, lapatinib, vandetanib). No relevant differences were found in IC50 between GL-1 and GL-40 for any of the PKIs tested.

Conclusion/Discussion

Several PKIs inhibited lymphoid cell proliferation with good direct activity shown for inhibitors of PLK1, Raf, FLT3, ALK, PIM-kinase, FAK, and aurora kinases and these require further (clinical) evaluation. PKIs targeting c-KIT, PDGFR, VEGFR showed variable antiproliferative effects, while PKIs targeting EGFR had little to no inhibitory effect. The PKIs tested showed similar IC50 in both the GL-1 and GL-40 cells, suggesting that P-gp overexpression has no role in tumor resistance to PKIs, guaranteeing their value in case of tumor resistance to cytostatic drugs.

Disclosures

Disclosures to report
P. Dubreuil has been involved in research on masitinib and AB Science.