2.3 – Cytotoxicity
ALK inhibitors (e.g., crizotinib) are used in clinical practice for various ALK expressing malignancies therefore we hypothesized that treatment with crizotinib would result in effective killing of ALKfus cells13. ALKfus+ Ba/F3 cells were plated in triplicate and cultured with varying doses of Crizotinib (SelleckChem) in cytokine-free media20. Cell viability and cell death were determined with the Promega Cell Titer Glow assay after 48 hours in culture on a BioTek Synergy H4 Hybrid plate reader via Gen5 2.1 Software; this was repeated for ALKfus+ cells in the presence of IL-3. Crizotinib-induced cytotoxicity in ALKfus+ Ba/F3 cells was enhanced in the absence of IL-3 compared to cells cultured in the presence of IL-3 (IC50 144nM vs. 1051nM for SPTBN1-ALK and 95nM vs. 1277nM for RANBP2-ALK ) (Fig. 2C).