2.2 – Fusion Functionality
To evaluate whether the ALK fusion transcripts generated oncoproteins, fusion transcripts were cloned from patient samples into the pCSII lentiviral vector (AddGene) under the EF1-Alpha promoter and tagged with GFP, which was used to transduce and transfect cell lines to produce model systems.
SPTBN1-ALK or RANBP2-ALK fusion transcripts were transfected into HEK293T cells (ATCC), and cells were maintained in DMEM supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin. IL-3 dependent Ba/F3 cells were transduced with SPTBN1-ALK orRANBP2- ALK fusion transcripts and maintained in RPMI 1640 supplemented with 10% FBS, 10 ng/mL IL3, L- glutamine, and penicillin/streptomycin. Cells were lysed with Triton X-Lysis buffer, underwent western blotting (Invitrogen iBind), stained with anti-ALK (Cell Signaling Technologies), and β–Actin antibodies (Cell Signaling Technologies). The presence of ALK protein was confirmed by western blot, depicted as bands at approximately 124kD and 175kD (SPTBN1-ALK and RANBP2-ALK, respectively) (Fig. 2A). The two protein species detected in the RANBP2-ALK lysates suggest that the ALK region of the fusion may be phosphorylated; this may suggest differential regulation of pathways involving ALK and would require further study.
The leukemic potential of the fusion transcripts were studied in a transformation assay. Transduced Ba/F3 cells were sorted to GFP homogeneity and growth kinetics post-IL-3 withdrawal were evaluated using Trypan blue staining and live cell counting with Countess II (Thermo Fischer Scientific).
Following IL-3 withdrawal from transduced Ba/F3 cells, cells expressing either SPTBN1-ALK or RANBP2-ALK had sustained growth and rapidly proliferated in cytokine-free media, whereas the parental line quickly died in the absence of cytokines, confirming the transformation potential of the fusion oncoprotein (Fig 2B). The presence of theSPTBN1-ALK led to greater cytokine-independent proliferation compared to that of RANBP2-ALK fusion.