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.