In vivo detection of the BCR/ABL1 protein

The BCR/ABL1 fusion protein is found in virtually all cases chronic
myeloid leukemia (CML) and a large proportion of acute
lymphoblastic leukemia (ALL). The fact that the BCR/ABL1 fusion
protein is crucial for the development of leukemia makes this
fusion protein an attractive target for therapy development. We
have developed a strategy for the in vivo detection of the BCR/ABL1
fusion protein, in which the presence of the BCR/ABL1 fusion
protein is detected intracellularly and if the fusion protein is
present an arbitrary action is initiated in the cell (e.g. mark the
cells or selectively kill the cells). Our BCR/ABL1 detection
strategy is based on protein-protein interactions. Two detection
proteins are expressed in the cells: 1) protein A, a GAL4-DNA
binding domain/BCR interacting protein fusion protein
(GAL4DBD-BAP-1) and 2) protein B, a GAL4-activation domain/ABL
interacting protein fusion protein (GAL4AD-CRKL). Only when
BCR/ABL1 is present in the cell, do protein A, protein B, and
BCR/ABL1 form a trimeric complex which activates the transcription
of reporter genes under the control of GAL4-upstream activating
sequence (UAS). A proof of principle for the strategy was
implemented in the yeast system. We did not use full length BAP-1
or CRKL but only those portions of the proteins that directly
interacted with BCR or ABL, respectively. We showed in the yeast
two hybrid system, that the C-terminus of BAP-1(amino acids
617-879) binds to full length BCR. The site of interaction of CRKL
and ABL was confirmed to be the N-terminal SH3 domain (SH3n) of
CRKL as described in the literature. Yeast cells (strain CG1945)
transformed with a protein A expressing plasmid (pGBT9-BAP), a
protein B expressing plasmid (pGAD424-CRKLSH3n), and a BCR/ABL
expressing plasmid (pES1/BCR-ABL) showed expression of the reporter
genes HIS3 and LACZ. The expression of the HIS3 reporter gene was
assayed by growth of the yeast cells on medium lacking histidine.
The expression of the LACZ gene was verified by a
beta-galactosidase filter assay. Yeast cells that were transformed
with the pES1 plasmid without the BCR/ABL1 coding region did not
show activation of the reporter genes. Several other negative
controls demonstrated the specificity of the assay. Thus the method
was able to clearly distinguish between BCR/ABL expressing cells
and cells did not express BCR/ABL1. We then adapted this system for
use in mammalian cells. The open-reading frames encoding the
proteins A and B were recloned into mammalian expression vectors.
The human embryonal kidney cell line HEK293 and the murine myeloid
progenitor cell line 32D which had been stably transfected with a
BCR/ABL expressing plasmid were tested. The firefly luciferase gene
and the yellow fluorescent protein (eYFP) were used to evaluate the
whole cell population and single cell, respectively. Unfortunately,
the system failed to work in the mammalian cell lines tested. Even
though the detection system did not work in mammalian cells, most
likely due to the cytoplasmic localization of the BCR/ABL1 fusion
protein, it should still be a viable strategy for the detection of
leukemia-associated fusion protein, which localize to the nucleus
(i.e AML-ETO). This strategy could be adapted for purging the bone
marrow of leukemia patients using therapeutically more useful
effector genes like suicide genes, which encode pro-drug converting
enzymes (e.g. HSV thymidine kinase), or markers that can easily be
assayed (e.g. YFP).