Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells.
OBJECTIVE
This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells.


METHODS
shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining.


RESULTS
Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells.


CONCLUSIONS
These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.
Myssina
Svetlana
S
Section of Experimental Haematology, Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Glasgow, UK.
Helgason
G Vignir
GV
Serrels
Alan
A
Jørgensen
Heather G
HG
Bhatia
Ravi
R
Modi
Hardik
H
Baird
Janet W
JW
Mountford
Joanne C
JC
Hamilton
Ashley
A
Schemionek
Mirle
M
Koschmieder
Steffen
S
Brunton
Valerie G
VG
Holyoake
Tessa L
TL
D017304:Annexin A5 / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D053148:Caspase 3 / Q000096:biosynthesis / Q000235:genetics; D000069439:Dasatinib; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus; D010677:Philadelphia Chromosome; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013844:Thiazoles / Q000494:pharmacology*; D013997:Time Factors
D017304:Annexin A5; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D012313:RNA; D016044:Fusion Proteins, bcr-abl; C505407:CASP3 protein, human; D053148:Caspase 3; D000069439:Dasatinib