Chronic Myeloid Leukemia (CML) represents 15-20% of all cases of leukemia, and is expected to increase by 39% in the next decade. This disease is triggered by a translocation of genetic material between chromosome 9 and 22, giving rise to a fusion gene BCR-ABL, which codifies an oncoprotein (BCR-ABL) responsible for the malignant transformation.

As resistance to conventional therapeutics (imatinib) raises, new drugs and therapeutics that may give answer to drug resistance are desired. Therefore knockdown of BCR-ABL with gene silencing agents such as small interfering RNA (siRNA) alone or combined with imatinib are seductive strategies.

The main goal of this work are: to evaluate the ability of a selected siRNA sequence (designed to specifically block BCR-ABL) to reduce mRNA and protein levels and consequently to promote human CML cell death; to demonstrate the cytotoxic activity of BCR-ABL siRNA against an imatinib resistant cell line; and to exploit possible synergies between gene silencing approaches and imatinib.

K562 cells (a human CML cell line) and IRK562 (an imatinib resistant human CML cell line established in our laboratory, characterized by a 2.4 fold increase on BCR-ABL mRNA levels and an imatinib IC50 18.3 fold higher than that observed for non-resistant K652 cells) were transfected with BCR-ABL siRNA complexed in transferrin-associated lipoplexes (ternary complexes). Levels of mRNA were evaluated by quantitative real time RT- PCR, protein levels by Western Blot and inhibition of cell proliferation by the Alamar Blue assay.

In synergistic studies cells were transfected with siRNA (0.2 μM) combined with different doses of imatinib. Different types and treatment schedules were evaluated (namely concomitant or sequential exposure of gene silencing agent and imatinib, using different incubation times). Cell viability was assessed 48h after starting the treatment.

Results indicate a 67% reduction on mRNA levels upon treatment with complexed BCR-ABL siRNA (0.2 μM). These results were correlated with a decrease of 30% on the protein levels. Approximately 70% cytotoxicity was achieved, this effect being specific and dose dependent. Treatment of IRK562 cells with the BCR-ABL siRNA (0.2 μM) resulted in 100% cell death. A 3 fold decrease in the imatinib IC50 was observed when combining siRNA and imatinib.

Therefore it can be concluded that gene silencing of the oncogene BCR-ABL with siRNA is a promising strategy which can be used alone or in combination with conventional drugs such as imatinib to overcome drug resistance.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA