Imatinib Increases Cytotoxicity of Melphalan and Their Combination Allows An Efficient Killing of CML Cells.

Abstract 4254

BCR/ABL-positive cells are relatively resistant to chemotherapy and, in order to evaluate the effect of Imatinib (IM) in reverting drug-resistance, we evaluated on K562 the toxicity of 1 h exposure to cytosine arabinoside (ARA-C) 20 μM, hydroxyurea (HU) 100 μM, and melphalan (MEL) 20 μM, after a pre-treatment of 24 h with 1 μM IM. The doses of the drugs were similar to that achieved in the plasma after standard chemoterapeutic treatment. Cell viability was evaluated by ATP-lite at 24, 48 and 72 hs from beginning of drug-free condition. The combinations of IM plus MEL induced the highest cytotoxicity (P<0,001 at 24, 48 and 72 hs vs MEL alone) indicating that pre-treatment with IM increased K562 exposition to the genotoxic damage of MEL. We next analyzed effects on cell cycle and DNA damage by alkaline comet assay induced by this drug combination and we observed that DNA damage peaked at 48 h with IM/MEL combination. In addition, flow cytometry analysis showed that IM/MEL combination reduced the cell accumulation in G2/M phase induced by MEL (P<0.001 vs MEL), thus reducing the ability to DNA repair and recovery.

These data indicate that inhibition of BCR/ABL activity by IM increased cell cytotoxicity of MEL by reducing the effectiveness of the DNA-repair pathways and decreasing the time for DNA repair at the G2/M checkpoint..

To ascertain that these results were linked to BCR/ABL inhibition, TonB.210, a cell line where the BCR-ABL expression is inducible by doxycycline (DOX), were treated in the same conditions. Only TonB.210 cultured with DOX were insensitive to MEL while IM/MEL combination reverted these drug resistance (P<0,001).

In the final step we studied the sequential association IM/MEL on the proliferative potential of myeloid progenitors of 6 CML patients at diagnosis. IM/MEL combination increased the reduction of the overall number of colonies in comparison to IM alone (P<0.05 vs IM). In addition, the analysis on CFU-GM and BFU-E colonies by qRT-PCR demonstrated that the IM/MEL combination led to the highest reduction in the number of BCR/ABL positive colonies (P<0.01 vs IM).

Therefore, our data indicate that the pre-inhibition of BCR/ABL activity by IM increases the toxicity of MEL and allows an efficient killing of human leukemic cells, thus suggesting new therapeutic combinations for CML patients