Combination of Imatinib with Cisplatin and Nutlin-3: Functional and Molecular Effects on Bcr-Abl Positive Cells.

Imatinib is highly effective in inducing remissions in chronic phase CML. However, complete eradication of the malignant clone by Imatinib monotherapy is rare. This prompted us to explore the efficacy of combination of Imatinib with the DNA damaging agent Cisplatin and with Nutlin-3, a compound which induces p53 accumulation by interfering with its binding to Mdm2. We used a Bcr-Abl positive cell line characterized by the loss of Imatinib sensitivity in the presence of optimal growth factor concentrations. Whereas combined treatment of Imatinib with either Cisplatin or Nutlin-3 in low and nontoxic doses induced ∼15–20% cell death, simultaneous treatment of all three compounds was highly effective (∼50% cell death) even in the presence of optimal growth conditions. Importantly, comparable effects were also seen in CFU assays performed with primary CD34 positive cells from 5 CML patients. To examine the molecular mechanisms of these effects we analyzed p53 dependent cellular response to Cisplatin in our cell line model in the presence or absence of Imatinib and/or Nutlin-3. The active Bcr-Abl kinase caused superinduction of p53 protein after DNA damage. We found both an upregulated p53 accumulation and enhanced p21 and Mdm2 RNA and protein levels upon Cisplatin treatment. This phenomenon could be reversed both by siRNA-mediated inhibition of Bcr-Abl expression and by Imatinib-induced inhibition of Bcr-Abl kinase activity: despite of optimal growth conditions inhibition of Bcr-Abl caused a significant reduction of p53 expression and activation. In the presence of Nutlin-3, p53 expression was significantly upregulated upon Cisplatin treatment both with and without Imatinib. However, Nutlin-3 was not capable to restore the Imatinib-induced blockade of the transcriptional activity of p53 on mdm2. The reduced p53 activation observed in Bcr-Abl positive cells treated with Imatinib was paralleled by a shift from cell cycle arrest to cell death both in the presence and absence of Nutlin selectively in Bcr-Abl positive cells rendering them hypersensitive to Cisplatin. In summary, combined treatment of Imatinib with low concentrations of a DNA damaging agent and a p53 activator is highly effective in vitro. Such combined treatment may prove to be clinically relevant for complete eradication of the malignant clone in CML, provided that conditions are found where it does not affect adversely normal hematopoietic cells in vivo.