Enhanced Cytotoxicity in Chronic Myeloid Leukemia Primitive Progenitors by the Combined Action of Imatinib and An HDM2-Inhibitor

In Chronic Myeloid Leukemia (CML) the 9;22 chromosomal translocation and corresponding BCR-ABL protein are present in the most primitive hematopoietic stem/progenitors (Lin−/CD38−/CD34+). These cells are refractory to the effect of BCR-ABL tyrosine kinase inhibitors. The mechanism of this resistance has not been fully elucidated but is clearly distinct from the mechanism of resistance in the more mature CML cells. The p53 gene is rarely mutated in the chronic phase of CML. BCR-ABL is able to positively affect p53 expression whose potential proapoptotic effect may be balanced by survival signals such as Bcl-XL and Stat signaling. However, BCR-ABL also positively regulates HDM2, the negative regulator of p53, which may be the alternative mechanism of counteracting the induced p53. In an effort to facilitate a cytotoxic effect directed against the refractory CML primitive stem/progenitor cells we elected to explore the role of stabilizing the p53 protein. Accordingly we tested a novel inhibitor of the HDM2-p53 interaction (MI-219; Ascenta), which interferes with unmutated p53 degradation. MI- 219 induced reproducible cytotoxicity in four CML blast-crisis cell lines with intact p53 (WDT2, WDT3, BV173 and BV173R) with an IC50 ~2 microM. The BV173R cell line which has the Imatinib resistant T315I mutation displayed a cytotoxic effect with the MI- 219 equal to its parental BV173 cell line (IC50 ~2 microM). Responses were associated with the induction of p53 protein, its targets p21^WAF1 and PUMA, and cleavage of PARP. The K562 cell line with mutated p53 did not respond to MI-219 as expected. MI-219 had a modest cytotoxic effect on magnetically separated (MACS) CD34+ cells from CML patients as a single agent (range of 30–50% cell death at 5 microM MI-219). Nevertheless, MI-219 markedly enhanced the cytotoxic effect of Imatinib on CD34+ cells, while as a single agent Imatinib induced 15–30% apoptosis. However the combination of 2 microM Imatinib and 5 microM MI-219 led to a cytotoxic effect averaging 76.4 ± 10.6% apoptosis. This enhanced cytotoxic effect was further noted in flow cytometrically sorted progenitor (Lin−/CD38+/CD34+) populations (~86.7% apoptosis). This combination equally induced apoptosis in primitive progenitor/stem cells (Lin−/CD38−/CD34+; ~83.0%), despite the minimal affect of each agent when given alone (Imatinib, ~20.8 % apoptosis; MI-219, ~36.9% apoptosis). This cytotoxic effect in primary CML cells was again associated with the induction of p53, p21^WAF1, and the cleavage of PARP. Here we demonstrate that an increased level of p53 bypasses T315I associated resistance to Imatinib, and in combination with Imatinib generates a substantial cytotoxic effect in early progenitors, which are otherwise refractory to the effect of either agent alone. Thus these observations propose that the combination of MI-219 an HDM2-inhibitor with Imatinib may facilitate the eradication of minimal residual disease present within the primitive Lin−/CD38−/CD34+ population of CML.