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B.Z. Carter et al.
   affected by the treatments (Figure 4B) at this time point. Although we did not observe decreased spleen leukemic hematopoiesis by imatinib (but rather some increase) or DS-5272, the combination statistically significantly reduced leukemia GFP+LSK (Figure 4C), while neither sin- gle agent alone, nor their combination, statistically signif- icantly altered the number of GFP–LSK in mouse spleen (Online Supplementary Figure S1B). In addition, the combi- nation also statistically significantly decreased GFP+CMP cells in spleen and tended to reduce leukemia WBC and mature myeloid cells (Figure 4C). Again, the treatments
did not affect GMP cells in the spleen.
Mice that received DS-5272 were treated for only two
weeks during the second half of the treatment (Figure 4A). Although DS-5272 by itself did not statistically significant- ly decrease CML cells in several cell populations, it greatly enhanced the activity of imatinib, especially in suppress- ing GFP+LSK cells, both in BM and spleen.
To further assess the anti-leukemia activity of each agent and the combination, we determined circulating leukemia at the end of and at 7 and 11 weeks after treat- ments by flow cytometry measurement of GFP+ total WBC and GFP+ neutrophils. As shown in Figure 5A, at the end of the treatments, all treated groups had statistically significantly fewer GFP+WBC and neutrophils compared with the control group (P<0.001), and the combination was statistically significantly more effective than imatinib (P<0.01 for both WBC and neutrophils) or DS-5272 alone (P<0.01 for WBC and P=0.0264 for neutrophils; left panel). At 7 weeks after treatments, only the combination group demonstrated statistically significant decreases in GFP+WBC (P=0.0134) and GFP+ neutrophils (P=0.0139) compared with the control group (middle panel). At 11 weeks after treatments, although all treatment groups still showed lower leukemia burden compared with controls, no statistical significance was reached (right panel). However, only 50% (7 of 14) of mice in the control group, compared to 60% (6 of 10) in the imatinib-treated group, 67% (10 of 15) in the DS-5272-treated group, and 71% (10 of 14) in the combination treatment group, remained alive at the end of treatment.
Treatments greatly improved overall survival in CML- bearing mice (P=0.0066) (Figure 5B). Although not statisti- cally significant, the median overall survival for the DS- 5272 or imatinib group was 211 and 249 days, respectively; both were markedly longer than that of the untreated con- trol group (141 days). The mice treated with the combina- tion had a median overall survival of 326 days, which was statistically significantly longer than that of the control (P=0.001), the imatinib-treated (P=0.039), and the DS-5272- treated (P=0.047) groups. The mice seemed to tolerate the treatments well. No obvious weight loss was observed in the treated mice (Online Supplementary Figure S2).
These data indicated that, like imatinib, inhibition of MDM2 by DS-5272 also had anti-leukemia activity in CML, and the combination had significantly more than each agent alone. The mice treated with the combination had deeper and longer lasting responses, suggesting that combined inhibition of MDM2 and BCR-ABL1 may target CML stem cells.
Combined inhibition of MDM2 and BCR-ABL1 targets chronic myeloid leukemia stem cells in BCR-ABL1 transgenic mice
and BCR-ABL1 targeted CML stem cells, we carried out another set of experiments (see Figure 4A). At the end of treatments, BM cells were obtained from each group for the secondary transplantation. Unfortunately, we did not obtain sufficient cells from the DS-5272 treatment group and the secondary transplantation was conducted using only cells from the untreated control and imatinib or ima- tinib/DS-5272 treated mice. The number of mice with evi- dence of engraftment in PB at 16 weeks was determined by flow cytometry. Engraftment was defined as GFP levels >1% (above the background levels in the PB of FVB/N mice without cell injection).
Figure 6A shows the percentage of GFP+ cells in PB sam- ples of mice injected with various amounts of cells from mice treated with different agents. Based on the engrafted and total mice transplanted in each group, we calculated the leukemia LT-HSC frequency. The CML LT-HSC fre- quency was statistically significantly different among the groups (P=0.0039) (Figure 6A), and statistically significant- ly decreased by the combination treatment compared with imatinib alone (P=0.0131) or the untreated control (P=0.0012). No statistically significant difference was observed between imatinib-treated and untreated groups (P=0.38), as expected. For every LT-HSC detected in the combination-treated group, three times as many cells were detected in the imatinib group, and four times as many cells were detected in the control group.
We also collected BM cells from mice that received 0.25x106 cells/mouse and determined GFP positivity by flow cytometry and BCR-ABL1 RNA levels by RT-PCR. As shown in Figure 6B, engraftment rates in BM were sim- ilar to those in PB samples (Figure 6A). The number of mice showing markedly lower BM BCR-ABL1 RNA levels (3 logs lower than that in controls) was consistent with the number of mice with GFP <1% in both BM and PB sam- ples (Figure 6B).
Discussion
Effectively treating patients with blast crisis CML, and eradicating CML stem cells to achieve cures and prevent disease progression to blast crisis, are two major chal- lenges facing CML therapy. We previously reported that combination of TKI with a MDM2 inhibitor enhanced apoptosis induction in not only proliferating but also qui- escent blast crisis CML progenitor cells in vitro.27 In the cur- rent study, we investigated if this combination strategy has the potential to target CML stem cells and improve cure rates in CML-CP.
Using an inducible transgenic Scl-tTa-BCR-ABL1 mouse model, we demonstrated that CML and CML stem/prog- enitor cells have increased p53 signaling and are more sen- sitive to BH3 peptide-induced apoptosis. This increase was further validated in samples obtained from patients with newly-diagnosed CML compared with normal BM controls, indicating oncogenic induction of p53. This observation supports the idea that CML cells have a propensity to death induction, and activation of p53 by MDM2 inhibition sensitizes CML to TKI. Indeed, we demonstrated that combined inhibition of MDM2 and BCR-ABL1 activated p53 signaling, targeted CML stem/progenitor cells, prolonged mouse survival, and decreased CML LT-HSC.
To determine whether combined inhibition of MDM2
In contrast to the report by Abraham et al.,24 we found
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