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MDM2 and BCR-ABL1 inhibition targets CML stem cell
    anti-apoptotic BCL-2 proteins enhances TKI activity in CML,17-19 and we demonstrated that BCL-2 is a key survival factor of CML stem cells, and targeting BCL-2 with ABT- 199, combined with a TKI, enhanced eradication of CML stem cells.20
Among its numerous tumor suppressor functions, p53 activates the expression of the pro-apoptotic BCL-2 pro- teins BAX, PUMA, NOXA, and BID triggering apoptosis.21- 23 Altered p53 and MYC transcriptional network in CML stem cells was recently reported, and targeting both p53 and MYC selectively eliminated CML stem cells.24 Activation of p53 by inhibition of SIRT1 or MDM2, in com- bination with TKI has been explored in CML.25,26 We report- ed that TKI in combination with the MDM2 inhibitor nut- lin3a enhanced apoptosis induction in proliferating and qui- escent blast crisis CML progenitor cells in vitro.27
The expression of p53 can be induced when cells are stressed, including those associated with oncogenic stimu- lation. Like other oncogenes, the hyper-proliferative signal from BCR-ABL1 can activate p53 and induce cell cycle block and senescence to counterbalance oncogenic stimulation signals. This may also contribute to CML stem cell mainte- nance. However, the role of p53 signaling proteins in BCR- ABL1 oncogene-driven CML/CML stem cells and the response of CML stem cells to the combined MDM2 and BCR-ABL1 inhibition have not been fully investigated.
Using an inducible, stem cell promoter (Scl)-driven trans- genic CML murine model (Scl-tTa-BCR-ABL1 mice),15,20,28,29 we here determine the expression of p53 and its signaling proteins in bone marrow (BM) cells and lineage-SCA-1+C- KIT+ (LSK) cells from CML and control mice, and in BM cells in CML mice treated with the MDM2 inhibitor DS- 5272, the TKI imatinib, or both, using novel CyTOF mass cytometry, which measures single-cell protein expression in phenotypically-defined cell populations. We also investigat- ed the anti-leukemia activity of combined MDM2 and BCR-ABL1 inhibition in this model.
Methods
Mouse model and cells
Mouse experiments were performed in accordance with MD Anderson Cancer Center Animal Care and Use Committee approved protocols. Scl-tTa-BCR-ABL1 FVB/N mice28,29 were pro- vided by Dr. R. Bhatia (University of Alabama at Birmingham, AL, USA). BM cells were collected from mice 3-4 weeks after tetracy- cline cessation (Tet-off) or from controls (Tet-on).
Human cells
Cells from newly diagnosed chronic phase CML (CML-CP) patients (Online Supplementary Table S1) and normal controls were obtained as described in the Online Supplementary Methods.
Real-time polymerase chain reaction
Real-time polymerase chain reaction (RT-PCR) was carried out as previously described20 using freshly isolated mouse or human BM cells. Primers are shown in Online Supplementary Table S2. The abundance of each transcript relative to that of Abl1 or ABL1 was calculated using the 2−DCt method, expressed as copies of each mRNA/1000 copies of Abl1 or ABL1.
Western blot
Western blot was performed as described previously.20 Antibodies against human p53 and BAX were purchased from Santa Cruz (Dallas, TX, USA).
Mass cytometry
Mouse BM cells were stained with metal-tagged antibodies for cell surface markers and intracellular proteins (Online Supplementary Table S3) and subjected to mass cytometry (CyTOF) analysis as previously described16,20,30 and as briefly described in the Online Supplementary Methods.
Mitochondrial priming
BH3 priming assay as previously described31 is described briefly in the Online Supplementary Methods.
In vivo experiments
GFP+ CML cells from donor mice as previously described15,20
were injected (0.6x106 cells/mouse) into FVB/N recipient mice (The Jackson Laboratory) irradiated at 900 cGy. After CML developed, assessed by flow cytometry measurement of GR-1 (LY6G)+ cells, mice were treated daily (oral gavage) with imatinib (100 mg/kg; vehicle: acidified water, pH 5.0) for four weeks, DS-5272 (50 mg/kg; vehicle: 0.5% w/v methylcellulose 400) for two weeks (ini- tiated two weeks after imatinib group), imatinib for two weeks and then plus DS-5272 for two additional weeks, or vehicle control (1:1 volume of each vehicle). Two sets of experiments were per- formed.
Experiment I: at the end of treatments, BM and spleen cells (n=3- 5/group) were collected and stained with a lineage cocktail and antibodies against SCA-1 (eBioscience, ThermoFisher Scientific), C- KIT (CD117), CD34, FcγRII/III, GR-1 (LY6G), and MAC-1 (CD11b) (all from BioLegend, San Diego, CA, USA) to measure leukemia LSK, myeloid progenitors, and myeloid cells as previously described.20 Peripheral blood (PB) leukemia burden was measured by total and GFP+ white blood cell (WBC) count (CD45+) and total and GFP+ neutrophils (Ly6G+) using flow cytometry. Mouse sur- vival was recorded.
Experiment II: BM cells were collected at the end of treatments for secondary transplantation as described previously.20 At 16 weeks, PB engraftment was determined, and the frequency of leukemia long-term hematopoietic stem cells (LT-HSC) was calcu- lated. Green fluorescent protein (GFP) positivity and BCR-ABL1 RNA levels in BM cells from mice that received 0.25x106 cells/mouse were determined.
Statistical analysis
Results are expressed as mean±standard error of the mean. P<0.05 was considered statistically significant (two-sided Student t- test). Correlation coefficient was determined by Pearson correlation analysis. Mouse survival was estimated by the Kaplan-Meier method and data were analyzed using the log-rank test. LT-HSC frequency was calculated using the Extreme Limiting Dilution Analysis software program (http://bioinf.wehi.edu. au/software/elda/).32
Results
BCR-ABL1 oncogene increases the expression of p53 and p53 targets
To determine p53 expression and p53 signaling in hematopoietic cells of CML mice and compare with those of controls, we collected BM cells from Tet-off CML and Tet-on control Scl-tTa-BCR-ABL1 FVB/N mice, and deter- mined the RNA levels of p53 and its target genes by RT- PCR. We found that BCR-ABL1 induction significantly increased Trp53 (p53) RNA and that of its target genes including Bax, Pmaip1 (Noxa), Mdm2, and Cdkn1a (p21) (Figure 1A) (P<0.001 for all genes analyzed), supporting oncogenic induction of p53.
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