Page 117 - Haematologica May 2020

MDM2 and BCR-ABL1 inhibition targets CML stem cell
We next stained BM cells from Tet-off and Tet-on Scl- tTa-BCR-ABL1 FVB/N mice with a panel of metal-tagged antibodies for cell surface and intracellular proteins (Online Supplementary Table S3) and performed CyTOF mass cytometry analysis. Following Cytofkit unsupervised sub- set detection based on RPhenoGraph clustering algo- rithms, expression of various proteins was determined in bulk CD45+ and LSK (cluster 9, pink circle) cells (see Figure 1B). We found increased levels of p53 and its targets, including NOXA, MDM2, and p21 in CD45+ and LSK cells from Tet-off BM cells compared with their Tet-on counter- parts, as displayed by RPhenoGraph (Figure 1B) as well as in a heat map showing the expression of each protein in each individual mouse (Figure 1C). We previously report- ed that BAX, another target of p53, was also higher in BM CD45+ and LSK cells from Tet-off CML mice compared to that in control mice.20
To demonstrate that increased expression of p53 and its target proteins also occurs in newly-diagnosed CML-CP patients, we first performed RT-PCR using RNA isolated from fresh BM samples of patients (n=5) (Online Supplementary Table S1) and normal controls (n=6). Although there were high individual variations, overall BM cells from CML patients expressed higher levels of RNA representing p53 signaling proteins compared with those from normal controls, with statistically significantly higher BAX (P=0.009) and markedly higher PMAIP1 (NOXA) (P=0.06) (Figure 2A).
We next isolated CD34+ cells from fresh BM or PB sam- ples of newly-diagnosed CML patients (n=7) (Online Supplementary Table S1) and fresh BM samples from nor- mal controls (n=5) and obtained sufficient material for determining p53 and BAX protein levels by western blot analysis. We found that CD34+ cells from CML-CP patient
A
B
Figure 2. Expression of p53 and target genes in samples from patients with chronic phase chronic myeloid leukemia (CML)-CP and normal bone marrow (BM) con- trols (NBM). RNA and protein lysates were prepared from freshly collected samples and CML samples were obtained from untreated newly diagnosed CML-CP patients. (A) RNA levels of TP53 and its target genes in CML patient samples (n=5) and NBM controls (n=6), determined by Taq-Man real-time polymerase chain reac- tion. (B) p53 and BAX protein levels in CD34+ cells of NBM (n=5) or of untreated newly diagnosed CML-CP patients (n=7) were determined by western blot analysis and the correlation of the two proteins in patient samples was shown. MWM: molecular weight markers. Error bars indicate standard error of the mean.
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