Activation of p53 Enhances Antileukemia Activity of Bcr-Abl Tyrosine Kinase Inhibitors in a Murine Model of CML

Abstract

Although CML patients under Bcr-Abl tyrosine kinase inhibitor (TKI) therapy have experienced high response rates and long-time survival, 50% or more patients relapse upon therapy cessation, largely because TKIs do not eliminate CML stem cells. We previously reported that inhibition of antiapoptotic Bcl-2 proteins synergized with TKIs to eradicate CML stem/progenitor cells. The tumor suppressor p53 induces apoptosis in part by modulating Bcl-2 family proteins. Although rarely mutated in CML, p53 activity is antagonized by Bcr-Abl tyrosine kinase-regulated MDM2. We hypothesize that activation of p53 by MDM2 inhibition will synergize with TKIs to eliminate CML cells. Using an inducible transgenic Scl-tTa- BCR-ABL mouse model, we first determined the expression of p53 and its target genes in bone marrow (BM) cells of Tet-on (control) and Tet-off (CML) mice. We found markedly increased RNA levels of p53 and its targets Bax, Noxa, and MDM2 in CML as compared to control mice. Using CyTOF mass cytometry and cytofkid automatic subset detection/clustering based on the expression of cell surface markers, we then determined p53 and its target proteins in bulk and Lin-Sca-1+cKit+ (LSK) populations at single cell levels and found overexpression of p53 and its targets such as Noxa in CML BM cells (CD45+) compared to controls. The differential expression was most pronounced in the LSK populations. For both control and CML mice, LSK cells expressed significantly higher p53 protein than their respective bulk cell population. We next evaluated the sensitivity of these cells to BH3 peptide priming. CML BM cells and more so LSK populations were more sensitive to BH3 peptides, especially to Noxa peptide than cells from control mice (P=0.044 in CD45+ and P=0.0005 in LSK populations) suggesting that CML cells are more primed to apoptosis stimuli. Finally, we tested the antileukemia activity of combined MDM2 and Bcr-Abl inhibition in mice engrafted with BM cells from Scl-tTa- BCR-ABL /GFP mice. Combined inhibition of Bcr-Abl tyrosine kinase (imatinib) and MDM2 (DS-5272) strongly reduced BM leukemic populations and LSK cells, increased Noxa, decreased leukemia burden, and significantly prolonged survival (P=0.0021 vs. control). Median survival days for control, imatinib, DS-5272, and the combination groups were 85, 110, 155, and 248; respectively.Our results demonstrate that CML cells/stem cells have increased p53 signaling and are highly primed to undergo apoptosis. Combined p53 activation and Bcr-Abl tyrosine kinase inhibition targets CML stem/progenitor cells and has the potential to significantly improve response and cure rates of CML. DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding. Seki:Daiichi Sankyo: Employment. Andreeff:Daiichi Sankyo: Consultancy.