Activation Of p53 By MDM2-Inhibition Sensitizes Quiescent Blast Crisis CML Stem/Progenitor Cells To Bcl-2 Inhibitor- and Tyrosine Kinase Inhibitor-Induced Cell Death
Authors: Carter Bing Z , Mak Po Yee , Mak Duncan H , Konopleva Marina , Cortes Jorge , Andreeff Michael 
Published: October 2019

Abstract
The transcriptional activation of pro-apoptotic Bcl-2 family members is a major mechanism of p53-mediated cell death. p53 can also directly contribute to mitochondrial mediated apoptosis by interacting with Bcl-2 family proteins. A number of anti-apoptotic Bcl-2 proteins such as Mcl-1 and Bcl-xL are regulated by the Bcr-Abl tyrosine kinase and confer apoptosis resistance to CML cells. It is therefore not surprising that p53 gene alterations may play a role in the disease progression of CML. The reported p53 mutation rate of 30% suggests potential therapeutic benefit of p53 activation by MDM2 inhibition alone or as sensitization of CML to therapeutic agents.
Tyrosine kinase inhibitors (TKIs), the front-line therapy for patients with chronic phase CML, are less effective in patients with blast crisis (BC) CML and inactive against quiescent CML stem/progenitor cells. Here we examine the effects of nutlin3a, which activates p53 by inhibiting MDM2, and its combinations with the Bcl-2 inhibitor ABT-737 and the TKI nilotinib on the viability of proliferating and quiescent CD34+CML stem/progenitor cells obtained from patients with BC CML.
Mononuclear cells from patients with BC CML who were resistant to multiple TKIs were stained with the cell division tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM) derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells are distinguishable by flow cytometry (within 7 to 12 days), the cells were treated with nutlin3a, ABT-737, nilotinib, nutlin3a plus ABT-737, or nutlin3a plus nilotinib for 48 hours with or without MSC co-culture. The cells were then stained with a CD34 antibody, and apoptosis (annexin V staining) was determined by flow cytometry. Apoptosis in proliferating and quiescent progenitor cells was defined as percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbrightcells, respectively.
Nutlin3a alone was able to decrease viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50 = 2.50 ± 0.92 μM and 2.54 ± 0.60 μM, respectively) and quiescent (IC50 = 3.70 ± 1.22 μM and 4.27 ± 0.77 μM, respectively) CD34+ CML stem/progenitor cells. Although not very active by itself, ABT-737, when combined with nutlin3a (n = 6), induced apoptosis synergistically in proliferating (CI = 0.24 ± 0.12) and more so in quiescent (CI = 0.09 ± 0.10) CD34+CML stem/progenitor cells, even when cells were co-cultured with MSCs (CI = 0.22 ± 0.09 for proliferating and CI = 0.03 ± 0.05 for quiescent stem/progenitor cells). The combination of nutlin3a with nilotinib (n = 4) was also highly synergistic in proliferating stem/progenitors (CI = 0.02 ± 0.04 without and CI = 0.05 ± 0.04 with co-culture) and in quiescent progenitors (CI = 0.07 ± 0.29 without and CI = 0.03 ± 0.06 with co-culture).
Treatment of primary CML blast cells with nultin3a increased p53, and pro-apoptotic Bax and Puma levels in all samples examined, indicating activation of transcriptional activity of p53. In addition, nutlin3a treatment decreased the protein levels of anti-apoptotic Bcl-xL and/or Mcl-1. Nilotinib also decreased the expression, both at the RNA and protein levels, of Mcl-1 and Bcl-xL, even in patients who had not responded to nilotinib treatment clinically.

Conclusions
Here we demonstrate that activation of p53 induces apoptosis in proliferating and quiescent BC CML stem/progenitor cells and sensitizes to Bcl-2 inhibitor- and TKI-induced cell death by, at least in part, regulating the expression of Bcl-2 family proteins. Results suggest a potential for utilizing this strategy in the treatment of BC CML, and also for the elimination of quiescent CML stem/progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.