MJ05, a Novel p53 Activating Compound, Effectively and Selectively Eliminates Human CML Stem/Progenitor Cells
Authors: Chu Su , Drummond Catherine , Li Ling , Ho YinWei , Lain Sonia , Bhatia Ravi 
Published: October 2019

Abstract
Tyrosine kinase inhibitors (TKI) are the mainstay of CML treatment but fail to eliminate leukemia stem cells (LSC), leading to high risk of disease recurrence when treatment is stopped. There is considerable interest in developing new strategies to target CML LSC. We have previously shown that p53 activation following SIRT1 deacetylase inhibition can inhibit growth and survival of TKI-treated CML LSC (Cancer Cell 2012, 21:266). We are therefore interested in investigating other strategies to activate p53 as potential approaches to target CML LSC. While conducting a screen of 20,000 small molecules for ability to activate p53-dependent transcription in TP53 wild-type ARN8 human melanoma cells we identified MJ05 amongst the top-ranking compounds. Importantly MJ05 did not activate p53-dependent transcription in T22 fibroblast cells. Increased p53 protein levels in ARN8 cells were seen within 6 hours and were accompanied by an increase in p21, pig3 and mdm2 mRNA and protein levels. Dependency on p53 was confirmed using p53-null and wild-type H1299 cells. Activation of p53 occurred without concurrent increase in DNA damage evidence by g-H2AX labeling, increased p53 Ser 15-phosphorylation, or inhibition of p53-HDM2 interaction. MJ05 treatment inhibited S-Phase progression of ARN8 cells without inhibition of ATM, ATR or DNA-PK phosphorylation, suggesting a unique mechanism of action. We tested the effect of MJ05 on primary normal or CML CD34+ cell by itself and in combination with TKI inhibitor Nilotinib, and compared with effects on Nutlin, a well studied inhibitor of p53-HDM2 interactions. Apoptosis was assessed by Annexin V labeling, proliferation by CFSE labeling, and colony forming cell (CFC) frequency, in methylcellulose progenitor assays. Treatment with MJ05 (5 and 10μM), with or without Nilotinib (1μM), for 72 hours in the presence of low concentrations of growth factor significantly and selectively increased apoptosis, inhibited proliferation and reduced colony CFC frequency in CML CD34+ cells compared to normal CD34+ cells. Combination of MJ05 with Nilotinib (1μM) resulted in significant increase in apoptosis of CML but not normal CD34+ cells. In contrast treatment with Nutlin (2 and 5 μM) resulted in similar increase in apoptosis in CML and normal CD34+ cells. We next evaluated the effect of treatment with MJ05 (10μM), Nilotinib and the combination for 72 hours on purified CML and normal CD34+CD38- stem/primitive progenitor cells and CD34+CD38+ committed progenitor cells. MJ05 significantly enhanced apoptosis of CML but not normal CD34+CD38- cells and CD34+CD38+ cells. Apoptosis was further enhanced by combination with Nilotinib (Table). MJ05 also resulted in significant reduction of proliferation in CML CD34+38+ and CD34+38- cells, with significantly less inhibition of proliferation of normal cells. MJ05 treatment markedly reduced CFU-GM and BFU-E generation from CML compared to normal CD34+CD38- and CD34+CD38+ cells. The combination with Nilotinib resulted in almost complete abrogation of CML CFC (Table). MJ05 resulted in significantly less inhibition of normal CFC, with greater effect on normal BFU-E compared to CFU-GM. Ongoing xenograft experiments are testing the effect of in vitro treatment with MJ05, Nilotinib or the combination on engraftment of CML and normal stem cells in NSG mice. Our studies indicate that MJ05, a unique, potent and selective p53 activating compound, is remarkably effective in inducing apoptosis and inhibiting growth of primitive CML stem/progenitor cells by itself and to an even greater extent in combination with Nilotinib. MJ05 treatment has significantly lesser effects on normal stem cells, and may offer a promising approach to selectively target CML LSC in combination with Nilotinib.Table 1NormalCMLUntreatedMj05+NilMj05+NilUntreatedMj05NilMj05+NilCD34+38+% Apoptosis4.5±0.19.0±1.14.13±018.8±7.17.5±0.815.9±4.412.3±1.723±7.4CFU-GM86±757.3±4.191±3.852±278.7±19.95±336±141±0BFU-E166±15.315±3.2155.7±9.314±1.595±19.30.7±0.721.3±5.50.3±03CD34+38-% Apoptosis4.2±0.26.1±0.34.2±0.15.3±0.423.0±1.230±4.114.6±3.850.1±7.2CFU-GM67.3±15.941.7±5.549±4.730.3±4.9192.7±50.66±2.372.67±48.70.7±0.7BFU-E58.7±20.211±2.526.3±2.97.6±0.9150.3±58.71±0.672±440.3±0.3

Disclosures:
No relevant conflicts of interest to declare.