Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia. However, ABL TKIs do not eliminate the leukemia stem cells (LSCs) in the bone marrow, which may represent the most important event in leukemia relapse after TKI discontinuation. Bone marrow is also considered a tissue with limited oxygen supply. Therefore, new approach against leukemia stem cells, which is presented in bone marrow under hypoxia, may improve the outcome of CML patients. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate limiting enzyme of the primary pathway for maintaining cellular NAD+ and that regulates intracellular adenosine triphosphate (ATP) levels in mammalian cells. It has been reported that NAMPT expression is upregulated in several human cancers.
Materials and methods: In this study, we established ABL TKI resistantin vitrocell line models (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R and Ba/F3 T315I) and used Ph-positive leukemia cell lines. We also investigated whether NAMPT inhibitor could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant under hypoxic condition.
Results: In chemical library screen of compounds, NAMPT inhibitor, CHS828 is selected the candidate drug for ABL TKI resistant cells under hypoxic condition. Drug repositioning and repurposing can be an important part of any drug discovery. Therefore, we examined NAMPT efficacy by using Ph-positive leukemia cell lines. We first investigated the NAMPT expression by microarray gene expression data from the online Gene Expression Omnibus (GEO). Gene expression of NAMPT is increased in CML patients compared to normal samples from the public microarray datasets of GSE13159. Protein expression of NAMPT is found in Ph-positive leukemia cell lines including ABL TKI resistant cells. In hypoxia, gene expression of NAMPT was increased in Ph-positive cells compare to normoxic condition. We next examined efficacy of NAMPT inhibitor, CHS828 in Ph-positive leukemia cell lines. We found that CHS828 treatment for 72 h decreased cell viability of Ph-positive cell lines in a dose dependent manner. We also found that CHS828 inhibited the proliferation of ABL TKI resistant cells (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R) and T315I mutant Ba/F3 cells under hypoxic condition. The rate of cell cycle progression was delayed and cells were blocked in G2/M phase. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. CHS828 inhibited constitutive NF-kB activity in the time and dose dependent manner. Intracellular ATP, which is the most important source of energy for cellular reactions, was drastically decreased after CHS828 treatment. NAD+ plays a vital role in diverse cellular processes that govern human health and disease. Intracellular NAD+ was decreased after CHS828 treatment. The wound healing effect was evaluated at 0, 24, 48, and 72 h by using NIH3T3 cells. In wound healing results, CHS828 treatment inhibited cellular migration of NIH3T3 cells. We next investigated the efficacy of ABL TKI and CHS828 against Ph-positive leukemia cell lines. ABL TKI and CHS828 treatment induced cellular growth inhibition compared with each drug alone. Caspase 3/7 activity was also increased after ABL TKIs and CHS828 treatment. ABL TKI and CHS828 reduced the colony formation ability of Ph-positive cells under hypoxic condition. Thein vivoefficacies of one of ABL TKI, ponatinib and CHS828 were evaluated in a mouse xenograft model. Ponatinib and CHS828 inhibited the growth of T315I mutant Ba/F3 cellsin vivomore than the control vehicle. We also found that co-treatment with ponatinib and CHS828 increased mouse survival. Combination treatment with ponatinib and CHS828 was also well tolerated, with no animal deaths in the treated mice.
Conclusion: The results of our study indicate that the ABL TKI and CHS828 may be a powerful strategy against Ph-positive cells including ABL TKI resistant cells and provide the promising clinical relevance as a candidate drug for treatment of CML stem cells of the bone marrow microenvironment under hypoxic condition.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
