Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

• Published in: Blood 2016-12, Vol.128 (22), p.1882-1882
• Main Authors: Michaels, Shawnya J., DeSouza, Ngoc, Shan, Yi, Li, Shaoguang, Bates, Saira
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.1882.1882

Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed. The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs). Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells. To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner. The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive. Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle