Targeting The Dopamine Receptor Signaling Limits The Self-Renewal Of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and Dopamine Receptor show differential activation in normal versus leukemia stem cells. Using a high-throughput discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells, the norvel dopamine receptor antagonist, thioridazine, was identified from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal (Cell 149, 1284, 2012). In the present study, we investigated the molecular mechanisms by which dompamine receptor antagonist, thioridizine, regulates the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or ponatinib-resistant Ph-positive acute lymphoblastic leukemia (n=2, Y253H/E255K/T315I BCR-ABL1 and T315I BCR-ABL1) into NOD/SCID/IL-2γc-/- mice. The cell fractions with CD34+CD38- CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of the domamine receptor antagonist on self-renewal and the relevance as a therapeutic target in ABL-tyrosine kinase-resistant BCR-ABL1 positive leukemia, we examined the activity of thioridazine against CD34+CD38-CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with thioridazine (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with thioridazine reduced the population of CD34+CD38- positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from thioridazine-treated donors. These results demonstrate the persistent effects of domapine receptor signaling inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of dompamine receptor pathway modulation on in vitro clonogenic growth. CD34+CD38-CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 5 μM of thiorizaxine for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Dopamine receptor pathway inhibition by thioridazine had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by thioridazine (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by thioridazine, NOD/SCID mice engrafted with T315I -BCR-ABL1-positive CD34+ CD19+ fractions were treated with thoridazine (20 mg/kg; p.o.) for 14 days. Thioridazine induced the expressions of p21Cip1 and reduced the expression of c-Myc, Oct-3/4, Sox-2, and Bcl-2. Our preclinical results indicate that thioridazine have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. It is expected that the domapine receptor antagonist may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells.