Targeting the Hedgehog Signaling Pathway Limits the Self-Renewal of BCR-ABL1 Positive Leukemia Cells: Molecular Mechanisms

Abstract 531

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 BMI-1 show differential activation in normal versus leukemia stem cells. Hedgehog signaling is increased in BCR-ABL1 positive stem and progenitor cells becoming more active with disease progression. We have previously shown that the combination with the hedgehog signaling pathway inhibitor, vismodegib and ABL tyrosine kinase inhibitor (ABL TKIs) inhibits the minimal residual cells in BCR-ABL1-positive leukemia cells (Blood: ASH Annual Abstracts, Nov 2011; 118: 63). In the present study, we investigated the molecular mechanisms by which vismodegib and LDE225 regulate 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 Ph-positive acute lymphoblastic leukemia (n=2, T315I BCR-ABL1 and WT-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 hedgehog inhibition on self-renewal and the relevance of the hedgehog pathway as a therapeutic target in BCR-ABL1 positive leukemia, we examined the activity of vismodegib and LDE225 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 vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with vismodegib or LDE225 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, subsequently treated with vismodegib or LDE225 for 28 days. 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 vismodegib or LDE225 treated donors. These results demonstrate the persistent effects of hedgehog inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of hedgehog 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 1 μM of vismodegib or 200 nM of LDE225 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. Hedgehog pathway inhibition by vismodegib and LDE225 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 vismodegib and LDE225 (p<0.001). To identify the mechanisms that limit the self-renewal of BCR-ABL1-positive cells by vismodegib and LDE225, NOD/SCID mice engrafted with WT-BCR-ABL1-positive CD34⁺ CD19⁺ fractions were treated with vismodegib (20 mg/kg; p.o.) or LDE225 (20 mg/kg; p.o.) for 14 days. Both vismodegib and LDE225 induced the expressions of p21^Cip1, pATM, pChk2 and γH2AX related with DNA damage response, and reduced the expression of Gli-1, Gli-2, Bcl-2, and cyclin D2. Our preclinical results indicate that vismodegib and LDE225 have potential as an important option for controlling the drug-resistant leukemia initiating cells in BCR-ABL1 positive leukemia. Although several hedgehog inhibitors have now entered clinical evaluation, it is expected that hedgehog inhibitors may become extremely useful therapeutic interventions in a number of hematological neoplasms, including BCR-ABL1 positive leukemia, where the persistence of cancer stem cells.