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. 2010 Dec 28;5(12):e15262.
doi: 10.1371/journal.pone.0015262.

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Tara L Lin  1 , Qiuju H Wang, Patrick Brown, Craig Peacock, Akil A Merchant, Sarah Brennan, Evan Jones, Karen McGovern, D Neil Watkins, Kathleen M Sakamoto, William Matsui
Affiliations

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Tara L Lin et al. PLoS One. .

Abstract

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.

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Conflict of interest statement

Competing Interests: Karen McGovern is an employee of Infinity Pharmaceuticals. William Matsui has served as a consultant to Infinity Pharmaceuticals. Kathleen M. Sakamoto has received research support from Abbott Laboratories and Genentech, Inc. The remaining authors have declared that no competing interests exist. The authors confirm that this does not alter adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Expression of Hh pathway components is common in precursor B-ALL cell lines.
Qualitative RT-PCR analysis of Hh pathway gene expression in precursor B-ALL cell lines. HFB, human fetal brain, used as positive control for all experiments. For each cell line, positive and negative control reactions were run with B-actin primers with (to demonstrate equal amounts of cDNA template across each cell line) and without (to rule out contamination with genomic DNA) reverse transcriptase. B-ACTIN- denotes PCR results from mock cDNA synthesis reactions lacking RT. Precursor B-ALL cell lines: HB 1119 (t11;19), Nalm6 (t2;6), REH (t12;21), RS4;11 (MLL gene rearrangement); SEM-K2 (MLL gene rearrangement), TOM-1 (Ph+). Electrophoresis was performed with each gene separately across the panel of cell lines. The separate gels are shown together for the purpose of clarity across cell lines in the figure.
Figure 2
Figure 2. Hh pathway is active and inhibited by the SMO-inhibitors cyclopamine and IPI-926.
Precursor B-ALL cell lines REH and RS4;11 were transiently transfected with a Gli-responsive luciferase reporter or pGL3-basic, a plasmid lacking GLI binding site, as a negative control. Relative luciferase levels were measured following 48 hours of treatment with vehicle control, purified, dual lipid-modified Shh ligand (Shh-NP), SHh antibody 5E1 (10 µg/ml), cyclopamine (5 µM) or IPI-926 (1 µM). Hh pathway stimulation with SHh (p<0.005) and inhibition with 5E1 (p<0.0001), cyclopamine (p<0.0005) and IPI-926 (p<0.0002) was statistically significant in both the REH and RS4;11 cell lines.
Figure 3
Figure 3. ALDH+ cells are enriched for self-renewal potential and targeted by IPI-926.
(A)ALDH+ and ALDHneg cells were isolated from the REH cell line by FACS using the Aldefluor reagent and DEAB control. The gated area represents the ALDH+ cells, measuring 1.18% of total REH population. (B) ALDH+ and ALDHneg cells from REH and RS4;11 were plated in methylcellulose to evaluate clonogenic growth. Colony formation was measured at 10–14 days. There was a 5-fold difference in colony number following initial plating for REH (p = 0.05) and RS4;11 (p<0.01). (C) A representative plate from initial plating was washed with media and cells resuspended prior to replating in methylcellulose for an additional 10-14 days. Secondary plating of ALDH+ and ALDHneg cells demonstrated an 8-fold difference in colony-formation between the ALDH+ and ALDHneg cell fractions for REH (p<0.002) and RS4;11 (p<0.001). (D)Unfractionated REH and RS4;11 cells were treated with cyclopamine (5 µM), IPI-926 (1 µM) or vehicle control for 10 days and the percentage of ALDH+ and ALDHneg cells was measured using the Aldefluor staining kit and FACS. The reduction in ALDHneg cells following treatment with cyclopamine and IPI-926 was statistically significant for REH (p<0.01) and RS4;11 (p<0.03).
Figure 4
Figure 4. Hh pathway regulates self-renewal in precursor B-ALL.
Clonogenic recovery of REH and RS4;11 cells following treatment with Hh pathway agonist and inhibitors. B-ALL cells were treated with Hh pathway modulators for 72 hours, washed free of drug and plated in quadruplicate in methylcellulose. At 10–14 days, colonies were counted (represented as initial plating). A representative plate was then washed and cells resuspended and replated. After an additional 10–14 days, colonies were counted (represented as secondary replating). Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups are expressed as % control ± SEM. (A)Colony-formation of REH and RS4;11 cells following treatment with the Hh pathway ligand SHhNP. Increased colony-formation was statistically significant in both cell lines at initial and secondary replating despite no further addition of drug (p<0.001 for all groups). (B) Colony-formation of REH and RS4;11 cells following treatment with Hh pathway inhibitors 5E1 (10 µg/ml), cyclopamine (5 µM), IPI-926 (1 µM). Inhibition of clonogenic growth at secondary replating for both REH and RS4;11 cells was statistically significant (p<0.02) with Hh pathway inhibition with 5E1, cyclopamine and IPI-926.
Figure 5
Figure 5. Engraftment of human leukemia cells in NOD/Scid mice.
Representative flow cytometry results of mouse bone marrow demonstrating engraftment of human leukemia. Mice were injected with REH cells and then treated with either vehicle control or IPI-926 for 21 days. Mouse bone marrow was harvested after development of symptoms and analyzed by flow cytometry for the presence of human leukemia cells. All mice in both treatment groups had engraftment of human leukemia cells that were mouse CD45 negative, human CD45 positive and human CD19 positive.
Figure 6
Figure 6. Prolonged overall survival of recipient mice of pre-treated IPI-926 REH cells.
NOD-Scid mice were injected by tail vein with REH cells that had been pre-treated with either IPI-926 or vehicle in vitro prior to injection. Mice were followed for progression of symptoms and overall survival, and recipient mice of pre-treated cells had statistically significantly longer overall survival (70 days versus 50 days, p<0.001).
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References

    1. Ingham P, McMahon A. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15:3059–3087. - PubMed
    1. Weng AP, Ferrando AA, Lee W, Morris JP, Silverman LB, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306:269–271. - PubMed
    1. Ji Z, Mei F, Johnson B, Thompson E, Cheng X. Protein kinase A, not Epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells. J Biol Chem. 2007;282:37370–37377. - PubMed
    1. Kawahara T, Kawaguchi-Ihara N, Okuhashi Y, Itoh M, Nara N, et al. Cyclopamine and quercetin suppress the growth of leukemia and lymphoma cells. Anticancer Res. 2009;29:4629–4632. - PubMed
    1. Warzecha J, Bonke L, Koehl U, Munkelt D, Göttig S, et al. The hedgehog inhibitor cyclopamine induces apoptosis in leukemic cells in vitro. Leuk Lymphoma. 2008;49:2383–2386. - PubMed

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