Regeneration-associated WNT signaling is activated in long-term reconstituting AC133bright acute myeloid leukemia cells

Abstract

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Figure 1

Human AC133⁺ cells are strongly expanded in AML. Representative dot plots of the immunophenotype analysis from the BM of (A) a healthy donor and (B) a patient with AML (AML No. 42 in Table W1). The CD45/CD133.1 co-staining was gated on BM MNCs; percentages on total cellularity are shown for gated normal and AML populations. (C) Flow cytometry analysis of the CD133.1 antigen in BM MNCs of healthy donors (n = 10) and AML patients (n = 25). The IQR for each sample group is indicated in the dot. Mann-Whitney U test was used to calculate the P value (α = 0.001).

Figure 2

Schematic visualization of dysregulated networks in KEGG database and representation of WNT gene expression profiles. (A) Visualization of WNT signaling pathway in the KEGG database. Highlighted genes are differentially expressed and red-starred pathways are overrepresented in AC133⁺ AML cells. (B) Heat map of the differentially expressed WNT-associated genes. The color bar under the patient sample dendrogram identifies AML samples (red) and healthy controls (blue). The names, accession numbers, as well as P values for the differentially overexpressed and underexpressed genes are shown in the right panels.

Figure 3

Altered WNT signaling in AC133⁺ AML cells. (A) Detection with padlock probe and target-primed rolling circle amplification of individual WNT10B transcripts on BM slides from AML patients. Green RCPs represent WNT10B transcripts, and red RCPs represent β-actin transcripts in consecutive sections. Cell nuclei are shown in blue. Images were acquired with x20 magnification. Scale bar, 10 µm. (B) The quantification of RCPs was done on three x20 images of BM biopsies of AML patients. For quantification, the numbers of RCPs of each image were counted digitally using CellProfiler software. The average of RCPs for each sample was calculated and it is reported as aratio between β-actin and WNT RCPs. (C) Immunoblot analysis of ABC, β-catenin (as detected with the N-terminal pan-β-catenin antibody), WNT10B, and Pygopus 2 protein expression in AC133⁺ cell fractions from 18 patient samples and 1 healthy donor introduced as control. GAPDH, loading control. HD, healthy donor; *therapy-related secondary AML.

Figure 4

β-Catenin activation in the subpopulation of AC133^bright AML cells expressing WNT10B. (A) Representative immunostaining micrographs show green fluorescence of cells expressing AC133 in a BM section of AML No. 9 (Table W1). Cell nuclei are shown in blue. Scale bar represents 10 µm. (B) Co-staining of BM from AML No. 9 adjacent serial section for expression of ABC (green) and WNT10B (red). Cell nuclei are shown in blue (DAPI). (C) False color maps of ABC/WNT10B double positive cells (blue) were obtained using an automatic threshold based on the moments algorithm implemented in the ImageJ program. DAPI staining was used to identify nuclei. Images obtained crossing ABC masks with WNT10B signals were used to count the percentage of ABC/WNT10B double positive cells over the total number of cells. The macro was validated against a trained experimenter over a sample of 830 total cells from eight different images. Differences in results were restricted to less than 0.01%. (D) Morphologic detail of cells showing intense specific staining for ABC (top panels) and WNT10B (bottom panels). All images were acquired with a x40 objective. Scale bars represent 10 µm. Representative images of at least three serial slides from five randomly selected patients.

Figure 5

AC133⁺ A46 cells express and release WNT10B. Dot plots of the immunophenotype analysis from AML No. 46 BM MNCs at diagnosis and after selection. (A) Patterns of CD38/CD133.1 (top left), CD117/CD45 (top right), and CD34/CD38 (bottom left) co-staining were gated on BM AML cells before selection. Representative CD34 and CD38 expression on Ficoll-selected MNCs (bottom center) and AC133sorted cells before culture (bottom right) is shown. Percentages on total cellularity are shown for gated AML populations. (B) Immunostaining assessment for WNT10B in AC133⁺ populations from A46 (top panels) or healthy donor (bottom panels). Representative images of at least five serial slides. Blue, nuclei; red, WNT10B; merge, WNT10B/DAPI. All images were acquired with a x40 objective. Scale bars represent 10 µm. (C and D) TOPFlash reporter assay showing luciferase expression driven by eight TCF/lymphoid-enhancing factor (LEF) binding sites. (C) Positive control was obtained by CM of pBA-WNT10B-transfected H293T cells. Expression of WNT10B was evaluated in pBA-WNT10B-transfected H293T by real-time PCR (left). TOPFlash reporter assay shows luciferase expression induced in Super 8x TOPFlash-H293T cells by pBA-WNT10B H293T CM (right). (D) TOPFlash reporter assay showing dose-dependent luciferase expression induced in Super 8x TOPFlash-H293T cells by A46 CM. Significance was evaluated by the unpaired Student's t test: *P < .05; **P < .001. Data represent the mean ± SD of triplicate reactions and are representative of three independent experiments.

Figure 6

AC133⁺ A46 cell transplantation in Rag2^-/-γc^-/- mice. (A) Overview of the experimental design: 1 x 10⁶ AC133⁺ A46 cells were injected into sublethally irradiated Rag2^-/-γc^-/- mice through the tail vein. Three weeks after transplantation, BM cells were collected and analyzed by flow cytometry. (B) Expression profiles of three recipient mice (M1–M3) are shown. hCD45⁺ population was identified in BMs of mice, and within hCD45⁺, the expression patterns of hCD34 and hCD133.1 (AC133) were analyzed. Cells were separated in subpopulations according to the expression of hCD34 and hCD133.1 and then analyzed for hCD38 and hCD133.1 expressions.

Figure 7

A46 AML cells induce ectopic gene expression and secondary body axis formation upon transplantation in zebrafish embryos. (A) Fluorescence microscopy of a live zebrafish embryo at 30% of epiboly (lateral view) transplanted at 3 hpf at the animal pole region with A46 cells previously blue-stained with Hoechst 33342. (B–D) Dorsal side view of 70% epiboly-stage embryos hybridized with a gsc-specific probe. Embryos have been injected with (B) normal AC133⁺ cells as control or (C and D) A46 AML cells. The arrowheads indicate the gsc endogenous signal, while arrows specify the position of zebrafish cells expressing ectopic gsc. (E) Bright-field microscopy of a 24-hpf zebrafish embryo injected with A46 AML cells (lateral view). The arrowhead and the dotted line indicate the secondary trunk/tail induced by A46 cells. (F, G) The embryo in E has been hybridized with a probe specific for the notochord and tail bud marker ntl. (F) The probe labels the notochord (n) in the endogenous trunk and the chordoneural hinge (cnh) in both tails (G, higher magnification). (H) Tail of a 24-hpf embryo hybridized with ntl-specific probe. The endogenous (n) and ectopic (*n) ntl signals run parallel along the axis of the embryo, indicating the presence of additional axial structures. (I) Dorsal view of a 24-hpf embryo that developed an ectopic head on the side of the endogenous one, as indicated by the expression of the brain marker gene pax2a. The dotted lines indicate the main (M) and secondary (S) axes. The optic stalk (OS) in close vicinity to the eye (e), the midbrain-hindbrain boundary (MHB), and the otic vesicles (OV) of the embryo are stained with the pax2a riboprobe, as well as several areas of the ectopic head (the asterisks indicate the two clearly recognizable additional otic vesicles). The image is composed of different pictures corresponding to several focal planes, since the embryo is not flat, and a single focal plane cannot comprise all the labeled structures belonging to the main and secondary axes. Scale bars represent 125 µm (A–D), 150 µm (E), 40 µm (F), 15 µm (G and I), or 25 µm (H).