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. 2013 Mar 7;121(10):1824-38.
doi: 10.1182/blood-2012-02-412890. Epub 2013 Jan 8.

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling

Bin Zhang  1 , Min Li, Tinisha McDonald, Tessa L Holyoake, Randall T Moon, Dario Campana, Leonard Shultz, Ravi Bhatia
Affiliations

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling

Bin Zhang et al. Blood. .

Abstract

Tyrosine kinase inhibitors (TKIs) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSCs, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSCs from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSCs) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony-forming ability and engraftment potential in immunodeficient mice. We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-cadherin-mediated adhesion to MSCs was associated with increased cytoplasmic N-cadherin-β-catenin complex formation as well as enhanced β-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated β-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-cadherin and the Wnt-β-catenin pathway in protecting CML LSCs during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSCs to TKI treatment and suggest new _targets for treatment designed to eradicate residual LSCs in CML patients.

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Figures

Figure 1
Figure 1
MSCs protect CML CD34+CD38− and CD34+CD38+ cells from TKI treatment. Primary CML and normal (NL) progenitor (CD34+) cells were stained with CFSE. CFSE+ primitive cells (Lin−CD34+CD38−) and committed cells (Lin−CD34+CD38+) were sorted by flow cytometry and cultured for 96 hours with or without MSCs and were treated with IM (5 μm), nilotinib (Nil; 5 μm), or dasatinib (Das; 0.15 μm) or were left untreated. The percentages of apoptotic primitive cells (A) and committed cells (B) were assessed on the basis of Annexin V+ labeling. A proliferation index was calculated based on reduction in CFSE levels for CML and NL CD34+CD38− cells (C) and CD34+CD38+ cells (D) treated with IM, nilotinib, or dasatinib compared with controls (Ctrls) with and without MSCs. (E) Representative flow cytometry plots and (F) graph showing apoptosis (Annexin V+ cells) within undivided (CFSE bright) and dividing (CFSE dim) CML CD34+CD38− cells treated with IM with and without MSCs. (G) Percentages of undivided (CFSE bright) CML CD34+CD38− and CD34+CD38+ cells after culture with or without IM treatment and with or without MSCs. (H) CML CD34+CD38− and CD34+CD38+ cells cultured for 96 hours with or without MSCs with or without treatment with IM (5 µm), nilotinib (5 µm), or dasatinib (0.15 µm) were plated in methylcellulose progenitor assays, and colony-forming capacity (CFC) frequencies were determined. (I) Representative flow cytometry plot and (J) graph showing CD34+ expression after culture of CML CD34+ cells with or without IM treatment and with or without MSCs. ns, not significant. n = 3. *P < .05.
Figure 2
Figure 2
MSCs protect CML LSCs capable of engrafting NSG mice from TKI treatment. CML CD34+ cells (2 × 106 cells per mouse) from 3 patients were cultured for 96 hours with and without MSCs and with or without IM and transplanted into NSG mice. Mice were euthanized after 4 and 10 weeks, and marrow contents of femurs, spleen cells, and blood cells were obtained. (A) Flow cytometry plots and (B) graphs showing human cell engraftment in peripheral blood (PB), BM, and spleen at 4 weeks (4w) posttransplantation. (C) BCR-ABL mRNA levels in marrow cells obtained from mice at 4 weeks posttransplantation. (D) Flow cytometry plots and (E) graphs showing human cell engraftment in PB, BM, and spleen at 10 weeks posttransplantation. (F) BCR-ABL mRNA levels in marrow cells obtained from mice at 10 weeks (10w) posttransplantation. ns, not significant. n = 5. *P < .05; ***P < .001.
Figure 3
Figure 3
Role of N-cadherin in MSC-mediated protection of CML stem/progenitor cells from TKI treatment. (A) CML CD34+ cells cocultured on MSCs without IM (left) and with IM (right) adhered to and migrated underneath (right, arrowhead) MSCs. (B) The fraction of MSC-adherent CML CD34+ cells with or without IM. (C) Representative flow cytometry plots and (D) graphs showing apoptosis of CML CD34+ cells (C) and CD34+CD38+ and CD34+CD38− cells (D) cultured without MSCs in a Transwell insert above MSC layers or in contact with MSCs in the absence or presence of IM (n = 3). (E) Representative flow cytometry plots showing N-cadherin expression on CML and normal CD34+, CD34+CD38−, and CD34+CD38+ cells. (F) N-cadherin mRNA levels in CML and normal CD34+CD38− and CD34+CD38+ cells (n = 3 CML patients and 3 healthy controls). (G) Representative flow cytometry plots showing N-cadherin expression on CML CD34+ cells cultured with and without IM and with or without MSCs. (H) N-cadherin mRNA levels in CML CD34+ cells cultured with and without IM in the presence or absence of MSCs (left), in adherent (adh) and nonadherent suspension (susp) cells, and in cells treated with an N-cadherin blocking (NCDH) and NCDH control peptide (right) (n = 3). (I) Reduced adhesion of CML CD34+ cells to MSCs in the presence of NCDH peptides. (J) Apoptosis of IM-treated CML CD34+ cells cocultured with MSCs in the presence of NCDH or control peptides (n = 3). (K) Apoptosis of IM-treated CML CD34+CD38− and CD34+CD38+ cells cocultured with MSCs and N-cadherin blocking or isotype control antibodies (n = 3). ns, not significant. *P < .05; **P < .01.
Figure 4
Figure 4
Enhanced Wnt–β-catenin signaling in TKI-treated CML stem/progenitor cells cocultured with MSCs. (A) Western blotting for P-Crkl, N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34+ cells cultured with and without MSCs and with and without IM treatment. (B) Western blotting for N-cadherin, β-catenin, P-GSK3β (S9), and actin in CML CD34+ cells and MSC-adherent and MSC-nonadherent CML CD34+ cells with and without IM treatment, and in CML CD34+ cells cocultured with MSCs and IM and NCDH or control peptide as shown. (C) Immunofluorescence microscopy of CML CD34+ cells labeled with antibodies to N-cadherin (green) and β-catenin (red) following culture as shown. Nuclei were labeled with 4,6 diamidino-2-phenylindole (DAPI; blue). Results shown are representative of 100 cells analyzed per slide. (D) N-cadherin and β-catenin protein–protein interactions were evaluated by using Duolink in situ PLA technology. Protein interactions are visualized as red dots. (E) Cytoplasm (cyt) and nuclear (nucl) fractions from CML CD34+ cells cultured with and without MSCs were analyzed by western blotting for β-catenin, α-tubulin, and sp1. (F) Wnt–β-catenin–related transcriptional activity was evaluated by using an improved TOPFlash reporter system in CML CD34+ cells cultured as shown. (G) Q-PCR analysis for mRNA expression of the Wnt–β-catenin _target genes cyclin-D1, c-Myc, and peroxisome proliferator-activated receptor delta (PPARD) in CML CD34+ cells cultured as shown. (H) Western blotting for β-catenin, N-cadherin, P-GSK3β (S9), and actin in normal CD34+ cells cultured with and without MSCs. (I) Q-PCR analysis for mRNA expression of N-cadherin, β-catenin and Wnt–β-catenin _target genes cyclin-D1, c-Myc, and PPARD in normal CD34+ cells cultured as shown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant. n = 3. *P < .05; **P < .01; ***P < .001.
Figure 5
Figure 5
Microarray assay of gene expression in CML CD34+ cells cocultured with and without MSCs and with or without IM. (A) The number of differentially expressed genes when comparing the different treatments and (B) the interactions between MSCs and IM in determining gene expression in CML CD34+ cells are shown. GSEA showed increased expression of gene sets related to HSC self-renewal and quiescence, cytokine signaling, adhesion, metabolism, and cell cycle regulation in MSC-cocultured CML CD34+ cells with or without IM (C) and enrichment of cadherin (D) and Wnt–β-catenin (E) –related gene sets.
Figure 6
Figure 6
Role of Wnt–β-catenin signaling in MSC-mediated protection of CML stem/progenitor cells from TKI treatment. (A) Apoptosis of CML CD34+ cells treated with IM (5 µM), ICG001 (5 µM), or IM plus ICG001 in the absence and presence of MSCs. Cell cycle of CML CD34+ cells treated with IM, ICG001, or IM plus ICG001 in the absence (B) and presence (C) of MSCs. (D) CML CD34+ cells were exposed to CM from Wnt1-transfected cells and Wnt reporter activity measured after 2 days. (E) Flow cytometry plots and (F) graph showing apoptosis of CML CD34+ cells cocultured with Wnt1-CM with or without IM treatment. (G) β-catenin protein expression and (H) nuclear localization of β-catenin in IM-treated CML CD34+ cells after addition of Wnt receptor antagonist DKK1(1 µg/mL). Results shown are representative of 100 cells analyzed per slide. (I) Apoptosis of IM-treated CML CD34+ cells cocultured with MSCs in the presence and absence of DKK1. (J) Proposed Wnt–β-catenin and N-cadherin interactions in CML CD34+ cells treated with TKI in the presence of MSCs. TKI treatment stabilizes β-catenin by reducing β-catenin phosphorylation, increasing N-cadherin–mediated adhesion to MSCs, and enhancing N-cadherin–β-catenin interaction. Wnt proteins secreted by MSCs activate β-catenin signaling in MSC-adherent CML stem/progenitor cells, leading to enhanced nuclear translocation of β-catenin and transcription of _target genes. Complex formation with N-cadherin in MSC-adherent CML stem/progenitor cells may protect β-catenin from degradation and provide a β-catenin pool that can be activated by exogenous Wnt ligands. MSC-induced N-cadherin and Wnt–β-catenin signaling protects and preserves CML stem/progenitor cells from TKI treatment. ns, not significant. n = 3. *P < .05.
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