The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML

Abstract

Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Because selective eradication of LSCs could offer substantial therapeutic benefit, there is interest in identifying the signaling pathways that control their development. We studied LSCs in mouse models of acute myelogenous leukemia (AML) induced either by coexpression of the Hoxa9 and Meis1a oncogenes or by the fusion oncoprotein MLL-AF9. We show that the Wnt/beta-catenin signaling pathway is required for self-renewal of LSCs that are derived from either hematopoietic stem cells (HSC) or more differentiated granulocyte-macrophage progenitors (GMP). Because the Wnt/beta-catenin pathway is normally active in HSCs but not in GMP, these results suggest that reactivation of beta-catenin signaling is required for the transformation of progenitor cells by certain oncogenes. beta-catenin is not absolutely required for self-renewal of adult HSCs; thus, targeting the Wnt/beta-catenin pathway may represent a new therapeutic opportunity in AML.

Figure 1. β-catenin is activated in developing leukemia cells and L-GMP derived from KLS-HoxA9/M and GMP-MLLAF9

a, Immunophenotypic analyses of GFP+ cells from bone marrow of mice transplanted with pre-leukemia KLS-HoxA9/M or GMP-HoxA9/M cells at 8 days, 1 month or 4 months (leukemia) post-transplantation. b, The heat map displays the top 30 probe sets for genes that show increased expression in the HSC population (KLS cells) and L-GMP using Comparative Marker Selection and permutation testing (200 probe sets passed a cutoff of P < 0.002). Note expression of Ptgs-1 (Cox-1) and Ptger1 (prostaglandin E receptor 1). c, Immunoblot analysis for active (dephosphorylated) β-catenin in normal GMP or L-GMP derived from GMP-MLLAF9 or KLS-HoxA9/M mediated leukemias. d, Immunofluorescence assessment for active β-catenin in c-Kit^high or c-Kit^low populations isolated from mice 1 month after injection of pre-leukemia KLS-HoxA9/M cells. (active β-catenin: red, Hoechst: blue; active β-catenin/Hoechst: merge).

Figure 2. Constitutively active β-catenin cooperates with HoxA9/M to induce AML from GMP cells

a, Constitutively active βcat* was transduced into pre-leukemia KLS-HoxA9/M or GMP-HoxA9/M cells and protein levels were assessed by immunoblot analysis. b, Survival curves of mice receiving KLS or GMP cells transduced with active βcat* (controls), GMP-HoxA9/M, GMP-HoxA9/M cells transduced with active βcat*, or KLS-HoxA9/M. 5 × 10⁵ cells were transplanted into sublethally irradiated recipients (n=10 in each group). P determined using the log-rank test

Figure 3. Pharmacological inhibition of β-catenin impairs LSC function

a, Immunoblot analysis of β-catenin levels in c-Kit^high cells sorted from bone marrow at 1 month post-injection of mice with KLS-HoxA9/M cells which were subsequently grown in methylcellulose with and without exposure to indomethacin for 3 weeks (25,26). b, Immunophenotypic analysis of bone marrow GFP+ cells after injection of KLS-HoxA9/M cells and treatment with either vehicle or indomethacin (repeated with 2 independent clones) for 7 days. Treatment began at day 3 post-injection and mice were sacrificed at day 10 post-transplantation (22,26). c,d Survival curves of mice (5 mice for each group) injected with the indicated number (10 – 10⁴) of GFP+ marrow cells sorted from control or Indomethacin (Indo)-treated mice that had received pre-leukemia KLS-HoxA9/M cells (c) or leukemic GMP-MLLAF9 cells (d) (repeated with 2 independent clones).

Figure 4. Deletion of β-catenin impairs the development of KLS-HoxA9/M and GMP-MLLAF9 induced AML

a, GFP + cells in bone marrow of mice injected with floxed β-catenin (βcat^loxp/loxp) or β-catenin-deficient (βcat^−/−) KLS-HoxA9/M cells at 18 hr, 1 month, 4 months post-transplantation. HoxA9/M-βcat^loxP/loxP KLS cells were transduced with a retroviral vector encoding Cre recombinase to generate βcat ^−/− KLS-HoxA9/M cells or an empty vector to generate control βcat^loxp/loxp KLS-HoxA9/M cells. After selection, 1 × 10⁶ infected cells were transplanted into sublethally irradiated recipients (n=5 in each group). Mice were sacrificed at the indicated time points to assess for GFP+ cells. b, Survival curves of mice transplanted with βcat^−/− KLS-HoxA9/M cells, βcat^loxp/loxp KLS-HoxA9/M cells, and empty vector or Cre-infected WT KLS-HoxA9/M (KLS cells were sorted from wild-type C57BL/6 mice and transformed with HoxA9/M to assess the effect of Cre on KLS-HoxA9/M leukemia development; n=10 for each group). c, Survival curves of mice transplanted with βcat^−/− GMP-MLLAF9 cells and βcat^loxp/loxp GMP-MLLAF9 cells (n=10 for each group). P determined using the log-rank test