The Canonical Wnt/β-Catenin Pathway Is Active in Pre-B Acute Lymphoblastic Leukemia (ALL) Cells and Augments Their Growyh and Survival.

Wnt signalling is known to play an important regulatory role in hematopoietic progenitors/stem cells during both fetal and adult development. Recently this pathway has been implicated in several hematological malignancies, however, its precise role in pathogenesis of pre-B ALL remains unknown. In this study we examined the expression of genes required for Wnt signalling in 12 ALL cases, normal bone marrow mononuclear cells, bone marrow stroma and the effect of Wnt-3a stimulation on leukemic cell growth and survival. RT-PCR analysis revealed expression of Wnt family members 2b, 5a, 10b and 16b in most ALL cases. Primary BM stroma expressed Wnt-5a alone while BM mononuclear cells expressed Wnts 3a, 5a, 10b and 16b. Wnt receptors Fz-7 and 8 were widely expressed on ALL cases while Fz-3 and 4 showed restricted expression. Together this suggests that Wnt signalling pathway may be active in pre-B ALL cells. To characterise functional consequences of Wnt pathway activation we investigated the effects of Wnt-3a on proliferation, survival and cell cycle status of blasts from three pre-B ALL cell lines, one newly derived stromal dependent cell line (STYG) and one primary patient sample under 3-day serum-free culture conditions. Exposure to Wnt-3a resulted in a mean 5.3-, 5.2-, 1.7-, 2.7- and 5.1- fold stimulation of 3H-thymidine incorporation in NALM6, Reh, LK63, STYG and primary ALL cells, respectively. Cell cycle analysis based on propidium iodide staining confirmed an increased proportion of cells progressing from G_0/1 through to S/G2/M phases. Wnt3a augmented survival in all leukemic cells by 18.6% (range 11.7% – 24.6%, p<0.001) in comparison to untreated cells under serum-free conditions as measured by Annexin V/PI staining. Immunofluorescence microscopy revealed elevated nuclear b-catenin levels in Wnt-3a stimulated cells, confirming activation through the canonical pathway. Our results suggest that Wnt proteins may exert a proliferative/survival effect on leukemic cells through an autocrine/paracrine regulatory loop.