Aberrant Regulation Of Wnt/Beta-Catenin Pathway Mediators In Chronic Myelogenous Leukemia Stem Cells

Abstract Background Recent research suggests that self-renewing leukemia stem cells (LSC) with increased beta-catenin expression are involved in chronic myelogenous leukemia (CML) progression. We investigated whether aberrant regulation of beta-catenin destruction complex genes contributed to the enhanced self-renewal potential of CML LSC. Methods FACS Aria purified normal and CML hematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP) and lineage positive cells were transduced for 48 hours with lentiviral luciferase GFP and transplanted intrahepatically into newborn RAG2−/−gama−/− mice. At 8 to 12 weeks human CD45+ cells were FACS-purified and transplanted into secondary recipients to establish a bioluminescent CML LSC model. RT-PCR for BCR-ABL was used to confirm CML engraftment. Wnt mediator mutation analysis was performed on cDNA via fluorescent denaturing high performance liquid chromatography (DHPLC) technology and SURVEYOR mismatch cleavage analysis both with the WAVE-HS System (Transgenomic, Gaithersberg, MD). Aliquots of PCR product (3-15 ul) from all samples were scanned for mutations by DHPLC and confirmed by Surveyor mismatch cleavage, and identified with bidirectional sequence analysis on an ABI 3100 sequencer using BigDye V3.1 terminator chemistry. Quantitative RT-PCR was also performed on CML progenitors using destruction complex gene specific primers. FACS analysis was performed with the aid of a FACS Aria to analyze changes in Wnt signaling pathway mediators. Results Advanced phase CML was typified by expansion of a GMP population with aberrantly activated beta-catenin expression, enhanced in vitro replating capacity as well as serial transplantation potential in a CML LSC bioluminescent imaging model suggesting that the GMP population was enriched for LSC (Figure 1). A targeted Wnt mutation analysis revealed a mutation in a key component of the beta-catenin destruction complex - glycogen synthase kinase 3beta (GSK) in progenitors from three of six blast crisis CML samples analyzed. Decreased GSK expression was confirmed via FACS analysis using a GSK specific antibody in three separate experiments with CML blast crisis progenitors (Figure 2). Some CML blast crisis progenitors also demonstrated a decrease in axin 2 by quantitative RT-PCR. Conclusions Altered expression of Wnt signaling pathway regulators, such as GSK3, may result in increased LSC self-renewal capacity and may represent novel therapeutic targets for advanced phase CML. Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 2. GSK FACS Analysis. Figure 2. GSK FACS Analysis.