Granulocyte–Macrophage Progenitors as Candidate Leukemic Stem Cells in Blast-Crisis CML

TLDR

CML progression to blast crisis is driven by self‑renewing leukemic stem cells, and in normal hematopoietic stem cells this self‑renewal is mediated by the beta‑catenin signaling pathway. The study aimed to determine whether leukemic stem cells in CML also depend on beta‑catenin signaling for self‑renewal. Researchers isolated hematopoietic stem cells and progenitor subsets from CML and normal marrow, quantified BCR‑ABL, beta‑catenin, and LEF‑1 transcripts by qRT‑PCR, visualized nuclear beta‑catenin via confocal microscopy and a TCF/LEF reporter, and assessed self‑renewal by in‑vitro replating assays while inhibiting beta‑catenin with axin lentiviral transduction. Granulocyte‑macrophage progenitors from blast‑crisis and imatinib‑resistant CML displayed elevated nuclear beta‑catenin, formed self‑renewing colonies, and their self‑renewal was diminished by axin, indicating beta‑catenin activation enhances leukemic stem cell activity.

Abstract

The progression of chronic myelogenous leukemia (CML) to blast crisis is supported by self-renewing leukemic stem cells. In normal mouse hematopoietic stem cells, the process of self-renewal involves the beta-catenin-signaling pathway. We investigated whether leukemic stem cells in CML also use the beta-catenin pathway for self-renewal.We used fluorescence-activated cell sorting to isolate hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from marrow during several phases of CML and from normal marrow. BCR-ABL, beta-catenin, and LEF-1 transcripts were compared by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay in normal and CML hematopoietic stem cells and granulocyte-macrophage progenitors. Confocal fluorescence microscopy and a lymphoid enhancer factor/T-cell factor reporter assay were used to detect nuclear beta-catenin in these cells. In vitro replating assays were used to identify self-renewing cells as candidate leukemic stem cells, and the dependence of self-renewal on beta-catenin activation was tested by lentiviral transduction of hematopoietic progenitors with axin, an inhibitor of the beta-catenin pathway.The granulocyte-macrophage progenitor pool from patients with CML in blast crisis and imatinib-resistant CML was expanded, expressed BCR-ABL, and had elevated levels of nuclear beta-catenin as compared with the levels in progenitors from normal marrow. Unlike normal granulocyte-macrophage progenitors, CML granulocyte-macrophage progenitors formed self-renewing, replatable myeloid colonies, and in vitro self-renewal capacity was reduced by enforced expression of axin.Activation of beta-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells.