Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.
Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Li K, Manz MG, Keating A, Sawyers CL, Weissman IL
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Activation of beta-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells.
Inhibitors of cellular signaling targets: designs and limitations.
Kinases carry out the reversible phosphorylation of proteins and lipids, and are responsible for direct or indirect control of almost every signaling pathway in cells, leading to responses such as proliferation, differentiation, metabolism, transport, and gene expression. It is estimated that the human genome may contain about 1000 kinases. To date, approx 500 protein kinases have been identified, of which about 400 are serine/threonine kinases and approx 100 are tyrosine kinases. Because of their central role in cellular signaling, as well as their primary role in disease progression, protein kinases are attractive therapeutic targets. Seven molecules targeting protein kinases, imatinib mesylate (Gleevec) and trastuzumab (Herceptin), an inhibitor of BCR-abl and an antibody against ERB-2, respectively, are currently marketed as anti-oncolytics. In addition, six molecules that block kinase activity are currently being investigated in Phase III clinical trials, whereas as many as 30 candidates targeting kinases are in early clinical development (Phase I or Phase II).Many kinases are activated by more than one stimulus and many proteins are phosphorylated by multiple kinases; therefore the complexity and redundancy of these reactions imposes major limitations on the development of “selective inhibitors” of kinases. The objective of this chapter is to provide an understanding of the process to evaluate potentially useful therapeutic compounds directed toward kinases. Areas that will be highlighted include target validation (demonstration that the target kinase is involved in disease), target enablement (the design of a systematic screening approach to evaluate kinase inhibitors through a series of cell-free and cell-based assays), and hit-to-lead determination (the iterative process of compound evaluation culminating in the selection of a clinical candidate). In addition, limitations are discussed in order to give the investigator guidance in developing, selecting and/or using pharmacological kinase inhibitors.
A bull in the Spanish countryside near Madrid