ABSTRACT
Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.
Subject(s)
Alternative Splicing/genetics , Glycogen Synthase Kinase 3/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplastic Stem Cells/enzymology , Animals , Base Sequence , Blast Crisis/enzymology , Blast Crisis/pathology , Glycogen Synthase Kinase 3 beta , Granulocyte-Macrophage Progenitor Cells/pathology , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mice , Molecular Sequence Data , Stem Cell TransplantationABSTRACT
Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.
Subject(s)
Cell Differentiation/drug effects , Erythroid Precursor Cells/enzymology , Erythroid Precursor Cells/pathology , Janus Kinase 2/antagonists & inhibitors , Polycythemia Vera/enzymology , Polycythemia Vera/pathology , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Amino Acid Substitution , Animals , Base Sequence , Erythroid Precursor Cells/drug effects , Female , Humans , Janus Kinase 2/genetics , Male , Mice , Middle Aged , Molecular Sequence Data , Phenylalanine/genetics , Protein Kinase Inhibitors/chemistry , Signal Transduction/drug effects , Stem Cell Transplantation , Valine/geneticsABSTRACT
To examine the mechanism by which growth-stimulated pancreatic beta-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and betalox5, a cell line derived from human beta-cells which progressively dedifferentiated in culture. MIN6/betalox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that betalox5 expresses a dominant transacting factor(s) that represses beta-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary beta-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in beta-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the beta-cell differentiated state.
