Tiffany Holmes

Glycogen Synthase Kinase-3β Inhibition Preserves Hematopoietic Stem Cell Activity and Inhibits Leukemic Cell Growth

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase‐3β (GSK‐3β) regulates the degradation of β‐catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK‐3β inhibition activates β‐catenin in cord blood CD34+ cells and upregulates β‐catenin transcriptional targets c‐myc and HoxB4, both known to regulate HSC self‐renewal. GSK‐3β inhibition resulted in delayed ex vivo expansion of CD34+ cells, yet enhanced the preservation of stem cell activity as tested in long‐term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long‐term cultures treated with GSK‐3β inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK‐3β inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK‐3β inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK‐3β may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK‐3β inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK‐3β may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

GSK‐3β Inhibition Promotes Engraftment of Ex Vivo‐Expanded Hematopoietic Stem Cells and Modulates Gene Expression

Glycogen synthase kinase‐3β (GSK‐3β) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK‐3β, 6‐bromoindirubin 3′‐oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non‐obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo‐expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine‐induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK‐3β provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation. STEM CELLS 2011;29:108–118

The Role of Glycogen Synthase Kinase-3β in Normal Haematopoiesis, Angiogenesis and Leukaemia

Glycogen synthase kinase 3 beta (GSK-3beta) was one of the first kinases identified and studied, initially for its role in the regulation of glycogen synthesis. Over the past decade, interest in GSK-3beta has grown far beyond glycogen metabolism, and this is due in large measure to the critical role that GSK-3beta plays in the regulation of many other cellular processes, particularly cell proliferation and apoptosis. GSK-3beta has been shown to regulate the proteolysis and sub-cellular compartmentalization of a number of proteins directly involved in the regulation of cell cycling, proliferation, differentiation and apoptosis. GSK-3beta also regulates the degradation of proteins that regulate gene expression and thus affects a variety of important cell functions. Specifically, GSK-3beta controls the degradation of beta-catenin, the main effector of Wnt that regulates haematopoiesis and stem cell function. In this case GSK-3beta is a negative regulator of Wnt. In contrast, GSK-3beta positively regulates NF-kappaB, another important biochemical pathway also involved in the regulation of multiple aspects of normal and aberrant haematopoiesis. GSK-3beta regulates degradation of IkappaB, a central inhibitor of NF-kappaB. In this way, GSK-3beta acts to control the resistance of leukaemic cells to chemotherapy through the modulation of NF-kappaB, a critical factor in maintaining leukaemic cell growth. In addition, GSK-3beta regulates the pro-inflammatory activity of NF-kappaB. As GSK-3beta is a pleiotropic regulator, inhibitors may increase the range of novel anti-leukaemic and anti-inflammatory drugs that control immune response.

Ex vivo expansion of cord blood progenitors impairs their short‐term and long‐term repopulating activity associated with transcriptional dysregulation of signalling networks

Cord blood (CB) has been established to be an alternative source of haematopoietic stem/progenitor cells (HPC) for transplantation. The number of HPC per CB unit is limited, which results in engraftment delay. Ex vivo expansion of HPC improvement must overcome this.

GSK-3β inhibition promotes early engraftment of ex vivo-expanded haematopoietic stem cells.

Umbilical cord blood (UCB) is a source of stem cells used for allogeneic transplantation, in addition to bone marrow and peripheral blood. Limited numbers of stem cells in a single UCB unit is associated with slow haematopoietic recovery and high risk of graft failure, particularly in adult patients. UCB stem cells can be expanded ex vivo; however, rapid differentiation reduces their regenerative potential. We have recently shown that Wnt/β‐catenin signalling is down‐regulated in ex vivo‐expanded stem cells; therefore, we propose that re‐activation of Wnt signalling using GSK‐3β inhibition may act to improve regenerative potential of these ex vivo‐expanded stem cells.