Alla Dolnikov

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

Using Small Molecule GSK3β Inhibitors to Treat Inflammation

Glycogen Synthase Kinase 3 beta (GSK3β) is a serine-threonine kinase originally identified for its role in the conversion of glucose to glycogen. Pharmacological inhibition can be achieved by drug binding to ATP or magnesium binding sites on the enzyme. Pharmaceutical companies have developed several small molecule GSK3β inhibitors for diabetes research. Additionally, GSK3β inhibitors are being clinically tested as therapeutics for neurological diseases, however, the mechanisms of involvement are unclear. Several studies have shown that the therapeutic effect of GSK3β inhibition is associated with the inhibition of inflammation. Similarly, the mechanisms underlying the anti-inflammatory function of GSK3β inhibition are not well understood. GSK3β inhibition attenuates activation of the pro-inflammatory transcription factor NFκB, and activates the immuno-modulatory transcription factor β-catenin. GSK3β inhibition has also been shown to induce secretion of the anti-inflammatory cytokine IL-10. In addition, pharmacological inhibition of GSK3β suppressed alloreactive T-cell responses. The combined anti-proliferative and anti-inflammatory properties of small molecule inhibitors of GSK3β make them an attractive treatment modality towards the control of inflammation.

Glycogen synthase kinase−3β inhibitors suppress leukemia cell growth

OBJECTIVE The objective of this study was to investigate the effect of small molecule inhibitors of glycogen synthase kinase-3β (GSK-3β) on leukemia cell growth and survival. MATERIALS AND METHODS Analysis of cytotoxicity and cell proliferation was conducted using the MTS assay, cell-cycle analysis, and division tracking. Apoptosis was investigated by Annexin-V/7-aminoactinomycin D and caspase-3 expression. The effect of GSK-3β inhibitors was also tested in vivo in an animal model of leukemia. Gene expression analysis was performed to identify the genes modulated by GSK-3β inhibition in leukemia cells. RESULTS GSK-3β inhibitors suppress cell growth and induce apoptosis in seven leukemia cell lines of diverse origin, four acute myeloid leukemia, one myelodysplastic syndrome, and one acute lymphoblastic leukemia samples. GSK-3β inhibitors are cytotoxic for rapidly dividing clonogenic leukemia blasts, and higher doses of the inhibitors are needed to eliminate primitive leukemia progenitor/stem cells. Slow cell-division rate, low drug uptake, and interaction with bone marrow stroma make leukemia cells more resistant to apoptosis induced by GSK-3β inhibitors. Global gene expression analysis combined with functional approaches identified multiple genes and specific signaling pathways modulated by GSK-3β inhibition. An important role for Bcl2 in the regulation of apoptosis induced by GSK-3β inhibitors was defined by expression analysis and confirmed by using pharmacological inhibitors of the protein. In vivo administration of GSK-3β inhibitors delayed tumor formation in a mouse leukemia model. GSK-3β inhibitors did not affect hematopoietic recovery following irradiation. CONCLUSIONS Our data support further evaluation of GSK-3β inhibitors as promising novel agents for therapeutic intervention in leukemia and warrant clinical investigation in leukemia patients.

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.

A retroviral library genetic screen identifies IRF-2 as an inhibitor of N-ras-induced growth suppression in leukemic cells

Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras-induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras-induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras-induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.

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.

The Role of IRF1 and IRF2 Transcription Factors in Leukaemogenesis

Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.

Identification of plasma Complement C3 as a potential biomarker for neuroblastoma using a quantitative proteomic approach

UNLABELLED The majority of patients diagnosed with neuroblastoma present with aggressive disease. Improved detection of neuroblastoma cancer cells following initial therapy may help in stratifying patient outcome and monitoring for relapse. To identify potential plasma biomarkers, we utilised a liquid chromatography-tandem mass spectrometry-based proteomics approach to detect differentially-expressed proteins in serum from TH-MYCN mice. TH-MYCN mice carry multiple copies of the human MYCN oncogene in the germline and homozygous mice for the transgene develop neuroblastoma in a manner resembling the human disease. The abundance of plasma proteins was measured over the course of disease initiation and progression. A list of 86 candidate plasma biomarkers was generated. Pathway analysis identified significant association of these proteins with genes involved in the complement system. One candidate, complement C3 protein, was significantly enriched in the plasma of TH-MYCN(+/+) mice at both 4 and 6weeks of age, and was found to be elevated in a cohort of human neuroblastoma plasma samples, compared to healthy subjects. In conclusion, we have demonstrated the suitability of the TH-MYCN(+/+) mouse model of neuroblastoma for identification of novel disease biomarkers in humans, and have identified Complement C3 as a candidate plasma biomarker for measuring disease state in neuroblastoma patients. BIOLOGICAL SIGNIFICANCE This study has utilised a unique murine model which develops neuroblastoma tumours that are biologically indistinguishable from human neuroblastoma. This animal model has effectively allowed the identification of plasma proteins which may serve as potential biomarkers of neuroblastoma. Furthermore, the label-free ion count quantitation technique which was used displays significant benefits as it is less labour intensive, feasible and accurate. We have been able to successfully validate this approach by confirming the differential abundance of two different plasma proteins. In addition, we have been able to confirm that the candidate biomarker Complement C3, is more abundant in the plasma of human neuroblastoma patient plasma samples when compared to healthy counterparts. Overall we have demonstrated that this approach can be potentially useful in the identification of biomarker candidates, and that further validation of the candidates may lead to the discovery of novel, clinically useful diagnostic tools in the detection of sub-clinical neuroblastoma.