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. 2008 Oct 30;455(7217):1205-9.
doi: 10.1038/nature07284. Epub 2008 Sep 17.

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy

Zhong Wang  1 , Kevin S Smith, Mark Murphy, Obdulio Piloto, Tim C P Somervaille, Michael L Cleary
Affiliations

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy

Zhong Wang et al. Nature. .

Abstract

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.

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Figures

Figure 1
Figure 1. Sensitivity of MLL leukaemia cell lines to GSK3 inhibition
a, The growth of human leukaemia cell lines was assessed after 3 days culture in the presence of the indicated concentrations of GSK3-IX (left panel) or roscovitine (right panel). The results are expressed as the cell numbers relative to those without drug treatment, and represent the mean of three independent experiments (± s.e.m.). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. b, Human leukaemia cell lines were cultured in the absence or presence of 10 μM SB216763 for 2 days. The results of a representative experiment are expressed as the fold change in cell number compared to day 0 (± s.e.m., n = 3). c, Human leukaemia cell lines were cultured in the presence of 5 μM SB216763 for 24 h, and BrdU incorporation was quantified by fluorescence-activated cell sorting (FACS) analysis.
Figure 2
Figure 2. Sensitivity of MLL-transformed mouse B and myeloid progenitors to GSK3 inhibition
a, The growth of myeloid progenitors transformed by various oncogenes was assessed after 3 days of culture in the presence or absence of a GSK3 inhibitor. The results of a representative experiment are expressed as the fold change in cell number compared to day 0 (± s.e.m., n = 3). b, c, The morphological features of MLL–ENL transformed myeloid progenitors were assessed after 4 days of culture in the presence or absence of GSK3 inhibitor. Original magnification, ×40. The bar graph (c) indicates the mean number of cells with the indicated morphological features (n = 3). d, The growth of B lymphoid progenitors transformed by E2A–HLF and BCL2 (BiEH), TEL–AML1 or MLL–AF4 oncogenes was assessed after 3 days of culture in the absence or presence (5 μM or 10 μM) of SB216763. The results of a representative experiment are expressed as the fold change in cell number compared to day 0 (± s.e.m., n = 3).
Figure 3
Figure 3. Genetic ablation of GSK3-β hypersensitizes MLL-transformed cells to pharmacological GSK3 inhibition
a, Western blot analysis demonstrates the amounts of GSK3 protein isoforms in wild type or Gsk3b−/− myeloid progenitors transformed by the indicated oncogenes. b, Wild type (+/+) or Gsk3b−/− (−/−) myeloid progenitors transformed by various oncogenes were incubated in the presence of the indicated concentrations of SB216763. Cell numbers were enumerated on day 2 and expressed as the fold change compared to day 0 (± s.e.m. of triplicate analyses). c, Western blot analysis demonstrates the relative amounts of β-catenin after treatment with the indicated concentrations (μM) of inhibitor in wild type (WT) or Gsk3b−/− myeloid progenitors transformed by MLL–ENL. d, Gsk3b−/− myeloid progenitors transformed by MLL oncogenes were stably transduced with Flag–GSK3-β (+) or vector (−), and then incubated in the presence or absence of 5 μM SB216763. Cell numbers were enumerated on day 2 and expressed as the fold change compared to day 0 (± s.e.m. of triplicate analyses).
Figure 4
Figure 4. Compound genetic deficiency of GSK3-α and GSK3-β impairs the growth and leukemogenicity of MLL-transformed cells
a, Western blot analysis was performed on wild type (WT) and Gsk3b−/− myeloid progenitors transformed by the indicated oncogenes (top) and transduced by lentiviral vectors lacking (−) or expressing (+) Gsk3a shRNA . b, Myeloid progenitors transformed by the indicated oncogenes were acutely transduced with lentiviral vectors lacking or expressing Gsk3a shRNA and then plated in methylcellulose medium. Colonies were enumerated after 5 days, and the mean (± s.e.m.) numbers of three independent determinations are expressed relative to vector alone. c, Proliferation of myeloid progenitors (WT, Gsk3b−/− or Gsk3b−/− Gsk3aKD) transformed by MLL–ENL (left panel) or NUP98–HOXA9 (right panel) was assessed at the indicated days in liquid culture (± s.e.m. of triplicate analyses). d, The morphological features of MLL–ENL transformed myeloid progenitors with the indicated genotypes were assessed after 4 days of culture. The bar graph indicates the mean number of cells with the indicated morphological features (n = 3). e, Survival curves are shown for cohorts of mice transplanted with cells (WT, Gsk3b−/− or Gsk3b−/− Gsk3aKD) stably transduced with MLL–ENL (10 mice each). f, Cell numbers were determined after 3 days of culture in the indicated concentrations of lithium chloride. g, Survival curves show significantly different latencies (P < 0.001) for the development of acute leukaemia in cohorts of mice transplanted with MLL–AF4 leukaemia cells (5 × 104) and maintained on normal or lithium carbonate (0.4%) laced chow as indicated.
Figure 5
Figure 5. GSK3 maintains MLL transformation through suppression of p27Kip1
a, Human leukaemia cell lines (RCH-ACV and KP-L-RY) were treated with 10 μM SB216763 for the indicated times and protein levels were assessed by western blot analysis. b, Murine myeloid progenitors transformed by the indicated oncogenes were cultured in the presence (+) or absence (−) of SB216763 (10 μM) for 24 h in liquid culture, and then subjected to western blot analysis. c, Western blot analysis demonstrates the p27Kip1 (upper panel) or β-tubulin (lower panel, loading control) protein levels in MLL–ENL transformed myeloid progenitors stably transduced with lentiviral vectors expressing shRNAs specific for p27Kip1 (denoted as sh1 to sh3). d, Myeloid progenitors transformed by MLL-ENL and stably transduced with lentiviral vectors lacking (−) or expressing one of three different p27Kip1 shRNAs were cultured for 3 days in the presence or absence of 10 μM SB216763. Viable cell numbers are expressed relative to the numbers obtained with lentiviral vector transduced cells ± s.e.m. of triplicate analyses).
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