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. 2010 Jun 15;17(6):597-608.
doi: 10.1016/j.ccr.2010.04.024.

GSK-3 promotes conditional association of CREB and its coactivators with MEIS1 to facilitate HOX-mediated transcription and oncogenesis

Zhong Wang  1 , Masayuki Iwasaki, Francesca Ficara, Chenwei Lin, Christina Matheny, Stephen H K Wong, Kevin S Smith, Michael L Cleary
Affiliations

GSK-3 promotes conditional association of CREB and its coactivators with MEIS1 to facilitate HOX-mediated transcription and oncogenesis

Zhong Wang et al. Cancer Cell. .

Abstract

Acute leukemias induced by MLL chimeric oncoproteins are among the subset of cancers distinguished by a paradoxical dependence on GSK-3 kinase activity for sustained proliferation. We demonstrate here that GSK-3 maintains the MLL leukemia stem cell transcriptional program by promoting the conditional association of CREB and its coactivators TORC and CBP with homedomain protein MEIS1, a critical component of the MLL-subordinate program, which in turn facilitates HOX-mediated transcription and transformation. This mechanism also applies to hematopoietic cells transformed by other HOX genes, including CDX2, which is highly expressed in a majority of acute myeloid leukemias, thus providing a molecular approach based on GSK-3 inhibitory strategies to target HOX-associated transcription in a broad spectrum of leukemias.

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Figures

Figure 1
Figure 1. Global gene expression changes of MLL cells in response to GSK-3 inhibition
The dataset of gene expression differences resulting from GSK-3 inhibitor treatment (10 μM SB216763 for 20 hours) was used for GSEA. Enrichment plots are shown for selected down-regulated gene sets identified by GSEA (Supplemental Table 2). See also Supplemental Tables S1 and S2.
Figure 2
Figure 2. HOX-induced proliferation is generally sensitive to GSK-3 inhibition
(A) Mouse myeloid progenitors immortalized by HOXA9 + MEIS1 were cultured in the presence or absence of 10 μM SB216763. Results show mean cell numbers expressed as fold-change compared to day 0 (error bars indicate ±SEM, n = 3). (B) Survival is shown for mice transplanted with HOXA9/MEIS1 leukemia cells (50,000 cells/mouse) and maintained on regular chow or chow containing 0.4% lithium with saline water (n=10 each cohort; p<0.01). Acute leukemia was confirmed by peripheral blood leukocyte counts and/or necropsy. (C) Western blot analysis (upper panel) was performed using an anti-GSK-3 antibody on HOXA9/MEIS1 leukemia cells transduced by lentiviral vectors expressing GSK-3 or GSK-3β shRNAs (#1 or #2 for each). Migrations of GSK-3 isoforms are indicated. Bar graph shows the growth of transduced cells in the presence or absence of 5 μM SB216763 for 3 days. Results are shown as relative cell proliferation compared to cell numbers in the absence of inhibitor (error bars indicate ±SEM, n=3). See also Figure S1.
Figure 3
Figure 3. GSK-3 regulates HOX/PBX/MEIS transcription complex activity through MEIS1 and CREB association
(A) Mouse myeloid progenitors immortalized by the indicated genes were cultured in the presence or absence of 10 μM SB216763. Cell numbers are expressed as fold-change compared to day 0. Representative experiments are shown (n = 3 each). (B) Colony forming ability is shown for myeloid progenitors immortalized by various oncogenes (indicated below) following 5 days culture in the presence or absence of SB216763. Results are shown relative to mean colony numbers without drug set at 100%. (C) Colony morphologies are shown for the experiment in panel B. Scale bar = 100 μm. (D) HoxB1 ARE reporter activity was assessed following co-transduction with constructs encoding the proteins indicated below the panel in the presence or absence of 10 μM SB216763. Luciferase activity was normalized to β–galactosidase activity. Results are shown as fold-change compared to control. (E & F) Activity of the HOXA9 reporter gene (E) or Top-Flash WNT reporter gene (F) was assessed in the presence or absence of 10 μM SB216763. Results of a representative experiment are displayed as fold change compared to control. All error bars indicate ±SEM, n=3. See also Figure S2.
Figure 4
Figure 4. CREB affects MLL and HOX-induced cell proliferation and sensitivity to GSK-3 inhibition
(A) Mouse myeloid progenitors immortalized by MLL-AF6 were transduced with retrovirus expressing CREB or empty vector. Mice (n=5 each cohort) transplanted with the transduced cells were monitored for leukemia-free survival (p<0.01). (B) MLL-AF6 or HOXA9/MEIS1 (HM) leukemia cells were stably transduced with CREB (+) or vector (−), and then incubated in the presence or absence of 10 μM SB216763. Cell numbers were enumerated on day 2 and expressed as relative change compared to no SB216763 treatment. Right panel shows protein levels detected by western blot analysis. (C) MLL-AF6 or HOXA9/MEIS1 leukemia cells were stably transduced with lentiviral vector expressing CREB shRNAs (#1 or #2), and then incubated in the presence or absence of 5 μM SB216763. Cell numbers were enumerated on day 2 and displayed as relative change compared to no SB216763 treatment. Right panel shows CREB protein levels in knockdown cells. (D) Myeloid progenitors immortalized by the indicated oncogenes were transduced with retroviral vectors expressing the various CREB proteins or empty vector (−). The cells were then plated and colonies enumerated after 5 days culture, and displayed relative to empty vector. Right panel shows CREB protein levels in transduced cells. HM, HOXA9/MEIS1; MA6, MLL-AF6; EH, E2A-HLF; NA9, NUP98-HOXA9. All error bars indicate ± SEM of triplicate analyses. See also Figure S3.
Figure 5
Figure 5. Association of CREB and MEIS1 is phosphorylation dependent
(A) IP-western blot analysis was performed on 293T cells co-transduced with FLAG-CREB and HA-MEIS1, or AML cells transformed by HOXA9 + HA-MEIS1 in the presence or absence of 20 μM SB216763. Cell lysates were immunoprecipitated with anti-HA antibody conjugated beads, followed by western blot analysis using anti-FLAG (CREB), anti-HA (MEIS1), anti-CREB or anti-PBX1b antibodies. (B) Human leukemia cell lines (indicated at the top) were cultured overnight in the presence (+) or absence (−) of SB216763 (20 μM), and cell lysates were subjected to immunoprecipitation with anti-MEIS monoclonal antibody conjugated beads, then subjected to western blot analysis with rabbit polyclonal antibodies specific for the indicated endogenous proteins. (C) HA-MEIS1 was co-expressed with FLAG-tagged CREB proteins (indicated at top) in the presence or absence of 20 μM SB216763. Cell lysates were used for immunoprecipitation with anti-HA or IgG conjugated beads, then subjected to western blot using anti-FLAG (CREB) or anti-HA (MEIS1) antibodies. (D) HA-MEIS1 was co-expressed with CBP, with or without FLAG-tagged CREB (indicated at top) in the presence or absence of 20 μM SB216763. Cell lysates were used for immunoprecipitation with anti-HA conjugated beads, then subjected to western blot using anti-CBP, anti-FLAG (CREB) or anti-HA (MEIS1) antibodies. (E) HA-MEIS1 was co-expressed with FLAG-tagged CREB and V5-tagged TORC1 in the presence or absence of 20 μM SB216763. Cell lysates were immuno-precipitated with anti-HA conjugated beads, then subjected to western blot using anti-V5 (TORC1), anti-FLAG (CREB) or anti-HA (MEIS1) antibodies. (F) Left panel: FLAG-tagged CREB (wt or mutant as indicated at top of panel) proteins were expressed in 293T cells, immunoprecipitated with anti-FLAG antibody conjugated beads, and subjected to western blot to detect the presence of endogenous CBP. Right panel: FLAG-tagged CREB was expressed in 293T cells in the absence (−) or presence (+) of SB216763, immunoprecipitated with anti-FLAG antibody conjugated beads, and subjected to western blot analysis to detect the co-precipitation of endogenous CBP. (G) FLAG-tagged CREB (or mutant CREB proteins indicated at top of panel) were expressed in 293T cells in the absence (−) or presence (+) of SB216763 and immunoprecipitated with anti-FLAG antibody conjugated beads. The precipitate was washed and subjected to western blot to detect CREB phosphorylation status using phospho-specific antibodies. (H) HA-MEIS1 was co-expressed with FLAG-tagged CREB in the presence of TORC2 shRNA constructs (#1, #2). Cell lysates were used for immunoprecipitation with anti-HA conjugated beads, then subjected to western blot using anti-TORC2, anti-FLAG (CREB) or anti-HA (MEIS1) antibodies. (I) HA-MEIS1 (WT) or HA-MEIS1 C-terminal deletion mutant (ΔCT) was co-expressed with FLAG-tagged CREB. Cell lysates were immuno-precipitated with anti-HA conjugated beads, then subjected to western blot using anti-FLAG (CREB) or anti-HA (MEIS1) antibodies. (J) Gal4-MEIS1 CT activity was assessed following co-transduction with CREB or TORC1 in the presence or absence of different GSK-3 inhibitors. (K) Gal4-MEIS1 CT or mutant reporter activities were assessed following co-transduction with CREB. (L) Gal4-MEIS1 CT reporter activity was assessed following co-transduction with TORC1 or CREB shRNA constructs (#1, #2) indicated below the panel. Results are shown as fold-change compared to control. For panels J-L, luciferase activity was normalized to β–galactosidase activity. Error bars indicate ±SEM.
Figure 6
Figure 6. FOS is a critical downstream effector of HOX/PBX/MEIS and CREB to mediate GSK-3 inhibitor sensitivity
(A) Human MLL-AF4 leukemia cell line RS 4;11 was cultured in the presence or absence of GSK-3 inhibitors (SB216763 or SB415296 for 20 hours). FOS transcripts were then measured by qRT-PCR, and expressed relative to untreated cells. (B) Expression of FOS in wild type (WT) or GSK-3β null (GSK-3β−/−) cells transformed by E2A-HLF was measured by qRT-PCR, and displayed relative to wild type cells. (C) FOS reporter was co-transfected with HOXB1, PBX1b and MEIS1a (H/P/M) or CREB in the presence (+) or absence (−) of SB216763. Luciferase activity was normalized with β–galactosidase activity. Results are displayed as fold change compared to control. (D) Myeloid progenitors immortalized by HOXA/MEIS1 were cultured for 24 hrs in the absence (−) or presence (+) of SB216763. ChIP was performed using antibodies against CREB or HA (MEIS1). Relative occupancy values were normalized against those obtained with IgG. (E) MLL-AF6 transformed mouse myeloid progenitor cells were stably transduced with a FLAG-FOS construct. Over-expression of FOS is shown by western blot using anti-FOS antibody. Sensitivities of cells to different concentrations of SB216763 are displayed compared to untreated cells. (F) MLL-AF6 transformed mouse myeloid progenitors stably transduced with vector (−) or FOS shRNAs (#1 or #2) and employed for studies in panel G were assessed for FOS transcript expression by qRT-PCR. Results are displayed relative to vector-transduced cells. (G) Cells transformed by the indicated oncogenes (top of panels) were transduced with FOS knockdown constructs and cultured in the indicated concentrations of SB216763. Cell numbers were enumerated and displayed as relative change compared to untreated cells. All error bars indicate ± SEM of triplicate analyses. See also Figure S4.
Figure 7
Figure 7. Schematic model depicting role of GSK-3 in promoting HOX-mediated transcription through CREB phosphorylation
GSK-3 activity promotes conditional association of CREB and its co-activators with MEIS1 to facilitate HOX-mediated transcription and oncogenesis, which are compromised following GSK-3 inhibition in leukemia cells.
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