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. Author manuscript; available in PMC: 2014 Dec 9.
Published in final edited form as: Cancer Cell. 2013 Nov 27;24(6):10.1016/j.ccr.2013.10.022. doi: 10.1016/j.ccr.2013.10.022

Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia

Erich Piovan 1,2,3,#, Jiyang Yu 4,5,#, Valeria Tosello 1,6, Daniel Herranz 1, Alberto Ambesi-Impiombato 1, Ana Carolina Da Silva 1, Marta Sanchez-Martin 1, Arianne Perez-Garcia 1, Isaura Rigo 1, Mireia Castillo 7, Stefano Indraccolo 2, Justin R Cross 8, Elisa de Stanchina 9, Elisabeth Paietta 10,11, Janis Racevskis 10,11, Jacob M Rowe 12, Martin S Tallman 13, Giuseppe Basso 14, Jules P Meijerink 15, Carlos Cordon-Cardo 7, Andrea Califano 1,4,5, Adolfo A Ferrando 1,7,16
PMCID: PMC3878658  NIHMSID: NIHMS537876  PMID: 24291004

SUMMARY

Glucocorticoid resistance is a major driver of therapeutic failure in T-cell acute lymphoblastic leukemia (T-ALL). Here we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy and effectively reverses glucocorticoid resistance in vitro and in vivo.

INTRODUCTION

Glucocorticoids are small lipophilic compounds derived from cortisol, a natural adrenal hormone that signals via the glucocorticoid receptor alpha, a nuclear receptor ligand-activated transcription factor encoded by the NR3C1 gene (Schlossmacher et al., 2011). Glucocorticoids play a fundamental role in the treatment of all lymphoid tumors because of their capacity to induce apoptosis in lymphoid progenitor cells (Inaba and Pui, 2010; Pui et al., 2011; Pui et al., 2012). The importance of glucocorticoid therapy in lymphoid malignancies is underscored by the strong association of primary glucocorticoid resistance with poor prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, prednisone poor response, defined as failure to show effective cytoreduction after 7 days of glucocorticoid therapy is strongly associated with increased risk of relapse and therapeutic failure (Dordelmann et al., 1999; Inaba and Pui, 2010) and in vitro resistance to glucocorticoids is associated with unfavorable prognosis in this disease (Hongo et al., 1997; Inaba and Pui, 2010; Klumper et al., 1995).

Primary glucocorticoid resistance is particularly frequent in T-ALL (Hongo et al., 1997; Inaba and Pui, 2010; Klumper et al., 1995), leading us to hypothesize that activation of one or more oncogenic signaling pathways implicated in T-cell transformation could be driving primary glucocorticoid resistance in T-ALL directly by interfering with glucocorticoid receptor function or indirectly via inhibition of glucocorticoid induced apoptosis. In this context, the AKT1 gene emerged as a plausible candidate as PI3K-AKT activation plays a major role in the pathogenesis of T-ALL, particularly in leukemias harboring mutations and deletions in the PTEN tumor suppressor gene (Palomero et al., 2007). Moreover, in silico analysis of signaling factors modulating transcriptional signatures associated with glucocorticoid resistance in T-ALL pointed to a potential role of AKT1 as driver of glucocorticoid resistance in T-ALL. These results together with the association of mutational loss of PTEN and increased AKT1 phosphorylation with primary glucocorticoid resistance in the clinic (Bandapalli et al., 2013; Morishita et al., 2012) and the availability of PI3K-AKT specific inhibitors in clinical trials for the treatment of human cancer, prompted us to analyze the mechanistic role of AKT1 in the control of glucocorticoid resistance in T-ALL.

RESULTS

AKT1 binds to the NR3C1 glucocorticoid receptor protein

Activation of gene expression by glucocorticoids is a multistep process that requires effective release of the glucocorticoid receptor from heat shock protein complexes, translocation to the nucleus and formation of a multiprotein transcriptional complex on the promoter of glucocorticoid target genes (Heitzer et al., 2007). To test if AKT1 can interact with and inhibit the glucocorticoid receptor protein, we transfected 293T cells with plasmid constructs driving the expression of Flag-tagged AKT1 and HA-tagged NR3C1 and isolated glucocorticoid receptor-containing protein complexes via immunoprecipitation using an anti-HA antibody. Western blot analysis demonstrated the presence of Flag-AKT1 in HA-NR3C1 immunoprecipitates, suggesting that AKT1 can interact with NR3C1 in vivo (Figure 1A). Reciprocal immunoprecipitation experiments confirmed the association between Flag-AKT1 and HA-NR3C1 (Figure 1B). Moreover, immunoprecipitation of NR3C1 protein complexes from the T-ALL cell lines DND41 and CCRF-CEM demonstrated that endogenous NR3C1 and AKT1 can interact in T-ALL lymphoblast cells (Figure 1C and Figure S1A). In addition, immunofluorescence analysis showed colocalization of NR3C1 and AKT1 in DND41 and CCRF-CEM cells (Figure 1D and Figure S1B). Finally, glutathione-S-transferase (GST)-pulldown assays showed that recombinant GST-NR3C1 fusion protein can directly interact with His-tagged AKT1 in vitro (Figure 1E).

Figure. 1. AKT1 interacts with the glucocorticoid receptor protein and regulates NR3C1 S134 phosphorylation.

Figure. 1

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(A) Western blot analysis of AKT1 and activated AKT1 after NR3C1 immunoprecipitation in 293T cells expressing Flag-tagged AKT1 and HA-tagged NR3C1. (B) NR3C1 western blot analysis after AKT1 immunoprecipitation in 293T cells expressing Flag-tagged AKT1 and HA-tagged NR3C1. (C) Western blot analysis of AKT1 after NR3C1 protein immunoprecipitation in DND-41 T-ALL cells. (D) Immunofluorescence colocalization analysis of AKT (green) and NR3C1 (red) proteins in DND41 cells. Cell nuclei stained with TO-PRO3 are shown in blue. Scale bar: 10 μm. (E) Analysis of AKT1-NR3C1 interaction via Western blot analysis of protein complexes recovered after NR3C1-GST pull down of recombinant His-tagged AKT1. (F) Partial alignment of the glucocorticoid receptor protein sequences flanking S134. (G) Western blot analysis of NR3C1 phosphorylation using an antibody against the AKT phospho-motif in NR3C1 protein immunoprecipitates from U2OS cells expressing MYR-AKT1 together with HA-NR3C1 or HA-NR3C1 S134A. (H) In vitro kinase analysis of AKT1 phosphorylation of recombinant NR3C1 (GST-NR3C1) and NR3C1 S134A mutant (GST-NR3C1 S134A) proteins. Top panel shows P-32 autoradiography after SDS-PAGE and corresponding protein loading is shown at the bottom. (I) ESI-MS/MS spectrum of monophosphorylated peptide STpS134VPENPK (S132 to K140) obtained after trypsin digestion of NR3C1 isolated from cells expressing constitutively active AKT1. (J) Collision induced dissociation of the molecular ion, [M+2H]2+ at m/z 519.72 (M = 1037.42 Da) corresponding to S134. Characteristic b- and y-fragment ions including y7 which contains pSer and features the loss of 98 Da (elimination of phosphoric acid) are shown. (K) Western blot analysis of NR3C1 phosphorylation using an anti AKT phospho-motif antibody in NR3C1 immunoprecipitates from CCRF-CEM cells expressing HA-NR3C1 treated with vehicle or the MK2206 AKT inhibitor. See also Figure S1 and Table S1.

AKT1 phosphorylates S134 in the NR3C1 protein

AKT1 kinase substrates are typically phosphorylated by AKT at RXRXXS/T motifs (Mok et al., 1999; Ozes et al., 1999; Zimmermann and Moelling, 1999). Phospho-AKT motif scanning analysis of NR3C1 revealed a potential AKT phosphorylation motif 131RSTS134 (Figure 1F and Figure S1C), suggesting that the glucocorticoid receptor could be an AKT1 substrate phosphorylated at serine 134. To test this possibility, we expressed HA-tagged wild type NR3C1 (HA-NR3C1) or an HA-tagged form of the glucocorticoid receptor with a serine to alanine substitution at position 134 (HA-NR3C1 S134A) in cells infected with retroviruses expressing MYR-AKT1. Protein immunoprecipitation of NR3C1 with an antibody against HA and subsequent Western blot analysis with an antibody recognizing the phospho-RXXS/T AKT phosphorylation motif showed the presence of a HA-NR3C1 phospho-AKT band in cells expressing the wild type glucocorticoid receptor, but not in cells expressing the HA-NR3C1 S134A mutant (Figure 1G). Consistently, in vitro kinase assays in which we analyzed the capacity of the AKT1 kinase to phosphorylate the wild type or S134A glucocorticoid receptor proteins demonstrated that AKT1 can effectively phosphorylate recombinant wild type NR3C1 protein in vitro, but not the serine 134 to alanine NR3C1 mutant protein (Figure 1H). Mass spectrometry analysis of HA-NR3C1 protein isolated from MYR-AKT1 expressing cells demonstrated the presence of serine phosphorylation at position 134 of the glucocorticoid receptor (Figure 1I,J), which was effectively abrogated (control S134P:non-P = 0.47; MK2206 S134P:non-P = 0.03; Table S1) upon treatment with MK2206, a highly potent and selective inhibitor of AKT (Hirai et al., 2010). Finally, and most notably, western blot analysis with the AKT phosphorylation motif antibody showed decreased AKT phosphorylated NR3C1 in NR3C1 immunoprecipitates from CCRF-CEM T-ALL cells upon AKT inhibition with MK2206 (Figure 1K).

AKT signaling inhibits NR3C1 nuclear translocation following glucocorticoid treatment

After establishing the interaction and phosphorylation of the glucocorticoid receptor by AKT1 we sought to elucidate the relevance of the NR3C1 S134 phosphorylation for glucocorticoid receptor function. Glucocorticoid induced cytoplasmic-nuclear shuttling is strictly required for glucocorticoid receptor activity, which functions as a ligand activated transcription factor (Heitzer et al., 2007). U2OS cells, which express undetectable levels of endogenous NR3C1 (Figure S2A), showed cytoplasmic localization of retrovirally expressed HA-tagged glucocorticoid receptor protein, which was completely relocalized to the nucleus upon dexamethasone treatment (Figure 2A). Notably, expression of MYR-AKT1 in these cells resulted in impaired nuclear relocalization of NR3C1 after dexamethasone treatment (Figure 2B). In addition, and in contrast with wild type glucocorticoid receptor, the NR3C1 S134A mutant protein showed increased nuclear localization in basal conditions and effective nuclear relocalization upon dexamethasone treatment (Figure 2C), even upon expression of MYR-AKT1 (Figure 2D). Next we analyzed the capacity of the MK2206 AKT inhibitor to modulate glucocorticoid induced translocation of NR3C1 to the nucleus in T-ALL cells. CCRF-CEM and MOLT3, two PTEN null, glucocorticoid resistant T-ALL cell lines expressing high levels of AKT activation, showed cytoplasmic localization of NR3C1 in basal conditions, which was only partially relocalized to the nucleus upon dexamethasone treatment (Figure 2E, Figure S2B). Inhibition of AKT with MK2206 effectively enhanced glucocorticoid-induced translocation of the NR3C1 protein to the nucleus in these cells (Figure 2E, Figure S2). Notably, similar results were obtained in primograft T-ALL lymphoblasts in which inhibition of AKT with MK2206 increased the nuclear translocation of the NR3C1 protein following glucocorticoid treatment (Figure 2F).

Figure. 2. AKT1-mediated S134 phosphorylation of the NRC3C1 protein impairs dexamethasone-induced glucocorticoid receptor nuclear translocation.

Figure. 2

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(A-D) Confocal microscopy analysis and quantitation of the distribution of NR3C1 cellular localization in U2OS cells expressing HA-NRC31 (A), HA-NRC31 and MYR-AKT1 (B), HA-NRC31 S134A (C), or HA-NRC31 S134A and MYR-AKT1 in basal conditions (DMSO) and after dexamethasone (Dexa; 1 μM) stimulation. Scale bar for all panels: 20 μm. (E) Cellular localization analysis of NR3C1 via nuclear and cytoplasmic cell fractionation and analysis of AKT1 signaling in cell lysates from CCRF-CEM T-ALL cells treated with vehicle only (DMSO), dexamethasone (Dexa; 1 μM), the MK2206 AKT inhibitor (1 μM) and MK2206 plus dexamethasone (1 μM each). (F) Cellular localization analysis of NR3C1 via Western blot analysis of nuclear and cytoplasmic cell fractions in cell lysates from primograft T-ALL lymphoblasts. Tubulin and MAX proteins are shown as controls for cytosolic and nuclear fractions. C: cytoplasmic fraction; N: nuclear fraction. Data in A-D are represented as mean ± SD. See also Figure S2.

Transcriptionally, MYR-AKT1 impaired the capacity of NR3C1 to activate a luciferase reporter construct under the control of a synthetic glucocorticoid response element (Figure 3A). Consistently, AKT activation via MYR-AKT1 expression or PTEN knockdown in DND41 and KOPTK1 T-ALL cells impaired key effector responses mediating glucocorticoid induced cell death including glucocorticoid receptor autoupregulation (Geng et al., 2008) and induction of BCL2L11, a proapototic factor essential for glucocorticoid induced cell death (Wang et al., 2003) (Figure 3B,C; Figure S3A,B). Consistently, glucorticoid induced apoptosis was significantly blunted in DND41 and KOPTK1 MYR-AKT expressing cells compared with controls (Figure S3C). To better analyze the potential impact of mutational loss of PTEN in T-ALL on the global transcriptional program induced by glucocorticoids, we performed microarray gene expression analysis of DND41 shRNA control and PTEN shRNA knockdown cells treated with dexamethasone. In agreement with a global inhibition of glucocorticoid receptor activity by AKT, microarray analysis of gene expression changes induced by dexamethasone treatment showed complete abrogation of glucocorticoid induced signatures upon activation of AKT1 via PTEN shRNA knockdown (Figure 3D). Moreover, GSEA analysis of genes regulated by glucocorticoids in primary leukemia samples (Schmidt et al., 2006) showed a marked enrichment in DND41 control cells treated with dexamethasone compared with dexamethasone-treated PTEN shRNA knockdown samples (Figure 3E).

Figure 3. AKT activation inhibits glucocorticoid-induced gene expression.

Figure 3

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(A) Luciferase reporter analysis of dexamethasone-induced glucocorticoid receptor transactivation in U2OS cells expressing MYR-AKT1 compared with GFP only expressing controls using a synthetic glucocorticoid response element reporter. (B) Western blot analysis of PTEN expression and AKT activation in DND41 T-ALL cells infected with lentiviruses expressing a PTEN shRNA construct (shRNA PTEN) or a control shRNA targeting the Renilla luciferase gene (shRNA LUC). (C) RT-PCR analysis of glucocorticoid regulated transcripts in control and PTEN knockdown DND41 cells treated with vehicle (DMSO) only or 1 μM dexamethasone (Dexa). (D) Heat map representation of the top differentially expressed genes between control DND41 cells treated with vehicle only (Control) vs. 1 μM dexamethasone (Dexa) and corresponding transcript levels in control and dexamethasone treated PTEN knockdown cells. The scale bar shows color coded differential expression with red indicating higher levels and blue lower levels of expression. (E) GSEA analysis of genes regulated by glucocorticoids in ALL patients undergoing glucocorticoid therapy in DND41 shRNA LUC dexamethasone treated cells compared with DND41 shRNA PTEN dexamethasone treated cells. Data in A and C are represented as mean ± SD. See also Figure S3.

Pharmacologic inhibition of AKT reverses glucocorticoid resistance in vitro and in vivo

To test the therapeutic role of AKT inhibition in the treatment of glucocorticoid resistant leukemias we first treated CCRF-CEM cells a PTEN-null glucocorticoid resistant T-ALL cell line with dexamethasone and MK2206. In these experiments AKT1 inhibition of CCRF-CEM lymphoblasts with MK2206 effectively restored glucocorticoid induced apoptosis and reversed glucocorticoid resistance in vitro (Figure 4A). Similar results were obtained in the glucocorticoid resistant MOLT3 and PF382 T-ALL cell lines (Figure S4A). Analysis of an extended panel of cell lines including B precursor ALL (Figure S4B), diffuse large B cell lymphoma (Figure S4C) and multiple myeloma (Figure S4D) lines showed additive effects with this combination, suggesting that the synergistic interaction between glucocorticoids and AKT inhibition may be most prominently relevant in the context of T-ALL.

Figure. 4. Pharmacologic inhibition of AKT with MK2206 reverses glucocorticoid resistance.

Figure. 4

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(A) Representative plots of apoptosis and cell viability quantification in CCRF-CEM T-ALL cells treated with vehicle only, MK2206 (100 nM), dexamethasone (Dexa; 1 μM) or dexamethasone plus MK2206 (Dexa + MK2206; 1 μM and 100 nM, respectively) in combination in vitro. (B) Tumor load quantification in vivo by bioluminescence imaging and analysis of luciferase activity and human CD45 expressing cells in the bone marrow of CCRF-CEM T-ALL xenografts treated with vehicle only, MK2206 (10 mg kg−1 via oral gavage twice a day), dexamethasone (5 mg kg−1 via intraperitoneal injection) or MK2206 (10 mg kg−1 twice a day) plus dexamethasone (5 mg kg−1). (C-D) Representative plots (C) and quantification (D) of cell viability in primograft T-ALL samples treated with vehicle only, MK2206 (100 nM-10 μM), dexamethasone (10 nM-1 μM) alone and dexamethasone (10 nM-1 μM) plus MK2206 (100 nM-10 μM) in combination. Percentages of viable (PI −), and non-viable (PI +) cells are indicated. Bar graphs represent mean ± SD. See also Figures S1 and S5.

Next we analyzed the effects of MK2206 and glucocorticoid in vivo in a xenograft model of glucocorticoid-resistant T-ALL. CCRF-CEM cells expressing the luciferase gene were injected intravenously in immunodeficient NOG mice and tumor engraftment was assessed by in vivo bioimaging at day 18. Animals harboring homogeneous tumor burdens were treated with vehicle only (DMSO), MK2206, dexamethasone or MK2206 plus dexamethasone for 3 days. In this experiment, animals treated with dexamethasone or MK2206 showed progressive tumor growth similar to that observed in vehicle-treated controls, while mice treated with MK2206 plus dexamethasone had significant antitumor responses (Figure 4B). Following these results we evaluated the response to the combination treatment MK2206 plus dexamethasone in primary T-ALL lymphoblasts. Towards this goal we established viable in vitro cultures of T-ALL leukemia primograft samples supported by bone marrow MS5 stroma cells expressing the Delta like 1 NOTCH1 ligand (Armstrong et al., 2009). Treatment of T-ALL leukemia cultures with MK2206 plus dexamethasone in combination showed significantly increased antileukemic effects compared with treatment with dexamethasone or MK2206 alone (Figure 4C,D and Figure S4E). Notably, the antileukemic effects of MK2206 plus dexamethasone were prominent in tumor samples with PTEN loss (TALL#6, TALL#9 and T-ALL#28), but were also realized in PTEN-expressing tumors with moderate levels of AKT1 activation (T-ALL #19, TALL#37 and T-ALL#39) (Figure S4F).

To further test the efficacy of this treatment combination in vivo we established leukemia xenografts in NOD rag gamma immunodeficient mice using three independent glucocorticoid resistant primograft T-ALL samples infected with lentiviruses expressing the luciferase gene. Animals harboring homogeneous tumor burdens by in vivo bioimaging were treated with vehicle only (DMSO), MK2206, dexamethasone or MK2206 plus dexamethasone. In this experiment, mice treated with dexamethasone or MK2206 showed progressive tumor growth similar to that observed in vehicle-treated controls, while mice treated with MK2206 plus dexamethasone showed significant antitumor responses (Figure 5). In these experiments analysis of AKT1 phosphorylation showed effective suppression of AKT1 signaling in mice treated with MK2206 and MK2206 plus dexamethasone in vivo (Figure S5A, B). Moreover, expression analysis of the TSC22D3 (GILZ) glucocorticoid receptor target (Ayroldi et al., 2007; D’Adamio et al., 1997; Riccardi et al., 1999) and PARP-1 cleavage, showed increased glucocorticoid response and enhanced apoptosis in leukemia infiltrated tissues of mice treated with MK2206 plus dexamethasone in combination, respectively (Figure S5A). Pharmacokinetic analysis of MK2206 and dexamethasone ruled out that this effect could be mediated by decreased clearance of dexamethasone as the pharmacokinetic profile of this glucocorticoid was identical in animals treated with dexamethasone alone and mice treated with MK2206 plus dexamethasone in combination (Figure S5C). In addition, even though we observed a modest decrease in MK2206 half life in mice treated with dexamethasone plus MK2206 (Figure S5C), AKT inhibitor levels obtained in animals treated with MK2206 as single agent already result in complete suppression of AKT activation in vivo (Figure S5A, B), supporting that this pharmacokinetic interaction does not account for the increased antileukemic responses observed in mice treated with dexamethasone plus MK2206.

Figure 5. Pharmacologic inhibition of AKT reverses glucocorticoid resistance in primary human T-ALL xenografts in vivo.

Figure 5

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(A-C) Representative examples of tumor load analysis via in vivo bioluminescence imaging in mice xenografted with three independent human T-ALLs treated with vehicle only, MK2206 (10 mg kg−1 twice a day), dexamethasone (5 mg kg−1) or MK2206 (10 mg kg−1 twice a day) plus dexamethasone (5 mg kg−1). (D-F) Quantitative analysis of tumor load and therapy response based on luciferase activity. (G-I) Representative images of spleens in primary T-ALL xenografted mice at the end of treatment. (J-L) Quantitative analysis of tumor burden estimated by spleen weight in T-ALL xenografted mice at the end of treatment. (M-O) Quantitative analysis of tumor burden estimated by luciferase counts in bone marrow in T-ALL xenografted mice at the end of treatment. Panels A, D, G, J and M correspond to T-ALL#27. Panels B, E, H, K, N correspond to T-ALL #19. Panels C, F, I, L and O correspond to T-ALL #9. Scale bar: 2 cm. Bar graphs in M-O represent mean ± SD. See also Figure S5.

Finally, we generated a mouse leukemia model in which glucocorticoid resistance is specifically driven by genetic loss of Pten using a well established retroviral transduction and bone marrow transplantation protocol (Chiang et al., 2008). In this model, transplantation of tamoxifen-inducible conditional Pten knockout (ROSA26Cre-ERT2/+;Ptenf/f) hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of the NOTCH1 receptor (NOTCH1 L1601P ΔPEST) resulted in the development of NOTCH1 driven T-ALL tumors as previously described (Chiang et al., 2008). Next we infected NOTCH1;ROSA26Cre-ERT2/+;Ptenf/f T-ALL lymphoblasts with a luciferase expressing retrovirus and transplanted them into secondary recipients which were treated with vehicle only or tamoxifen in order to generate Pten-non-deleted and Pten-deleted isogenic tumors, respectively. Treatment of Pten-non-deleted tumor bearing mice with dexamethasone showed a significant improvement in survival compared with vehicle only treated controls (Figure 6A). In contrast, and consistent with a role of Pten loss and AKT1 activation in promoting glucocorticoid resistance, all mice harboring Pten-deleted tumors failed to respond to dexamethasone treatment and showed no survival differences compared to vehicle treated controls (Figure 6B).

Figure 6. Pharmacologic inhibition of AKT reverses glucocorticoid resistance in a mouse model of glucorticoid resistant T-ALL.

Figure 6

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(A) Kaplan-Meier curve in mice treated with dexamethasone (Dexa) or vehicle (Control) after allograft transplantation of Pten-non-deleted (Ptenf/f) NOTCH1-induced T-ALL tumor cells. Arrows indicate drug treatment. (B) Kaplan-Meier curve in mice treated with dexamethasone (Dexa) or vehicle (Control) after allograft transplantation of Pten-deleted (Pten−/−) NOTCH1-induced T-ALL tumor cells. Arrows indicate drug treatment. (C, D) Representative images, changes in bioluminescence by in vivo imaging and analysis of treatment response in mice allografted with NOTCH1-induced Pten−/− mouse leukemia cells treated with vehicle only, MK2206 (10 mg kg−1 via oral gavage twice a day), dexamethasone (Dexa; 5 mg kg−1) or MK2206 (10 mg kg−1 twice a day) plus dexamethasone (5 mg kg−1) (Dexa + MK2206). (E) Kaplan-Meier overall survival curve in mice allografted with NOTCH1-induced Pten−/− mouse leukemia cells and treated with vehicle only (control), MK2206 (10 mg kg−1 via oral gavage twice a day), dexamethasone (Dexa; 5 mg kg−1) or MK2206 (10 mg kg−1 twice a day), plus dexamethasone (5 mg kg−1) (Dexa + MK2206). (F, G) Quantification of glucocorticoid-induced loss of viability in isogenic NOTCH1-induced Ptenf/f or Pten−/− mouse leukemia cells infected with retroviruses expressing the wild type (F) or the S134A mutant (G) glucocorticoid receptor NR3C1. Data in F and G are represented as mean ± SD. See also Figure S6.

To test the efficacy of MK2206 and glucocorticoids in combination we treated mice transplanted with NOTCH1-induced Pten-deleted murine tumors expressing luciferase in secondary recipients, with vehicle only (DMSO), MK2206, dexamethasone or MK2206 plus dexamethasone and monitored their response to therapy by in vivo bioimaging. Animals treated with dexamethasone or MK2206 in this experiment showed progressive tumor growth similar to that observed in vehicle-treated controls while mice treated with MK2206 plus dexamethasone showed significant antitumor responses (Figure 6C,D), which translated in significantly improved survival in this group (Figure 6E). As before, analysis of AKT1 phosphorylation showed effective suppression of AKT1 signaling in mice treated with MK2206 and MK2206 plus dexamethasone in vivo (Figure S6A), while GILZ expression analysis showed increased glucocorticoid response (Figure S6A) and PARP-1 cleavage demonstrated increased apoptosis in tumor cells from animals treated with MK2206 plus dexamethasone in combination (Figure S6A). Consistently, histological analysis of leukemia infiltrated bone marrow showed increased antileukemic effects (Figure S6B) and marked increased apoptosis (Figure S6C) in mice xenografted with NOTCH1-induced Pten−/− T-ALL cells treated with MK2206 plus dexamethasone in combination.

Finally, we analyzed the role of NR3C1 S134 phosphorylation in the therapeutic response to glucocorticoids and the effects of Pten loss in glucocorticoid therapy in this model. Retroviral expression of the glucocorticoid receptor enhanced the response of NOTCH1-induced Pten non-deleted leukemias to glucocorticoid treatment; an effect that was effectively abrogated upon Pten loss (Figures 6F and S6D). In contrast, expression of the AKT-resistant NR3C1 S134A mutant protein was equally effective at increasing the antileukemic effects of glucocorticoids in Pten non-deleted and Pten null lymphoblasts (Figures 6G and S6D). Overall, these results support that pharmacologic inhibition of AKT can effectively enhance glucocorticoid response and reverse glucocorticoid resistance in T-ALL.

DISCUSSION

Numerous mechanisms have been proposed to explain the lack of effective glucocorticoid induced apoptosis in prednisone poor responders (Bhadri et al., 2012). However, and despite much research over the last two decades, the molecular basis of primary glucocorticoid resistance in ALL remain poorly understood. Given that primary glucocorticoid resistance is a cell intrinsic property of leukemia lymphoblasts present before exposure to glucocorticoid therapy, we proposed that primary oncogenic signaling pathways involved in leukemia transformation could also function as negative upstream regulators of the glucocorticoid receptor driving this phenotype. The identification of AKT1 as a driver of glucocorticoid resistance in T-ALL is perfectly consistent with this model. Notably, direct activation of AKT1 can drive T-cell transformation (Kharas et al., 2010); activation of PI3K-AKT signaling as result of deletions and mutations in PTEN (Palomero et al., 2007) is highly prevalent in T-ALL; and PTEN mutations are associated with primary glucocorticoid resistance in the clinic (Bandapalli et al., 2013).

Mechanistically, we show that AKT1 can induce glucocorticoid resistance by phosphorylation of the glucocorticoid receptor in position S134, which impairs the nuclear relocalization of the glucocorticoid receptor protein and blocks transcriptional regulation of glucocorticoid target genes. However, it should be noted that additional mechanisms downstream of AKT1 in addition to direct inactivation of the glucocorticoid receptor can also promote resistance to glucocorticoid therapy by promoting cell growth, metabolism and survival in T-ALL. In this regard, mTOR phosphorylation by AKT impairs glucocorticoid induced apoptosis by increasing the expression of MCL1 (Wei et al., 2006). In addition, AKT phosphorylation inhibits BAD mediated apoptosis (Bornhauser et al., 2007; Datta et al., 1997; del Peso et al., 1997); AKT-mediated phosphorylation of XIAP prevents the autoubiquitination and subsequent degradation of this antiapoptotic factor (Bornhauser et al., 2007; Dan et al., 2004); and increased metabolism induced by AKT activation can antagonize metabolic inhibition induced by glucocorticoids (Beesley et al., 2009). The convergent effects of direct (shown here) and indirect mechanisms downstream of AKT1 antagonizing the antileukemic effects of glucocorticoids further support the role of the PI3K-AKT pathway as therapeutic target for the reversal of primary glucocorticoid resistance in T-ALL.

EXPERIMENTAL PROCEDURES

Patient samples

T-ALL samples were provided by Columbia Presbyterian Hospital, the Eastern Cooperative Oncology Group (ECOG), University of Padova and Hospital Central de Asturias with informed consent and analyzed under the supervision of the Columbia University Medical Center IRB committee.

Inhibitors and Drugs

MK2206 or 8-[4- (1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3 (2H)-one hydrochloride [1:1] was from Selleck Chemicals LLC. Dexamethasone and 4-Hydroxytamoxifen were from Sigma-Aldrich.

In vitro Kinase Assays

Flag-tagged recombinant GST-NR3C1 and GST-NR3C1 S134A mutant proteins were incubated with recombinant active His-AKT1 protein (Millipore) in kinase buffer (Cell Signaling) containing γ-32P-ATP and analyzed by autoradiography after SDS-PAGE.

Mass Spectrometry

HA-NR3C1 protein was immunoprecipitated from U2OS cells stably expressing HA-tagged human NR3C1 and Myr-AKT with anti-HA antibody conjugated beads (Sigma), electrophoresed on a 3-8% Tris-Acetate gel, stained with Simply Blue Stain (Invitrogen), excised, reduced with DTT, alkylated with iodoacetamide and digested with trypsin and analyzed for phosphorylated peptides by nanoLC-ESI-MS/MS. MS/MS spectra were processed using ProteinLynx from the MassLynx 4.0 software and searched against the Swiss-Prot protein database using Mascot (www.matrixscience.com) with differential modifications for Ser/Thr/Tyr phosphorylation (+79.97).

To test the effects of AKT1 inhibition in NR3C1 S134 phosphorylation we treated U2OS cells stably expressing HA-tagged human NR3C1 and the constitutively active Myr-AKT with MK2206 or DMSO (10 μM, 5 hours). We immunoprecipitated HA-NR3C1 protein with anti-HA antibody conjugated beads (Sigma). Immunopurified HA-NR3C1 was then electrophoresed on a 4-12% Bis-Tris gel, stained with Simply Blue Stain (Invitrogen), digested with trypsin and analyzed for the phosphorylation status at residue S134 by microcapillary LC-MS/MS.

Immunofluorescence

We analyzed U2OS cells stably expressing wild type NR3C1 or the S134A NR3C1 by NR3C1 immunofluorescence (1:500; Santa Cruz Biotechnology), followed by Alexa Fluor 594 (1:1000; Invitrogen) staining and confocal microscopy. In colocalization studies, T-ALL cell lines were immunostained with rabbit antibodies against NR3C1 (1:300; Santa Cruz Biotechnology) and a mouse antibody against AKT (1:100)( Cell Signaling, 2920) using anti-rabbit Alexa Fluor 594 (1:500; Invitrogen) and anti-mouse Alexa Fluor 488 (1:500; Invitrogen) as secondary antibodies. We stained nuclei with TO-PRO3 reagent (Invitrogen) and visualized them by confocal imaging on a Zeiss LSM510-NLO microscope.

Luciferase Reporter Assays

We performed NR3C1 reporter assays using the Cignal GRE Reporter (luc) Kit (SABiosciences).

Microarray Expression Analysis of Glucocorticoid response

DND41 shRNA LUC and DND41 PTEN shRNA cell were treated with vehicle only (DMSO) or dexamethasone (1μM) for 24 hours in triplicate. RNA was isolated, labeled and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina) using standard procedures. Raw gene expression data was log transformed and quantile normalized using MATLAB. Differentially regulated genes in DND41 shRNA LUC vs DND41 PTEN shRNA upon dexamethasone treatment with a fold change > 2 were selected by t-test. Glucocorticoid regulated transcripts (t-test p<0.01 and fold change >1.25) in primary ALL were identified from processed expression data obtained from peripheral blood lymphoblasts from 13 pediatric ALL patients after 24 hours of treatment with glucocorticoids (Schmidt et al., 2006). Enrichment of these glucocorticoid signature genes in shRNA PTEN vs shRNA LUC dexamethasone treated samples normalized to DMSO controls was analyzed by GSEA using the t-test metric comparing and 1,000 permutations of the genes.

Mice

Animal procedures were approved by the Columbia University IACUC. ROSA26Cre-ERT2/+ mice expressing a tamoxifen-inducible form of the Cre recombinase from the ubiquitous Rosa26 locus (Guo et al., 2007) and Pten conditional knockout mice (Ptenf) have been previously described (Trotman et al., 2003). To generate NOTCH1-induced T-ALL tumors in mice we performed retroviral transduction of bone marrow cells with an activated form of the NOTCH1 oncogene (NOTCH1 L1601P ΔPEST) and transplanted them via intravenous injection into lethally irradiated recipients as previously described (Chiang et al., 2008). Primary human T-ALL xenografts experiments were generated via intravenous injection of human T-ALL lymphoblasts into NOD rag gamma mice. We evaluated disease progression and therapy response by in vivo bioimaging with the In Vivo Imaging System (IVIS, Xenogen), human CD45 analysis by flow cytometry and analysis of luciferase activity in bone marrow and spleen.

Statistical Analyses

We performed statistical analysis by Student’s t-test and Wilcoxon signed-rank test. We considered results with p<0.05 as statistically significant. Survival in animal experiments was represented with Kaplan-Meier curves and significance was estimated with the log-rank test (Prism GraphPad).

Supplementary Material

01

HIGHLIGHTS.

  • AKT1 directly phosphorylates the NR3C1 glucocorticoid receptor protein.

  • AKT mediated S134 phosphorylation blocks nuclear translocation of NR3C1.

  • PTEN loss and AKT1 activation induce glucocorticoid resistance in T-ALL.

  • Pharmacologic inhibition of AKT1 reverses glucocorticoid resistance in T-ALL.

SIGNIFICANCE.

Improving the outcomes of patients with acute lymphoblastic leukemia (ALL) will require the identification of specific molecular mechanisms driving resistance to therapy and developing new pharmacologic strategies to neutralize them. Primary resistance to glucocorticoids, defined as failure to achieve robust cytoreduction after a week of glucocorticoid therapy, is of particular importance as it is associated with very poor prognosis. Here we show that AKT1 can drive resistance to glucocorticoids via direct inhibition of the glucocorticoid receptor. Consistently, pharmacologic inhibition of AKT effectively reverses glucocorticoid resistance. These results highlight the potential role of AKT1 as therapeutic target in the treatment of glucocorticoid resistant T-ALL.

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health (grant R01CA129382 to A.A.F.; U24 CA114737 to E.P.; RC2 CA148308 to A.C. and A.F.), the Stand Up To Cancer Innovative Research Award (A.A.F.), the Chemotherapy Foundation (A.A.F.), the Leukemia & Lymphoma Society Scholar Award (A.A.F.), a Leukemia & Lymphoma Society SCOR Grant (A.A.F.), and the ECOG Leukemia Tissue Bank. We are grateful to A. Kung for the FUW-luc vector, J. Aster for the MigR1-NOTCH1 L1601PΔP vector, P. P. Pandolfi for the Ptenf conditional knockout mouse, T. Ludwig for the ROSA26Cre-ERT2/+ mouse, S. Minuzzo for generating T-ALL xenografts, L. Xu for help in animal procedures, M. A. Gawinowicz from the Proteomics Share Resource at the Herbert Irving Comprehensive Cancer Center at Columbia University for assistance in mass spectrometry analysis and R. Baer for helpful discussions and revision of the manuscript.

Footnotes

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ACCESION NUMBERS

Microarray gene expression data is available in Gene Expression Omnibus (accession numbers GSE41062, GSE32215, GSE2677 and GSE10609).

SUPPLEMENTAL INFORMATION

Supplemental information includes Supplemental Experimental Procedures, seven figures and one table.

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