AKT regulates NPM dependent ARF localization and p53mut stability in tumors

Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53mut stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53mut status.

position 48. Peptide synthesis and immunizations were carried out by Eurogentec. The anti phospho-Ser48-NPM used in this study was affinity purified against the phosphopeptide. Secondary antibodies anti-mouse and anti-rabbit HRP conjugates were purchased from Pierce and the Jackson Laboratory.
Fluorescent tagged antibodies for use on the Licor Odyssey western blot imaging system were purchased from Licor and Invitrogen. Fluorescent conjugated secondary antibodies for immunofluorescence were purchased from Invitrogen.
Nuclear lysates were prepared as described previously [6] with additional modifications. Briefly, 3-5 x 10 6 cells were trypsinised and harvested by centrifugation (500 x g, 5 min) , washed twice in TBS and resuspended in 1-2 ml ice cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) by gently pipetting in a 1 ml tip. The cells were left on ice for 15 min to swell, after which 75 µl of 10 % NP-40 was added. The tube was vigorously vortexed for 10 sec and centrifuged at 500 x g for 2 min. The supernatant, which constitutes the cytoplasmic fraction, was removed. The nuclear pellet was re-suspended in 150 µl ice-cold lysis buffer (150 mM NaCl, 20 mM Hepes pH 7.5, 0.5 mM EDTA, 0.5% (v/v) NP-40, 10 mM sodium β-glycerophosphate, 50 mM NaF, 1 mM Na 3 VO 4 , 5 mM sodium pyrophosphate and 'Complete' proteinase inhibitor cocktail EDTA free (1 tablet/10 ml lysis buffer) and the sample sonicated. The nuclear extract was centrifuged (20,817 x g, 15 min, 4 0 C) and the supernatant containing the nuclear extract was used as an in-put for immunoprecipitation or added to SDS-PAGE sample buffer as described above.
Primary antibody detection was achieved with HRP conjugated secondary antibodies (Pierce) and exposure to X-Ray film (Kodak). For blots which were quantified, samples were run by western blot and transferred onto Nitrocellulose membrane (Biorad). Membranes were blocked in Licor blocking buffer and incubated with primary antibody overnight followed by incubation with fluorescently conjugated secondary antibodies. Membranes were scanned on the Licor Odyssey infrared scanner and signal intensity determined using the Odyssey software (V3.0). Signals were normalized to GAPDH as a loading control. All quantification was done on the same nitrocellulose membrane without stripping.
In-Vitro Kinase assay: Endogenous AKT was immunoprecipitated from T24 cells, glycine eluted and combined with immunoprecipitated HA-NPM, HA-NPM-S48A or anti HA-IP from non-transfected cells as indicated in 1 x kinase buffer (Cell Signaling). The reaction was incubated with cold ATP (20 µM) and radio-labeled gamma 32 P ATP (2 µM) at 37°C. The reaction was heat inactivated in the presence of denaturing SDS-PAGE sample buffer, separated by SDS-PAGE, western blotted and exposed to a phosphor screen. The membrane was additionally probed by standard western blot.
Purification of p14 ARF from HeLa Nuclear extracts: Fractionation of HeLa cells was performed as previously described [7]. Briefly, 20 grams of HeLa cell pellets (Cilbiotech, Mons, Belgium) were Protein Labeling Mix 35S, PerkinElmer). Cells were metabolically labeled for 1 Hr before being washed twice with complete media containing unlabeled Met/Cys. Cells were chased for 30 min-4 Hrs in complete media at 37ºC before being washed twice with PBS and lysed by scrapping in 1 % (v/v) NP-40 lysis buffer. p53 was immunoprecipitated as outlined above. Samples were run on a 10 % NuPage Bis Tris gel. Gels were dried before exposure to a phosphor screen. Additionally samples were probed by western blot.
Immunofluorescence and Immunohistochemistry: For analysis of cells using the In Cell Analyzer 1000, automated epifluorescence microscope (GE Healthcare), cells were plated into 96-well plates at a density of 10,000 cells/well and incubated overnight at 37°C with 5% CO 2 in a humidified incubator. Cells were exposed to inhibitor 24 hours prior to fixation. Cells were fixed with 4 % paraformaldehyde and permeabilized with 1 % (v/v) TritonX-100 and blocked with a 1 (w/v) % solution of BSA in PBS.
Cells were incubated with primary antibody as indicated (1:1000 dilution), overnight at 4°C. Primary antibody was detected using Alexafluor conjugate secondary antibodies (Invitrogen) at 1:1000 dilution. For frozen tissue sections, slides were fixed in acetone for 10 minutes at room temperature. Slides were dried, washed in PBS and non-specific binding blocked with 3% (v/v) normal bovine serum (NBS), PBS, 0.1 % (v/v) Triton-X 100 for 20 minutes. Slides were incubated with primary and secondary antibodies as outlined above before image acquisition.
Xenograft tumors and tissue microarrays (Biomax) were formalin fixed and paraffin embedded prior to sectioning and staining. Sections were re-hydrated sequentially from xylene -ethanol -water prior to antigen retrieval by boiling in 10 mM sodium citrate and blocked in TNB (Perkin Elmer). Endogenous peroxidise was blocked with 0.3 % Hydrogen Peroxide (H 2 O 2 ) prior to all immuno-peroxidase staining protocols. Non-specific binding of secondary antibody was blocked using 3 % normal serum from the animal of origin of the corresponding secondary antibody. Slides were incubated in primary antibody (1:100) overnight at 4°C. Secondary antibodies were detected using Avidin Biotin Complex (ABC) reagent (Vector labs), followed by the chromogen 3,3'-Diaminobenzidine (DAB) reagent (Vector labs) as per the manufacturer's instructions. Sections were counterstained with heamatoxylin and imaged under a light microscope (Nikon) or the ScanScope digital slide scanner (Aperio). Immunohistochemical staining was quantified by H-score. Staining intensity was grouped into four categories and a numerical multiplier assigned (bracketed); no stain (0) low intensity (+1), moderate intensity (+2) and high intensity (+3). The percentage of cells, within each staining intensity, was multiplied by the multiplier to give a total H-score for comparison. Scoring was completed on multiple representative fields of view from each sample (n=3). For total scoring of p Ser48NPM slides were scanned with the ScanScope digital slide scanner and total signal intensity and total area of positive staining from the DAB stain quantified by the scanscope software and grouped into scores of no stain, low intensity, moderate intensity and high intensity and scored as above. Further characterization of Pancreatic Ductal Adenocarcinoma was performed under guidance of a pathologist and specific cytoplasmic/nuclear staining was scored by H-Scare as outlined above.
Clonogenic survival curves are representative of independent replicate experiments.
3D colony growth assay: 3D colony assay of the KPC mouse derived cells was adapted from a previously described protocol for 3D culture of mouse pancreatic cells [9]. Cells were resuspended at a density of 2.    Immunoprecipitates and whole cell lysates were probed with the indicated antibodies.