Background
Acute myeloid leukemia (AML) is a quickly progressive malignant disease of the myeloid lineage of hematopoietic cells, where overall three-year survival is below 20% for patients above 65 years [
1]. As elderly patients do not tolerate the intensive chemotherapy and stem cell transplantation of current treatment regimes [
2], the development of less toxic and more specific targeted therapy is necessary. Small-molecule MDM2 inhibitors like nutlin-3 have emerged as a potent and promising treatment option for cancers harboring wild type TP53, including AML [
3‐
5], and the oral formulation of nutlin-3, RG7112, has completed the first early phase clinical trials for both solid cancers and hematological malignancies [
6‐
8]. Intriguingly, these small-molecule p53 activators have demonstrated selective toxicity for cancer cells versus normal cells [
4,
9], and may also induce reversible cell cycle arrest of normal cells to protect them from adverse effects of conventional chemotherapy [
10].
While nutlin-3 initially was thought to exert its anti-cancer activity specifically through inhibition of the p53-MDM2 interaction, recent studies have demonstrated dual-targeting and p53 independent effects of nutlin-3 [
11‐
13]. The efficacy of nutlin-3 and other MDM2 inhibitors in hematological malignancies seems however largely to depend on the expression and activation of wild type p53 [
4,
9,
14,
15]. In addition to TP53 mutational status, several other molecular mechanisms have been shown to affect the sensitivity to MDM2 targeted therapy, including FLT3 and NPM1 mutational status [
15‐
17], E2F-1 transcriptional activity [
18], overexpression of MDMX [
19], and MDM2 levels [
4,
9]. The observed resistance to nutlin-3 in cohorts of AML patients could be explained by the extensive heterogeneity and range of molecular abnormalities of the disease [
2,
4]. For instance, aberrant recruitment of histone deacetylases (HDACs) and overexpression of heat shock proteins (Hsps) have been shown to be involved in the molecular pathogenesis and therapy response of AML [
20,
21], and could therefore be considered as potential therapeutic targets to combine with MDM2 inhibition. Inhibitors of HDACs and Hsp90 have been found to enhance p53 acetylation and inhibit MDMX, and synergize with nutlin-3 to induce p53-mediated apoptosis [
22‐
24]. The direct effect of nutlin-3 on regulation of histones and heat shock proteins has however not been determined.
In this study, we aimed to investigate mechanisms underlying the anti-leukemic activity of nutlin-3. We examined the functional role of p53 acetylation in nutlin-sensitivity, and hypothesized that nutlin-induced acetylation of other proteins than p53 would be of importance for the anti-leukemic effect of nutlin-3. Combining immunoprecipitation of acetylated proteins with quantitative proteomics, we identified novel targets of nutlin-induced acetylation, and investigated their participation in the nutlin-mediated response in AML cell lines and primary AML cells.
Discussion
Small-molecule MDM2 antagonists like nutlin-3 have demonstrated beneficial effects in cellular and preclinical models of various cancer types, including AML [
5]. This type of non-genotoxic specific targeted therapy holds promise for the treatment of AML patient groups lacking satisfactory treatment options due to toxicity and complications associated with current treatment regimes [
2]. A better understanding of the molecular mechanisms behind the anti-cancer activity of these compounds is however needed for further development of this type of therapy. The identification of molecular targets that could affect the sensitivity to the drug may be of importance for classification of patient groups that would benefit from the therapy, and for designing combinational therapy in order to overcome resistance, lower doses, and reduce side effects.
It is well established that expression and activation of p53 is a major determinant in nutlin-induced apoptosis [
3,
4,
9]. Previous studies have also shown that nutlin-3 enhances the acetylation of p53 in different human cancer cell lines [
22,
23]. Our results confirm the universality of nutlin-induced p53 acetylation in both AML cell lines and other human cancer cell lines, and furthermore demonstrate that the increase in p53 acetylation is independent of a simultaneous increase in total p53. The experiments applying a p53 acetylation defective mutant clearly illustrate that in addition to expression of p53, the modulation status of p53 is of great importance in nutlin-sensitivity. However, it should be taken into consideration that this mutant also is resistant to MDM2 mediated ubiquitination, resulting in higher expression levels of this mutant compared to wild type p53. Importantly, the p53 6KR mutant shows intact p53 transcriptional activity, but without the inhibitory regulation of MDM2 [
35]. Acetylation of p53 has been shown to be essential for its activation and regulation of different processes [
36‐
38], and to play an important role in therapy response [
39,
40]. Meanwhile, high expression level of p53 is associated with poor prognosis and resistance to therapy in AML [
41]. The possibility that the high levels of p53 is a consequence of modifications like acetylation, and that also p53 acetylation status in primary AML samples could provide information about nutlin-sensitivity need to be examined in future experiments. There are several possible explanations regarding the molecular mechanisms behind nutlin-induced p53 acetylation; Disruption of MDM2-p53 interaction could prevent MDM2 mediated ubiquitination or deacetylation of p53 [
42,
43], or nutlin-3 could prevent MDM2 from interacting with and inhibiting acetyl transferases important for p53 acetylation and activity [
44,
45]. These and other possible molecular mechanisms need to be further explored.
In general, protein lysine acetylation has been shown to play an important role in regulation of cellular function and cancer cell signaling, also in AML [
46,
47]. In addition to inhibiting MDM2-p53 interaction and modulating p53, nutlin-3 may affect several other proteins, either as a consequence of p53 transcription-dependent or -independent effects [
48], changed interactions between MDM2 and other proteins than p53 [
11,
28], or direct effect of nutlin-3 interaction with other proteins than MDM2 [
12]. Accordingly, we wanted to examine if nutlin-3 could enhance the acetylation of other proteins than p53. The methodology using SILAC in combination with an anti-acetyl-lysine antibody and mass spectrometry analysis has previously successfully been applied to identify and quantify alterations in acetylated proteins in cells treated with HDAC inhibitors, and both histones and heat shock proteins were identified as lysine acetylated [
49,
50]. The novel observation that nutlin-3 enhances the acetylation of histones, could add information regarding the molecular mechanisms behind the synergism of nutlin-3 and HDAC inhibitors [
22,
23]. While acetylation of histones is important for their transcriptional activity [
20], acetylation of heat shock proteins have been shown to inhibit their chaperone activity and promote their export and extracellular location [
51,
52]. This could explain the decrease in total levels of Hsp27 and Hsp90 as a consequence of nutlin-induced acetylation of these proteins. The combination of HDAC and Hsp90 inhibitors has demonstrated synergism in leukemia, but antagonism in other tumor models [
53]. Also the combination of HDAC inhibitors and nutlin-3 has shown contradictory results in different experimental settings [
22,
23,
54,
55]. As for p53, there are several possible mechanisms behind nutlin-induced acetylation of histones and heat shock proteins, including alterations in interaction between MDM2, histones and heat shock proteins or between MDM2 and components involved in regulating the acetylation of these proteins; further investigations are therefore warranted.
p53 and p53 acetylation seemed to be of importance for nutlin-mediated regulation of total and acetylated levels of heat shock proteins (Figure
6B). Nutlin-induced acetylation of Hsp90 occurred also in cells without p53, while downregulation of total levels of Hsp90 and Hsp27 was dependent of wild type p53. Previous studies using another MDM2 inhibitor have also shown downregulation of other heat shock proteins in wild type p53 cancer cells in response to treatment [
56]. Cells transfected with a p53 acetylation defective mutant demonstrated increased levels of MDM2 and acetylated Hsp90 by the transfection itself, but no effects on regulation of total or acetylated heat shock proteins in response to nutlin-treatment. In future perspectives, it would be interesting to perform similar experiments with acetylation defective heat shock protein mutants to investigate the role of heat shock protein acetylation in nutlin-induced p53 acetylation.
Sensitivity to both MDM2 and Hsp90 inhibitors is influenced by different molecular mechanisms in AML [
15,
17]. As high expression of heat shock proteins has been associated with poor prognosis and therapy resistance in AML [
21,
57], and different heat shock proteins may interact with and inhibit p53 [
29,
31], we wanted to examine if total levels of different heat shock proteins in AML patient samples could affect the sensitivity to nutlin-3. We did not find any significant correlations between nutlin-sensitivity and concentration of intracellular levels of different heat shock proteins in 40 primary AML samples. However, when the sample cohort was divided into sensitive and non-sensitive patient samples, there was a trend towards higher expression of heat shock proteins in the least sensitive patient samples, although the differences were not significant. Considering the fact that samples with TP53 mutations may respond differently to nutlin-3 compared samples with wild type p53, we also included analyses on the patient set including only samples with wild type TP53 (
n = 31), with similar results. The number of patient samples is however relatively low; a larger number of patient samples should therefore be included to determine if there are significant differences in heat shock protein levels in nutlin-sensitive versus non-sensitive samples. It would also be of interest to correlate levels of acetylated heat shock proteins and levels of induction of acetylated heat shock proteins in response to nutlin-3 with nutlin-sensitivity in primary AML samples.
To examine the functional effect of heat shock protein inhibition on nutlin-sensitivity, we chose to combine nutlin-3 with the Hsp90 inhibitor geldanamycin. The combination of nutlin-3 with Hsp90 inhibitors has previously demonstrated synergism in solid tumors [
24], while nutlin-3 and geldamamycin exhibited various effects in classical Hodgkin’s lymphoma depending on TP53 mutational status [
58]. Determination of drug interaction by Bliss independence analysis assumes that the two drugs act through independent mechanisms [
34]; nutlin-3 acts as an MDM2 inhibitor, and geldanamycin binds to and inhibits Hsp90 (although the may converge on the same pathway and indirectly affect the same down stream targets). Based on Bliss independence analysis with observed higher actual than expected response for both MOLM-13 cells and 9 out of 10 responsive primary AML samples, we propose that nutlin-3 and geldanamycin would kill cells independently of each other in a synergistic manner. Possible mechanisms may include enhanced Hsp90 inhibition and p53 activation [
33]. As Hsp90 has a wide range of client proteins, additional molecular mechanisms behind the observed synergism behind nutlin-3 and Hsp90 inhibitors have been proposed [
24]. To eliminate potential off-target effects of geldanamycin, the use of short hairpin RNAs (shRNAs) for stable and specific knockdown of Hsp90 in combination with nutlin-3 could be an option in future experiments. Inhibition of Hsp90 has been shown to induce Hsp27, possibly contributing to antagonizing the anticancer activity of Hsp90 inhibitors [
53]. In contrast, inhibition or knock down of Hsp27 also inhibits Hsp90 [
59]. Hence, in future studies, it would be interesting to combine nutlin-3 with shRNAs or small molecule oligonucleotides against Hsp27.
In our proteomics approach, we restricted the study to alterations in the lysine acetylome in the whole cell lysate compared to more extensive analysis of the proteome. As no other isolations or fractionations into for example nuclear and cytoplasmatic fractions were performed, a limitation of this procedure may be that only the most abundant proteins were detected. Further investigations could therefore include studying nutlin-induced acetylation and modulation of other less abundant proteins as well.
Methods
Cell lines and primary AML cells
The human AML cell lines MOLM-13 and HL60, and the human osteosarcoma cell line SAOS-2 and the human lung cancer cell line H1299 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), while the human AML cell line OCI-AML3 was purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Cell lines were cultured according to manufacturer’s procedure. For patient material, all studies were performed in accordance with the Helsinki declaration and approved by the regional Ethics Committee (REK Vest;
http://helseforskning.etikkom.no, Norwegian Ministry of Education and Research). Samples were collected after informed consent, and mononuclear cells were isolated and stored frozen in liquid N
2 as previously described [
60]. Normal peripheral blood lymphocytes were obtained from healthy blood donors (Blodbanken, Haukeland University Hospital, Bergen, Norway). Primary AML cells and normal peripheral blood lymphocytes were cultured in StemSpan SFEM™ (StemCell Technologies Inc., Vancouver, BC, USA).
Compounds
Nutlin-3 (Cayman Chemical Company, Michigan, USA) and geldanamycin (Sigma-Aldrich, Inc., St Louis, MO, USA) were dissolved in DMSO, and stored at −80°C. When used in cell culture work, the final concentration of DMSO did not exceed 0.1%.
Western blotting
Western blotting was performed as previously described [
22]. The following antibodies were used; p53 (Bp53-12), Mdm2 (SMP-14) (Santa Cruz Biotechnology, CA, USA), Mdm2 (2A10), Mdm2 (IF2), anti-Hsp27 (G3.1) (Calbiochem, San Diego, CA, USA), p21 (SX118) (BD Biosciences, San Jose, CA, USA), phospho-p53 (Ser15), phospho-p53 (Ser20), ac-p53 (Lys382) (Cell Signaling Technologies, Beverly, MA, USA), anti-Histone H2B, anti-Hsp90 (Millipore, Temecula, CA, USA), anti-acetyl-Histone H2B (Lys120) (Upstate cell signaling solutions, Lake Placid, NY, USA), anti-acetyl-Hsp90 (Lys294) (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA), secondary horse radish peroxidase conjugated mouse and rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA), actin (AC-15) (Abcam plc, Cambridge, UK). Bands were quantified using region of interest analysis on Kodak Molecular Imaging Software version 5.0.1 (Carestream Health, Rochester, NY, USA). Fold induction are given in arbitrary units and are defined as protein of interest/actin following normalization of control.
Flow cytometry
Flow cytometric analysis was performed as previously described [
22], using antibodies against Hsp90 α/β (F-8) PE (Santa Cruz Biotechnology, CA, USA) and Hsp27 (G3.1) PE (Enzo Life Sciences, Farmingdale, NY, USA).
Plasmids and transfections
p53 cDNA constructs of p53 FL and p53 6KR were previously described [
35]. Transfections were performed using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, GmbH, Mannheim, Germany) according to the manufacturer’s procedure as previously described [
61].
Cell viability and proliferation assays
Evaluation of apoptosis, viability and proliferation in cell lines and primary AML cells after drug treatment was accomplished using Hoechst 33342 (Invitrogen, Carlsbad, Ca, USA), the viability/proliferation reagent WST-1 (Roche Diagnostics GmbH, Mannheim, Germany),
3H-thymidine (Amersham International, Amersham, UK) incorporation assay, APOTEST-FITC kit (Nexins Research, Kattendijke, The Netherlands) or Alexa Fluor 488 Annexin V/ Dead Cell Apoptosis Kit (Molecular Probes, Invitrogen, Eugene, Oregon, USA) as previously described [
22].
Immunoprecipitation
Approximately 50 million cells were lysed in Triton® X-100 lysis buffer containing 150 mM NaCl, 50 mM Tris HCl pH 8.0, 1% Triton® X-100 (Plus one, Pharmacia Biotech, Uppsala, Sweden), Complete mini Protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany), 5 mM NaF, 1 mM Na-orthovanadate, 10 mM nicotinamide and 1 μM TSA, and immunoprecipitation was carried out using μMACS ProteinG Microbeads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s procedure. The cell lysate was pre-cleared with μMACS Protein G MicroBeads to remove unspecific binding to the beads followed by a pre-clear using an unspecific antibody (Chromatographically purified Rabbit IgG, Invitrogen, Camarillo, CA, USA) and μMACS Protein G MicroBeads to remove unspecific binding to the immunoglobulines, before new μMACS Protein G MicroBeads and anti-acetyl-lysine antibody (4G12) (Millipore, Billerica, MA, USA) were added to the pre-cleared lysate for immunoprecipitation of acetylated proteins. Proteins were eluted in 95°C SDS loading buffer and loaded directly on to a gel for electrophoresis.
Stable isotope labeling with amino acids in cell culture (SILAC), mass spectrometry and analysis of mass spectrometry data
MOLM-13 cells were grown in SILAC RPMI media with 10% dialyzed FBS, 1% penicillin, 0.1 mg/ml L-Lysine-2HCL and 0.1 mg/ml mg L-Arginine-HCl, or 0.1 mg/ml
13C
6 L-Lysine-2HCl and 0.1 mg/ml mg
13C
615N
4 L-Arginine-HCl (Pierce SILAC Protein Quantification Kit – RPMI 1640, Thermo Scientific, Pierce Protein Research Products, Rockford, IL, USA) for six passages [
25], and incorporation efficiency was determined by mass spectrometric analysis. Cell lysates were mixed at a ratio of 1:1 (5 mg protein of each sample) before immunoprecipitation procedures were performed. Eluted proteins from the immunoprecipitation were separated by one-dimensional gel electrophoresis and stained with Coomassie Blue (Pharmacia Biotech, Uppsala, Sweden). The gel was sliced into 13 gel pieces prior to reduction, alkylation, trypsin digestion and analysis by nano-LC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled to an ESI-Orbitrap (LTQ Orbitrap XL, Thermo Scientific, Bremen, Germany) mass spectrometer as previously described [
62]. The peptides were identified and quantified using the MaxQuant and Perseus software (version 1.2.2.5 and 1.2.0.16) [
63] with the following settings: carbamidomethyl (C) as fixed modification, and oxidation (M), acetylation (K) and acetylation (protein N-term) as variable modifications. FDR was 1%, MS tolerance was 10 ppm and MS/MS tolerance was 0.7 Da. Only proteins with more than 1 peptide were included in the analysis. All ratios are given as normalized values and are tested with Benjamini-Hochberg FDR test (
p < 0.05) using significance B.
Analysis of intracellular levels of heat shock proteins
Intracellular levels of heat shock proteins Hsp27 (phospho-Ser82), Hsp27 (phospho-Ser15), Hsp40, Hsp60, Hsp70 and Hsp90α were determined using the Hsp/Chaperone 8-plex MultiBead kit (Assay Designs, Inc., Ann Arbor, MI, USA) according to manufacturer’s instructions as previously described [
17].
Statistical analysis
In cell viability and proliferation assays, triplicates were analyzed for each sample, and results given as means +/− standard error of mean. Statistical significance of differences in averages was determined using a two-tailed Student’s t-test. For statistical comparison between different patient groups, we used Mann–Whitney U-test. Correlation analysis was performed using Pearson’s correlation, and synergism was calculated by Bliss Independence analysis. For all statistical analysis, p < 0.05 was considered significant. Graphs and calculations were obtained using GraphPad Prism® 5.0 (GraphPad Software, La Jolla, CA, USA). Results from flow cytometric analysis were visualized using TMEV microarray software suite version 4.3.01 (Dana-Farber Cancer Institute, Boston, MA, USA).
Competing interests
The authors have no competing interests.
Authors’ contributions
IH, FSB, SL, ØB and BTG designed the study. IH, HR, HKF, and RH performed experiments. IH, JAO, HR and BT analyzed data. SL contributed with reagents. IH, JAO, BT and BTG wrote the manuscript. EMC critically revised the manuscript. All authors revised the manuscript. All authors read and approved the final manuscript.