Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rγnull xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.
Elderly acute myeloid leukemia (AML) patients do not tolerate the intensive combination chemotherapy or bone marrow transplantation required to treat their disease, resulting in an overall survival rate <10%.1, 2, 3 Thus, the necessity for more specific targeted and less toxic therapy is critical for these patients.
Although mutations in TP53 occur in <10% of patients with AML, overexpression of its main negative regulator, MDM2, is frequently observed.4 Furthermore, aberrant recruitment of histone deacetylases (HDACs) is detected in AML, leading to myeloid differentiation block and leukemic blast accumulation.5
Nutlin-3 is a small-molecule antagonist of MDM2, which has been found to bind specifically to the p53-binding pocket of MDM2, activate the p53 pathway in cancer cells with wild type p53, and inhibit tumor growth in a non-genotoxic manner in xenografted tumor mice.6 p53-mediated effects of nutlin-3, such as induction of cell cycle arrest and apoptosis, have been demonstrated in cancers characterized by non-mutated TP53, including AML.7 Valproic acid (VPA) is a well-tolerated anticonvulsant drug that has been used clinically for more than three decades.8 Recently, it has also been shown to have HDAC inhibitor activity, and to induce differentiation and apoptosis in AML progenitors and blasts, with a limited toxicity profile in elderly AML patients.9, 10, 11, 12
Both VPA and nutlin-3 affect the regulation of p53; nutlin-3 by inhibiting MDM2 and VPA by inhibiting HDACs that participate in p53 deacetylation and destabilization.6, 13 We therefore hypothesized that concomitant inhibition of MDM2 and HDACs would additively induce p53-mediated apoptosis and inhibit tumor growth. The effects of the combinational treatment on apoptosis, p53 and target gene induction, and inhibition of proliferation were tested in vitro in AML cell lines and primary AML cells, and sensitivity towards the combinational treatment was correlated to clinical parameters. To evaluate the combinatorial treatment in vivo, we established a luciferase expressing MOLM-13luc AML xenograft model in NOD-scid IL2rγnull mice. Subsequent, bioluminescent imaging permitted non-invasive spatio-temporal detection of disease development and therapy response monitoring.
Materials and methods
Cell lines, primary AML cells, normal peripheral blood lymphocytes and cord blood cells
Cell lines and cell culture conditions are described in Supplementary information. AML samples were collected from patients after informed consent and approval by the regional Ethics Committee (REK Vest; http://helseforskning.etikkom.no; Norwegian Ministry of Education and Research). Normal peripheral blood lymphocytes were obtained from healthy blood donors (Blood bank, Haukeland University Hospital, Bergen, Norway), whereas normal umbilical cord blood was obtained from healthy individuals after caesarian section (Women's Clinic, Haukeland University Hospital, Bergen, Norway). Preparation and culturing of cells is described in Supplementary Information.
Clinical parameters of AML patients
Clinical parameters including FAB classification, cell surface markers, karyotype, resistance, survival and FLT3/NPM1 mutational status were routinely analyzed and collected. RNA extraction and p53-mutational analysis was performed as previously described.14
Nutlin-3 and -3a (Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in DMSO and stored at −80 °C. When used in cell culture work, the final concentration of DMSO did not exceed 0.1%. For animal experiments, nutlins were administered orally (p.o.) as a suspension in vehicle consisting of 2% hydroxypropyl cellulose and 0.5% tween 80 (Sigma, St Louis, MO, USA) in sterile water. VPA (Orfiril, Desitin Arzneimittel GmbH, Hamburg, Germany) (100 mg/ml in solution) was stored at −80 °C for cell culture work. For animal experiments, VPA was administered by intraperitoneal injection.
p53 flow cytometric analysis, detection of apoptosis by annexin V-propidium iodide (Annexin-PI) and detection of Lysotracker Red stained cells are described in detail in Supplementary Information.
Transmission electron microscopy (TEM)
For evaluation of autophagic features, cells were prepared for and analyzed by transmission electron microscopy as previously described.16
Cell viability and proliferation assays
Evaluation of apoptosis and inhibition of proliferation in cell lines and primary AML cells after drug treatment was performed using different assays; Hoechst 33342, Annexin-PI, 3H-thymidine incorporation, ATP and Alamarblue assay, for details see Supplementary Information.
Transfections of luciferase expressing construct and sorting of cells
The luciferase expressing construct L192 and the TetActivator were transfected into Phoenix cells, and retroviral infection was performed as previously described.17 Sorting of highly bioluminescent MOLM-13luc cells is described in Supplementary Information.
MOLM-13 xenograft model and optical imaging
The animal experiments were approved by The Norwegian Animal Research Authority and performed in accordance with The European Convention for the Protection of Vertebrates Used for Scientific Purposes. For all experimental details, see Supplementary Information.
For details on statistical analysis, see Supplementary Information.
Combinational therapy of nutlin-3 and VPA induces apoptosis synergistically in the wild type TP53 AML cell line MOLM-13
We were particularly interested in whether nutlin-3, an MDM2 antagonist,6 and VPA, a known class I and II HDAC inhibitor affecting p53 acetylation,13 could work in tandem to elicit enhanced apoptotic effect on AML cells expressing wild type p53. To determine synergistic effects of VPA and nutlin-3 on cell viability, MOLM-13 cells were treated with increasing doses of VPA (0–2 mM) or nutlin-3 (0–20 μM) alone or in combination at a fixed ratio (1:100) for 72 h (nutlin-3 for the last 24 h) and analyzed by Annexin-PI. The dose-effect curve for each drug was determined and combination index values were calculated according to the Chou–Talalay method,18 whereas effect on cell viability was expressed as fraction of cells affected. The results are presented in Figure 1a, indicating strong synergism between the two drugs along the entire dose-response curve. The apoptotic effect of VPA (50–1000 μM, 24, 48 and 72 h) and nutlin-3 (0.5–10 μM, last 24 h) alone and in combination MOLM-13 cells, was also investigated by DNA-specific staining with Hoechst 33342 (Supplementary Figure 1A). Although induction of apoptosis by VPA alone was minimal (P>0.05), nutlin-3 mediated effective apoptosis (>50%) at 10 μM. At lower concentrations, particularly the combination of VPA at 500 μM and nutlin-3 at 5 μM demonstrated super-additive induction of apoptosis in comparison with single treatments.
Repeat studies employing nutlin-3 (5 μM) and VPA (500 μM) exhibited significantly better effect of the combination on viability (Hoechst 33342) compared with either agent alone (P<0.001) at 24, 48 and 72 h (Figure 1b). Furthermore, Bliss Independence analysis19 of the data revealed synergistic apoptosis induction with higher actual response than expected response for the combination of nutlin-3 (5 μM) and VPA (500 μM) at all time points (P<0.0001; Supplememtary Figure 1B). The effect of the combinational therapy on apoptosis induction in MOLM-13 cells is clearly visualized by staining with Hoechst 33342 and microscopic imaging of fragmented/condensed cell nuclei, whereas no effects are seen in normal peripheral blood lymphocytes (Supplementary Figure 1C). Finally, the remarkable efficacy and synergy of the combination upon apoptosis in wild type TP53 AML cells was reaffirmed by Alamar Blue and ATP cell viability assays, with Bliss Independence analysis also confirming synergy with these assays (Supplementary Figure 1D).
Synergistic apoptosis induction by nutlin-3 and VPA involves super additive induction of p53, acetylated p53 and p53 target genes
As VPA's effect increases with time and concentration,20 we incubated MOLM-13 cells for 72 h (500 μM) to visualize protein induction. Nutlin-3 was added for the last 24 h at a lower concentration than for cell viability assays (2.5 μM) to avoid massive apoptosis. As expected, nutlin-3 induced p53, acetylated p53 and subsequently MDM2 and p21, whereas VPA, as observed in cell viability studies, demonstrated negligible effect. The combination, however, super additively induced p53 and acetylated p53 and most strikingly p21 and MDM2 (Figure 1c; western blots). Also primary AML cells showed increased induction of p53 and target genes with the same treatment conditions (significant increase in p21 and MDM2 induction; P<0.05), as analyzed by flow cytometric analysis of p53, p21 and MDM2 (Figure 1d). To determine the role of p53 in the combinational therapy, we transfected MOLM-13 cells with shp53 (providing a 70% knockdown of p53; data not shown) and compared the effects of the combinational treatment between MOLM-13 cell lines (wild type p53 and shp53) with Annexin-PI co-staining and flow cytometric analysis. Although MOLM-13 cells with wild type p53 responded to treatment with nutlin-3 (5 μM), VPA (500 μM) or the combination, those transfected with shp53 demonstrated only diminutive effects upon viability (Figure 1e). These results suggest that regulation of p53 is central to the effect of combinational therapy of nutlin-3 and VPA.
Combinational therapy of nutlin-3 and VPA induces autophagy in MOLM-13 cells
We assessed modulation of autophagy markers LC3B and p62 in MOLM-13 cells treated with VPA (500 μM, 72 h), nutlin-3 (2.5 μM, last 24 h) or the combination of both (Figure 1c), demonstrating decreased expression of the full-length LC3B isoform and p62. In addition, flow cytometric analysis of MOLM-13 cells treated with VPA (500 μM, 48 h), nutlin-3 (5 μM, last 24 h) or the combination showed significantly increased number of acidic organelles (Lysotracker Red) with the combination compared with either agent alone (P<0.001 and <0.01, respectively) (Figure 1f). Corresponding transmission electron microscopy micrographs confirmed autophagy induction in cells treated with nutlin-3 and the combination by detection of autophagosomes.
Similar results are shown for Lysotracker Red at 72 h (Supplementary Figure 2A), and in more detailed transmission electron microscopy micrographs, demonstrating accumulation of autophagosomes in the combination therapy compared with either agent alone (Supplementary Figure 2B).
Combinational therapy of nutlin-3 and VPA induces apoptosis synergistically in primary AML cells
Primary AML cells from 31 heterogeneous AML patient samples (Supplementary Table 1), peripheral blood lymphocytes, and cell lines MOLM-13 (wild type TP53, length mutated FLT3), OCI-AML3 (wild type TP53, wild type FLT3), HL-60 (deleted TP53, wild type FLT3) and NB4 (mutated TP53, wild type FLT3), exhibited various sensitivities to the combination treatment in vitro as determined by Annexin-PI staining (Figure 2a). MOLM-13 cells with wild type TP53 and mutated FLT3 demonstrated synergistic effects of the combination treatment also with this assay, whereas the AML cell lines HL-60, NB4 and OCI-AML3 were more resistant. Of the 10 most sensitive patient samples, 50% were FLT3 wild type, demonstrating the potential of the combinational treatment also for patients irrespective of FLT3 status. The frequency of TP53 mutation in AML is exceptionally low and in this patient set only 2 of the 31 (6.5%) samples harbored TP53 mutations, which were among the most resistant samples. However, no significant correlations were found between the patient clinical parameters and sensitivity towards the combination. Pooling of the patient data (Annexin-PI) demonstrated significant reduction in mean viability for the combinational treatment versus nutlin-3 or VPA alone (P<0.001; Figure 2b) and synergism, as calculated by Bliss Independence (P<0.05; Figure 2c). Analysis of the 28 responding AML patient samples, showed higher actual than expected response for 24 of the 28 patient samples (Figure 2d), reflecting synergistic effect of the combinational treatment on viability in most of the patient samples. The superior effect of the combinational treatment compared with either agent alone in primary AML cells is exemplified in Supplementary Figure 3A. Analysis of viability by Hoechst 33342 showed similar results (P<0.05; Supplementary Figure 3B).
Inhibition of proliferation by combinational therapy of nutlin-3 and VPA correlates with CD34 expression of AML blasts
Primary AML cells (n=56) exhibited various sensitivities to the combination treatment of nutlin-3 (5 μM) and VPA (500 μM) for 48 h (nutlin-3 added for the final 24 h) in vitro also in 3H-thymidine incorporation proliferation assay (Figure 3a). Further examination of sensitivity to the combination and patient clinical parameters revealed a significant Pearson correlation between sensitivity towards the combination treatment (defined by 3H-thymidine incorporation) and percentage CD34 expression (Pearson r=0.3211, P=0.02). Additionally, when patients were divided into populations of CD34low or CD34high (high as defined by >20% of blast cells stained CD34 positive by flow cytometry), the combination of nutlin-3 and VPA resulted in a significant increase in sensitivity in CD34low leukemic cells (P<0.05, n=53; Figure 3b).
Effects of the combination therapy on normal CD34+ cord blood cells and on clonogenicity of leukemic progenitors
CD34+ cells isolated from umbilical cord blood from three different healthy donors were assayed (Annexin-PI) for their sensitivity to nutlin-3 (5 μM), VPA (500 μM) or the combination, for 48 h (nutlin-3 added for the final 24 h). Contrasted against similar results from sensitive AML patient samples (n=10) and the whole cohort of AML patient samples (n=31), where significant differences in means for viability between the combination of nutlin-3 and VPA versus either agent alone (P<0.001; Supplementary Figure 4) were determined, CD34+ cells were only found to be more sensitive to the combination when compared with nutlin-3 alone (P<0.5), suggesting VPA as the main mediator of toxicity in normal hematopoietic progenitors. In clonogenicity assay, AML patient cells sensitive to the combination demonstrated decreased numbers of clonogenic progenitors (mean=31.9% of control; n=3) in comparison with nutlin-3 (54.8%) and VPA (60.9%) cultures (data not shown). The efficacy of nutlin-3 in insensitive patient cells (n=4) was also relatively high (62.4%), whereas VPA stimulated growth of clonogenic progenitors in these samples (129.1%), annulling the effect of nutlin-3 in the combination therapy (92.7%). These data suggest that VPA may have different effects in leukemic stem and progenitor cells compared with the major bulk of AML blasts.21
Combination of nutlin-3 and VPA significantly inhibits disease development in an in vivo MOLM-13 AML xenograft model
We next asked the question if the combination of nutlin-3 and VPA could be effective in an in vivo model of AML with wild type TP53. We thus evaluated the combination in a bioluminescent orthotopic (Supplementary Figure 5),22 and also in a subcutaneous, xenograft model of MOLM-13, employing either nutlin-3 or nultin-3a respectively (details of regimes outlined in Supplementary Materials and methods). Although the limited treatment regime used in the orthotopic model abrogated survival studies, quantification of bioluminescence allowed for direct comparison between treatment groups, and perhaps more significantly, permitted visualization of systemic anti-leukemic efficacy (Figure 4).
Although demonstrating minimal in vitro efficacy in MOLM-13 cells, VPA treated animals showed averaged lower bioluminescence (P=0.1, 0.14 and 0.22 for weeks 1–3 respectively; Figure 4b). Nutlin-3 demonstrated a significant cytoreductive effect upon MOLM-13luc xenografts, particularly on day 14 (P=0.014 versus control; Figures 4a and b). Remarkably, co-treatment of MOLM-13luc leukemic mice with the combination resulted in considerable inhibition of systemic, leukemic progression as evidenced by significantly reduced whole-body bioluminescence at all time points (P=0.02, 0.0002 and 0.028 versus controls respectively; Figures 4a and b).
A second subcutaneous MOLM-13 model in NOD-scid mice (n=5 per group) that did not require a myeloreductive-conditioning regime for xenograft, employing enatiomerically pure nutlin-3a (125 mg/kg) and higher doses of VPA (350 mg/kg; details of regimes outlined in Supplementary Materials and methods) was performed (Figure 4c). Although single agent treatments reduced tumor growth appreciatively in comparison with controls (Figure 4c) the combination group significantly impaired tumor growth in comparison with single agent arms (Figure 4c) and vehicle controls. Sacrifice of animals upon reaching tumor volumes of ethical limit revealed significant increase in survival of the combination animals in comparison with controls (P=0.007) and nutlin-3a (P=0.013) or VPA (P=0.041) treated mice (Figure 4d). Upon termination of the study, (28 days) three of five animals receiving the combination treatment still had tumors below ethical limits when all other animals in the study had been euthanized.
We hypothesized that judicious co-treatment of VPA and nutlin-3 would additively enhance p53 acetylation and induce apoptosis in wild type p53 AML cells.12, 13, 23, 24 The synergy observed in MOLM-13 wild type p53 cells in three independent assays (Figure 1 and Supplementary Figure 1) was remarkable.
The importance of p53 modulation and p53 response in this particular treatment was demonstrated by super-additive induction of p53, acetylated p53 and p53 target genes, in addition to abrogated effect of the combination by shRNA against p53 (Figures 1c–e). Mechanisms involving hyperacetylation of p53 by co-treatment of HDAC inhibitors with nutlin-3 are supported by Palani et al.25 Increased induction of autophagy by the combination therapy was also noted (Figures 1c and f, Supplementary Figure 2), possibly as a result of increased p53. However, p53 has been shown to both induce and inhibit autophagy depending on location in the cell.26 In addition, autophagy may represent both a survival and a cell death mechanism;27 the role of autophagy in the response to the treatment therefore needs to be further explored. Although these results strongly indicate a central role for p53 in this combination they do not rule out the possibility of p53 independent effects.
High levels of MDM2 expression have also been associated with higher sensitivity towards nutlin-3.7, 28 As MDM2 has been shown to recruit HDAC1 to p53,29 a possible hypothesis that MDM2 inhibition by nutlin-3 may further prevent HDAC activity upon p53 by reducing the recruitment of HDAC1 to p53 is therefore plausible. Also in p53 independent models, the two drugs may have similar effects, as both compounds have been shown to enhance the levels of p73 and p21.12, 30, 31 In our study, the combination had no effect in cell lines with deleted or mutated p53, whereas there was an effect in patient samples with mutated p53, although these were mainly among the less sensitive samples (Figures 2 and 3). Further examination of mechanisms underlying the synergistic interaction of the combination in both p53 dependent and independent models is warranted.
In vitro results demonstrated an overall synergistic induction of apoptosis in primary AML cells (n=31) (Figure 2). Given the heterogeneity of AML and our cohort, it is not surprising that some of the patients responded to the therapy, whereas some were more resistant. As for any targeted therapy, patient stratification is critical to identifying those that would benefit best from the combination treatment. 3H-thymidine proliferation assay revealed a significant correlation of drug efficacy to CD34low cellular expression (Figure 3), suggesting that patients of a more differentiated phenotype may respond best to the combination of nutlin-3 and VPA. Also patient samples with poor risk cytogenetics seemed to respond well to the treatment (4 of 10 most sensitive patient samples), suggesting that the combination therapy may be of benefit to both standard risk patients and poor prognosis patients. In contrast to the recent findings of Long et al.,32 we did not find any significant correlation between FLT3 mutational status and sensitivity for either nutlin-3 or VPA alone, or for the combined treatment. The role of these and other molecular response predictors33, 34 need to be examined in a larger number of samples, ideally in future clinical trials where these therapeutics are evaluated. Although the combination therapy elicited a toxic effect on normal CD34+ progenitors in vitro (Supplementary Figure 4), the concentration of VPA used in our experiments is equivalent to serum levels normally well tolerated in vivo.8 Furthermore, nutlin-3 exerted minimal toxicity on these cells in vitro, also observed by Kojima et al.7 Ongoing (Nutlin; ClinicalTrials.gov NCT00623870) and future clinical trials are however needed in order to determine the clinical safety of the therapy.
To truly evaluate the combination of nutlin-3 and VPA we developed an orthotopic model of wild type TP53 AML that would permit spatio-temporal monitoring of disease pathology and systemic drug efficacy by optical imaging.22 Thus, this orthotopic model represents a more clinically relevant preclinical paradigm. Irradiated NOD-scid and NOD-scid β2mnull models for the xenotransplantation of human AML cell lines,35, 36 resulted in a variable or non-leukemic state when transplanted with MOLM-13 (Supplemental Figure 4A). Subsequent xenotransplantation of luciferase expressing MOLM-13 cells in NOD-scid IL2rγnull,37 resulted in systemic leukemia with reproducible disease latency (Supplemental Figure 4). Optical imaging demonstrated significantly reduced photon counts for mice receiving the combination of nutlin-3 (200 mg/kg) and VPA (50 mg/kg), and to a lesser extent nutlin-3, for the duration of this regime (Figures 4a and b), with relapse to higher photon counts following treatment stop. Interestingly, combination treated mice showed statistically lower numbers of photon counts than controls even at day 21, 9 days after last treatment, which was not observed for nutlin-3 only treated animals. This is in contrast to resumed tumor growth following cessation of nutlin-3a treatment reported in a responsive lymphoma xenograft model, suggesting that continuous dosing is requisite for efficacy.38 In a further subcutaneous in vivo model of MOLM-13 (Figures 4c and d), mice were treated with enatiomerically pure nutlin-3a (125 mg/kg) and VPA at high dose (350 mg/kg). In this study the combination significantly inhibited tumor growth in comparison with controls and the nutlin-3a and VPA only treatment arms (Figure 4c). Furthermore, upon termination of the study on day 28, 60% of the combination treated animals were still alive and within ethical limits, whereas all animals in the other groups had been humanely euthanized (Figure 4d). These results suggest that combination of HDAC inhibitors with MDM2 antagonist is beneficial in the treatment of AML expressing wild type p53, and is to the best of our knowledge the first report of nutlin-3 efficacy in an AML preclinical model.
In conclusion, our results demonstrate that combined therapy of VPA and nutlin-3 activates p53 mediated apoptosis synergistically in vitro and in vivo in AML, and propose concomitant inhibition of HDACs and MDM2 as a novel promising non-genotoxic therapeutic option in AML.
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This study was supported by The Norwegian Cancer Society (Kreftforeningen), The Western Norway Regional Health Authority (E.Mc.C. and B.T.G.) and Bergen Research Foundation. We thank Lena F Hansen, Lene M Vikebø, Michaela Popa, Maren Boge, Kjetil Jacobsen, Jørn Skavland, Bjarte S Erikstein, Andre Sulen, Paulina Ruurs, Lasse Evensen, Marianne Enger, Randi Hovland, Harald Valen, Lars Helgeland, Edith Fick, Line Bjørge, Liv Cecilie Vestrheim and Reidar Myklebust for discussion, expert advice and technical assistance. The optical imaging and transmission electron microscopy was performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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McCormack, E., Haaland, I., Venås, G. et al. Synergistic induction of p53 mediated apoptosis by valproic acid and nutlin-3 in acute myeloid leukemia. Leukemia 26, 910–917 (2012). https://doi.org/10.1038/leu.2011.315
- valproic acid
- in vivo imaging
- acute myeloid leukemia
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