Background
Acute lymphoblastic leukemia (ALL) is a heterogeneous group of malignant disorders derived from B- or T-cell lymphoid progenitor cells. ALL is ranked as the fifth most common childhood cancer and accounts for a large proportion of cancer-associated deaths in children every year [
1]. Over the past 50 years, advances in chemotherapy regimens have increased the cure rate for children with newly diagnosed ALL in the developed world to approximately 85% [
1]. However, the remaining approximately 15% of children with ALL are not expected to survive because of relapse [
2]. The problems of relapse, morbidity, and mortality are even more pronounced in adult patients with ALL. Novel treatments are desperately needed in order to improve survival in patients with ALL that is refractory to treatment or relapses after an initial response.
ALL has been shown to be associated with genetic and epigenetic alterations [
3], and progress in elucidating the pathogenesis of ALL has revealed a large number of potential targets for anticancer therapy. For example, the discovery that Bcr-Abl is expressed in approximately 30% of cases of ALL in adults has been successfully translated into treatment with small molecule tyrosine kinase inhibitors (e.g., imatinib and bosutinib) [
4]. The ETV6-RUNX1 fusion gene is found in approximately 25% of cases of ALL in children [
5]. Chatterton et al. reported that 325 genes were hypermethylated and downregulated and 45 genes were hypomethylated and upregulated in pediatric B-cell ALL [
6]. Epigenetic alteration indicates that targeted therapy against ALL is promising. Excitingly, vorinostat, a pan-histone deacetylase inhibitor, and more recently romidepsin, a bicyclic pan-histone deacetylase inhibitor, have been approved by the US Food and Drug Administration for treatment of relapsed or refractory cutaneous T-cell lymphoma [
7].
Reversible protein acetylation is an important posttranslational modification that regulates the function of histones and many other proteins [
8]. Histone acetylation is mediated by histone acetyl transferases (e.g., p300, CBP, and p/CAF in mammalian cells), while acetyl groups are removed by histone deacetylases [
9]. Recently, the histone deacetylase sirtuin 1 (SIRT1) has been shown to be important in leukemia. Sirtuin 1 (SIRT1) is a stress-response and chromatin-silencing factor belonging to the class III histone deacetylases family, which is involved in various nuclear events such as transcription, DNA replication, and DNA repair [
10]. SIRT1 has been shown to inhibit the maturation of preadipocytes [
11] and promote resistance to conventional chemotherapeutic agents [
12,
13]. Additionally, mammalian SIRT1 is a key regulator of cancer cell survival in the face of cellular stresses. SIRT1 and other sirtuins were found to regulate cell survival during stress through deacetylation of key cell cycle and apoptosis regulatory proteins, including p53 [
14,
15], Ku70 [
16], and forkhead transcription factors [
10]. Of importance, SIRT1 is highly overexpressed in several types of tumors [
17]. Recently, SIRT1 has been demonstrated to promote Bcr-Abl-driven leukemogenesis and the survival of chronic myelogenous leukemia stem cells [
18,
19].
In the present study, we initially discovered that SIRT1 level was higher in primary ALL cells than in control cells. We then hypothesized that inhibition of SIRT1 by its specific small molecule inhibitor Tenovin-6 induces apoptosis in ALL cells by releasing the expression of tumor suppressor genes such as p53. We tested this hypothesis in ALL cell lines (REH and NALM-6) and in primary cells from 41 children with ALL and 2 adult patients with ALL. Our findings suggest that Tenovin-6 may be a promising agent for ALL therapy.
Methods
Reagents
Tenovin-6 was purchased from Cayman Chemical (Ann Arbor, MI). Antibodies against SIRT1 (H-300), p53 (DO-7), cyclin D1 (C-20), Mcl-1 (S-19), and proliferating cell nuclear antigen (PCNA) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against PARP (clone 4C10-5), caspase-3, XIAP, and anti-CD19 conjugated with phycoerythrin were from BD Biosciences (San Jose, CA). Antibodies against K382-acetyl-p53 and c-Myc were from Cell Signaling Technology (Beverly, MA). Anti-SIRT2 was purchased from Atlas Antibodies. The CD133 MicroBead Kit including anti-CD133 conjugated with APC was from Miltenyi Biotec, Inc. (Shanghai, China). Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies were from Pierce Biotechnology (Rockford, IL).
Cell culture
REH and NALM-6 cells from American Type Culture Collection (Rockville, MD) were cultured in RPMI 1640 (Invitrogen, Shanghai) supplemented with fetal calf serum (FCS; Kibbutz Beit, Haemek, Israel) and 100 units/mL penicillin and streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2.
Primary cells from patients with ALL
Peripheral blood or bone marrow samples from 43 patients with ALL (Children with ALL, 41 cases; Adult patients with ALL, 2 cases), acute myelogenous leukemia (AML; 4 cases), Lymphoma (1 case), and 5 healthy adult donors were obtained from the Sun Yat-sen Memorial Hospital of Sun Yat-sen University and Guangdong Provincial People’s Hospital. This study was approved by the Sun Yat-sen University Ethics Committee according to institutional guidelines and the Declaration of Helsinki principles, and written informed consent to participate in this research and written informed consent to publish the resultant results were obtained from all the patients involved or their legal guardians for children under the age of 16. The clinical information for the 48 patients is in Table
1.
Table 1
Clinical characteristic of patients with leukemia
1 | 11/M | 216.45 | ALL-L2, B | BCR/ABL (+, 72%) | Initial | 5.44 |
2 | 4/M | 1.09 | ALL-L2, B | Neg | Initial | 14.65 |
3 | 3/F | 4.27 | ALL-L2, B | Neg | Initial | 8.05 |
4 | 10.6/M | 17.66 | ALL-L2, B | Neg | Initial | 4.7 |
5 | 0.6/M | 23.16 | ALL-L2, B | Neg | Initial | 2.72 |
6 | 10/F | 531 | ALL-L2, T | Neg | Initial | 7.49 |
7 | 2.3/F | 14.97 | ALL-L2, B | MLL (+, 82%) | Initial | 7.15 |
8 | 10/M | 22.98 | ALL-L2, T | Neg | Initial | 10.49 |
9 | 10.6/M | 17.66 | ALL-L2, B | Neg | Initial | 2.03 |
10 | 2.4/F | 24 | ALL-L2, B | Neg | Relapsed | 5.9 |
11 | 10/M | 43.8 | ALL-T | Neg | Relapsed | 4.5 |
12 | 1.9/M | 44.2 | ALL-L2, B | Neg | Initial | 3.03 |
13 | 3.5/F | 5.2 | AML-M0 | Neg | Initial | 8.15 |
14 | 1.6/F | 5.1 | AML-M7 | Neg | Initial | 3.08 |
15 | 3/M | 4.27 | ALL-L2, B | Neg | Initial | 2.88 |
16 | 2/M | 58.9 | ALL-L1, B | unknown | Initial | 4.03 |
17 | 13/M | 5.66 | ALL-L2, B | Neg | Relapsed | 6.98 |
18 | 9.5/M | 10.59 | ALL-L2, B | Neg | Initial | 7.21 |
19 | 0.7/M | 43 | ALL-L2, B | Neg | Initial | 13.82 |
20 | 7/M | 6.55 | ALL-L2, B | Neg | Relapsed | 17 |
21 | 0.2/M | 54.83 | ALL-L2, B | MLL (+, 86%) | Initial | 4.83 |
22 | 0.5/M | 208.85 | ALL-L2, B | Neg | Initial | 3.8 |
23 | 0.8/F | 137.41 | ALL-L2, B | MLL (+, 96%) | Initial | 7.24 |
24 | 11/M | 24.06 | ALL-L2, B | Neg | Initial | 3.91 |
25 | 11/M | 56.8 | ALL, mature B | Neg | Initial | 4.26 |
26 | 2/F | 281.31 | ALL-L1, T | Neg | Initial | 4.13 |
27 | 10/F | 34.7 | AML-M3b | PML-RARa (+, 35%) | Initial | 7.83 |
28 | 3/F | 8.07 | ALL-L2, B | TEL/AML1 (+, 85%) | Initial | 3.58 |
29 | 14.2/M | 104 | ALL-L2, B | BCR/ABL (+, 82%) | Initial | 3.75 |
30 | 12/M | 384 | ALL-L2, B | Neg | Initial | 8.16 |
31 | 4/M | 2.01 | ALL-L2, B | TEL/AML (+, 75%) | Initial | 6.15 |
32 | 12/M | 189 | ALL-L2, T | Neg | Initial | 7.07 |
33 | 1.5/F | 4.7 | ALL-L2, B | Neg | Initial | 4.74 |
34 | 2/F | 26.7 | ALL | Neg | Initial | 5.32 |
35 | 9/F | 3.8 | ALL-L2, B | Neg | Initial | 12.48 |
36 | 4.2/M | 17.4 | ALL-L2, B | Neg | Initial | 6.2 |
37 | 6/F | 52.53 | ALL-L2, B | TEL/AML (+) | Initial | 3.35 |
38 | 8/M | 8.95 | ALL-T | Neg | Initial | 4.31 |
39 | 8/M | 34.3 | ALL-L2, B | Neg | Initial | 4.03 |
40 | 5/M | 26.46 | ALL-L2, B | Neg | Initial | 12.56 |
41 | 9/M | 3.29 | ALL-L2, B | Neg | Initial | 10.2 |
42 | 1/M | 21.6 | AML | Neg | Initial | 14.75 |
43 | 12/M | 82.79 | ALL-L1, T | Neg | Initial | 6.65 |
44 | 4/M | 6.62 | Lymphoma | Unknown | Initial | 4.81 |
45 | 0.8/F | 137.41 | ALL-L2 | MLL (+, 96%) | Initial | 13.22 |
46 | 11/M | 24.06 | ALL-L2 | Neg | Initial | 16.38 |
47 | 55/F | 62.75 | ALL | Neg | Initial | ND |
48 | 28/F | 2.65 | ALL | Neg | Initial | ND |
Mononuclear cells were isolated by Histopaque gradient centrifugation (density 1.077; Sigma-Aldrich, Shanghai) [
20-
22]. Contaminating red cells were removed by incubation in 0.8% ammonium chloride solution for 10 min. After a washing, cells were suspended in RPMI 1640 medium supplemented with 10% FCS. All drug treatments started after the cells were precultured in fresh medium for 24 hours.
For separation of stem/progenitor cells of ALL, the mononuclear cells were mixed with MicroBeads conjugated to monoclonal anti-human CD133 antibodies (isotype: mouse IgG1, clone AC133) and loaded onto a MACS column with separator according to the instructions from Miltenyi Biotec Inc [
20]. After removing from the magnetic field, the magnetically retained CD133+ cells were eluted as the positively selected cell fraction. The purity was examined with a flow cytometer after staining of CD133-APC.
Cell viability assay
Cell viability was evaluated by MTS assay (CellTiter 96 AQueous One Solution reagent, Promega, Shanghai) as described previously [
20-
22]. The IC
50 was determined by curve fitting of the dose–response curve.
Soft agar clonogenic assay in ALL cell lines
ALL cell lines were treated with Tenovin-6 or diluent (DMSO, control) for 24 hours, washed with PBS, and seeded in Iscove's medium containing 0.3% agar and 20% FCS in the absence of drug treatment [
20-
22].
The colony-forming capacity of normal bone marrow cells and primary ALL blast cells was analyzed by use of methylcellulose medium (Methocult H4434, Stem Cell Technologies) according to the manufacturer's instructions. Tenovin-6 was added to the initial cultures at a concentration of 1 μM to 10 μM. After 14 days of culture, the number of colony-forming units was evaluated under an inverted microscope according to standard criteria [
20-
22].
Reverse transcription and quantitative real-time PCR
Total RNA from cultured cells was extracted using Trizol reagent (Invitrogen, Shanghai). Two micrograms of RNA was processed directly to cDNA by reverse transcription with SuperScript III following the manufacturer’s instructions (Invitrogen, Shanghai). PCR primers for each gene were designed using real-time PCR primer design; sequences used in this study were as follows: p53, forward 5’-GTGGAAGGAAATTTGCGTGT-3’, reverse 5’-TGGTGGTACAGTCAGAGCCA-3’; p21, forward 5’-GACTCTCAGGGTCGAAAACGG-3’, reverse 5’-GCGGATTAGGGCTTCCTCTT-3’; Noxa, forward 5’-GCAAGAACGCTCAACCGAG-3’, reverse 5’-TTGAAGGAGTCCCCTCATGC-3’; Puma,forward 5’-ACCTCAACGCACAGTACGAG-3’, reverse 5’-CGGGTGCAGGCACCTAATTG’; Bax, forward 5’-GAACCATCATGGGCTGGACA’, reverse 5’-GCGTCCCAAAGTAGGAGAGG’; c-myc, forward 5’-CAGCGACTCTGAGGAGGAAC-3’, reverse 5’-TCGGTTGTTGCTGATCTGTC-3’; cyclin-D1, forward 5’-GCTGTGCATCTACACCGACA-3’, reverse 5’-CCACTTGAGCTTGTTCACCA-3’; LEF1, forward 5’-CGAATGTCGTTGCTGAGTGT-3’, reverse 5’-GCTGTCTTTCTTTCCGTGCT-3’; 18 s, forward 5’-AAACGGCTACCACATCCAAG-3’, reverse 5’-CCTCCAATGGATCCTCGTTA-3’. We used SYBR Premix Ex Taq (Perfect Real-time; Takara Bio) for qRT-PCR with Applied Biosystems 7500 Real-time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The specificity of PCR products was checked on agarose gel. Expression levels of 18S rRNA were used as an endogenous reference.
Western blotting analysis
Whole cell lysates prepared in RIPA (radioimmunoprecipitation) assay buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethanesulfonyl fluoride, 20 mM sodium fluoride, 0.2 mM sodium orthovanadate, and Complete Protease Inhibitor Mix, one tablet per 50 ml) [
20-
22]. Cytoplasmic and nuclear fractions were prepared as described previously [
20-
22]. Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membranes, which were then incubated with the primary antibodies. After incubation with appropriate secondary antibodies, the immunoblots were developed using SuperSignal Western blotting kits (Pierce Biotechnology) and exposed to X-ray film according to the manufacturer’s protocol. Western blots were stripped between hybridizations with stripping buffer [10 mM Tris–HCl (pH 2.3) and 150 mM NaCl].
Flow cytometry analysis of cell cycle
After drug treatment, cells were collected and fixed overnight in 66% cold ethanol at −20°C. The cells were then washed twice in cold PBS and labeled with propidium iodide for 1 hour in the dark. Cell cycle distribution was determined by use of a FACSCalibur flow cytometer with CellQuest software [
20-
22].
Measurement of apoptosis
Apoptosis was evaluated with an AnnexinV-fluoroisothiocyanate apoptosis detection kit according to the instructions of the manufacturer (Sigma-Aldrich, Shanghai) and analyzed with use of a FACSCalibur flow cytometer and CellQuest software as previously described [
20-
22].
Electrophoretic mobility shift assay
The WT-TCF probe was prepared by annealing 5’-TGCCGGGCTTTGATCTTTG-3’ and 5’-AGCAAAGATCAAAGCCCGG-3’ deoxyoligonucleotides [
23]. Double-stranded probes were end-labeled using biotin. EMSA was performed with use of the Light Shift Chemiluminescent EMSA kit (Pierce Biotechnology) according to the manufacturer's instructions [
20].
Statistical analysis
Data from all the experiments are expressed as mean ± 95% CI unless otherwise stated. GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA) was used for statistical analysis. Comparisons among multiple groups involved one-way ANOVA with post-hoc intergroup comparison with the Tukey test. P < 0.05 was considered statistically significant.
Discussion
Novel targeted therapy for ALL is desperately needed. In the present study, we showed that Tenovin-6, an inhibitor of the class III histone deacetylase sirtuin, was effective as a single agent and in combination with frontline chemotherapeutics against ALL cells. Tenovin-6 treatment activated p53 and induced cell growth inhibition and apoptosis in ALL cell lines and primary ALL cells. Furthermore, we found that Tenovin-6-induced inhibition of SIRT1/2 activity decreased Wnt/β-catenin signaling and eliminated ALL stem/progenitor cells.
SIRT1 deacetylates histone and nonhistone proteins that are involved in many cellular functions. Although the role of SIRT1 in tumorigenesis remains controversial [
30-
33], SIRT1 expression was shown to be significantly elevated in a number of human cancers, including acute myeloid leukemia [
34], prostate cancer [
35], colorectal cancer [
36], skin squamous cell carcinoma [
37], chemoresistant leukemia [
38], and CD133-positive glioblastoma stem cells [
39]. In accord with these findings, our results showed that the expression of SIRT1 was elevated in primary ALL cells compared with control. Of note, SIRT1 has been demonstrated to promote the development of chronic myelogenous leukemia [
18,
19].
A number of nonspecific and specific inhibitors of SIRT1 have been discovered, including nicotinamide, sirtinol, splitomicin, HR73, cambinol [
32], the tenovins [
24], and the indole derivative EX527. Two of these inhibitors, cambinol [
32] and tenovin [
24], were tested in animal models of cancer and showed great antitumor effect against Burkitt lymphoma and melanoma, respectively. In an
in vitro peptide deacetylase activity assay, Tenovin-6 was shown to inhibit the activity of SIRT1 and SIRT2 with IC
50 values of 21 μM and 10 μM, respectively. Our results in the current study demonstrated that treatment of ALL cells with Tenovin-6 at even 1 μM led to hyperacetylation and activation of p53 within approximately 2 to 6 hours.
Results of the present study indicated that Tenovin-6 treatment inhibits growth and induces apoptosis both in ALL cell lines and in primary ALL cells at micromolar concentrations, however, many ALL cells were sensitive to the agents, while several cells were resistant (Figure
3C). We assume that the sensitivity correlates with the p53 mutation status or with the SIRT1/2 expressions, this remains to be further investigated. Of importance, Tenovin-6 is synergistic with the conventional chemotherapeutic agents etoposide and cytarabine and also active against primary cells from patients with relapsed ALL. Tenovin-6 disturbed the cell cycle distribution in ALL cells by restricting the cells in G
1 phase. The inhibitory effect of Tenovin-6 on cell growth and survival may be explained by the activation of p53 and elevation of p21 after Tenovin-6 treatment.
Cancer stem cells are resistant to chemotherapy and believed to be the source of relapse of tumor. Using phenotypically defined stem/progenitor cells and functional assay, we first showed that Tenovin-6-induced inhibition of SIRT1/2 eliminated ALL stem/progenitor cells. The CD133 + CD19- fraction in ALL cells represents the stem/progenitor cells of ALL. We then found that Tenovin-6 induced marked apoptosis in ALL stem/progenitor cells. Furthermore, Tenovin-6 significantly inhibited the colony-forming capacity of ALL cells (Figure
3D & E).
Tenovin-6-mediated decrease in β-catenin, a key regulator of self-renewal of cancer stem cells may be involved in the elimination of ALL stem/progenitor cells. Our data demonstrated that Tenovin-6 remarkably lowered the levels of total and active β-catenin and blocked the downstream signaling. The underlying mechanism may be associated with Tenovin-6-induced Dvl inhibition and p53 activation. SIRT1 can form a complex with β-catenin and Dvl [
26]. Tenovin-6-induced Dvl inhibition is postulated to reduce the stability of the complex. p53 can negatively regulate β-catenin level [
40]. Activation of p53 by Tenovin-6 may thus reasonably explain the decrease in β-catenin.
In summary, we found that the novel sirtuin inhibitor Tenovin-6 is effective in killing pre-B ALL cells and eradicating ALL stem/progenitor cells (CD133 + CD19-). Tenovin-6 may represent an important therapeutic agent against pre-B ALL alone or in combination with standard chemotherapeutics and is therefore worthy of further clinical investigation in ALL.
Conclusions
In the present study, we initially found that the level of SIRT1, a class III histone deacetylase, was higher in primary ALL cells from patients than in peripheral blood mononuclear cells from healthy individuals. we found that the novel sirtuin inhibitor Tenovin-6 is effective in killing pre-B ALL cells and eradicating ALL stem/progenitor cells (CD133 + CD19-). Tenovin-6 may represent an important therapeutic agent against pre-B ALL alone or in combination with standard chemotherapeutics and is therefore worthy of further clinical investigation in ALL.
Acknowledgements
This study was supported by grants from the National Basic Research Program of China (973 Program grant no. 2009CB825506 to J. Pan), National Natural Science Funds (no. 81025021, no. 81373434, no. 91213304, no. 90713036, and U1301226 to J. Pan), the Research Foundation of Education Bureau of Guangdong Province, China (Grant cxzd1103 to J. Pan), and the Fundamental Research Funds for the Central Universities (to J. Pan).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YJ designed and performed experiments and wrote the manuscript; QC performed experiments and wrote the manuscript; CC provided patient specimens and reviewed the data; XD provided patient specimens and reviewed the data; BJ performed experiments; and JP directed the study, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.