Background
Acute myeloid leukemia (AML) is a serious disease of the hematopoietic system characterized by de-differentiation and uncontrolled proliferation of immature hematopoietic precursor cells in the bone marrow [
1]. Leukemia results from the accumulation of genetic and epigenetic alterations during the multistep process of tumorigenesis, including activation of oncogenes and/or inactivation of tumor suppressor genes. Many transcription factors have been shown to play an important role in aggressive hematological tumors. Within the past decade evidence that post-translational modification of proteins, including phosphorylation [
2], acetylation [
3], ubiquitination [
4] and SUMOylation [
5,
6], plays a critical role in the initiation, progression and maturation of AML has accumulated.
Positive regulatory domain I-binding factor 1 and retinoblastoma-interacting zinc finger protein-1 (PRDM16) is a transcription factor [
7]. PRDM16 is also termed MEL1 because it shares 63 % sequence similarity with PRDM3/MECOM (MDS1 and EVI1 complex) [
8]. PRDM16 encodes two isoforms: the full-length PRDM16 (or MEL1) and the short-form sPRDM16 (or MEL1S) [
9]. PRDM16, but not sPRDM16, contains a 134-amino acid PR domain, which is highly homologous to the SET domain, a structural hallmark of histone methyltransferases [
7,
10]. PRDM16 plays an important role in palatogenesis [
11], maintenance of hematopoietic [
12] and neuronal stem cells [
13], and adipose tissue differentiation [
14‐
17]. It acts as an H3K9me1 methyltransferase, which is required to maintain the integrity of mammalian heterochromatin [
7,
9,
10,
18]. PRDM16 is reported to contribute to translocation-induced leukemia [
7,
9,
10,
18]. However, only the PR domain-negative isoform of sPRDM16 is potentially oncogenic in leukemia [
10], and the underlying molecular mechanisms are largely unknown.
Small ubiquitin-related modifier (SUMO) is a highly conserved ubiquitin-like protein which acts as a reversible and highly dynamic post-translational modifier of a large number of proteins [
19,
20]. SUMOylation is catalyzed by a multistep enzymatic cascade including activating (E1), conjugating (E2), and ligating (E3) enzymes. SUMOylation alters the localization, activity or stability of target proteins [
19], and is reversed by a family of Sentrin/SUMO-specific proteases (SENPs).
Accumulating evidence has shown that SUMOylation plays a wide range of roles in the regulation of growth and development of all eukaryotes, for example, influencing transcriptional regulation and genome integrity. Deregulation of SUMOylation has been found in many human diseases including cancer, seizures and Alzheimer’s diseases [
21]. Recently, sPRDM16 was reported to be SUMOylated [
22], and we have found PRDM16 to also be SUMOylated (data not shown). However, the role of PRDM16 SUMOylation in progression of AML is unknown.
In this study, we have explored sPRDM16 SUMOylation in AML in vivo and in vitro. We found that SUMOylation of sPRDM16 regulated expression of genes during AML differentiation, and promoted AML progression while inhibiting differentiation of AML cells.
Methods
Plasmids and antibodies
The plasmid MSCV-PRDM16 was purchased from Addgene (Cambridge, MA, USA). Plasmid FLAG-sPRDM16 was generated from MSCV-PRDM16 by standard molecular cloning methods. Plasmids HA-SUMO1, HA-UBC9, RGS-SENP1 and RGS-SENP1-mutant were designed and developed as previously described [
23]. Anti-Flag antibody was obtained from Sigma (clone M2, St. Louis, MO, USA); anti-HA antibody was purchased from COVANCE (Beijng, China); anti-RGS antibody was obtained from QIAGEN (Germantonw, MD, USA); and anti-SUMO1 and PRDM16 antibodies from Abcam (Cambridge, MA, USA).
Site-directed mutagenesis
The potential SUMOylation residues from lysine (K) to arginine (R) in sPRDM16 were mutated using the QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).
FLAG-sPRDM16-K568R was generated with the following primers:
Sense 5′-TTGCTGGTCAGGGCTGAGCCA -3′ and Antisense 5′-TGGCTCAGCCCTGACCAGCAA-3′.
Cell culture
HEK293T cells were cultured in DMEM (Hyclone) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Gibco). THP-1 cells were maintained at 37 °C with 5 %CO2 in RPMI-1640 Medium (Hyclone) supplemented with 0.05 mM 2-mercaptoethanol (Sigma) and 10 % fetal bovine serum (Gibco). Plasmids were transiently transfected into HEK293T cells using LipofectamineTM2000 (Invitrogen) according to manufacturer’s instructions.
Evaluation of cell adherence (morphological differentiation)
Differentiation of THP-1 cells to macrophage-like cells was assessed by measureing adherence to plastic cell culture wells. Log phase cells were centrifuged and resuspended at at 1 × l06 cell/ml in fresh RPMI1640 complete medium containing 3 nM PMA and incubated for 24 h. Nonadherent cells were collected from the supernatant after washing, then adherent cells were separated gently by cell scraper on ice. The adherent and non-adherent cells were counted, and the sum correlated well with the original number of cells plated. To evaluate of differentiation, control and treated cells were removed from the Petri dishes, pelleted by centrifugation, and resuspended in 1 ml fresh medium.
Western blot
Total protein was extracted from cells or tissues using radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 1 mM EDTA) with 1 % protease inhibitor cocktail. Equal amounts of protein extracts (40 μg) were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. Membranes were blocked with 5 % w/v non-fat dry milk dissolved in Tris-buffered saline plus Tween-20 (TBS-T; 0.1 % Tween-20; pH 8.3) at room temperature for 1 h, and incubated with primary antibodies at 4 °C overnight. After washing with TBS-T, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h at room temperature. Immunobands were visualized using enhanced chemiluminescence (ECL) kit (GE Healthcare, Waukesha, WI, USA) according to manufacturer’s instructions and exposed to X-ray films.
Immunoprecipitation
Cells were collected at 48 h after transfection and lysed using an ice-cold RIPA buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 1 mM EDTA) with 10 mM N-ethylmaleimide, 1 mM PMSF and protease inhibitors (Roche). Cell lysis was performed on ice for 20 min and the cell lysate was sonicated for 5 s three times. After centrifugation at 14,000 g for 10 min at 4 °C, the supernatants were added to the appropriate antibody coupled to 20 μl of anti-FLAG M2 agarose beads (Sigma). The bead suspension was incubated at 4 °C for 2 h on a rotating shaker. Beads were then washed 5 times with RIPA buffer, mixed with 15 μL 2× SDS sample buffer and boiled at 100 °C for 5 min. The samples were subjected to Western blot analysis.
Lentiviral transduction
To obtain lentivirus particles, 5 × 10
5 HEK293T packaging cells were plated in 60-mm culture dishes and transiently transfected with 2 μg of each lentivirus vector mixture (pCDH-sPRDM16 1 μg, psPAX2 0.75 μg, pMD2.G 0.25 μg) together with 5 μl Lipofectamine 2000 (Invitrogen). Supernatant containing lentivirus was collected 36 h after transfection, filtered using 0.45-mm filters and used immediately for infection. Logarithmically growing THP-1 cells were transduced with lentivirus as previously described [
24].
Cell proliferation assays
To assess cellular proliferation, cells were seeded in 24-well plates (2 × 105 cells in 1 ml medium per well) and counted each day using a hematocytometer and trypan blue staining to exclude dead cells. Colorimetric proliferation assays were performed in 96-well plates as 8-fold measurements. A WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay (Dojindo, Shanghai, China) was conducted using 2000 cells in 100 μl medium per well in 96-well plates. The cell counting kit-8 (CCK-8) assay was performed according to the manufacturers’ recommendations.
A soft agar suspension (0.35 % agar) containing colony-forming cells was plated over a soft agar underlay (0.6 % agar). THP-1 cell complete culture medium was added and changed twice a week. After 1000 cells were embedded in the soft agar in 6-well plates (end concentration 0.35 % agar in complete medium) and grown over 12 days, colony numbers were counted under microscope.
Flow cytometry
For flow cytometry, cells were fixed with 4 % paraformaldehyde in PBS for 10 min and then blocked with 1 % BSA in PBS for 10 min at room temperature. Fixed, blocked cells were incubated with APC-conjugated anti-CD11b monoclonal antibody (BD PharMingen) for 15 min on ice, and cells were washed 3 times with 1 % BSA in PBS. Staining was analyzed using a FACS Calibur instrument (BD PharMingen).
Animal systemic leukemia models
NOD.CB17-Prkdc
scid/J (NOD/SCID) mice were purchased from Shanghai SLAC laboratory Animal Co., Ltd. (Shanghai, China). Animals were maintained at the animal facility of Shanghai Jiao Tong University School of Medicine in accordance with the local regulations and handled under sterile conditions. The study protocol was approved by the Review Committee for the Use of Human or Animal Subjects of Shanghai Jiao Tong University School of Medicine. Transplantations were performed by intravenous injections of six to eight week old mice. THP-1 cells (1 × 10
7 cells per mouse in 200 ml saline vehicle) were injected into the tail vein to create the leukemia models 5 h after total body irradiation with 1.5 Gy using a 137Cs source to enhance angiogenic potential [
25]. After transplantation, the mice were monitored for leukemia symptoms, such as weight loss, hunch-back, and decreased activity. All procedures were carried in accordance with national and international laws and policies.
MRNA-sequencing and data analysis
RNAs from the THP-1 cell line with stable expression of sPRDM16-WT or sPRDM16–K568R or a mock-transfected cell line were purified using TrizolTM method and subsequently cleaned using RNAeasy Kit (Qiagen). The polyadenylated RNAs purified from the cells were used for the construction of a sequencing library using the ScriptSeq Complete Gold Kit (Epicentre, Illumina). Cluster generation and sequencing were carried out using standard procedures in Hi-Seq 2500 Illumina platform. We used a single-end sequencing protocol to generate a 50 nt read at each end. RNA-seq reads were aligned to the human genome using TopHat (Johns Hopkins University, Baltimore, MD, USA). Cufflinks was employed to normalize Data and perform relevant comparisons among the different samples. Gene ontology analysis was performed using DAVID GO analysis software to search for enriched pathways.
Quantitative real-time PCR analysis
Total RNA was extracted by Trizol kit (Invitrogen) and treated with DNase (Promega). Complementary DNA was reverse transcribed using M-MLV reverse transcriptase and random hexamers according to the manufacturer’s protocol (Takara). All experiments were performed with Power SYBR® Green PCR Master Mix (Applied Biosystems) using the LightCycler® 480 Real-Time PCR System (Roche). PCR was carried out in triplicate and standard deviations representing experimental errors were calculated. Differences in cDNA input were normalized to GAPDH. All data were analyzed by the LightCycler® 480 software (Roche). The following PCR primers were used:
hKLF10-forward-5′-ACTGCGGAGGAAAGAATGGA-3′, hKLF10-reverse-5′-CTGGGAGGAGTGCTGGGAAC-3′; hCCL5-forward-5′-GCTGTCATCCTCATTGCTAC-3′, hCCL5-reverse-5′-CATTTCTTCTCTGGGTTGGC-3′; IL6R-forward-5′-TGCCAGTATTCCCAGGAGTC-3′, IL6R-reverse-5′-GGCAGTGACTGTGATGTTGG-3′; hLIF-forward-5′-ACAGAGCCTTTGCGTGAAAC-3′, hLIF-reverse-5′-TGGTCCACACCAGCAGATAA-3′; hNUMB-forward-5′-CGATGACCAAACCAGTGACAG-3′, hNUMB-reverse-5′-AGAGGGAGTACGTCTATGACCG-3′; hBCL3-forward-5′-ACTGCCTTTGTACCCCACTC-3′, hBCL3-reverse-5′-GGTATAGGGGTGTAGGCAGGT-3′; hHDAC9-forward-5′-GGATCAAAGCTCTCCACCCC-3′, hHDAC9-reverse-5′-TGGGCTCAGAGGCAGTTTTT-3′.
GAPDH-forward-5′-AGAAGGCTGGGGCTCATTTG-3′, GAPHD-reverse-5′-AGGGGCCATCCACAGTCTTC-3′.
Results were expressed as relative expression, normalized to the internal control.
Statistical analysis
All data are presented as mean ± standard deviation (S.D.). Statistical analysis was performed using Student’s t-test and values of P ≤ 0.05 were considered statistically significant.
Discussion
In this study, we demonstrated that sPRDM16 promoted the proliferation and enhanced the self-renewal capacity, of THP-1, while inhibiting differentiation of these AML cells. We further confirmed that K568 is a bona fide sPRDM16 SUMOylation site. Accordingly, mutation of sPRDM16 SUMOylation site K568 partially abolished the influence of sPRDM16 on proliferation and differentiation of AML in vitro and in vivo. Furthermore, THP-1 cells overexpressing sPRDM16-K568R mutant displayed distinct a gene expression profile from wild type sPRDM16 following incubation with PMA. Our findings suggest that K568 SUMOylation plays an important role in the pathogenesis of AML.
PRDM16 has previously been reported to be involved in myeloid and lymphoid malignancies, and to play a role in the regulation of hematopoietic [
12], neuronal stem cell growth [
13], and differentiation of adipose tissue [
14,
17,
26]. It is widely believed that sPRDM16 is an oncogene [
27,
28]. Further, it is well documented that sPRDM16 promotes AML progression by regulating gene transcription through direct DNA binding and/or interaction with transcriptional co-factors and chromatin modifiers [
29]. Accumulating clinical evidence indicates that deregulation of sPRDM16 is closely associated with abnormal AML phenotypes [
9,
10,
28,
30], implicating sPRDM16 in AML pathogenesis. Consistent with these findings, we found that sPRDM16 promoted proliferation and enhanced self-renewal capacity, while inhibiting differentiation of THP-1 AML cells.
In addition, increasing evidence suggest that SUMOylation may have a major role in the evolution of the hematopoietic system and AML. Some studies have indicated that SUMO is an integral component of chromatin and regulates specific transcriptional programs [
31]. Recently, Guillaume Bossis
et al. reported that an important role of SUMOylation is to regulate the expression of specific genes involved in AML cell response to chemotherapeutic drugs. and inhibition of the SUMO pathway reduces AML cell growth in xenograft mice [
6]. The transcriptional activity of sPRDM3, a member of the PR domain family, is negatively regulated by SUMO1 in acute promyelocytic leukemia (APL) [
32]. Using various functional assays to compare overexpression of PRDM16-WT or sPRDM16-K568R in THP-1 cells, we demonstrated that sPRDM16 SUMOylation contributed to progression of AML. Furthermore, a SUMOylation mutant of sPRDM16 attenuated its ability to facilitate tumor growth and suppress the differentiation of THP-1 cells
in vitro. Animal systemic leukemia transplantation models further indicated that sPRDM16 SUMOylation may be a risk factor for leukemia
in vivo.
Despite the established role of sPRDM16 in leukemia development, the molecular mechanisms underlying sPRDM16 SUMOylation-mediated progression of AML remain elusive. In particular, very few downstream target genes of sPRDM16 have been identified. Our analysis of stable THP-1 cell lines generated by polyclonal lentiviral infection with sPRDM16–WT or sPRDM16–K568R revealed a distinct gene expression profile. mRNA-sequence data indicated no significant difference in gene expression between sPRDM16-WT and sPRDM16-K568R-THP-1 cells in the absence of PMA. Interestingly, following induction of differentiation by PMA, we found that 237 genes were differently expressed between sPRDM16–WT and sPRDM16–K568R-THP-1 cells. Consequently, we confirmed that KLF10, BCL3, HDAC9, CCL5, IL6R, LIF and NUMB, all of which are closely related to differentiation in hematopoietic and leukemic cells [
33‐
37], are downstream targets of sPRDM16, and are influenced by SUMOylation. KLF10 is a transcription factor that regulates differentiation of bone marrow-derived macrophages [
33,
38]. BCL3 plays a critical role in targeting the differentiation of myeloid progenitors [
34]. The chemokine CCL5 induces selective migration of monocytes and drives their differentiation [
36]. Numb plays critical roles in cell fate determination as an evolutionary conserved protein [
37,
39]. These results indicate that SUMOylation of sPRDM16 is an important mechanism by which sPRDM16 promotes the growth and proliferation of hematopoietic progenitors and leukemic cells.
Acknowledgments
This work was funded by grants from National Natural Science Foundation of China (NSFC 39870046, 81270605, 30971066, 81470324), Third Military Medical University Clinical and Science Great Fund Project (2012XLC03), Chongqing Postgraduate Education Reform Project (yjg123114), Chongqing Natural Science Fund Project (CSTC’2008BA5001), The Military Emphasis Medical Scientific Research Project Fund (BWS13C018) and Third Military Medical University Education Reform Project.
Competing interest
We have no conflict of interest to declare.
Authors’ contributions
SD carried out all the molecular biology, biochemistry and animal studies, participated in the sequence alignment, performed the statistical analysis and drafted the manuscript. JC conceived of the study, participated in the design and coordination, and helped to draft the manuscript. Both authors read and approved the final manuscript.