Oncogene (2015) 34, 3000–3010 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

Regulation of p21 by TWIST2 contributes to its tumor-suppressor function in human acute myeloid leukemia X Zhang1,2,6, W Ma1,2,6, J Cui1,3,6,7, H Yao1,3, H Zhou3, Y Ge1, L Xiao1, X Hu3, B-H Liu4, J Yang4, Y-Y Li4, S Chen2,3, CJ Eaves5, D Wu2,3,8 and Y Zhao1,2,8 TWIST2 has a dual function in tumors. Its implication in the initiation and metastasis of various solid tumors is well established, and its tumor-suppressor role in murine osteosarcoma cells has been reported recently. However, the function of TWIST2 and its underlying mechanisms in human normal and malignant hematopoiesis remain unclear. In the present study, we found that TWIST2 directly regulated p21 in human hematopoietic cells and whose silence promoted cell proliferation and cell cycle progression. Hypermethylation of TWIST2 occurred to 23 out of the 75 adult acute myeloid leukemia (AML) patients and resulted in the impaired expression of both TWIST2 and p21. Conversely, TWIST2 overexpression inhibited the growth of AML cells partially through its direct activation of p21 with intact HLH (helix-loop-helix) domain. The microarray data and gene expression validation showed that TWIST2 was sufficient to activate known tumor-suppressor genes, whereas suppress known oncogenes, which further supported its inhibitory effect against AML cells. Taken together, our data have identified a novel TWIST2-p21 axis that modulates the cell cycle of both normal and leukemic cells and demonstrated that the direct regulation of p21 by TWIST2 has a role in its tumor-suppressor function in AML. Oncogene (2015) 34, 3000–3010; doi:10.1038/onc.2014.241; published online 4 August 2014

INTRODUCTION Twist was firstly identified in Drosophila for its pivotal role in mesoderm specification,1 in which it could act together with Snail and Daughterless to govern a developmental network.2 It is believed that 425% transcriptional factors of Drosophila are directly regulated by Twist.3 In mammals, Twist family consists of two members, Twist1 and Twist2, which implicates in multiple biological processes, including the differentiation of muscle, bone and mesenchymal lineage.4–12 As both Twist1 and Twist2 make the cells resistant to tumor-initiation-induced apoptosis or senescence, they are classified as oncogenes.9,13,14 In addition, the critical role of TWIST proteins in promoting cancer cell growth, epithelial–mesenchymal transition and self-renewal has been reported in many documents.10–13,15 Nevertheless, Ishikawa et al.16 have reported that Twist2 is a tumor suppressor in murine osteosarcoma cells possibly through its induction of the expression of fibulin-5 (Fbln5, a known tumor suppressor). Moreover, Twist2 inhibits the expression of matrix metalloproteinase 9, which is implicated in the tumor invasion.16 Thus Twist2 has a dual function in malignant transformation. In line with this, the aberrant DNA methylation and gene expression of TWIST2 have been reported in human acute lymphoid leukemia (ALL) bearing RUNX1-ETV6, which results in the inactivation of TWIST2. The reexpression of TWIST2 inhibits the growth of ALL cells in vitro, though the molecular mechanisms remain unclear.17 Sharabi

et al.18 have reported that Twist-2 is exclusively expressed in myeloid progenitors but not in lymphoid progenitors and Twist-2deficient mice manifest myeloproliferative disease. However, the expression and function of TWIST2 in normal and leukemic (acute myeloid leukemia (AML)) hematopoietic cells are still unknown. To address these questions, we first analyzed the proliferation and cell cycle upon TWIST2 silence in human CD34-enriched hematopoietic cells in this study. Next, we analyzed the methylation status of TWIST2 in human AML cells and its effect on TWIST2 expression. Finally, we investigated the effect of TWIST2 on the growth of AML cells in vitro and in vivo and how TWIST2 functioned. Our data have identified a novel TWIST2-p21 axis that regulates the cell cycle of both normal and leukemic hematopoietic cells, which implicates TWIST2 as a novel tumor suppressor in human AML. RESULTS TWIST2 modulates the growth and cell cycle of human hematopoietic cells Recently, Twist2 was identified as a negative regulator of murine myeloid progenitor cells;18 however, the function of TWIST2 in human hematopoietic cells remained unknown. Herein, we validated two independent shRNA sequences to silence TWIST2 in CD34-enriched cells (Figure 1a). The silence of TWIST2 in these

1 Cyrus Tang Hematology Center, Soochow University, Suzhou, People's Republic of China; 2Collaborative Innovation Center of Hematology, Soochow University, Suzhou, People's Republic of China; 3The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostasis, Suzhou, People's Republic of China; 4Shanghai Center for Bioinformation Technology, Shanghai, People's Republic of China and 5Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. Correspondence: Dr D Wu, The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostatis, Suzhou, Jiangsu, People's Republic of China. or Dr Y Zhao, Cyrus Tang Hematology Center, Soochow University, 199 Ren’ai Road, Suzhou, Jiangsu 215123, People's Republic of China. E-mail: [email protected] or [email protected] 6 These authors contributed equally to this work. 7 Current address: Shanxi Academy of Medical Science, Shanxi Da Yi Hospital, Taiyuan, Shanxi 030032, People's Republic of China. 8 These two authors are co-senior authors. Received 14 October 2013; revised 8 June 2014; accepted 15 June 2014; published online 4 August 2014

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Figure 1. TWIST2 modulates the growth and cell cycle of normal hematopoietic cells. (a) Lentiviral vectors were used to deliver two independent shRNA sequences to CD34-enriched human hematopoietic cells from healthy donors, and then the transcript and protein expression of TWIST2 were assessed in the control and shRNA-delivered cells. (b) The proliferation of control and TWIST2-silenced cells was compared in liquid culture media supplied with a cocktail of cytokines, including Flt-3 ligand, stem cell factor, interleukin (IL)-3, IL-6 and granulocyte colony-stimulating factor. (c) CFC production of the control and TWIST2-silenced cells were compared. BFU-E, burst-forming uniterythroid; CFU-GM, colony-forming unit-granulocyte/macrophage; Mix, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte. (d) The cell cycle status of control and TWIST2-silenced cells were analyzed. Data are shown as mean ± s.e.m. from at least three independent experiments. *Mean Po0.05 and **mean P o0.01, which were estimated with t-test in a two-tailed fashion.

cells promoted the cell proliferation and the production of CFC (colony-forming cell) (Figures 1b and c); in addition, it promoted the cell cycle progression (Figure 1d). Next, we found that TWIST2 silence was able to significantly suppress the expression of p21 and enhance the expression of CCND1 but unable to alter the expression of p27 (Figure 2a). Similar data were obtained with western blotting to analyze these key cell cycle regulators as well (Figure 2b). Moreover, we found that p53 was unchanged upon TWIST2 silence, which suggested that the alteration of p21 was p53 independent. Chromatin immunoprecipitation (ChIP) revealed that TWIST2 directly bound p21 in CD34+ cells but not CCND1. A piece of sequence (about 2 kb away from the regulatory sequence of p21) and the irrelevant nuclear protein ZFX (zinc finger protein, X-linked) were served as the negative controls (Figure 2c). Conversely, the overexpression of TWIST2 enhanced the expression of p21 and inhibited the CFC production of CD34+ cells. The silence of p21 enhanced the CFC production of CD34+ cells (Figure 2d) and partially rescued the repressed CFC production upon TWIST2 overexpression (Figure 2e). Of note, the rescue of CFC production by p21 silence was pronounced for colony-forming unit-granulocyte/macrophage but not burst-forming unit-erythroid. Frequent hypermethylation of TWIST2 in human AML The aberrant expression of TWIST2 due to hypermethylation has been reported in both human CLL and ALL;17,19 however, whether TWIST2 is deregulated in human AML patients remained unclear. To address this, the methylation status of TWIST2 was analyzed © 2015 Macmillan Publishers Limited

with methylation-specific PCR in several AML cell lines and cells from 75 adult AML patients. The methylation with various degrees was detected with COBRA (Combined Bisulfite Restriction Analysis) in five out of six AML cell lines, including THP-1, Dami, HL60, NB4 and OCI-AML5 (data not shown). The representative sequence data of methylation status in THP-1 and Dami cells are shown (Figure 3a). Then we detected the methylation status of primary cells from AML patients and healthy donors. Totally, 75 patients diagnosed as M0 to M6 with the FAB (French–American–British) classification and 6 healthy donors were recruited. The hypermethylation of TWIST2 was detected in 23 out of the 75 patients (~30%); however, no methylation was detected in the cells from the healthy donors (n = 6, Figure 3a, Supplementary Figure S1A). The clinical characteristics of these 75 AML patients based on their methylation status were summarized (Supplementary Table S1). The hypermethylation occurred in all subtypes of patients except M5; and it happened in most M2 patients (12 out of 16) but very few M3 patients (1 out of 17). Next, we assessed the gene expression of TWIST2 in some AML patients (n = 30) divided into the methylated and unmethylated groups. As shown in Figure 3b, the expression of TWIST2 in the methylated subset of AML patients (n = 13) was significantly lower than that of the unmethylated subset of leukemia patients (n = 17) or the healthy donors (n = 6). We also found that the expression of p21 was lower in the methylated group compared with the unmethylated group (Supplementary Figure S2). In addition, the methylation-specific PCR products of those 13 TWIST2 methylated AML patients were sequenced. Based on their methylation Oncogene (2015) 3000 – 3010

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Figure 2. TWIST2 directly regulates the expression of p21 in hematopoietic progenitor cells. (a) The expression of p21, CCND1 and p27 were assessed in control and TWIST2-silenced cells. (b) The protein expression of p53, p21, p27 and CyclinD1 were analyzed. (c) CD34+ normal hematopoietic cells were analyzed by ChIP with a specific TWIST2 antibody. The p21 promoter region analyzed in this study (−800 to +200 bp) contained seven E-box consensus binding sites, Seq#1 (−532 ~ − 380) and Seq#2 (−55 ~ 140) were designed for ChIP analysis, meanwhile Seq#3 (1992 ~ 2112) about 2 kb away from the regulatory region was used as a negative control. A pair of primers was also designed to analyze the regulatory region of CCND1 with ChIP. ZFX was served as an irrelevant nuclear protein control. (d, e) The CD34+ cells were infected by control and TWIST2 lentiviral vectors, and then they were transfected with scramble and p21-specific siRNA, respectively. The expression of p21 and the CFC production were assayed. BFU-E, burst-forming unit-erythroid; CFU-GM, colony-forming unit-granulocyte/macrophage; Mix, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte. Data are shown as mean ± s.e.m. *Mean P o0.05 and **mean Po0.01, which were estimated with t-test in a two-tailed fashion from at least three independent experiments.

contents, they were divided into three subgroups as low (o 33%), median (34–66%) and high (467%) methylated groups. We found that the expression of TWIST2 was conversely correlated with the content of methylation (Figure 3c). Finally, THP-1, NB4 and Dami cells were treated with 5-aza-2′-deoxycytidine, and the expression of TWIST2 was restored along with the Oncogene (2015) 3000 – 3010

removal of methylation modifications (Supplementary Figure S1B); moreover, the TWIST2 protein expression was also enhanced in THP-1 cells upon 5-aza-2′-deoxycytidine treatment (Supplementary Figures S1C and D). Collectively, these data demonstrated that the hypermethylation impaired the expression of TWIST2 in human AML. © 2015 Macmillan Publishers Limited

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Figure 3. TWIST2 is hypermethylated in human AML patients. (a) DNA samples were modified with bisulfite, and then a CG-enriched regulatory region of TWIST2 was amplified with methylation-specific PCR. Some PCR products were subcloned for sequence analysis; the methylation status of normal bone marrow, THP-1 cells, Dami cells and 3 AML samples was shown. Each row represented a randomly selected clone. The unmethylated CpG sites were marked as open circles and the methylated CpG sites as filled circles. (b) RNA extracts were prepared from normal adult bone marrow (n = 6) and AML patients (n = 30). The gene expression of TWIST2 was measured with quantitative RT–PCR and compared among the groups of normal bone marrow, methylated (n = 13) and unmethylated (n = 17) subsets of AML patients. (c) The methylation-specific PCR products of these 13 methylated samples were subcloned and sequenced to determine the content of methylation. They were divided into three subgroups (low, o33%; median, 34 ~ 66%; and high, 467%) according to their methylation content, and then the expression of TWIST2 was compared among these subgroups. Data are shown as mean ± s.e.m. P-value was estimated with t-test in a twotailed fashion.

Overexpression of TWIST2 inhibits the growth of AML cells We constructed and validated a lentiviral vector to deliver TWIST2 to AML cells (Supplementary Figures S3A to D). TWIST2 significantly suppressed the proliferation and CFC capacities of Dami and THP-1 cells (Figures 4a and b); similar results were obtained with other AML cell lines, including SHI-1, NB4, HL60 and HEL cells (Supplementary Figure S3E). Conversely, the silence of TWIST2 promoted the proliferation and enhanced the CFC production of both Dami and THP-1 cells (Supplementary Figure S4), though the extent of growth enhancement upon TWIST2 silence was not as evident as the inhibition induced by TWIST2 overexpression (Figures 4a and b). To assess the in vivo effect of TWIST2 on AML cells, the control and TWIST2-expressed THP-1 cells (1 × 107) were injected subcutaneously to the nude mice. TWIST2 protected the mice from tumor formation evidently compared with the control mice within 30 days postinjection (Figure 4c; Venus group, 11/15; TWIST2 group, 2/16). The green fluorescent protein signal of the tumors was readily detected, and the hematoxylin and eosin staining showed that leukemic cells were inside the tumors (data not shown). Similar in vivo results were obtained with Dami cells as well (Venus group, 7/7; TWIST2 group, 0/8; 2 × 106 cells/mouse). Finally, we delivered TWIST2 to primary cells from four AML patients, and the CFC production was inhibited by TWIST2 ranging from 45% to 86% (Figure 4d). These data supported the notion that TWIST2 had the anti-leukemia effect, which was in line with the data by Thathia et al.17 © 2015 Macmillan Publishers Limited

TWIST2 inhibits the growth of AML cells via its direct activation of p21 Previously, only Thathia’s group reported that TWIST2 inhibited the proliferation of leukemic cells; however, the molecular mechanism remained unresolved. To address this, we reasoned that as a bHLH (basic helix-loop-helix) protein TWIST could regulate the gene expression through the dimmer formation depending on HLH domain and the DNA binding with its basic region or the interaction with other transcriptional factors through its N-terminus or Twist-box in the C-terminus to modulate their transcriptional functions, such as p53 and nuclear factor-κB.20–22 To determine whether dimmer formation, DNA binding, N-terminus or Twist-box was required for the inhibitory effect of TWIST2, we constructed a HLH-deficient mutant of TWIST2 (F86P), a basic region defect mutant (b−), an N-terminus truncated mutant (ΔN) and a C-terminus truncated mutant (ΔC), which were verified with western blotting (Supplementary Figures S5A and B). We first found that F86P and b− mutants did not have the suppressive effect on the cell growth and CFC production as wild-type TWIST2, while ΔN and ΔC had similar inhibitory effects as the wild-type TWIST2 (Figures 5a and b). TWIST2 also inhibited the colonyformation ability in soft agar and the migration capacity of THP-1 cells; however, F86P and b− almost completely diminished these effects (Supplementary Figures S5C and D). Next, F86P and b− had similar tumorigenic capacity and tumor growth rate compared with the control vector (Figure 4c, Supplementary Figure S5E). Oncogene (2015) 3000 – 3010

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Figure 4. TWIST2 inhibits the growth of AML cells. (a, b) Cell proliferation in liquid culture and CFC in methylcellulose-based medium were compared between Venus (control) and TWIST2-transduced Dami and THP-1 cells. (c) Equal amount of Venus and TWIST2-transduced THP-1 cells (1 × 107) were injected subcutaneously into nude mice. A representative photograph of tumor growth of the Venus and TWIST2 expressed cells in nude mice is shown, and the fluorescence was monitored with IVIS II imaging system as well. (d) The Venus and TWIST2transduced cells from AML patients (n = 4) were analyzed with the CFC assay. Data are shown as mean ± s.e.m. *Mean P o0.05 and **mean Po0.01, which were determined with t-test in a two-tailed fashion.

Finally, F86P did not have the inhibitory effect as the wide-type TWIST2 against primary AML cells, while b− partially retained the inhibitory effect as the wide-type TWIST2 (Figure 5d). These data indicated that TWIST2 mainly exerted its inhibitory effect through the formation of HLH dimmer and the direct transcriptional regulation of target genes. As p21 and CCND1 had altered expression upon TWIST2 knockdown in the normal hematopoietic cells, we first assessed their expression in AML cells upon TWIST2 overexpression. TWIST2 elevated the expression of p21, whereas suppressed the expression of CCND1 in primary AML cells and THP-1 cells (Figures 6a and 7b). Accordingly, the altered protein expression of p21 and CyclinD1 upon TWIST2 expression was confirmed with THP-1 and Dami cells. Meanwhile, the decrease of p27 was not as evident as p21, and p53 was not changed upon TWIST2 (Figure 6b). In the ChIP analyses, p21 but not CCND1 was directly bound by TWIST2 (Figure 6c). However, F86P was not able to bind p21 (Supplementary Figure S6A), which was consistent with the proliferation and CFC production data. We also detected the increased transcription activity of p21 promoter in TWIST2expressed cells but not the F86P-expressed cells compared with the control cells (Supplementary Figure S6B). There are seven E-box consensus binding sites within the p21 regulatory sequence subcloned this study. To determine which E-box was critical for the activation upon TWIST2, the E-boxes were deleted sequentially to generate various reporter vectors (Δ1 ~ Δ6). The reporter assay indicated the importance of the E-box at − 156, which could be the putative TWIST2-binding site (Supplementary Figure S7). A consistent G0/G1 arrest and according decrease of G2/S/M phase cells were observed with Dami and THP-1 cells (Figure 6d, left panel); importantly, the similar cell cycle alteration was observed with three primary AML samples as well (Figure 6d, right panel). Oncogene (2015) 3000 – 3010

However, F86P mutant did not cause the cell cycle alteration with Dami, THP-1 and primary AML cells, which confirmed the importance of dimmer formation of TWIST2 to inhibit the growth of AML cells. Finally, the silence of p21 led to partial restoration of CFC production upon TWIST2 overexpression in THP-1 cells (Supplementary Figures S8A and B). Global gene expression data support the tumor-suppressor role of TWIST2 in leukemia Though the novel TWIST2-p21 axis has a critical role in the regulation of the proliferation and cell cycle of AML cells, it is still appealing to obtain detailed insights of TWIST2 acting as a novel tumor suppressor in leukemia. Herein, we collected the control and TWIST2-expressed THP-1 cells to generate global gene expression data (GSE57347). Totally, 662 differentially expressed genes were identified by the criteria that over two-fold expression alteration and a P-value o0.05 were considered as significant difference (Figure 7a and Supplementary Table S2). Initially, we found that the genes previously reported to be related with TWIST-mediated tumor initiation, metastasis and senescence suppression, such as YB-1, AKT2, BMI-1 and p16 were not altered upon TWIST2 overexpression.9,13,14,23–25 Conversely, a group of known tumor-suppressor genes were upregulated upon TWIST2 overexpression, including H19, BTG2, BIN1, KLF6 and DUSP5;26–31 and a group of oncogenes were downregulated upon TWIST2 overexpression, such as N-MYC, HOXB9, BCL11A and CCND1.32–37 Totally 16 genes were selected to perform quantitative reverse transcriptase–PCR (Q-RT–PCR) for the validation of microarray analysis, and 14 of those were agreeable with microarray data (Figure 7b). Five out of 14 those genes were further validated with Dami and NB4 cells (data not shown). The expression of CCND1, © 2015 Macmillan Publishers Limited

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Figure 5. The HLH domain is required for TWIST2 to inhibit the growth of AML cells. (a, b) Cell proliferation and CFC production of Venus (control), TWIST2, b−, F86P, ΔN and ΔC transduced THP-1 cells are shown. (c) 1 × 107 Venus, TWIST2, b− and F86P transduced cells were injected subcutaneously to nude mice, and then the formation of tumors was observed. The tumor-free survival of the mice within 1 month was analyzed with Kaplan–Merier method. (d) CFC production of Venus, TWIST2, b− and F86P transduced cells from two individual AML patients is shown. Data are shown as mean ± s.e.m. P-values for Venus, b− and F86P cells against TWIST2-expressed cells were estimated with t-test in a two-tailed fashion. *Mean P o0.05 and **mean P o0.01.

BIN1, DUSP5, DUSP6 and CDKN1A (p21) were analyzed with control and TWIST2-expressed primary AML cells as well (n = 3, Figures 6a and 7c), and the expression of BIN1, CCND1, DUSP5 and p21 were significantly different between the control and TWIST2-expressed cells. Conversely, p21 and BIN1 were downregulated and CCND1 was upregulated upon TWIST2 silence in normal hematopoietic cells (Figures 1a and 7d). Using THP-1 cells, we also demonstrated that the dimmer formation of TWIST2 was required for the altered expression upon TWIST2 (Supplementary Figure S6C). In addition, we performed the pathway enrichment analysis, and ‘AML’ pathway (hsa05221) was significantly enriched in these differentially expressed genes (Supplementary Table S3). Finally, we also found that 14 leukemia-related pathways were enriched by performing GSEA (gene set enrichment analysis), which are summarized in Supplementary Table S4. Overall, the genetic profiling supported the implication of TWIST2 in human leukemia; meanwhile, it also indicated that the identification of TWIST2-p21 axis in this study did not exclude the involvement of other factors in TWIST2-mediated growth arrest. DISCUSSION TWIST belongs to bHLH proteins and many bHLH transcriptional factors, such as E2A family, stem cell leukemia factor (SCL/Tal1), Lyl-1 and the HLH Id family have critical role in normal hematopoiesis.38–44 The regulatory role of TWIST in the development of immune cells has been reported;20,45 however, its functional role in normal hematopoiesis has not been revealed © 2015 Macmillan Publishers Limited

until Twist-2 was identified as a negative regulator of myelopoiesis.18 In the present study, we demonstrate that TWIST2 regulates the proliferation and cell cycle of the normal hematopoietic cells; thus in murine and human hematopoietic cells Twist2/TWIST2 has similar function. The aberrant expression of many bHLH proteins leads to malignant transformation.38–44 Recently, hypermethylation of TWIST2 has been reported to occur in human RUNX1-ETV6+ ALL.17 In this study, we find that TWIST2 hypermethylation happens in nearly 1/3 adult AML patients and results in its impaired expression, which implicates the aberrant expression of TWIST2 in human AML. We utilize various models to demonstrate that TWIST2 overexpression is sufficient to inhibit the proliferation of AML cells, which is in line with what Thathia et al.17 have found with RUNX1ETV6+ ALL cells. In addition, we identify a novel TWIST2-p21 axis that has a critical role in controlling cell cycle of normal and AML cells. Finally, we generate genetic profiling data, which help to investigate the network governed by TWIST2. Thus we conclude that TWIST2, a novel human hematopoietic regulator, is frequently inactivated in human AML and has a role of tumor suppressor in this disease. This novel function of TWIST2 is opposite to the known function of TWIST1 in hematological malignancies. For example, Cosset et al.46 have found that TWIST1 is expressed in human chronic myeloid leukemia, which is an independent risk factor for refractory treatment of Imatinib mesylate. The expression of TWIST1/miR10a/b axis in human myeloplastic syndrome is Oncogene (2015) 3000 – 3010

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Figure 6. TWIST2 activates p21 and arrests AML cells in G0/G1 phase. (a) The expression of p21 and CCND1 was assessed in Venus (control) and TWIST2-transduced primary AML samples (n = 3). (b) Western blotting was used to compare the protein expression of p21, CyclinD1, p27 and p53 between Venus and TWIST2-transduced cells. (c) TWIST2-expressed THP-1 cells were analyzed with ChIP, three PCR products were used to monitor the binding of p21 by TWIST2 and ZFX was used as an irrelevant nuclear protein control. (d) The cell cycle analyses for Venus, TWIST2 and F86P expressed THP-1, Dami and primary AML cells are displayed. *Mean P o0.05, which was estimated with t-test in a two-tailed fashion.

elevated, which causes the resistance of apoptosis in these malignant cells.47,48 Twist-1 has been identified as a key target for TrkC to promote the survival and proliferation of leukemic cells as well.49 Though TWIST1 and TWIST2 are similar in terms of protein sequence and have similar function in many scenarios, accumulating data suggest that they have diverse functions as well. For example, Sharabi et al.18 have found that Twist1 is highly expressed in hematopoietic stem cell enriched subset, while Twist2 is exclusively expressed in myeloid progenitors but not the lymphoid progenitors; which indicates the unequal function of Twist1 and Twist2 in hematopoietic stem cells and progenitor cells. In another case, mutations of TWIST1 and TWIST2 in human cause Saethre–Chotzen syndrome and Setleis Syndrome, respectively.50–52 In our hands, we found that both TWIST1 and TWIST2 were expressed in normal CD34+ cells and various AML cell lines. Similarly to TWIST2, TWIST1 also bound the regulatory region of p21. However, the overexpression of TWIST2 was not sufficient to alter the expression of TWIST1 in THP-1 cells Oncogene (2015) 3000 – 3010

(Supplementary Figure S9). It is very interesting to investigate whether and how these TWIST proteins interact in the same cell. In the work by Ishikawa et al.,16 the genetic profiling has been generated to delineate the mechanism of how Twist2 inhibits osteosarcoma cells. Fbln5, a reported tumor suppressor is upregulated upon Twist2 overexpression.16 However, these data may not necessarily be suitable to understand the tumorsuppressor role of TWIST2 in human leukemia because of the different cell types. In the present work, our genetic profiling data indicated the implication of TWIST2 in human leukemia and strongly supported the tumor-suppressor role of TWIST2 in human AML. Nevertheless, it will be informative to obtain the profiling data with normal hematopoietic progenitor cells, in which TWIST2 is expressed at an endogenous level. This would provide other insights beyond TWIST2-p21 axis to understand how TWIST2 modulates these cells, and ultimately, how its aberrant expression contributes to the hematological transformation. Indeed, in this study we have observed that the silence of p21 only partially © 2015 Macmillan Publishers Limited

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Figure 7. Microarray data support the notion that TWIST2 is a novel tumor suppressor. (a) Four independent paired THP-1/Venus and THP-1/ TWIST2 cells were used for gene expression analysis using whole human genome 44 K microarray (Agilent Technologies). In all, 662 genes were identified to be differentially expressed significantly (4two-fold change and P o0.05), which were displayed in the heat map. (b) Fourteen genes indicated by microarray data were chosen to perform Q-RT–PCR using THP-1/Venus and THP-1/TWIST2 cells. (c) BIN1, DUSP5 and DUSP6 were further assessed with Venus and TWIST2-transduced AML cells (n = 3). (d) The expression of BIN was quantitatively measured with control (shNC) and TWIST2-silenced (shRNA#1 and shRNA#2) normal bone marrow cells. *Mean Po 0.05, which was estimated with t-test in a two-tailed fashion.

rescues the growth inhibition induced by TWIST2, and the restoration sometimes is cell-type specific (Figure 2e). Given that TWIST2 is able to regulate multiple oncogenes and tumor suppressors, these efforts will not only provide new clues to interpret how TWIST2 inhibits leukemic cells but also an opportunity to pursue novel therapeutic targets for the disease management.

MATERIALS AND METHODS Cells and cultures THP-1, Dami, HL60, NB4, HEL and 293T cells were purchased from the cell bank of Chinese Academy (www.cellbank.org.cn); SHI-1 was originally maintained in the laboratory of Dr Suning Chen. These cells were maintained either with RPMI1640 or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Bone marrow cells from human AML patients and healthy donors were collected from the First Affiliated Hospital of Soochow University with written informed consent approved by the Ethical Committee of Soochow University. The cells were used for the DNA/ RNA extraction and lentiviral transduction experiments. After gradient centrifuge with Lympholyte-H cell separation media (Cedarlane Laboratories, Burlington, NC, USA), the yielded nucleated cells were either cyropreserved or purified with human CD34 EasySep kit (Stem Cell Technologies, Vancouver, BC, Canada). Totally, 85 AML patients and 6 healthy donors were recruited in the present study. The clinical characteristics were summarized in Supplementary Table S5. Cells from 75 AML patients were analyzed for the methylation status of TWIST2, and among which 30 were further analyzed for the expression of TWIST2. © 2015 Macmillan Publishers Limited

DNA methylation detections Genomic DNA was isolated with DNA purification Kit (Purelink Genomic DNA Kit, Life Technologies, Grand Island, NY, USA), and then 500 ng DNA were treated with bisulfate following purification with the Methycone conversion kit (Life Technologies). The bisulfate-treated DNA was used for the amplification of CG-enriched region of TWIST2 promoter; the primer information is in Supplementary Table S6. The amplified PCR products were subject to COBRA or sequence analysis to determine the content of DNA methylation.

RNA extractions and Q-RT–PCR Total RNA was extracted with PicoPure RNA extraction kit (Arturus, Mountainview, CA, USA) and reverse transcribed with SuperScript III (Life Technologies). Q-RT–PCR was performed using SYBR Green PCR MasterMix (Applied Biosystems, Foster City, CA, USA) with 7500 real-time PCR system (Applied Biosystems). The gene-specific primers are summarized in Supplementary Table S6.

Microarray analysis Four biological replicates of control and TWIST2 lentiviral vectortransduced THP-1 cells were harvested for microarray analysis using Agilent whole human genome oligo-chips (4 × 44 K) in Shanghai Biotechnology Corporation. The gene expression data has been assigned an accession ID as GSE57347. Data were normalized with quantile normalization, and the differentially expressed transcripts were determined based on Student’s t-test (Po 0.05) and fold change (42). All differentially expressed genes were clustered by Hierarchical Clustering method, their enrichment in KEGG pathways were also assessed. GSEA was executed with the computational method GSEA version 2.0.53,54 Oncogene (2015) 3000 – 3010

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3008 Lentiviral vector construction, virus production and transduction A lentiviral vector was originated from Dr Christopher Baum (Hannover Medical School, Hannover, Germany). The Twist2 cDNA and its various mutants were subcloned into the lentiviral vector. At the C-terminus of TWIST2 and its mutant, a single FLAG was fused. The two mutants of TWIST2 (TWIST2F86P and TWIST2b−) were generated with site-directed mutagenesis Kit (Beyotime, Shanghai, China), and the resulting point mutants were mutated at F86P and R74A/E75A/R76A/R78A, respectively. The other two mutants of TWIST2 (TWIST2ΔN(1–28) and TWIST2ΔC(141–160)) were generated with the PCR method. The efficacy of these mutants was described previously by others.5 All vectors were verified with sequencing. The viral production was performed with a standard method as described previously,55 and the expression of TWIST2 and its mutants were verified by western blotting with an antibody against TWIST2. Normal hematopoietic cells and AML blast cells were infected by concentrated lentivirus as previous description.55 The fluorescent cells were purified with a cell sorter (Aria III, Becton Dickinson, Franklin Lakes, NJ, USA), and 1000 transduced cells were plated into methylcellulose media (MethoCult H4230, Stem Cell Technologies) plus a cocktail of cytokines, including stem cell factor (50 ng/ml), interleukin-3 (20 ng/ml), interleukin-6 (20 ng/ml), granulocyte macrophages colony-stimulating factor (20 ng/ml), granulocyte colony-stimulating factor (20 ng/ml) and erythropoietin (3 IU/ml); the colonies were classified and numerated 14–16 days later. Lentiviral vectors to silence TWIST2 together with the negative control (shNC) were from GenePharma Co, Ltd (Shanghai, China). The sequences to silence TWIST2 were shRNA#1, 5′-GCAAGAAGTCGAGCGAAGA-3′ and shRNA#2, 5′-GCTGAGCAAGATCCAGACG-3′. The scramble and p21-specific siRNA were also from GenePharma, and the sequence against p21 was 5′-GTCACTGTCTTGTACCCTTGT-3′. Double-stranded RNA was delivered to hematopoietic cells using spermine-introduced pullulan (a gift from Dr Jian Liu and Dr Haiyan Xu, Chinese Academy of Medical Sciences and Peking Union Medical College) as described previously.56 The transfection efficiency was as high as 85% for both THP-1 and CD34+ cells.

Xenoengraftments with nude mice Six-to-eight-week-old nude mice were purchased from Shanghai Laboratory Animal Center, the Chinese Academy of Science (SLACCAS, Shanghai, China). Virus-transduced cells were injected subcutaneously, and then the growth of the tumors was observed closely. The tumors were analyzed with luminescence detector (IVIS Lumina II, Caliper Life Sciences, Hopkinton, MA, USA) and then dissected for hematoxylin and eosin staining. This study was approved by the Animal Experimental Committee of Soochow University and performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Migration assay Leukemic cell migration was carried out using transwell chamber. In brief, 2 × 105 cells were seeded into the upper chamber (Corning Costar, Corning, NY, USA) in serum-free media. After 20 h incubation at 37 °C, the migrated cells on the lower chamber were fixed with 100% methanol and stained with Wright–Giemsa solution (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Six random fields were imaged by Olympus FSX-100 microscope (Olympus, Tokyo, Japan), and the cells were counted to calculate the relative migration.

Soft agar assay The soft agar assay was performed following the previous description.57 Briefly, various lentiviral vector-infected cells were added to growth medium with 0.2% agar and layered onto 1% agar beds in 35-mm dishes. Cells were fed with 1 ml of medium every 3 days. The colonies were stained with 0.005% crystal violet and counted 14 days later.

Cell cycle analysis In all, 1 × 106 cells were collected and washed with phosphate-buffered saline (PBS) thoroughly and then resuspended with remaining PBS. The cells were fixed with 70% prechilled ethanol and stored in freezer overnight. The cells were treated with RNaseA at 37 °C for 30 min and then stained with propidium iodide (Sigma, St Louis, MO, USA) at room temperature in dark for 15 min. The cells were washed and resuspended with PBS for flow cytometry analysis (Calibur, Becton Dickinson). Oncogene (2015) 3000 – 3010

The distribution of the cell cycle status was re-analyzed with the FlowJo software (Ashland, OR, USA).

Western blotting Cell lysis was prepared with the protein lysate buffer purchased from (Beyotime) supplemented with 100 mM phenylmethanesulfonylfluoride, and then the protein samples with same amount (15 μg/lane) were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to the Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) using Bio-Rad gel system (Bio-Rad, Hercules, CA, USA). The blotting was performed following the instructions of the suppliers of various antibodies, including anti-TWIST2 (ab57997, Abcam, Cambridge, MA, USA), anti-CCND1 (no. 2926, Cell Signaling Technology, Danvers, MA, USA), anti-p27 (no. 2552S, Cell Signaling Technology), anti-p53 (K0181-3, MBL, Nagoya, Japan), anti-p21 (no. 2946, Cell Signaling Technology) and anti-ACTIN (AC40, Sigma). The blot was developed with chemiluminescence substrate (GE Healthcare Life Sciences, Piscataway, NJ, USA) automatically (Kodak Medical X-Ray Processor 102, Rochester, NY, USA).

ChIP assays ChIP was performed with the EZ-ChIP kit with the instruction of the manufacturer (Millipore). Briefly, 2 × 106 cells were collected and washed with PBS thoroughly. The cells were fixed with formaldehyde; and DNA fragments ranging from 200 to 1000 bps were yielded with sonication. RNA polymerase antibody, TWIST2 antibody, ZFX antibody (PAB20245, Abnova, Walnut, CA, USA), TWIST1 antibody (ab175430, Abcam) and the immunoglobulin G isotype antibody were used to incubate with the lysate. The DNA fragment was then purified with phenol extraction and column cleansing provided by the kit. The DNA samples were quantified, and the same amount of them were analyzed with gene-specific primers, which are summarized in Supplementary Table S6.

Reporter gene expression analysis The promoter region of p21 was obtained with PCR and subcloned into pGL3 Basic to generate pGL3-p21. A serial of p21 mutants were generated with PCR methods and sub-cloned to pGL3 as well. In all, 3 μg pGL3-p21 or p21 mutant mixed with 0.3 μg RL-TK were delivered to cells using the electroporation method with Nucleofector device (Lonza, Basel, Switzerland) following the instruction of the manufacturer. Forty-eight hours later, the cells were collected, and the luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) with Luminoskan Ascent reader (Thermo Scientific, Waltham, MA, USA). After normalizing the activity of the Firefly luciferase reporter to that of the Renilla luciferase reporter, the relative transcription activities were calculated and compared.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was funded by National Natural Science Foundation of China (no. 31170755), National Key Scientific Project of China (973 Program no. 2011CB933501), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, no. 20113201120017), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Province’s Key Medical Center (ZX201102) and National Public Health Grand Research Foundation (No.201202017).

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