Oncogene (2008) 27, 6749–6760 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE C/EBPa and C/EBPe induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion

H Matsushita1, H Nakajima2, Y Nakamura3, H Tsukamoto3, Y Tanaka1, G Jin1, M Yabe1, S Asai1, R Ono4, T Nosaka4, K Sugita5, A Morimoto6, Y Hayashi7, T Hotta8, K Ando8 and H Miyachi1

1Department of Laboratory Medicine, Tokai University School of Medicine, Kanagawa, Japan; 2Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 3Teaching and Research Support Center, Tokai University School of Medicine, Kanagawa, Japan; 4Department of Microbiology, Mie University Graduate School of Medicine, Mie, Japan; 5Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi, Japan; 6Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto, Japan; 7Department of Hematology/Oncology, Gunma Children’s Medical Center, Gunma, Japan and 8Department of Hematology, Tokai University School of Medicine, Kanagawa, Japan

CCAAT/ bindingproteins (C/EBPs) have an Introduction important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid Recurrent translocations involvingthe mixed lineage leukemia (AML). Their involvement in myelomonocytic leukemia (MLL) gene located on 11q23 leukemia, however, is still unclear. Therefore, the expres- frequently occur in various hematological malignancies sion and function of C/EBPs in myelomonocytic cells with includingacute myeloid leukemia (AML). In the MLL-fusion were investigated. Retinoic acid (RA) translocations, truncated N-terminal MLL with induced monocytic differentiation in the myelomonocytic AT hooks and MT domain are fused in-frame to one of cell lines with MLL-fusion genes, THP-1, MOLM-14 and more than 40 translocation partners to produce proteins HF-6 cells, accompanied by monocytic differentiation with novel properties. The translocation partners share with the upregulation of C/EBPa and C/EBPe. Mono- little , but the resultant chimeric cytic differentiation by RA treatment was confirmed in proteins are thought to alter the transcription on MLL primary AML cells usinga clonogenicassay. When the target genes (Li et al., 2005). activity of C/EBPa or C/EBPe was introduced into HF-6 Rearrangements involving 11q23 or MLL are found in cells, their cellular growth was arrested through differ- both AML and acute lymphoid leukemia (ALL) cases. In entiation into monocytes with the concomitant marked AML, they occur more frequently (5–30%) in the downregulation of . Cebpe mRNA was upregulated subtypes with monocytic components, such as M4, M5a by the induction of C/EBPa-ER, but not vice versa, thus and M5b, based on the French–American–British classi- suggesting that C/EBPe may have an important function fication (Schoch et al., 2003; De Braekeleer et al., 2005). in the differentiation process. Introduction of Myc Consistent with this, in vivo studies in mice have revealed isoforms into HF-6 cells partially antagonized the that MLL-fusion genes caused AML, which was arrested C/EBPs effects. These findings suggest that the ectopic at the late myelomonocytic stage, regardless of the expression of C/EBPe, as well as C/EBPa, can induce the developmental level of the initiatingcells (Cozzio et al., monocytic differentiation of myelomonocytic leukemic 2003). AML with MLL-fusion genes has been reported to cells with MLL-fusion gene through the downregulation of have poorer prognosis with combination chemotherapy Myc, thus providinginsight into the development of novel and stem cell transplantation (the median overall therapeutic approaches. survival: 8.9 months) (Schoch et al., 2003). Therefore, Oncogene (2008) 27, 6749–6760; doi:10.1038/onc.2008.285; the development of novel therapies against AML with published online 8 September 2008 MLL-fusion genes should be investigated. CCAAT/enhancer bindingproteins (C/EBPs) are a Keywords: ; mixed lineage family of transcription factors that have an important leukemia gene; monocytic differentiation; CCAAT/enhan- function in the regulation of cellular proliferation and cer bindingprotein- a; CCAAT/enhancer bindingprotein- e differentiation. In the hematopoietic system, C/EBPa and C/EBPe are required for granulocytic commitment of multipotent myeloid progenitors and terminal differ- entiation of granulocytes, respectively (Yamanaka et al., Correspondence: Dr H Matsushita, Department of Laboratory 1997; Zhang et al., 2004). The expression and function Medicine, Tokai University School of Medicine, 143 Shimokasuya, of C/EBPa and C/EBPe are often altered in AML Isehara, Kanagawa 259-1193, Japan. E-mail: [email protected] leukemogenesis. in CEBPA are found in Received 8 May 2007; revised 10 June 2008; accepted 8 July 2008; about 10% of AML cases (Pabst et al., 2001a; published online 8 September 2008 Gombart et al., 2002). The expression of C/EBPa in Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6750 AML cells is inhibited by AML1-ETO or internal were positive for a-naphthyl butyrate staining, which tandem duplication of fms-like tyrosine kinase 3 (FLT3- was specific for the monocytic lineage (Figure 1c), and ITD), and the ectopic expression of C/EBPa induces negative for naphthol AS-D chloracetate and myeloper- granulocytic differentiation in the leukemic cells with oxidase stainings, which were specific for the myeloid these mutated genes (Pabst et al., 2001b; Zheng et al., lineage (data not shown). The expression of the 2004). PML-RARa in acute promyelocytic leukemia monocytic differentiation markers CD11b and CD36 (APL) inhibits the expression of C/EBPe, and the were upregulated with RA treatment in THP-1 and ectopic expression of either C/EBPa or C/EBPe induces MOLM-14 cells (Figure 1d). granulocytic differentiation in vivo (Truong et al., 2003). The effects of ATRA or 9-cis RA treatment on These studies suggest that the altered expression and primary AML cells harboring MLL-fusion were also function of C/EBPa and C/EBPe are involved in subsets analysed. Three cases of primary AML cells were sorted of AML, and that the induction of C/EBPa and C/EBPe out accordingto their surface markers before clonogenic can thus induce the differentiation of leukemic cells. assay (Supplementary Table 1). Small colony-forming Retinoic acid (RA) is known to induce terminal units or clusters from leukemic cells (L-CFU as the differentiation of normal myeloid progenitors, and abbreviation includingboth of them) were observed on is widely used to induce the differentiation of leukemic days 11–14. L-CFU was classified in the followingthree cells in APL patients. The application of the differentia- types, granulocyte/macrophage-L-CFU (L-CFU-GM), tion activity of RA to myelomonocytic cells with granulocyte-L-CFU (L-CFU-G) and macrophage-L- MLL-fusion genes has also been explored (Hemmi CFU (L-CFU-M), accordingto the cellular contents and Breitman, 1985; Iijima et al., 2004). However, the (Figure 2a). ATRA or 9-cis RA induced L-CFU-M previous studies did not clarify the importance of the formation, mainly small monocytic clusters, in place of C/EBPs function in their RA-induced monocytic L-CFU-GM and L-CFU-G (Figure 2b). An immuno- differentiation. This study demonstrated that the growth phenotypic analysis of L-CFU was performed in case 1, arrest and monocytic differentiation of myelomonocytic and uncovered the decrease of total cellular number and cells with MLL-fusion gene is induced by ectopic the increase of CD36-positive cells (Figure 2c). Taken expression of either C/EBPa or C/EBPe alone, as well together, these findings suggest that ATRA and 9-cis as by RA. These findings may lead to a novel RA treatment inhibited the proliferation of AML cells therapeutic approach in AML with MLL-fusion genes with MLL-fusion genes, accompanied by monocytic by enhancingC/EBPs functions. differentiation.

C/EBPs were upregulated in RA-induced monocytic Results differentiation of myelomonocytic cells with MLL-fusion genes RA inhibited the proliferation and induced the differentiation To gain insight into the molecular mechanisms of of myelomonocytic cells with MLL-fusion genes RA-induced growth arrest and monocytic differentiation To see the potential commitment of cell lineage in of myelomonocytic cells with MLL-fusion genes, the myelomonocytic cells with MLL-fusion genes, human expression of C/EBPs was analysed. HF-6 cells weakly myelomonocytic cell lines with MLL-AF9, THP-1 and expressed the N-terminally truncated (30 kDa isoform: MOLM-14 cells, were treated with RA. HF-6 cells, a p30) isoforms of C/EBPa, and lesser extent, the murine cell line established by the introduction of functional isoform (42 kDa isoform: p42) of C/EBPa, MLL-SEPT6 derived from t(X;11)(q24;q23) into mur- and did not express C/EBPe. RA upregulated C/EBPa; ine hematopoietic cells were also used (Ono et al., 2005). 9-cis RA induced them more than ATRA, and 10À6 M HF-6 cells were assumed to be arrested at the was more effective, when compared with 10À8 M. The myelomonocytic stage, based on their pale cytoplasm p42 isoform of C/EBPa was upregulated more intensely occasionally accompanied by a few vacuoles and than the p30 isoform, which was reported to antagonize azurophilic granules, weakly-positive for myelomono- the p42 isoform (Pabst et al., 2001a) (Figure 3a). RA cytic markers Mac-1, Gr-1 and c-Kit, and negative also upregulated the p32 and p30 isoforms of C/EBPe, reactions for Sca-1 and CD34. When all of these cell and lesser extent, the p27 isoform (Figure 3b). lines were treated with all-trans RA (ATRA) or 9-cis The upregulation of Cebpa and Cebpe mRNAs in RA, their proliferation was suppressed in a dose- HF-6 cells by 10À6 M RA was detected in an analysis dependent manner (Figure 1a). HF-6 and THP-1 cells usingquantitative reverse transcription (RT)–PCR completely died within 7 days after 10À6 M of RA (either (Figure 3c). The induction of the p42 isoform of ATRA or 9-cis RA) treatment. However, the inhibitory C/EBPa and the p32 isoform of C/EBPe by RA was effect of RA on the proliferation of MOLM-14 cells was also observed in THP-1 and MOLM-14 cells (Figures 3d partial. The cells gradually increased in number, and and e) (Lee et al., 2002). However, the induction of the p30 repeated exposure to fresh media with 10À6 M of RA isoform of C/EBPa, and the p30 and p27 isoforms of completely inhibited their growth (data not shown). C/EBPe were not apparent in either of these two cell lines. Morphologically, monocytic differentiation was ob- Although only two kinds of MLL-fusion genes were served in all the cell lines followingRA treatment examined, the findings indicated that myelomonocytic (10À6 M) (Figure 1b). HF-6 cells differentiated by RA cells with MLL-fusion genes were committed to the

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6751 control ATRA 9-cis RA control 10-8M (10-6 M) (10-6 M) 10-7M 10-6M cell count HF-6 (X105/ml) ATRA 9-cis RA 20 20

HF-6 10 10 0 0 THP-1 012345 012345

8 8

THP-1 4 4

0 0 0246 0246 MOLM-14 30 30 20 MOLM-14 20 10 10 0 0 0246 0246 THP-1 6 6 CD11b CD36 Kasumi-1 4 4 40 60 2 2 30 0 0 40 0246 0246

20 (%) days (%) 20 10

0 0 control ATRA 9 cis RA control ATRA 9 cis RA 10-6M 10-6M 10-6M 10-6M

Control MOLM-14 CD11b CD36 60 60 ATRA 40 10-6M 40 (%) (%) 20 20 9-cis RA -6 0 0 10 M control ATRA 9 cis RA control ATRA 9 cis RA 10-6M 10-6M 10-6M 10-6M Figure 1 Effects of retinoic acid (RA) on myelomonocytic cells with MLL-fusion genes. (a) RA-induced growth inhibition of myelomonocytic cells with MLL-fusion genes. Kasumi-1 cells were used as a negative control. (b) RA-induced monocytic differentiation of myelomonocytic cells with MLL-fusion genes. HF-6 cells were cultured with RA for 2 days, and THP-1 and MOLM- 14 cells for 5 days. All these cells exhibited an extended, less basophilic cytoplasm with few granules and an irregular shaped-nucleus with indentation after RA treatment. A number of vacuoles were also recognized in RA-treated HF-6 cells. May–Giemsa staining. (c) a-naphthyl butyrate (a-NB) stainingand inhibition with sodium fluoride (NaF) in HF-6 cells treated with RA. Magnification: Â 400 in (b) and (c). (d) The induction of CD11b and CD36 in THP-1 and MOLM-14 cells by 5-day treatment with 10À6 M RA. The analyses were performed usingflow cytometry. monocytic lineage and differentiated by RA treatment, (C/EBPa-ER and C/EBPe-ER, respectively), 4-Hydro- with the concomitant upregulation of C/EBPs. xytamoxifen (4-HT) could induce C/EBPs activity (Fukuchi et al., 2006; Nakajima et al., 2006). Their ectopic expression in HF-6 cells was confirmed by Induction of C/EBPs activity inhibited the cellular growth immunoblotting(Figure4a). HF-6 cells with empty and promoted the monocytic differentiation in HF-6 cells vector (HF-6/pMY) proliferated, but the cellular growth To investigate whether the induction of C/EBPs activity was completely inhibited by the expression of C/EBPa- could promote the monocytic differentiation and ER or C/EBPe-ER (Figure 4b). HF-6/pMY cells had maturation in myelomonocytic cells with MLL-fusion morphologically blastic features, whereas HF-6/ genes, inducible forms of C/EBPa or C/EBPe were C/EBPa-ER and HF-6/C/EBPe-ER cells showed retrovirally introduced into HF-6 cells. Because C/EBPa monocytic differentiation after 1 day with or without and C/EBPe were fused to the estrogen 4-HT stimulation as they did with RA treatment

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6752

L-CFU-M L-CFU-GM L-CFU-G

100

75 L-CFU-M 50 L-CFU-GM L-CFU-G

Percentage (%) Percentage 25

0 RA RA RA ATRA ATRA cis cis cis control control control 9- 9- 9-

Case 1 Case 2 Case 3 (6.5X104/dish) (2X103/dish) (3.5X104/dish)

4 /plate) 5 3 CD36 positive

2

1 Cell number (X10 Cell number 0 controlATRA 9-cis RA Figure 2 Retinoic acid (RA) inhibited the colony formation and induced monocytic differentiation of primary acute myeloid leukemia (AML) cells with MLL-fusion genes. Primary AML cells with MLL-fusion genes were sorted out before clonogenic assay, according to the expression of their surface markers (Supplementary Table 1). Colony-formation units from AML samples (L-CFU) with or without 10À6 M of all-trans RA (ATRA) or 9-cis RA were analysed on days 11–14 in triplicate, except for case 2 (one plate per group). (a) Representative pictures of macrophage-L-CFU (L-CFU-M), granulocyte/macrophage-L-CFU (L-CFU-GM) and granulocyte-L- CFU (L-CFU-G). Magnification: Â 100 (L-CFU-M), Â 40 (L-CFU-GM and L-CFU-G) (b) ATRA- and 9-cis RA-induced monocytic differentiation of primary AML cells with MLL-fusion genes. The results of the average number of total L-CFU without RA treatment are shown as 100% in each case. Note that the percentage of L-CFU-M markedly increased after treatment with ATRA or 9-cis RA, whereas those of L-CFU-GM and L-CFU-G decreased. (c) RA inhibited the proliferation of AML-derived cells and induced CD36-positive cells in case 1. All the cells in the plates for clonogenic assay were collected and counted. Their expression of CD36 was analysed by flow cytometry, and the absolute number of CD36-positive cells was calculated.

(Figure 4c). To quantify the monocytic differentiation of lated nucleus with coarse chromatin formation. An HF-6 cells by inducingC/EBPs activity, the cells were intermediate form has morphological features between counted by classifyingthem into three forms; an those of an immature and mature form: a basophilic immature form is monoblastic cells with a basophilic cytoplasm and an indented nucleus with rather fine cytoplasm and a round nucleus composed of fine chromatin formation. The mature forms were not chromatin formation. A mature form is similar to induced in HF-6/pMY cells, but were induced mature macrophages, which has an extended, clear in HF-6/C/EBPa-ER and HF-6/C/EBPe-ER cells, cytoplasm with vacuolation and an indented or lobu- regardless of 4-HT treatment (Figure 4d). Analysis by

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6753 a HF-6 b HF-6 ATRA 9-cis RA 9-cis RA ATRA 10-8M1010-6M10-8M -6M 10-6 M 10-6 M C/EBPα h: 0 164816 48 1648 16 48 C/EBPε h: 0 122448122448 p42 P32/p30 p27 p30 β-actin β-actin

c d THP-1 600 C/EBP α 12 h 1 d 3 d 5 d ε C/EBP C/EBPα CCA9CA9 CA9 CA9 400 p42

(%) p30 200 C/EBPε p32/p30 p27 0 0h 12h 24h 12h 24h β-actin ATRA 9-cisRA 10-6M 10-6M

e MOLM-14 1 d 3 d 5 d C/EBPα C A 9 A 9 A 9 p42

p30 C/EBPε p32/p30 p27

β-actin

Figure 3 Retinoic acid (RA) induced the expression of CCAAT/enhancer bindingproteins (C/EBPs) in myelomonocytic cells with MLL-fusion genes. Immunoblotting revealed the induction of (a) the p42 isoform of C/EBPa, and (b) p32 and p30 isoforms of C/EBPe in HF-6 cells with RA. (c) Quantitative reverse transcription (RT)–PCR also revealed the upregulation of C/EBPa and C/EBPe mRNAs in HF-6 cells with RA (10À6 M). The mRNA content was measured relative to that of murine Gapdh. The relative ratios in regard to the data before RA treatment (at 0 h) are presented in percentages. Upregulation of the p42 isoform of C/EBPa and p32/p30 isoform of C/EBPe by RA was also recognized in (d) THP-1 and (e) MOLM-14 cells usingimmunoblotting.C: control, A: treatment with 10À6 M of ATRA, 9: treatment with 10À6 M of 9-cis RA, d: days.

flow cytometry showed marked induction of Mac-1 and not increased in HF-6/pMY cells (6.59%), but was Gr-1 in HF-6/C/EBPa-ER and HF-6/C/EBPe-ER induced by C/EBPa or C/EBPe activation in HF-6 cells cells on day 2 (Figure 4e). Their intensities were (44.81 and 26.46%, respectively, Figure 4f). higher in the cells expressing C/EBPa-ER than Taken together, these findings suggested that the C/EBPe-ER, and enhanced with 4-HT treatment (data induction of either C/EBPa or C/EBPe activity by not shown). Because the empty vector did not lead to itself could induce the monocytic differentiation and any changes in HF-6 cells as mentioned above, the growth in HF-6 cells. inhibition and monocytic differentiation were thought to be specific for the expression of C/EBPa-ER or C/EBPe-ER. Changes of mRNA expression in HF-6 cells induced To evaluate the contribution of apoptosis in growth by C/EBPs activation inhibition of HF-6 cells by induction of C/EBPs activity, To observe the expression of the genes associated the percentage of annexin V-positive/propidium iodide- with cellular proliferation and differentiation in negative cells was determined in HF-6/C/EBPa-ER HF-6/C/EBPa-ER or HF-6/C/EBPe-ER cells, their or HF-6/C/EBPe-ER cells cultured with 4-HT. After expression levels were determined usingquantitative 18 h-incubation with 1 mM of 4-HT, this cell fraction was RT–PCR (Figure 5).

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6754 We found that the ectopic expression of either with 4-HT, respectively) and upregulated Sfpi1, the C/EBPa-ER or C/EBPe-ER activity markedly decreased murine homologof PU.1 (454±151 and 345±134% at Myc expression (21±3and13±3%, at 12-h incubation 4-h incubation with 4-HT, respectively). Although

a α ε b P P ) P B B 3 F /E /E /ml C 5 n -G -C - o S ti S S c E E 2 e RE R R f -I -I -I in Y Y o MY M M N p p p (Kd) 1 100 * 0 75 cell count (X10 024 α ER ** * days α β-actin pMY /4-HT(-) pMY /4-HT(+) α GFP C/EBPα-ER /4-HT(+) α β-actin C/EBPε-ER /4-HT(+)

c pMY C/EBPα-ER C/EBPε-ER

4-HT(-)

4-HT(+)

100 4-HT+ d mature f at sorting (18h-incubation) intermediate 80 HF6/pMV/oh/pos.002 HF6/pMV/18h/pos.002 104 104 immature 0.88% 0.33% 3 3 60 10 10 ) 102 102 (% FL2-H FL2-H 40 101 101 11.19% 6.59% pMY 100 100 100 101 102 103 104 100 101 102 103 104 20 FL4-H FL4-H HF6/CEBPa/0h/neg.004 HF6/CEBPa/18h/pos.004 4 0 104 10 1.19% 2.81% 4-HT : - --+++- 103 103 α 2 2 HF-6 pMY C/EBPα C/EBPε C/EBP 10 10 FL2-H FL2-H -ER -ER -ER 101 101 14.54% 44.81% 100 100 e at sorting (day 0) 4-HT+ (day 2) 100 101 102 103 104 100 101 102 103 104

60 30 PI FL4-H FL4-H 50 25 40 20 HF6/CEBPe/0h/pos.006 HF6/CEBPe/18h/pos.006 30 15 4 Mac-1 10 104 20 10 Counts 10 Counts C/EBP ε 3 1.80% 2.89% 5 10 103 0 0 -ER 0 1 2 3 4 0 1 2 3 4 2 10 10 10 10 10 10 10 10 10 10 10 102 FL2-H Mac 1-P Mac 1-P FL2-H 1 1 50 25 10 10 40 20 0 14.49 26.46% 10 100 30 15 100 101 102 103 104 100 101 102 103 104 Gr-1 20 10 Counts 10 Counts 5 FL4-H FL4-H 0 0 100 101 102 103 104 100 101 102 103 104 Annexin V Gr1-P Gr1-P pMY C/EBPα-ER C/EBPε-ER

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6755 the changes were mild, the ectopic expre- was examined. Retroviral vectors expressingthe two ssion of C/EBPa and C/EBPe similarly downregulated major isoforms Myc1 and Myc2 (pMYpuro-Myc1 Hoxa7 and Hoxa9, which are known to be related to and pMYpuro-Myc2, respectively) were constructed chimeric MLL-induced leukemogenesis, (Ayton and and infected to HF-6 cells to generate HF-6/Myc1 and Cleary, 2003), and upregulated Cdkn1a encoding HF-6/Myc2 cells. Overexpression of Myc isoforms in p21WAF1 (data not shown). Cebpe was upregulated by these cells was confirmed by immunoblotting the induction of C/EBPa-ER with 4-HT (593±69% at (Figure 6a). HF-6/Myc1 and HF-6/Myc2 cells had 4-h incubation with 4-HT), but the induction of morphologically blastic features similar to the original C/EBPe-ER with 4-HT could not upregulate Cebpa HF-6 cells (Figure 6b). The expression intensity of (102±2 and 134±7% at 4- and 12-h incubation with Mac-1 and Gr-1 was slightly decreased in these cells, 4-HT, respectively). when compared with original HF-6 cells and those These results suggested that (i) C/EBPa or C/EBPe- with the empty vector (HF-6/pMYpuro) (Figure 6c). induced monocytic differentiation may be related to the The proliferation rates of HF-6/Myc1 and HF-6/Myc2 downregulation of Myc and upregulation of Sfpi1, and cells were similar to those of HF-6 cells (data not (ii) the expression profiles with the ectopic expression of shown). C/EBPa or C/EBPe closely resembled each other, except C/EBPa-ER or C/EBPe-ER was then infected into for upregulation of Cebpe mRNA by C/EBPa. HF-6/Myc1 and HF-6/Myc2 cells, and their expression was confirmed with immunoblotting(Figure6d). Although the intermediate forms of monocytic lineage Overexpression of Myc partially antagonized the cells were increased in HF-6/Myc1 cells expressing C/EBPs functions in HF6 cells C/EBPa-ER or C/EBPe-ER, no mature forms were As Myc was markedly downregulated in HF-6 cells by induced in all these cells (Figure 6e). C/EBPa-ER or C/EBPs induction, the ability of Myc to inhibit C/EBPe-ER slightly upregulated the intensities of Mac-1 C/EBPs-induced monocytic differentiation of HF-6 cells and Gr-1 in HF-6/Myc1 and HF-6/Myc2 cells, but

Myc /Gapdh Sfpi-1 /Gapdh

600 100 400 (%)

(%) 50 200

0 0 Cebpa /Gapdh Cebpe /Gapdh pMY 0h pMY 4-HT / 4h 600 pMY 4-HT / 12h C/EBPα-ER 0h α 400 C/EBP -ER 4-HT / 4h (%) C/EBPα-ER 4-HT / 12h ε 200 C/EBP -ER 0h C/EBPε-ER 4-HT / 4h C/EBPε-ER 4-HT / 12h 0 Figure 5 Changes in the mRNA expression of HF-6 cells induced by CCAAT/enhancer binding proteins (C/EBPs) activation. The expression levels of Myc, Sfpi1, Cebpa and Cebpe mRNA in HF-6/C/EBPa-ER or HF-6/C/EBPe-ER cells were analysed by quantitative reverse transcription (RT)–PCR. Total RNA was extracted before or after 4 and 12-h incubation with 1 mM of 4-HT. The mRNA content was measured relative to that of murine Gapdh. The relative ratios in regard to the data from the HF6/empty vector (pMY) before 4-HT treatment are presented in percentages.

Figure 4 CCAAT/enhancer bindingproteins (C/EBPs) inhibited the proliferation and induced the monocytic differentiation of HF-6 cells. (a) Detection of C/EBPa-ER and C/EBPe-ER in HF-6 cells. They were recognized by anti-ER antibody. Anti-GFP antibody was used for detection of viral integration in HF-6 cells. *: the specific bands, **: nonspecific bands. (b) Growth inhibition of HF-6 cells by the induction of C/EBPs activity. The cells were sorted with green fluorescent on day 0, and sequentially analysed the proliferation with or without 1 mM of 4-Hydroxytamoxifen (4-HT). pMY: pMY-IRES-GFP, used as a control vector. (c) Morphological changes of HF-6 cells by induction of C/EBPs activity. Cytospin slides were prepared on day 1. As a result of C/EBPs expression, the cytoplasm became less basophilic and obtained a number of vacuoles, and the nucleus became indented or lobulated. May–Giemsa staining, magnification: Â 400. (d) The differentiation count of HF-6 cells expressingpMY, C/EBP a-ER or C/EBPe-ER on day1. They were cultured with or without 4-HT for 24 h. (e) The expression of Mac-1 and Gr-1 in HF-6 cells induced by C/EBPs activity. Evaluation was done at sorting(day 0) and 2-day incubation with 1 mM of 4-HT (day 2). Dotted lines: with isotype controls. (f) Induction of apoptosis in HF-6 cells by C/EBPs activation. Annexin-positive/propidium iodine (PI)-negative cells were counted as apoptotic cells at sortingand after 18-h incubation with 1 mM of 4-HT.

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6756 the intensities were not as high as those observed These assays were also performed usingproliferating in HF-6/C/EBPa-ER or HF-6/C/EBPe-ER cells HF-6/Myc1 and HF-6/Myc2 cells introduced with (Figures 4e and 6f). Both of the cells expressing C/EBPa-ER or C/EBPe-ER after 1-week culture. The C/EBPa-ER or C/EBPe-ER could proliferate under sustained expression of C/EBPa-ER or C/EBPe-ER in 4-HT treatment, although the partial growth inhibition these cells was confirmed usingquantitative RT–PCR. was still stronger in HF-6/Myc1 than in HF-6/Myc2 These cells showed a blastic appearance, lower inten- (Figure 6g). sities of Mac-1 and Gr-1 and increased proliferation

abHF-6/pMYpuro HF-6/Myc1 HF-6/Myc2 HF-6/pMYpuro HF-6/Myc2 HF-6/Myc1 HF-6

Myc1 c HF-6 HF-6/pMYpuro HF-6/Myc1 HF-6/Myc2 Myc2 HF6/Gr1F/Mac1P.003 HF6/P2/Gr1F/Mac1P.006 HF6/P2/Gr1F/Mac1P.012 HF6/P2/Gr1F/Mac1P.009 WB: α Myc 104 104 104 104 94.2% 90.8% 61.4% 61.8% 103 103 103 103 102 102 102 102 1 1 1 WB: α β-actin Mac1-P 10 Mac1-P 101 Mac1-P 10 Mac1-P 10 0 0 0 10 100 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

Mac-1 Gr1-F Gr1-F Gr1-F Gr1-F Gr-1

d Myc2 Myc1 e intermediate immature -ER -ER -ER -ER 100 α ε ε α 80 60 (%) C/EBP C/EBP pMY pMY C/EBP C/EBP 40 α ER * * 20 ** ** 0 ε ε

Myc1 α α α Myc Myc2

α β-actin Myc2/pMY Myc1/pMY Myc2/C/EBP Myc1/C/EBP Myc2/C/EBP Myc1/C/EBP

f Myc1 Myc2 40 40 30 30 Mac-1 20 20 Counts 10 10 Counts 0 0 pMY 100 101 102 103 104 100 101 102 103 104 Mac1-P Mac1-P C/EBPα-ER 40 40 C/EBPε-ER 30 30 Gr-1 20 20 Counts 10 Counts 10 0 0 100 101 102 103 104 100 101 102 103 104 Gr1-P Gr1-P

g HF-6/Myc1 HF-6/Myc2 15 /ml) /ml)

5 20

5 pMY 10 C/EBPα-ER 10 ε 5 C/EBP -ER

0 0 cell count (X10 024cell count (X10 024 days days

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6757 rates, in comparison to the cells immediately after on the developmental level of the cell, rather than the infection. The expression levels of Myc and Sfpi1 types of differentiation inducers. mRNA in HF-6/Myc1 and HF-6/Myc2 cells were equal, This study showed that the induction of Sfpi1, the regardless of the ectopic expression of C/EBPs. mouse homologof PU.1, was commonly observed in the The induction of Cebpe mRNA was not observed in C/EBPs-induced monocytic differentiation of HF-6 HF-6/Myc1 or HF-6/Myc2 cells expressingC/EBP a-ER cells. This may be a key step in the differentiation, (data not shown). because PU.1 is crucial for the monocytic development. Taken together, these data suggested that the ectopic Although C/EBPa functions against PU.1 and induces expression of Myc overcame the growth arrest, while granulocytic differentiation in granulocyte–macrophage also partially inhibitingthe monocytic differentiation of progenitors (Dahl et al., 2003), there have been several HF-6 cells induced by the C/EBPa or C/EBPe activity. reports that show positive regulation of PU.1 by C/EBPa in other cell components. C/EBPa can bind and activate the PU.1 enhancer (Kummalue and Discussion Friedman, 2003). PU.1 mRNA can be upregulated by C/EBPa in the granulocytic differentiation of 32Dcl3 This study demonstrated that the induction of C/EBPa cells (Wang et al., 1999). C/EBPs and PU.1 coopera- or C/EBPe activity by itself could inhibit the cellular tively regulate neutrophil esterase and growth and induce the monocytic differentiation of eosinophilic granule gene expression (Oelgeschlager myelomonocytic cells with MLL-fusion genes. In addi- et al., 1996; Gombart et al., 2003). Moreover, C/EBPs, tion, the downregulation of Myc induced by C/EBPs namely C/EBPb, are upregulated and bind to PU.1 activity appears to have an important function in their promoter to induce the expression of PU.1 mRNA in monocytic differentiation. ATRA-induced granulocytic differentiation of APL Many previous studies have shown that C/EBPa and cells (Mueller et al., 2006). These reports as well as the C/EBPe have an important function in granulocytic data presented here suggest that C/EBPs induce PU.1, differentiation. However, this study showed that human and that C/EBPs and PU.1 function cooperatively in and murine myelomonocytic cells with MLL-fusion late granulocytic and monocytic differentiation after the genes ceased their proliferation and were differentiated granulocyte–macrophage progenitor stage, including into the monocytic lineage following treatment with myelomonocytic cells with MLL-fusion genes. RAs, potent differentiation inducers for the granulocytic The gene expression profiles were similar between lineage, accompanied by the upregulation of C/EBPa C/EBPa- and C/EBPe-induced HF-6 cells in this study. and C/EBPe. In addition, the ectopic expression of This implies a common mechanism in monocytic C/EBPs induced HF-6 cells into monocytes. Consistent differentiation of HF-6 cells by C/EBPs. C/EBPe with this, C/EBPa and C/EBPe are also reported to induction by the ectopic expression of C/EBPa-ER induce monocytic differentiation: C/EBPa regulates suggests that C/EBPe probably have an important the monocyte-colony-stimulatingfactor receptor gene function in monocytic differentiation of HF-6 cells. and CD14 (Zhang et al., 1996; Pan et al., 1999), and Another possible explanation is that C/EBPa and contributes to monocytic commitment of primary C/EBPe share the common targets in growth arrest myeloid progenitors by directly activating PU.1 (Wang and differentiation. C/EBPa is not only a transcriptional et al., 2006). C/EBPe also induces the gene expression of factor, but functions through protein–protein interac- monocyte-colony-stimulatingfactor receptor (Williams tion. The target molecules in this interaction include the et al., 1998), and is required for the development and proteins related to cell cycle progression, such as cyclin- function of mature macrophages (Tavor et al., 2002). dependent kinase 2 (CDK2) inhibitor p21, pRB, , The expression of chemokines MIP-1g and MCP-3 is CDK2 and CDK4 (Schuster and Porse, 2006). C/EBPe defective in macrophages from C/EBPe-deficient mice downregulates CDK4/6, cyclin D2/A/E, Bcl-2 and Bcl-x (Kubota et al., 2000). This study indicates that HF-6 (Nakajima et al., 2006), and has a direct interaction with cells are presumably arrested at the monoblastic stage E2F (Gery et al., 2004; Walkley et al., 2004). These based on both morphological and immunophenotypical interaction and downregulation are supposed to cause analysis. It may be presumed that the differentiation cellular growth arrest, thus resulting in the induction of inducers includingRAs and C/EBPs are shared in both apoptosis as well as monocytic differentiation, as a monocytic and granulocytic differentiation, and that the common pathway. These effects on cellular growth may cell lineage commitment and differentiation is dependent be more potent than the induction of monocytic

Figure 6 The overexpression of Myc rescued the growth arrest and partially inhibited differentiation of HF6 cell induced by CCAAT/enhancer bindingproteins (C/EBPs) activation. ( a) The detection of the overexpressed Myc1 and Myc2 in HF-6 cells by immunoblotting. (b) Morphology of HF-6 cells with indicated viruses. May–Giemsa staining, magnification: Â 600. (c) Flow cytometric analysis of HF-6/Myc1 and HF-6/Myc2. The intensity of Mac-1 and Gr-1 was slightly decreased in HF-6/Myc1 and HF-6/Myc2. (d) Detection of the overexpressed C/EBPs in HF-6/Myc1 and HF-6/Myc2. *: C/EBPa-ER, **: C/EBPe-ER. (e) The differentiation count of HF-6/Myc1 and HF-6/Myc2 cells expressingC/EBP a-ER or C/EBPe-ER on day 2. They were cultured with 1 mM of 4-HT. (f) Flow cytometric analysis of Mac-1 and Gr-1 in HF-6/pMYpuro, HF-6/Myc1 and HF-6/Myc2 cells expressing C/EBPa-ER or C/EBPe-ER. The analyses were performed on day 2. Dotted lines: with isotype controls. (g) The proliferation of HF-6/Myc1 and HF-6/Myc2 expressingC/EBP a-ER or C/EBPe-ER. The cells were cultured with 1 mM of 4-HT.

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6758 differentiation as observed in HF-6 cells, when the the murine cell line HF-6 cells might be more strictly reinforcement of C/EBPs function is applied as an committed to the monocytic lineage than the human cell antileukemic therapy. However, mutated C/EBPa with- lines MV4;11 and MOLM-14 cells. out transcriptional activity induces differentiation of In summary, myelomonocytic cells with MLL-fusion APL cells in vivo but mutated C/EBPe cannot (Lee et al., gene were observed to cease the cellular growth and 2006), suggesting that C/EBPa and C/EBPe do not differentiate into monocytes by RA treatment concomi- actually share the same molecular pathway of differ- tantly with upregulation of C/EBPa and C/EBPe or by entiation. induction of C/EBPa or C/EBPe activity. The down- The crucial role of Myc in C/EBPs-induced monocytic regulation of Myc is crucial, and C/EBPe may therefore differentiation of HF-6 cells was also demonstrated in have an important function in C/EBPs-induced differ- this study. Myc induces transformation of hematopoie- entiation. This is the first report to show the monocytic tic cells cooperatively with MLL-ENL in vitro (Schreiner differentiation of myelomonocytic cells with MLL- et al., 2001). Myc inhibits the expression of C/EBPa by fusion gene induced by ectopic expression of C/EBPe bindingits promoter region(Freytagand Geddes, 1992). by itself, as well as C/EBPa. These findings may lead to In contrast, C/EBPa negatively regulates c-Myc through the development of novel C/EBPs-modulatingthera- the c-Myc promoter (Johansen et al., 2001). C/EBPe peutic approaches against AML with MLL-fusion also upregulates the Myc-antagonist Mad1 and down- genes. regulates c-Myc through repression of -mediated transcription (Gery et al., 2004; Walkley et al., 2004). Partial inhibition of C/EBPe-induced differentiation by Materials and methods c-Myc is also observed in 32D cells (Nakajima et al., 2006). These observations suggest that Myc contributes Reagents to leukemogenesis by MLL-fusion genes through All-trans RA, 9-cis RA and 4-HT (Sigma, St Louis, MO, USA) inhibition of C/EBPa expression, and that overexpres- were resuspended in ethanol. Puromycin and blasticidin-S sion of C/EBPs downregulates Myc and induces (Sigma) were diluted in distilled water. All the aliquots were 1 differentiation. The present findings that C/EBPs stored at À20 C. induced monocytic differentiation of HF-6 cells are consistent with the underlyingmechanism reported in Cell lines previous studies. The THP-1 and Kasumi cells were obtained from RIKEN BioResource Center (Ibaragi, Japan) and American Type However, in contrast to Myc, ectopic expression of Culture Collection (ATCC) (Manassas, VA, USA), respec- internal tandem duplication of FLT3 (FLT3-ITD) tively. MOLM-14 cells were provided from Cell Biology cannot inhibit C/EBPs-induced monocytic differentia- Institute, Research Center, Hayashibara Biochemical Labora- tion in HF-6 cells (H Matsushita et al., unpublished tories (Okayama, Japan). They were maintained in RPMI 1640 data). FLT3-ITD contributes to the cellular prolifera- supplemented with 10% fetal bovine serum, 1 U/ml penicillin tion of AML cells through several signal pathways G, 1 mg/ml streptomycin. HF-6 cells were maintained in the includingthe JAK/STAT, phosphoinositol 3-kinase and same growth medium supplemented with murine interleukin-3 MAPK pathways. These observations suggest that (10 ng/ml final concentration). The packaging cell line growth arrest and differentiation induction by C/EBPa PLAT-E, a generous gift from Dr Toshio Kitamura (Institute and C/EBPe are achieved through molecules functioning of Medical Science, University of Tokyo, Tokyo, Japan), was maintained in Dulbecco’s modified Eagle’s medium in the cell cycle rather than signals for cellular supplemented with 10% fetal bovine serum, 1 mg/ml puromy- proliferation related to FLT3-ITD. cin, 10 mg/ml blasticidin-S, 1 U/ml penicillin G and 1 mg/ml Human AML cells with MLL-fusion genes, MV4; streptomycin. All cells were cultured at 37 1C and 5% CO2. 11 and MOLM-14 cells, have also been reported to be differentiated by the induction of C/EBPa, however, Patient samples the committed cell lineage was granulocytic, but not Three cases of AML samples with 11q23 abnormalities were monocytic (Radomska et al., 2006). This difference of used (Supplementary Table 1). The AML cells were purified the committed cell lineage could be dependent on the from bone marrow or peripheral blood usingFicoll as methods used for determination of the lineage. They mononuclear cells, and were stored in a liquid nitrogen tank. evaluated only nitroblue tetrazolium reduction activity The studies were conducted accordingto the guidelinesof the in MV4;11 with C/EBPa, and their granulocytic changes revised Helsinki protocol, after informed consent from all were not typical and definite. It is sometimes difficult to patients’ parents was obtained. determine whether the lineage is monocytic or granulo- cytic in the differentiated cells from leukemic cells with Myeloperoxidase and esterase staining The Esterase stainingkit, Esterase AS-D stainingkit and New conventional methods includingcytochemical studies PO-K stainingkit (Muto Pure Chemicals, Tokyo, Japan) were and immunophenotyping, because the differentiation used for a-naphthyl butyrate, naphthol AS-D chloracetate and processes share the same mechanisms and phenotypes in myeloperoxidase staining, respectively, according to the various degrees. Especially, these cells are neither manufacturer’s protocol. normal granulocytes nor monocytes, and may not have the typical features of either of them. The other Plasmids possibility is that this discrepancy could be due to pMY-IRES-GFP/C/EBPa-ER and pMY-IRES-GFP/C/EBPe- differences in species or the developmental level, that is, ER were generated by ligating either the C/EBPa-ER

Oncogene Monocytic differentiation of MLL-AML by C/EBPs H Matsushita et al 6759 or C/EBPe-ER fragment into pMY-IRES-GFP, respectively 50- and 30-MLL probe (Supplementary Table 1). A clonogenic (Kitamura et al., 2003; Fukuchi et al., 2006; Nakajima et al., assay was performed as described previously (Kawada et al., 2006). Two murine Myc isoforms (582–1946 for Myc1 and 1999), with or without 10À6 M of ATRA or 9-cis RA. 627–1946 for Myc2 in NM_010849, respectively) were amplified L-CFU-G, L-CFU-GM and L-CFU-M from primary AML by RT–PCR and ligated into pMYpuro to generate pMYpuro- cells were counted on days 11–14. Myc1 and pMYpuro-Myc2 (Kitamura et al., 2003). Quantitative RT–PCR Transfection, retroviral production and infection Total RNA was extracted with Isogen (Nippon Gene, Tokyo, These procedures were described previously (Nakajima and Japan). cDNA was synthesized with SuperScript First-Strand Ihle, 2001). Synthesis System for RT–PCR (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed with SYBR Premix Assay for cell proliferation and apoptosis Ex Taq (Perfect Real Time) (Takara Bio, Shiga, Japan) in Cellular proliferation and apoptosis were analysed usingthe LightCycler ST300 (Roche Diagnostics, Indianapolis, IN, Cell CountingKit-8 (Dojindo Laboratories, Kumamoto, USA). The relative levels of gene expression were calculated Japan) and Annexin V-Biotin Kit (Beckman Coulter, Full- usingstandard curves generatedby the serial dilution of the erton, CA, USA), respectively. Streptavidin-allophycocyanin PCR products. The mRNA content was measured relative to (APC) (BD Pharmingen, San Diego, CA, USA) was used to that of murine Gapdh. All the samples were independently detect annexin V-biotin in an analysis of apoptosis. analysed at least three times for each gene. The primer pairs are shown in Supplementary Table 2. Flow cytometry The cells were preincubated in phosphate-buffered saline Immunoblotting supplemented with 0.2% human g-globulins (Sigma) for The procedures were described previously (Nakajima and Ihle, 15 min at 4 1C. They were then incubated with the monoclonal 2001). The applied primary antibodies included rabbit serum antibodies for 30 min at 4 1C. The applied monoclonal against C/EBPa (14AA), C/EBPe (C-22), -a antibodies included anti-PE-conjugated anti-mouse CD117 (ER) (MC-20) (Santa Cruz Biotechnology, Santa Cruz, CA, (c-Kit), CD11b (Mac-1), and Ly-6C (Gr-1), FITC-conjugated USA), green fluorescent protein (Invitrogen) and Myc anti-mouse CD34, Ly-6A/E (Sca-I) and Gr-1, APC-conjugated (Upstate, Lake Placid, NY, USA), and monoclonal antibodies anti-human CD34, PE-conjugated anti-human CD11b and against b-actin (AC-15) (Sigma). FITC-conjugated anti-human CD33 and CD36 (BD Pharmin- gen). An analysis was performed using FACSCaliber (Becton Dickinson, Franklin Lakes, NJ, USA). Acknowledgements

Clonogenic assay We thank Dr Toshio Kitamura (Institute of Medical Science, To delete normal hematopoietic stem cells and increase the University of Tokyo, Tokyo, Japan) for providingPLAT-E purity of AML cells, AML samples were sorted out using cells. This study was supported in part by Research and Study FACS/Vantage (Becton Dickinson) according to the expres- Program of Tokai University Educational System General sion of their surface marker. All of the primary AML samples Research Organization and a Grant-in Aid for Scientific did not express CD34 (Supplementary Table 1). The purity of Research (C) No. 19591139 from the Ministry of Education, AML progenitors was evaluated by a FISH analysis using Culture, Sports, Science and Technology of Japan.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene