Leukemia (2011) 25, 921–931 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE

CD52 as a molecular target for immunotherapy to treat acute myeloid leukemia with high EVI1 expression

Y Saito, S Nakahata, N Yamakawa, K Kaneda, E Ichihara, A Suekane and K Morishita

Department of Medical Science, Division of Tumor and Cellular Biochemistry, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

Ecotropic viral integration site 1 (EVI1) is an oncogenic Human CD52 is a glycosylphosphatidylinositol-anchored transcription factor in human acute myeloid leukemia (AML) antigen expressed on the cell surfaces of normal and malignant with chromosomal alterations at 3q26. Because a high expres- .9–11 Although the function of CD52 is largely sion of EVI1 in AML cells predicts resistance to unknown, CD52 is a favored target for therapy and chemotherapy with a poor outcome, we have searched for 12–15 molecular targets that will treat these patients with AML. In this immunosuppression before bone marrow transplantation. study, we determined that CD52, which is mainly expressed on (CAMPATH-1H; Genzyme, Cambridge, MA, lymphocytes, is highly expressed in most cases of AML with a USA) is a recombinant humanized High high EVI1 expression (EVI1 ). CAMPATH-1H, a humanized targeting the CD52 antigen.16,17 The binding of CAMPATH-1H monoclonal antibody against CD52, has been used to prevent to CD52 on lymphocytes induces complement-dependent graft-versus-host disease and treat CD52-positive lymphopro- cytotoxicity (CDC),18,19 antibody-dependent cell-mediated liferative disorders. Here, we investigated the antitumor effect 20–22 23,24 of CAMPATH-1H on EVI1High AML cells. CAMPATH-1H signifi- cytotoxicity (ADCC) and direct cytotoxicity. Here, we cantly inhibited cell growth and induced apoptosis in CD52- examined the cytotoxic effects of CAMPATH-1H on EVI1High positive EVI1High leukemia cells. Furthermore, CAMPATH-1H leukemia cells in vitro and the effects of CAMPATH-1H induced complement-dependent cytotoxicity and antibody- High treatment on tumor growth in immunodeficient NOD/Shi-scid/ dependent cellular cytotoxicity against CD52-positive EVI1 IL-2Rgnull (NOG) mice xenografted with EVI1High leukemia leukemia cells. After an intravenous injection of CAMPATH-1H into NOD/Shi-scid/IL-2Rc;null mice with subcutaneous engraft- cells. Because CAMPATH-1H showed a direct cytotoxic effect, ment of EVI1High leukemia cells, tumor growth rates were CDC and ADCC activity, as well as an in vivo antitumor effect significantly reduced, and the mice survived longer than those against EVI1High AML cells, CAMPATH-1H is likely to be a good in the phosphate-buffered saline-injected control group. Thus, therapeutic agent for AML with EVI1High expression. CAMPATH-1H is a potential therapeutic antibody for the treatment of patients with EVI1High leukemia. Leukemia (2011) 25, 921–931; doi:10.1038/leu.2011.36; published online 11 March 2011 Materials and methods Keywords: CD52; CAMPATH-1H; EVI1; acute myeloid leukemia Cell lines UCSD/AML1 (refs 25, 26) cells derived from human AML were cultured in RPMI 1640 medium (Wako, Osaka, Japan) supple- Introduction mented with 10% fetal bovine serum and 1 ng/ml granulocyte– macrophage colony-stimulating factor. Seven cell lines, Murine ecotropic viral integration site 1 (EVI1) was first identified U937 (ref. 27), K562 (ref. 28), KG-1 (ref. 29), HEL (ref. 30), as a leukemia-associated activated by murine retroviral HL60 (ref. 31), THP-1 (ref. 32) and HNT-34 (ref. 33), were 1,2 integration. A high expression of the human homologue EVI1 is purchased from the RIKEN Cell Bank (Tsukuba, Japan). MOLM1 found in 5–10% of patients with acute myeloid leukemia (AML), (ref. 34) was purchased from the Hayashibara Institute but 3q26 abnormalities near the EVI1 gene are only (Okayama, Japan). Kasumi-3 (refs. 35, 36) was kindly provided 3–5 detected in 1–3% of all AML cases. Because AML patients with by Dr Asoh (Hiroshima University, Hiroshima, Japan). K051 and High a high EVI1 expression (EVI1 ) had a significantly reduced K052 (ref. 37) were kindly provided by Dr Nomura (Nippon overall survival, overexpression of the EVI1 gene is thought to be Medical School, Tokyo, Japan). NH was kindly provided by Dr 6,7 a poor prognostic factor for AML patients. Patients with Suzukawa (University of Tsukuba, Tsukuba, Japan). FKH-1 (ref. 38) High EVI1 AML often do not respond to chemotherapy and and OIH-1 (ref. 39) were kindly provided by Dr Hamaguchi 8 hematopoietic cell transplantation; therefore, identifying novel (Musashino Red Cross Hospital, Tokyo, Japan). U937, K562, HL60, High molecular targets in EVI1 AML is of particular importance. In THP-1, K051, K052, NH, HNT-34, MOLM1 and Kasumi-3 were this study, we identified CD52 as a new surface molecule highly cultured in RPMI 1640 medium (Wako) supplemented with 10% High expressed on most EVI1 leukemia cells by DNA microarray, fetal bovine serum. The UCSD/AML1, MOLM-1, HNT-34 and reverse-transcription polymerase chain reaction (RT-PCR) and Kasumi-3 cell lines each have chromosome 3q26 abnormalities with flow cytometry analyses. an EVI1High, whereas U937, K562, KG-1, HEL, HL-60, THP-1, K051, K052, NH, FKH-1 and OIH-1 do not have 3q26 abnormalities and Correspondence: Dr K Morishita, Division of Tumor and Cellular show a low EVI1 expression (Supplementary Table 1). Biochemistry, Department of Medical Science, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan. E-mail: [email protected] Patient samples Received 11 October 2010; revised 15 December 2010; accepted 21 Leukemia cells were obtained from the peripheral blood (PB) of December 2010; published online 11 March 2011 AML patients who had a blast population of more than 80% in CD52 is a molecular target of AML with EVI1High expression Y Saito et al 922 their PB at diagnosis before chemotherapy (Supplementary 1 Â 105 U937 cells were plated in 96-well flat-bottomed plates and Table 2). After Ficoll–Hypaque (Sigma, Saint Louis, MO, USA) were infected with either an EVI1 retroviral supernatant separation of the PB, the purity of the blast cells was confirmed (pGCDMsam-EVI1-IRES-EGFP) or a mock retroviral supernatant by flow cytometry using immunofluorescence staining of (pGCDMsam-IRES-EGFP) with 100 ng/ml polybrene for 24 h. After phycoerythrin-conjugated CD11b and CD33 (Supplementary 7 days, green fluorescent protein-positive U937 cells were sorted Figure S2). The study was approved by the Institutional Review with a JSAN cell sorter (Bay Bioscience, Kobe, Japan). Board of the Faculty of Medicine, University of Miyazaki. Informed consent was obtained from all blood and tissue donors according to the Declaration of Helsinki. Establishment of stable UCSD/AML1 cell lines expressing shEVI1 A DNA-based small hairpin (sh) RNA expression vector Antibodies (pSIREN-retroQ-ZsGreen plasmid; Takara-Bio, Inc.) was used The human monoclonal antibody CAMPATH-1H that recog- in the EVI1 knockdown experiment. The following sequence nizes CD52 was obtained from Bayer Schering Pharma AG was cloned into the BamHI–EcoRI site of the plasmid to create (Berlin-Wedding, Germany). The antibody against caspase-3 an shRNA against human EVI1: (ref. 22) 50-GATCGCTCTAAGG was commercially obtained from Cell Signaling Technologies CTGAACTAGCAGTTCAAGAGACTGCTAGTTCAGCCTTAGATT (Beverly, MA, USA; catalog no. 9251). TTTTG-30. A pSIREN-retroQ-ZsGreen-shLuc plasmid containing shRNA against luciferase (Takara-Bio Inc.) was used as a control. Retroviral particles were generated using the p10A1 Oligonucleotide microarray packaging vector (Takara-Bio Inc.) and transient transfection of The protocol used for the sample preparation and microarray the 293T cell line, which was carried out with a Hilymax processing is available from Affymetrix (Santa Clara, CA, USA). liposome transfection reagent (Dojindo, Kumamoto, Japan). For Briefly, at least 5 mg of purified RNA was reverse transcribed retroviral infection, 1 Â 106 UCSD/AML1 cells were placed in using Superscript II reverse transcriptase (Life Technologies, 6 cm dishes containing 5 ml of retroviral supernatant with Grand Island, NY, USA) with the primer T7-dT24 containing a 100 ng/ml polybrene for 24 h. ZsGreen-positive UCSD/AML1 T7 RNA polymerase promoter. After a second strand of cDNA cells were sorted with a JSAN cell sorter (Bay Bioscience) was synthesized with RNase H, Escherichia coli DNA poly- 2 weeks after viral infection. The repression of EVI1 expression merase and E. coli DNA ligase, the cDNA was transcribed was confirmed by RT-PCR as described above. in vitro to produce biotin-labeled cRNA with a MEGAscript High Yield Transcription Kit (Ambion, Austin, TX, USA) as recommended by the manufacturer. After the cRNA was linearly Flow cytometry analysis amplified with T7 polymerase, the biotinylated cRNA was The cells were stained with the biotinylated CAMPATH-1H on cleaned with an RNeasy Mini Column Kit (Qiagen, Valencia, ice for 15 min. They were washed and then labeled with a CA, USA), fragmented to 50–200 nucleotides, and then phycoerythrin-labeled streptavidin antibody on ice for 15 min. hybridized to the Affymetrix U133 Plus 2.0 After washing, the treated cells were analyzed on a FACScan Array. The stained microarray was scanned with a GeneArray (Becton Dickinson, San Jose, CA, USA). Scanner (Affymetrix) and the intensity of the signal was calculated with Affymetrix software, Microarray Suite 5.0. All data were scaled with the global scaling method to adjust the Cell growth inhibition assay target intensity to 300. Then, we chose those with at least Leukemia cells (2 Â 105 cells per ml) were incubated with a 10-fold increase in the expression of the EVI1High AML cells various concentrations of CAMPATH-1H in complete medium Low compared with low levels of EVI1 (EVI1 ) AML cells with a at 37 1Cin5%CO2. The cell growth was evaluated with the statistical significance of P less than 0.01 by the t-test. Trypan blue exclusion assay. The live cells were enumerated after Trypan blue staining using microscopy. Reverse-transcription polymerase chain reaction The levels of CD52, EVI1 and b-actin mRNA in the AML cells Apoptosis assay were measured by RT-PCR. Briefly, total RNA was extracted Apoptosis assays were performed using the Apoptosis Detection using Trizol (Invitrogen, Carlsbad, CA, USA), and 1 mg of total (MBL, Nagoya, Japan) according to the manufacturer’s RNA was reverse-transcribed to obtain first-strand cDNA using protocol. UCSD/AML1, HNT-34 or PT8 cells (2 Â 105 cells per an RNA-PCR kit (Takara-Bio Inc., Tokyo, Japan). cDNA ml) were incubated with CAMPATH-1H (10 mg/ml) in complete fragments were amplied by PCR using specic primers. The medium at 37 1Cin5%CO2 for 48 h. The cells were washed primers used were as follows: CD52 forward, 50-CATCAGCCTC and resuspended in binding buffer at a concentration of 1 Â 106 CTGGTTATGG-30, reverse, 50-AAATGCCTCCGCTTATGTTG-30; cells per ml. The suspension was then incubated with 5 mlof EVI1 forward, 50-CACATTCGCTCTCAGCATGT-30, reverse, Annexin-V and 5 ml of propidium iodide for 15 min at room 50-ATTTGGGTTCTGCAATCAGC-30; and b-actin forward, 50-G temperature in the dark. Samples were analyzed using a ACAGGATGCAGAAGGAGATTACT-30, reverse, 50-TGATCCA FACScan (Becton Dickinson). Annexin-V-positive cells were CATCTGCTGGAAGGT-30. considered apoptotic.

Establishment of stable U937 cell lines expressing EVI1 Cytotoxicity assays pGCDMsam-EVI1-IRES-EGFP was kindly provided by The CDC and ADCC activities of CAMPATH-1H were measured Dr A Iwama (Chiba University, Chiba, Japan). To produce by a lactate dehydrogenase (LDH)-releasing assay using a recombinant retrovirus, the plasmid DNA was transfected into cytotoxicity detection kit (Roche, Indianapolis, IN, USA). 293gp cells along with the vesicular stomatitis virus G expression EVI1High AML cells (1 Â 104) were incubated with various plasmid by CaPO4 co-precipitation. For retroviral transduction, concentrations of CAMPATH-1H and human serum as the

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 923 source of complement at a dilution of 1:6 (for the CDC assay) or with medium and complement alone) and M is the maximum with human PB mononuclear cells as effector cells (effector: release of target cells (activity released from target cells lysed target, 50:1 for the ADCC assay) in supplemented RPMI 1640 at with 2% Triton X-100); ADCC % specific lysis ¼ 100 Â 37 1C in 96-well flat-bottomed plates. After an additional (EÀSEÀST)/(MÀSE), where E is the experimental release (super- incubation for 4 h at 37 1C, the extent of cell lysis was natant activity from target cells incubated with antibody and determined by measuring the amount of LDH released into effector cells), SE is the spontaneous release in the presence of the culture supernatant. The maximum LDH release was effector cells with antibody (supernatant activity from target determined for cells lysed with 2% Triton X-100. The percentage cells incubated with effector cells), ST is the spontaneous release of specific lysis was calculated according to the following of target cells (supernatant activity from target cells incubated formula: CDC % specific lysis ¼ 100 Â (EÀS)/(MÀS), where with medium alone) and M is the maximum release of E is the absorbance of the experimental well, S is the absorbance target cells (activity released from target cells lysed with 2% in the absence of monoclonal antibody (cells were incubated Triton X-100).

Figure 1 CD52 is highly expressed in EVI1High myeloid leukemia cells. (a) The expression profiles of CD52 mRNA by DNA microarray. RNA samples from eight EVI1Low and four EVI1High AML cell lines were used for determining the gene expression profiles. (b) Semiquantitative RT-PCR analysis for EVI1 and CD52 is shown for 11 EVI1Low and four EVI1High AML cell lines (top panel), as well as primary AML cells from three patients with EVI1Low and from nine patients with EVI1High expression (bottom panel). The expression of b-actin is shown at the bottom as a control.

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 924 Xenograft tumors Results Six- to eight-week-old female NOG mice were given a single subcutaneous injection of 5 Â 106 UCSD/AML1 cells suspended EVI1High myeloid leukemia cells express high levels of in 100 ml phosphate-buffered saline (PBS) and mixed with an CD52 equal volume of Matrigel (BD Matrigel, BD Biosciences, To search for novel molecular targets in refractory myeloid Bedford, MA, USA). Xenografts were allowed to establish to leukemia with EVI1High, we initially analyzed the gene an average size of 50–100 mm3, after which mice were expression profiles of 12 human myeloid cell lines using an randomized into two groups. The groups of mice were given oligonucleotide microarray (Human Genome U133 Plus 2. 0 either PBS or CAMPATH-1H at a dose of 100 mg weekly Array; Affymetrix) containing 38 500 genes. Four cell lines with intravenously for 4 weeks. Tumor volumes were derived as the chromosome 3q26 abnormalities (UCSD/AML1, HNT-34, product of the length, width and height of the tumor measured Kasumi-3 and MOLM-1) expressed EVI1High, and eight myeloid once a week with a caliper. A Kaplan–Meier survival analysis cell lines without chromosome 3q26 abnormalities (HEL, was performed using StatView (SAS Institute, Cary, NC, USA). HL-60, K052, THP-1, FKH-1, K051, NH and OIH-1) expressed EVI1Low (Figure 1a). When the expression profiles between EVI1High and EVI1Low leukemia cell lines were compared, we Data analysis detected 26 genes that were upregulated over 10-fold in A faculty statistician analyzed the data. P-values were calcu- EVI1High leukemia cells when compared with EVI1Low leukemia lated using the Student’s t-test for a comparison of independent cells (Po0.01) (Table 1). Among these genes, nine genes encode data sets. Differences were considered statistically significant if membrane containing extracellular domains. These the P-value was less than 0.05. genes include the CD52 antigen (CAMPATH-1 antigen).

Table 1 Highly expressed genes in EVI1High AML cells compared with EVI1Low AML cells

Gene symbol Description Fold change Localization

EVA1 Epithelial V-like antigen 1 54.98 Membrane/extracellular MMRN1 Multimerin 1 49.34 Membrane/extracellular LOC284262 Hypothetical protein LOC284262 47.46 Unknown EHD2 EH-domain containing 2 44.45 Membrane/nucleus SETBP1 SET-binding protein 1 40.48 Nucleus CALCRL Calcitonin receptor-like 38.95 Membrane/extracellular CLEC7A C-type lectin domain family 7, member A 32.33 Cytoplasm/membrane PTPRM Protein tyrosine phosphatase, receptor type, M 26.07 Membrane/extracellular LCK -specific protein tyrosine kinase 24.61 Kinase/cytoplasm ITGA6 Integrin, alpha 6 23.83 Membrane/extracellular CD52 CD52 antigen (CAMPATH-1 antigen) 19.35 Membrane/extracellular DEPDC2 DEP domain containing 2 18.45 Intracellular PTRF Polymerase I and transcript release factor 17.28 Intracellular S100Z S100 calcium-binding protein, zeta 16.21 Intracellular RASGEF1B RasGEF domain family, member 1B 14.88 Intracellular GPR56 G-protein-coupled receptor 56 13.98 Membrane/extracellular PHLDA2 Pleckstrin homology-like domain, family A, member 2 13.88 Intracellular CD300A CD300A antigen 13.08 Membrane/extracellular TRPS1 Trichorhinophalangeal syndrome I 12.55 Nucleus DPP4 Dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2) 12.07 Membrane/cytoplasm TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8 11.89 Membrane/extracellular FLJ11996 Hypothetical protein FLJ11996 11.48 Unknown GNGT2 Guanine nucleotide-binding protein (G protein), gamma-transducing 11.03 Membrane/cytoplasm activity polypeptide 2 MFAP3L Microfibrillar-associated protein 3-like 10.98 Membrane/extracellular SYTL4 Synaptotagmin-like 4 (granuphilin-a) 10.27 Secretion Abbreviations: AML, acute myeloid leukemia; EVI1, ecotropic viral integration site 1. A list of genes that were selected by expression levels greater than 100 and an over 10-fold increased expression in EVI1High AML cells than that in EVI1Low AML cells with significant differences (Po0.01). Fold changes were calculated by the mean expression values of EVI1High AML cells compared with EVI1Low AML cells.

Figure 2 EVI1-dependent expression of CD52 in AML cells. (a) The expression of CD52 on various leukemic cells by flow cytometry using CAMPATH-1H. Cells were stained with phycoerythrin-labeled CAMPATH-1H, followed by analysis in an FACS. Figures show representative FACS histogram profiles of normal lymphocytes (PBL) and B lymphoid leukemia cell lines (BALL1 and RAMOS) in the upper panel, EVI1Low AML cell lines (U937, NH and K562) in the second panel, EVI1High AML cell lines (UCSD/AML1, HNT-34, Kasumi-3 and MOLM1) in the third panel and primary leukemic cells from patients with EVI1Low (PT1) or EVI1High (PT8, PT9 and PT11) AML in the bottom panel. Open histograms represent cells stained with isotype IgG controls and filled histograms indicate cells stained with CAMPATH-1H. (b, c) The induction of CD52 expression by a forced expression of EVI1 in U937 with EVI1Low expression. Panel b shows the expression level of CD52 in parental, mock or EVI1 transfectant U937 cells by RT-PCR, and panel c shows the CD52 expression by FACS analysis. (d, e) The introduction of the shEVI1 expression vector in UCSD/ AML1 cells with EVI1High expression decreases CD52 expression. Panel d shows the CD52 mRNA level in parental, control shLuc or shEVI1 transfectant UCSD/AML1 cells by RT-PCR, and panel e shows the CD52 expression by FACS analysis.

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 925

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 926 A microarray analysis of the samples from the EVI1High AML Because a humanized anti-CD52 antibody, CAMPATH-1H, has cells versus the EVI1Low AML cells confirmed upregulation of already been developed for immunosuppression before bone CD52 in the EVI1High AML cells (Supplementary Figure S1). marrow transplantation and for gene- for

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 927 lymphoproliferative disorders, we further analyzed whether the (Figure 2d). FACS analysis showed that the percentage of CD52- CD52 antigen is a novel target in EVI1High AML. positive cells in AML1/shEVI1 cells was considerably lower In the next experiment, CD52 expression was examined in 15 (24.0%) than that in AML1/shLuc (45.1%) cells with decreased myeloid leukemia cell lines and primary leukemia cells from 12 fluorescence (Figure 2e). Thus, the CD52 expression in myeloid patients with AML using RT-PCR. We included three additional leukemic cells is partly dependent on the expression of EVI1, cell lines without chromosome 3q26 abnormalities (U937, K562 and CD52 may be one of the downstream target genes regulated and KG-1) in the analysis. The CD52 expression levels in four directly or indirectly by EVI1. cell lines with EVI1High expression (UCSD/AML1, HNT-34, Kasumi-3 and MOLM-1) were higher than that in the other 11 cell lines with EVI1Low expression (Figure 1b, top). In primary CAMPATH-1H induced a direct cytotoxic effect on High leukemia cells, EVI1 and CD52 were more highly expressed in EVI1 AML cells nine patients with chromosome 3q26 abnormalities than that in Because CAMPATH-1H was highly reactive with the surface of High three patients without chromosome 3q26 abnormalities EVI1 AML cells, it may be a good therapeutic candidate for (Figure 1b, bottom). Thus, CD52 may be a potential marker EVI1-positive AML. We initially tested the growth inhibitory and Low for EVI1High myeloid leukemia cells. direct cytotoxic effects of CAMPATH-1H on an EVI1 AML cell line(K562)andtwoEVI1High AML cell lines (UCSD/AML1 and HNT-34) with two B-ALL cell lines (BALL1 and RAMOS) as controls. CAMPATH-1H identifies the CD52 antigen on the After the cells were incubated with various concentrations of High surface of EVI1 myeloid leukemia cells CAMPATH-1H for 48 h, the cell viability was evaluated by Trypan Next, we determined the surface expression of the CD52 antigen blue staining (Figure 3a). Cell growth was inhibited in the three AML by FACS analysis using phycoerythrin-labeled CAMPATH-1H cell lines with EVI1High expression in a dose-dependent manner, and (Figure 2a). We used control PB lymphocytes from a healthy 51.5–78.9% of cells remained viable at 10 mg/ml CAMPATH-1H. In volunteer and two B lymphoid leukemia cell lines (BALL1 and contrast, over 90% of B-ALL cells were viable at this drug RAMOS) as positive controls and 63.6–89.7% of these cells concentration. In the next experiment, primary AML cells from a Low were labeled by CAMPATH-1H. In EVI1 myeloid leukemia patient with EVI1Low expression (PT1) and from three patients with cell lines (U937, NH and K562) and primary AML cells from a EVI1High expression (PT8, PT9 and PT11) were used to test the direct Low patient with EVI1 (PT1), 2.4–10.4% of the cells were labeled cytotoxic effect of CAMPATH-1H. Over 90% of PT1 cells were by CAMPATH-1H with a low signal intensity. In contrast, viable at all drug concentrations; however, only 52.5–57.5% of High 56.4–98.7% of EVI1 myeloid leukemic cells (UCSD/AML1, EVI1High AML cells from patients (PT8, PT9 and PT11) survived the HNT-34, Kasumi-3 and MOLM1) were robustly labeled by maximal dose of 100 mg/ml, suggesting that CAMPATH-1H directly CAMPATH-1H, yielding higher signal intensity than BALL1 and suppressed the survival of EVI1High AML cells. RAMOS cells. Moreover, CAMPATH-1H bound to the majority To confirm whether the cytotoxic effect of CAMPATH-1H is of leukemia cells from three AML patients with inv(3)(q21q26) dependent on apoptotic cell death, we treated two EVI1High (PT8 and PT9) or with t(3;21)(q26;q22) (PT11) at a high signal AML cell lines (UCSD/AML1 and HNT-34) and primary EVI1High High intensity similar to EVI1 cell lines. AML cells (PT8) with 10 mg/ml of CAMPATH-1H for 4 days and To determine whether the EVI1 transcription factor activates determined the cell viability and apoptosis. In all of the EVI1High CD52 gene expression, we introduced an EVI1 expression AML cells, the growth rate of CAMPATH-1H-treated cells was Low vector into an EVI1 leukemia cell line, U937, and an shRNA slower than that of control immunoglobulin G (IgG)-treated cells High targeting EVI1 (shEVI1) into an EVI1 leukemia cell line, (Figures 3b, d and f) and 15.5–40.1% of EVI1High AML cells UCSD/AML1. After introducing the EVI1 expression vector into underwent apoptosis 2 days after treatment (Figures 3c, d and f). U937 cells, the expression of EVI1 was clearly detected in Moreover, the amount of cleaved caspase-3 (18 kDa) was U937/EVI1 cells by RT-PCR, and the level of CD52 mRNA was increased in the EVI1High leukemic cells after 2 days of also increased in U937/EVI1 cells in comparison with parental CAMPATH-1H treatment (Figure 3h). Thus, our results indicate or mock vector-transfected cells (Figure 2b). FACS analysis that CAMPATH-1H effectively induces apoptosis in EVI1High showed that the population of CD52-positive cells was AML cells. increased (61.1%) in U937/EVI1 cells in comparison to U937/ mock cells (19.3%), with increased fluorescence intensity (Figure 2c). Moreover, after the introduction of shEVI1 or CAMPATH-1H-mediated CDC and ADCC against shRNA against luciferase (shLuc) as a control into UCSD/AML1 EVI1High AML cells cells, the expression level of CD52 mRNA was decreased in Because CAMPATH-1H exerts its antitumor effects on lympho- UCSD/AML1/shEVI1 cells, along with a decreased expression of cytes or lymphoid leukemia cells through immunological EVI1, when compared with parental or UCSD/AML1/shLuc cells mechanisms, such as CDC and/or ADCC, by virtue of its

Figure 3 Induction of direct cytotoxicity against EVI1High AML cells by CAMPATH-1H treatment. (a) Viable cells after treatment with the indicated concentration of CAMPATH-1H for 48 h were examined using Trypan blue dye exclusion and are shown as the percentages of the values obtained from the untreated parental cells. The left panel shows the percent viability of cell lines with EVI1Low expression (K562, RAMOS and BALL1; dot lines with open marks) and with EVI1High expression (UCSD/AML1 and HNT34; solid lines with closed marks). The right panel shows the percent viability of primary AML cells with EVI1Low expression (PT1; dot lines with open marks) and with EVI1High expression (PT8, PT9 and PT11; solid lines with closed marks). The experiments were performed in triplicate and repeated independently at least three times. (b, d, f) Viable cell numbers of EVI1High AML cells (UCSD/AML1, HNT-34 and PT8) were shown at the indicated time points after treatment with 10 mg/ml of CAMPATH-1H or with control IgG. Student’s t-test (Po0.05) was used for the statistical analysis between the control IgG- and CAMPATH-1H- treated AML cells. (c, e, g) CAMPATH-1H treatment induces the apoptosis of EVI1High AML cells. Following treatment with CAMPATH-1H for 48 h, cells were labeled with Annexin-V and propidium iodide, and the percent of apoptotic cells was determined using flow cytometry. The experiments were performed in triplicate and repeated independently at least three times. (h) Identification of cleaved caspase-3 after the treatment with CAMPATH-1H against EVI1High AML. EVI1High AML cell lines (UCSD/AML1 and HNT-34) were treated with CAMPATH-1H (10 mg/ml) for 48 h, and western blot analyses were performed by anti-caspase-3 antibody. Asterisks indicate nonspecific bands.

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 928 IgG Fc region, we initially investigated the CDC activity of death was observed in the PT1 and PT8 cells, but a moderate CAMPATH-1H on EVI1High AML cell lines (UCSD/AML1 and CDC effect was observed using the PT-9 and PT11 cells HNT-34) along with EVI1Low AML (K562) and B-ALL cell lines (Figure 4a, right panel). Thus, CAMPATH-1H does not appear (BALL1 and RAMOS) as controls. CAMPATH-1H treatment to have a significant CDC effect on EVI1High AML cells. induced a slight increase in cell death in the two B-ALL and two Next, the ADCC activity of CAMPATH-1H was examined EVI1High AML cell lines, although it had no effect on the EVI1Low against the same cell lines and primary AML cells as those in the K562 cell line (Figure 4a, left panel). Next, primary AML cells CDC. In the two B-ALL and two EVI1High leukemia cell lines, from a patient with EVI1Low (PT1) and from three patients CAMPATH-1H significantly increased the percentages of cell with EVI1High expression (PT8, PT9 and PT11) were used to test death in a dose-dependent manner, whereas EVI1Low K562 cells the effect of CAMPATH-1H on CDC. Only a low level of cell showed almost no response (Figure 4b, left panel). Treatment

Figure 4 The induction of CDC and ADCC activities by the treatment of CAMPATH-1H and in vivo antitumor activity against EVI1High AML cells in mouse xenograft models. (a) CAMPATH-1H-mediated CDC activity in various leukemia cells. The left panel shows the percent viability of cell lines with EVI1Low expression (K562, RAMOS and BALL1; dot lines with open marks) and cell lines with EVI1High expression (UCSD/AML1 and HNT34; solid lines with closed marks). The right panel shows the percent viability of primary AML cells with EVI1Low expression (PT1; dot lines with open marks) and AML cells with EVI1High expression (PT8, PT9 and PT11; solid lines with closed marks). After the cells were incubated with human serum complement and CAMPATH-1H at concentrations from 0.1 to 100 mg/ml, the extent of cell lysis was measured with an LDH- releasing assay and is shown as the percentage of the value obtained from the untreated parental cells. The experiments were repeated independently at least three times. (b) CAMPATH-1H-mediated ADCC activity in various leukemia cells. ADCC assays were performed with the same series of cells used in (a). After incubation of the cells with PB mononuclear cells from a normal donor and 0.1–100 mg/ml of CAMPATH-1H, ADCC activity was measured by LDH release. The results are the mean and standard deviations for each sample, which was independently repeated in triplicate. (c) CAMPATH-1H reduces tumor growth in NOG mice xenografted with EVI1High AML cells. CAMPATH-1H was administered at 4 mg/kg intravenously weekly for 4 weeks. Tumor growth was assessed by measuring the volume of each tumor at weekly intervals. Each group contained five mice. The graphs depict the average tumor volume for the PBS control group and the CAMPATH-1H-treated group±standard error (d) Kaplan–Meier survival plot of UCSD/AML1-bearing NOG mice. CAMPATH-1H was administered at 4 mg/kg intravenously weekly for 4 weeks. Kaplan–Meier survival curves are shown for PBS- and CAMPATH-1H-treated groups (n ¼ 5 per group).

Leukemia CD52 is a molecular target of AML with EVI1High expression Y Saito et al 929 with CAMPATH-1H also resulted in a significant increase in cell EVI1High expression displayed chromosomal abnormalities death in primary cells from three patients with EVI1High AML involving chromosome 7, such as t(3;7)(q26;q21), partial (PT8, PT9 and PT11), and no response was observed in the case deletion of chromosome 7 or monosomy 7. Patients with of the EVI1Low PT1 cells (Figure 4b, right panel). These results EVI1High AML exhibit specific clinical features, including suggest that CAMPATH-1H exerts a significantly stronger ADCC elevated platelet counts, association with monosomy 7 or effect than a CDC effect against EVI1High AML. chromosome 7 deletion, refractoriness to therapy and a poor prognosis.43 It has been suggested that the genomic instability and myelodysplasia with monosomy 7 occur as a consequence In vivo antitumor effect of CAMPATH against EVI1High of EVI1 activation after gene therapy for chronic granulomatous AML in a xenograft model disease.44 It seems likely that EVI1 and gene(s) on chromosome To investigate the effects of CAMPATH-1H on EVI1High AML 7 play a crucial role in regulating CD52 expression. We have in vivo, USCD/AML1 cells with EVI1High expression were previously shown that EVI1 is expressed on bone marrow stem subcutaneously inoculated into immunodeficient NOG mice. and progenitor cells and has an important function in maintain- After tumors reached a size of 50–100 mm3, 100 mgof ing hematopoietic stem cells.45 It is, therefore, tempting to CAMPATH-1H or PBS as a control was intravenously injected speculate that specific populations may express CD52 once a week for 4 weeks, and tumor growth and mouse survival along with EVI1. Future studies should examine the expression were monitored (Figures 4c and d). CAMPATH-1H significantly of CD52 on hematopoietic stem cells and its relevance to inhibited the tumor growth of UCSD/AML1 cells when leukemia development. It is also important to determine compared with the control (Po0.05) (Figure 4c). The median whether EVI1 directly activates the CD52 promoter. survival time of control mice was 75 days, and all control mice Although the mechanism of cytotoxicity of CAMPATH-1H is died within 100 days. In contrast, the median survival was not well understood, CAMPATH-1H had minimal direct prolonged to 127 days in mice injected with CAMPATH-1H, cytotoxicity, but exhibited significant CDC against chronic and four out of five mice were still alive more than 100 days lymphocytic leukemia and acute lymphocytic leukemia after inoculation. Taken together, these data suggest that cells.18,19 In our experiments, CAMPATH-1H lysed EVI1High CAMPATH-1H is a promising candidate therapeutic antibody AML cells more efficiently through direct cytotoxicity or ADCC for AML patients with EVI1High expression. than through CDC (Figures 3 and 4). CD52 is a 12-amino-acid glycopolypeptide containing a large glucose moiety, and CAMPATH-1H recognizes C-terminal amino acids and part of Discussion the glycosylphosphatidylinositol anchor of CD52.46,47 The amount of protein and/or post-translational modifications, such In this study, we found that CD52 was highly expressed in most as glycosylation, may be involved in differential immune EVI1High leukemia cells. Using the humanized anti-CD52 responses to CAMPATH-1H. monoclonal antibody CAMPATH-1H, we showed that CAM- We showed that EVI1High AML cells are good targets for the PATH-1H inhibited cell growth and induced apoptosis of cytolytic activity of CAMPATH-1H. In the standard schedule of EVI1High leukemia cells through direct cytotoxicity and/or CAMPATH-1H for allogeneic stem cell transplantation, the ADCC in vitro. Moreover, we showed the therapeutic efficacy treatment is not always efficient because the antibody adsorbs of CAMPATH-1H in an in vivo model of EVI1High leukemia onto T lymphocytes. Therefore, combinations of CAMPATH-1H using NOG mice. Although NOG mice lack functional T and with active chemotherapeutic agents such as daunorubicin, natural killer cells, murine neutrophils were considered to be etoposide and cytarabine need to be explored in future studies. effector cells mediating ADCC as well as direct cytotoxicity in Finally, these studies provide support for a clinical trial of our mouse model. Indeed, CAMPATH-1H efficiently inhibited CAMPATH-1H in patients with EVI1High AML and potentially in the growth of leukemia xenografts in an EVI1High AML xenograft patients with other AMLs that express CD52. model. These data suggest that the use of CAMPATH-1H may be effective in treating myeloid leukemia with EVI1High. Conflict of interest CD52 is a small glycosylphosphatidylinositol-linked protein of unknown function. CAMPATH-1H (Alemtuzumab), a huma- The authors declare no conflict of interest. nized antibody against CD52, is used to deplete leukemia cells from patients with relapsed/refractory chronic lymphocytic 15 leukemia. Recent studies have suggested the effectiveness of Acknowledgements CAMPATH-1H treatment in additional hematopoietic malig- nancies, including peripheral T-cell lymphoma, T-cell prolym- We gratefully acknowledge Genzyme for providing the 40–42 phocytic leukemia and cutaneous T-cell lymphoma. CAMPATH-1H antibody for the study. 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