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`NEOPLASIA
`
`Expression of tumor necrosis factor–related apoptosis-inducing ligand, Apo2L,
`and its receptors in myelodysplastic syndrome: effects on in vitro hemopoiesis
`Dae Young Zang, Ray G. Goodwin, Michael R. Loken, Eileen Bryant, and H. Joachim Deeg
`
`Tumor necrosis factor–related apoptosis-
`inducing ligand (TRAIL), a member of the
`tumor necrosis factor (TNF) family, binds
`to several cell-surface receptors with dis-
`tinct functions (agonistic receptors 1 and
`2 [TRAIL-R1, TRAIL-R2]; decoy receptors
`3 and 4 [TRAIL-R3, TRAIL-R4]). Expres-
`sion and function was characterized in
`patients with myelodysplastic syndromes
`(MDSs). While normal marrow showed
`negligible expression of TRAIL and recep-
`tors (except TRAIL-R3), TRAIL and all
`receptors were constitutively expressed
`Introduction
`
`in MDS marrow. Following TRAIL expo-
`sure, MDS marrow showed significant
`increases in apoptosis, whereas normal
`marrow, except for a subset of CD341
`precursors, did not (P5 .012). Marrow
`from 21 patients with MDS was then
`propagated in long-term cultures in the
`presence or absence of TRAIL. While in
`advanced MDS (refractory anemia with
`excess blasts in transformation [RAEB-T]
`and tAML [MDS transformed into AML]),
`colony numbers decreased in the presence
`of TRAIL (63.0% 6 10.4% of untreated group
`
`[100%]), numbers increased in patients with
`RA or RAEB (160.2% 6 90.5% of untreated
`group). TRAIL eliminated preferentially
`clonally abnormal cells as identified by
`chromosomal markers. Thus, TRAIL and
`receptor expression differed significantly
`between normal and MDS marrow, and
`TRAIL modulated in vitro hemopoiesis in
`MDS dependent upon disease stage but
`not, to a detectable extent,
`in normal
`marrow. (Blood. 2001;98:3058-3065)
`
`© 2001 by The American Society of Hematology
`
`The myelodysplastic syndromes (MDSs) comprise a heteroge-
`neous group of hemopoietic stem cell disorders usually character-
`ized by a cellular marrow with dysplastic features, peripheral blood
`cytopenias, and a variable incidence of transformation into acute
`myeloid leukemia (AML).1 MDS generally occurs in elderly
`the median age at diagnosis being 65 to 70 years.2
`patients,
`Allogeneic hemopoietic stem cell
`transplantation is currently
`considered the only curative treatment modality for MDS; how-
`ever, age and performance status of patients and lack of histocom-
`patible donors limit
`the applicability of this treatment. Also,
`regimen-related toxicity is a major problem in patients with MDS
`undergoing transplantation.3,4 It would be desirable, therefore, to
`develop new therapeutic strategies to improve or restore normal
`hemopoietic function.
`One mechanism invoked to explain the apparent discrepancy
`between cellular marrow and peripheral blood cytopenias in
`patients with MDS is programmed cell death (apoptosis), which
`occurs with increased frequency in MDS marrow.5,6 Several
`cytokines or ligands known to have proapoptotic properties, such
`as interleukin-1b (IL-1b), tumor necrosis factor a (TNFa), and
`Fas-ligand are up-regulated in many patients with MDS.5,7-9 We
`and others have shown that blockade of TNFa or Fas-ligand
`enhances hemopoietic colony transformation from MDS marrow in
`vitro and improves blood cell counts in vivo.5,7,10 However, it is
`clear from those studies that the regulation of hemopoiesis in MDS
`is complex and multiple factors are involved. The TNF-related
`apoptosis-inducing ligand (TRAIL), also known as Apo2 ligand
`
`(Apo2L), is a member of the TNF family and induces apoptosis
`preferentially in transformed and tumor cells but generally not in
`normal cells.11-13 However, these observations must be extrapolated
`with caution because TRAIL effects on normal cells, for example,
`in the brain, have been documented.14 In MDS marrow, normal
`precursors and clonally abnormal cells typically coexist. If expo-
`sure to TRAIL resulted in selective elimination of the abnormal
`clone, TRAIL might be a useful
`therapeutic agent for MDS,
`certainly for marrow purging (and autologous stem cell transplanta-
`tion) if not for in vivo treatment. The exact mechanism by which
`TRAIL eliminates tumor cells preferentially or selectively is not
`known. One possibility is differential expression of agonistic
`receptors 1 and 2 (TRAIL-R1 and -R2, also known as death
`receptors 4 [DR4] and DR5) and antagonistic or modulatory
`receptors, TRAIL-R3 and -R4, also known as decoy receptors 1
`(DcR1) and DcR2.15,16 An alternative mechanism would be differ-
`ential expression of, or functional defects in, cytoplasmatic inhibi-
`tors of apoptosis such as the Fas-associated death domain protein
`(FADD)17 or FADD-like IL-1b–converting enzyme (ICE)–
`inhibitory protein (FLIP).18
`The objective was to exploit the potential selective killing of
`clonal and, presumably, malignant cells by human TRAIL.
`Thus, we determined the effect of TRAIL on apoptosis and
`hemopoiesis in long-term marrow cultures (LTMC) of normal
`and MDS marrow, and characterized the expression of TRAIL
`and its receptors. We also characterized the expression of FLIP
`in marrow cells from patients with MDS and healthy donors.
`
`From the Clinical Research Division, Fred Hutchinson Cancer Research
`Center and the Department of Medicine, University of Washington; Immunex;
`and Hematologics, Seattle, WA.
`
`Supported by PHS grants HL36444, CA18029 and CA87948. H.J.D. is also
`supported by a grant from the Gabrielle Rich Leukemia Fund.
`
`Submitted March 14, 2001; accepted July 6, 2001.
`
`ligand with Genentech.
`
`Reprints: H. Joachim Deeg, Fred Hutchinson Cancer Research Center, 1100
`Fairview Ave N, D1-100, PO Box 19024, Seattle, WA 98109-1024; e-mail:
`jdeeg@fhcrc.org.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`Ray G. Goodwin owns stock in Immunex, which is codeveloping TRAIL/Apo2
`
`© 2001 by The American Society of Hematology
`
`3058
`
`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
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`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`TRAIL AND APOPTOSIS IN MDS
`
`3059
`
`Materials and methods
`
`Marrow cells
`
`Bone marrow (BM) samples were obtained from 44 patients with MDS
`(refractory anemia [RA], n 5 20; RA with ring sideroblasts [RARS], n 5 4;
`RA with excess blasts [RAEB], n 5 9; RAEB in transformation [RAEB-T],
`n 5 9; chronic myelomonocytic leukemia [CMML], n 5 2), 6 patients
`whose disease had transformed to AML, and 20 healthy volunteer donors.
`Patients and healthy donors had given informed consent according to the
`procedures approved by the Institutional Review Board of the Fred
`Hutchinson Cancer Research Center (FHCRC). Samples were processed as
`described19,20 for in vitro hemopoiesis, phenotyping, characterization of
`apoptosis, RNA extraction, and cytogenetic analysis with fluorescence in
`situ hybridization (FISH).
`
`Cell lines
`
`Human acute myelogenous leukemia cell line KG1, acute T-cell leukemia
`cell line Jurkat, and human colon cancer cell line Colo 205 were obtained
`from American Type Culture Collection (Rockville, MD).
`
`Antibodies and reagents
`
`The following monoclonal antibodies (mAbs) were obtained from commer-
`cial sources: fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-
`conjugated mouse anti–human CD3 (IgG2a), CD13 (IgG1), CD14 (IgG2a),
`CD34 (HPLC-2, IgG1), and CD45 (IgG1) (Becton Dickinson, San Jose,
`CA, or Caltag, San Francisco, CA); FITC or PE-conjugated mouse F(ab9)2
`IgG1 or IgG2a or IgG2b (Becton Dickinson) or mouse IgM (Catalag) as
`isotype controls; FITC or PE-conjugated goat F(ab9)2 anti–mouse IgG
`(H1L) or IgM (Caltag) as secondary antibodies; rabbit anti–mouse IgG to
`carboxy terminus human FLIP long form for Western blot (Calbiochem, La
`Jolla, CA). The mouse anti–human IgM STRO-1 mAb, which recognizes a
`stromal cell surface antigen,21 was obtained from Developmental Studies
`Hybridoma Bank (Iowa City, IA). The monoclonal mouse anti–human
`TRAIL (IgG1, M180, blocking; M184, nonblocking), TRAIL-R1 (IgG2a,
`M271), TRAIL-R2 (IgG1, M413), TRAIL-R3 (IgG1, M430), TRAIL-R4
`(IgG1, M444), as well as stem cell factor (SCF), and leucine zipper
`(LZ)–tagged soluble trimeric human TRAIL were supplied by Immunex
`(Seattle, WA). IL-1, IL-3, IL-6, granulocyte colony-stimulating factor
`(G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-
`CSF) were purchased from R & DSystems (Minneapolis, MN).
`
`Phenotyping of marrow cells
`
`Phenotypic analysis of marrow cells was carried out by flow cytometry as
`described.5 For determination of the expression of TRAIL and its receptors, 105
`cells were incubated with 10 mg/mL mAb against human TRAIL or TRAIL-R1
`to -R4 for 30 minutes on ice. After washes in phosphate buffered saline (PBS),
`cells were incubated for 30 minutes on ice with PE-conjugated (secondary) goat
`F(ab9)2 anti–mouse IgG. For 2-color immunofluorescence, FITC-conjugated
`mAbs for CD3, CD19 (for the lymphocyte gate), CD13 (for the granulocyte
`gate), CD14 (for the monocyte gate), and CD34 (for the hemopoietic progenitor
`cell gate) were incubated after staining with mAbs to human TRAIL and
`TRAIL-R1 to -R4, PE-conjugated (secondary) goat F(ab9)2 anti–mouse IgG.
`Blast cells were isolated on the basis of orthogonal light scatter and CD45
`labeling as described.22,23 Cells labeled with irrelevant isotype-matched mAb and
`incubated with the secondary detection antibody served as negative controls.
`Data on at least 10 000 events were collected using a FACScan (Becton
`Dickinson) and analyzed using CellQuest software (Becton Dickinson).
`
`Reverse transcription–polymerase chain reaction amplification
`
`RNA preparation and reverse transcription (RT) were carried out as
`described.5,18 Total RNA was prepared from cell pellets using the RNeasy
`Total RNA kit (Qiagen, Chatsworth, CA). Cells were lysed in buffer RLT (4
`M guanidinium isothiocyanate, 0.5% sarcosyl, and 0.1 M 2b-mercaptoetha-
`nol) and homogenized using a QIAshredder spin column. After centrifuga-
`
`tion, an equal volume of 70% ethanol was added to the lysate, and the
`mixture was applied onto RNeasy spin columns. Total RNA, which binds to
`the silica gel membrane under high salt-buffer conditions, was washed with
`buffers RW1 and RPE (supplied with the kit), and then eluted in
`DEPC-treated water. Finally, any remaining DNA was removed by
`digestion with RNase-free DNase. The concentration and purity of the RNA
`was determined by measuring the absorbance at A260/A280 and by gel
`electropheresis.
`To synthesize cDNA, 1 mg the total RNA (generally corresponding to
`’ 0.5 3 106 cells) was combined with 1 mL Oligo (dT)12-18 (500 mg/mL;
`Gibco/BRL, Gaithersburg, MD) and heated for 5 minutes to 70°C; the
`resulting heteroduplex was resuspended to 20 mL in RT buffer and
`incubated for 30 minutes at 37°C with 200 U Moloney murine leukemia
`virus (M-MLV) reverse transcriptase (Gibco/BRL). After adding a second
`100 U aliquot M-MLV, the reaction was continued for an additional 15
`minutes prior to heat-inactivating the enzyme for 2 minutes at 80°C. cDNA
`was stored at 270°C. Samples were then thawed in batches for polymerase
`chain reaction (PCR) amplification.
`The cDNA was amplified by PCR in a reaction mixture consisting of 50
`mM KCl, 20 mM Tris-HCl (pH 8.4), 0.1% Triton X-100, 1 mM DTT, 2.0
`mM MgCl2, all 4 dNTPs (each at 0.2 mM), 10 pmol of each oligonucleotide
`primer (see below), and 2.5 units Taq DNA Polymerase (Boehringer
`Mannheim). Amplification was performed in 0.5-mL Gene Amp tubes in a
`final volume of 50 mL. The PCR mixes were overlaid with mineral oil and
`amplified for 30 to 40 cycles (see below) denaturation (at 94°C for 1
`minute), annealing (at 55°C to 65°C [see below] for 1 minute, and extension
`at 72°C for 1 minute). PCR products were size-fractionated and analyzed by
`1.5% agarose gel electropheresis and normalized according to the amount
`of b-actin in the same cDNA sample. PCR product sizes were determined
`from comparison to the Ready-Load 100 base pair (bp) DNA standard
`ladder (Gibco/BRL).
`PCR reactions were performed using the following primers: b-actin (for-
`ward: 59-TCCTGTGGCATCCACGAAACT-39, reverse: 59-ATCGTCCACCG-
`CAAATGCTTC-39); TRAIL (forward: 59-GGAACCCAAGGTGGGTAGAT-
`39, reverse: 59-TCTCACCACACTGCAACCTC-39); TRAIL-R1 (forward: 59-
`CTGAGCAACGCAGACTCGCTGTCCAC-39, reverse: 59-TCCAAGGAC-
`ACGGCAGAGCCTGTGCCAT-39); TRAIL-R2 (forward: 59-GCCTCATGGA-
`CAATGAGATAAAGGTGGCT-39, reverse: CCAAATCTCAAAGTACGCA-
`CAAACGG-39); TRAIL-R3 (forward: 59-GAAGAATTTGGTGCCAATGCCA-
`CTG-39, reverse: 59-CTCTTGGACTTGGCTGGGAGATGTG-39); TRAIL-R4
`(forward: 59-CTTTTCCGGCGGCGTTCATGTCCTTC-39, reverse: 59-GTT-
`TCTTCCAGGCTGCTTCCCTTTGTAG-39); and long form of FLIP (forward:
`59-AATTCAAGGCTCAGAAGCGA-39, reverse: 59-GGCAGAAACTCTGCT-
`GTTCC-39). The sizes of the expected products were 304, 192, 506, 502, 612,
`453, and 226 bp, respectively. Human b-actin PCR-cycle conditions were 95°C
`for 45 seconds, 55°C for 1 minute, and 72°C for 45 seconds for 30 cycles. Human
`TRAIL-R1, -R2, -R3, and FLIPL conditions were 94°C for 1 minute, 55°C for 1
`minute, 72°C for 1 minute for 30 cycles. Human TRAIL-R4 conditions were
`95°C for 4 minutes 15 seconds, followed by 30 cycles of 95°C for 45 seconds,
`60°C for 45 seconds, and 72°C for 45 seconds for 30 cycles.18 Samples were
`resolved on 2% agarose gels and visualized with ethidium bromide.
`
`Western blot analysis
`
`Western blots were performed as previously described.18 Briefly, marrow
`mononuclear cells (MMNCs) from patients with MDS or from healthy
`donors were lysed in PBS containing 1% Nonidet P-40, 0.35 mg/mL PMSF,
`9.5 mg/mL leupeptin, and 13.7 mg/mL pepstatin A. The lysed cells were
`washed, and protein concentrations of the extracts were determined by
`colorimetric assay (Bio-Rad, Richmond, CA). Equal amounts of protein
`were separated by sodium dodecyl sulfate–polyacrylamide gel electrophore-
`sis (SDS-PAGE), transferred to polyvinylidine difluoride (PVDF; Bio-
`Rad), and blocked with 5% nonfat dry milk in 1 3 Tris buffered saline for 1
`hour. The membranes were then incubated with the FLIP antiserum (diluted
`1:1000) overnight, washed and incubated with an anti–rabbit horseradish
`peroxidase antibody (diluted 1:10 000; Amersham, Arlington Heights, IL)
`for 1 hour. Following several washes,
`the blots were developed by
`chemiluminescence reagent (Pierce, Rockford, IL).
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
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`
`3060
`
`ZANG et al
`
`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`replicates of 3. LZ-TRAIL (300 ng/mL) and M180 as a blocking Ab to
`TRAIL (300 ng/mL) were added to culture wells as indicated. Cultures
`were maintained at 33°C in a humidified air of 5% CO2 atmosphere. After 1
`week, nonadherent cells were collected, plates were washed twice with
`Ca11- and Mg21-free Hanks balanced salt solution, and 100 mL 0.05%
`trypsin was added to each well. Adherent cells were removed and combined
`with nonadherent cells and washed once. The cell mixture was then
`resuspended in 3.5 mL 2 3 Dulbecco medium containing 40% fetal calf
`serum, mixed with an equal volume of 0.06% agar (Difco, Detroit, MI) and
`aliquoted into 35 3 10 mm culture dishes (2 mL/dish). Ten mL of a
`cytokine mixture (including IL-1, IL-3, IL-6, G-CSF, GM-CSF, and SCF at
`concentrations of 10 mg/mL each) was added to each dish.27 Untreated cells
`served as controls. After 2 weeks, the plates were scored for colony
`numbers. Groups of at least 40 cells were counted as colonies.
`
`Statistical analysis
`
`The Mann-Whitney U test was used to compare the levels of expression of
`TRAIL and TRAIL receptors between normal and MDS marrow. A
`Wilcoxon signed-rank test was used to compare apoptosis percentages and
`colony numbers between untreated and LZ-TRAIL–treated normal and
`MDS marrow. Significant differences between data from specific groups
`were defined as those with P # .05.
`
`Results
`
`TRAIL-induced apoptosis in normal and MDS marrow
`
`In ancillary dose/response experiments, apoptosis was determined
`in the TRAIL-sensitive cell lines Colo 205, Jurkat, and KG1. The
`extent of apoptosis was assessed by Annexin V–staining and DNA
`histograms (data not shown). Based on those results, marrow from
`healthy donors and patients with MDS was exposed to TRAIL at
`concentrations as high as 1000 ng/mL for up to 24 hours. Results
`are summarized in Figures 1 and 2. In unfractionated mononuclear
`cells from normal marrow, even at the highest TRAIL concentra-
`tions and exposure for 24 hours, no increase in the proportion of
`apoptotic cells was detectable. In MDS marrow,
`in contrast,
`apoptosis increased above baseline already at TRAIL concentra-
`tions of 10 ng/mL and reached a maximum/plateau with a 100%
`increase at TRAIL concentrations of 300 ng/mL and exposure
`durations of 6 to 12 hours. An increase in apoptosis was observed in
`all subpopulations of cells studied (Figure 2):
`total marrow,
`24.8% 6 14.0% increased to 36.3% 6 18.0%, P 5 .011; CD31,
`34.3% 6 20.0% to 44.4% 6 24.9%, P 5 .002; CD141, 19.2% 6 17.2%
`29.7% 6 24.2%, P 5 .006; CD131,
`27.9% 6 24.5% to
`to
`37.1% 6 27.1%, P 5 .004; CD341, 33.3% 6 35.1% to 43.2% 6 30.7%,
`
`Figure 2. Apoptosis in subpopulations of normal and MDS marrow cells. Shown
`is the extent of apoptosis before (left column) and after TRAIL exposure (right
`column) obtained with 10 normal and 14 MDS marrow and exposure for 6-12 hours at
`300 ng TRAIL/mL. Shown are mean values; standard deviations ranged from 6 4%
`to 6 7% in normal, and 6 6% to 6 15% for MDS marrow.
`
`Figure 1. TRAIL-induced apoptosis in normal and MDS marrow cells. The extent
`of apoptosis in normal and MDS marrow cells exposed to TRAIL at 0 to 1000 ng/mL
`for 12 hours was assessed by Annexin V staining and by way of DNA histograms (see
`“Materials and methods”). Shown are the mean values for normal marrow (n 5 10)
`and for MDS marrow (n 5 22); standard deviations ranged from 6 6% to 6 8% for
`normal, and 6 8% to 6 14% for MDS marrow.
`
`Apoptosis assay
`
`Marrow cells from healthy donors and from patients with MDS were
`incubated in 48-well plates (5 3 105 cells/well) with progressively increas-
`ing concentrations of purified LZ–human TRAIL (0, 10, 30, 100, 300, 1000,
`and 3000 ng/mL) for 3, 6, 12, and 24 hours.12,13,18 Apoptotic changes were
`identified by staining with FITC-conjugated Annexin V (Immunotech,
`Westbrook, ME)24,25 and by means of DNA histograms to identify
`hypodiploid nuclei (sub–G1 peak) using flow cytometric analysis as
`described.26 To determine a possible effect of LZ-TRAIL on the marrow
`“microenvironment” and on stroma cells, adherent cell cultures were
`established.27 Adherent cell layers from marrow cells and STRO-1–positive/
`glycophorin-negative sorted cells were established in T25 tissue culture
`flasks (Corning, NY) using Iscoves modified Dulbecco medium (Gibco,
`Grand Island, NY) supplemented with 12.5% each of heat-inactivated horse
`and fetal calf serum (FCS), 0.4 mg/mL L-glutamine, 1 mM sodium
`pyruvate, 10-6 M hydrocortisone, 10-4 M 2-mercaptoethanol, 100 U/mL
`penicillin, and 100 mg/mL streptomycin.28 Flasks were demidepleted of
`cells at weekly intervals. After 4 weeks, nonadherent cells and monocytes
`were removed by 3 trypsinization passes, and negative selection by
`PE-conjugated anti-CD14 mAb. Apoptosis was determined as described.
`
`Cytogenetic and fluorescence in situ hybridization analysis
`
`To determine whether TRAIL-mediated apoptosis occurred preferentially
`in clonal cells (identifiable by a chromosomal marker), we performed
`cytogenetic and fluorescence in situ hybridization (FISH) analysis on
`marrow cells from 9 patients with MDS before and after exposure to
`LZ-TRAIL (300 ng/mL).29 We also determined the proportion of cytogeneti-
`cally abnormal cells among apoptotic (Annexin V–stained) and nonapop-
`totic (Annexin V–negative) marrow cells from 3 patients with MDS
`following sorting by flow cytometry after exposure to LZ-TRAIL
`(300 ng/mL).
`
`In vitro hemopoiesis
`
`Marrow cultures were carried out using the Dexter method.30,31 Stromal
`layers were established in T25 tissue culture flasks (Corning) using Iscoves
`modified Dulbecco medium. Flasks were demidepleted of cells at weekly
`intervals. At 2 to 3 weeks, the stromal cells were irradiated with 1800 cGy,
`removed from the flask by a brief exposure to 0.05% trypsin, and
`1.25 3 105 viable cells/mL were plated as a supportive layer in flat-
`bottomed 48-well plates (Costar, Cambridge, MA). After 1 to 5 days,
`MMNCs (0.5 3 106 cells/well) were layered onto the stromal layers in
`
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`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`TRAIL AND APOPTOSIS IN MDS
`
`3061
`
`Figure 3. TRAIL-induced apoptosis in MDS marrow.
`TRAIL-induced apoptosis in aberrant MDS blasts (A) and
`in CD341 precursors from MDS marrow (B). Panels in the
`left columns represent data before, and in the right
`columns, after TRAIL exposure. Shown are the gates
`used (top), scattergrams (middle), and DNA histograms
`(bottom) of gated cells. FSC indicates forward scatter;
`SSC, orthogonal scatter; FLI-FITC, fluorescence intensity
`after labeling with FITC-conjugated Annexin V; FL2-PI,
`fluorescence intensity after staining with propidium iodine;
`counts, relative number of cells. Percentage figures in the
`bottom panels indicate proportions of apoptotic cells.
`
`P 5 .043).
`between CD341
`difference
`no
`There was
`/CD331 and CD341/CD332 precursors. Two examples, MDS blasts
`and CD341 precursors, are shown in Figure 3. The most prominent
`increase in apoptosis was seen in MDS blasts (22.9% 6 12.4% to
`39.6% 6 16.1%). The increase in TRAIL-mediated apoptosis was
`prevented in the presence of the blocking anti-TRAIL mAb, M180 (data
`not shown). In normal marrow, no change in the proportion of apoptotic
`cells following LZ-TRAIL exposure was detected in the CD31, CD141,
`or CD131 gates. However, in the gate of CD341 precursors, the
`proportion of apoptotic cells was 4.3% 6 2.8% before and 7.3% 6
`4.3% after LZ-TRAIL exposure (n 5 7; P 5 .06). This finding suggests
`a physiologic role for TRAIL and its receptors in the regulation of
`normal hemopoiesis at a CD341 precursor stage as recently suggested
`by others.32
`Differences in regard to susceptibility to apoptosis were also
`observed among adherent cell layers, whereas TRAIL exposure
`failed to have a significant effect on adherent layers derived from
`normal marrow (10.2% apoptosis before TRAIL exposure, 14.4%
`after TRAIL exposure),
`the proportion of apoptotic cells in
`adherent layers derived from MDS marrow increased significantly
`after TRAIL exposure from 23.6% 6 7.1% to 47.4% 6 9.4%
`(P 5 .04). Apoptosis was limited to CD141 and other mononuclear
`cells; no apoptosis was observed in STRO2/1 glycophorin2/
`CD142 stromal cells.
`
`Surface expression of TRAIL and its receptors on normal and
`MDS marrow cells
`
`The higher TRAIL susceptibility of malignant cells may be due to
`differential expression of the agonistic (TRAIL-R1 and -R2) and
`decoy (TRAIL-R3 and -R4) receptors.15,16 We determined surface
`expression of TRAIL and its receptors on marrow cells from 21
`patients with MDS and from 10 healthy donors. Results are
`summarized in Figure 4A. In normal marrow, the expression of
`TRAIL and its receptors was negligible except for TRAIL-R3. On
`MDS marrow cells, in contrast, TRAIL and receptors R1, R2, R3,
`
`and R4 were constitutively expressed: TRAIL, 1.4% 6 0.6%
`(mean 6 SD) of cells in normal vs 7.9% 6 10.0% in MDS marrow
`(P 5 .011); TRAIL-R1, 1.4% 6 0.4% vs 16.3% 6 23.1% (P 5 .017);
`TRAIL-R2, 1.4% 6 0.6% vs 14.9% 6 21.2% (P 5 .001); TRAIL-R3,
`28.3% 6 20.7% vs 23.6% 6 17.2% (P 5 .495); TRAIL-R4,
`1.6% 6 0.6% vs 7.0% 6 11.3% (P 5 .043). TRAIL-R3 expression
`on normal cells was restricted to the granulocyte gate (CD131)
`(65.0% 6 25.8%). TRAIL-R2 expression in MDS marrow was
`significantly higher than in normal marrow in the lymphocyte gates
`(CD31 or CD191), in the monocyte gate (CD141), and in the
`granulocyte gate. In purified CD341 cells or blasts, a distinct
`pattern of expression was noted especially for TRAIL-R2. Among
`CD341 cells from MDS marrow, 17.9% 6 34.3% expressed
`TRAIL-R2 vs 0.2% 6 0.1% in normal marrow. Among MDS
`blasts, 50.5% 6 48.1% were TRAIL-R2–positive vs 1.4% 6 1.2%
`in normal marrow.
`To estimate TRAIL sensitivity on the basis of expression of
`TRAIL receptors, we calculated the ratio of agonistic (TRAIL-R1
`and -R2) to antagonistic (TRAIL-R3 and -R4) receptors. As shown
`in Figure 4B, in MDS marrow this ratio was significantly higher
`than in normal marrow (18.4% 6 20.3 vs 5.1% 6 6.3, P 5 .015).
`These data are compatible with the higher susceptibility to
`TRAIL-induced death observed in MDS marrow.
`
`Expression of mRNA for TRAIL and its receptors in
`marrow cells
`
`Estimation of mRNA expression was based on band intensities of
`electropheresed RT-PCR products. Although mRNA for TRAIL
`and all TRAIL receptors was detected in cells from both normal
`and MDS marrow, in agreement with the data on cell surface
`expression, the ratio of agonistic TRAIL-R1 and -R2 to decoy
`TRAIL-R3 and -R4 receptors was higher in MDS than in normal
`marrow (Figure 5). When only CD341 cells were considered, the
`ratio was again higher in CD341 cells from MDS marrow. Further,
`within MDS marrow, there was a higher ratio in blast cells than in
`
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`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`Figure 4. Surface expression of TRAIL and TRAIL receptors on marrow cells
`and ratio of surface expression of TRAIL-R1 and R2 to TRAIL-R3 and R4.
`(A) Surface expression of TRAIL and receptors R1 to R4 on marrow cells from 10
`healthy donors (Healthy) and 20 patients with MDS (MDS). Shown are results for total
`marrow, CD31, CD141, CD131, and CD341 cells (mean 6 1 SD); b-actin served as
`internal standard. (B) Ratio of surface expression (as shown in part A) of TRAIL
`receptors R1 and R2 (agonistic) over R3 and R4 (decoy) in marrow from 10 healthy
`donors and from 20 patients with MDS. Shown are the results (mean 6 1 SD) for total
`marrow and for CD31, CD141, CD131, and CD341 cells, respectively.
`
`Figure 5. Expression of TRAIL and TRAIL receptors R1-R4 mRNA in normal and
`in MDS marrow. Shown are examples for total mononuclear cells (MNC) and CD341
`precursors, and separately, MDS blasts sorted on the basis of CD45 expression and
`orthogonal light scatter as described previously; b-actin served as internal standard.5,22
`
`consistent with the notion that apoptosis is facilitated in blasts,
`thereby controlling the size of the clonal (blast) pool.
`
`Cytogenetics and FISH analysis
`
`the non–blast cell population. This suggests that blast cells might
`be particularly vulnerable to TRAIL-induced apoptosis, a fact that
`might explain the phenomenon that a clonal (blast) population can
`exist
`in MDS marrow for extended periods of time without
`expanding and developing into frank leukemia.
`
`To determine whether TRAIL-mediated apoptosis eliminated selec-
`tively or preferentially clonal (malignant) cells, we performed
`FISH analysis on the marrow from patients with MDS with a
`cytogenetic marker before and after TRAIL exposure. Based on
`results of ancillary time course studies, cells were exposed to
`
`Expression of apoptosis inhibitory protein FLIP
`
`In addition to the expression of agonistic and decoy receptors,
`differences in intracellular modification of TRAIL signals could
`explain differential effects in normal and malignant cells. FLIP, a
`cytoplasmatic inhibitor of apoptosis, can interfere with TRAIL-
`induced cell death.33 Griffith et al described a correlation between
`the level of expression of FLIP and sensitivity to TRAIL in a
`line.18 Thus, we compared the level of FLIP
`melanoma cell
`expression in normal and MDS marrow at the mRNA and protein
`levels. FLIP mRNA was present in both normal and MDS marrow,
`though quantitative differences were observed in MDS marrow.
`Differences in RNA levels for FLIP were reflected in differences in
`the protein levels as determined by Western blots (Figure 6). The
`ratio of mRNA expression of agonistic (R1 and R2) receptors to the
`cytoplasmatic inhibitor FLIP was higher in MDS marrow than in
`normal marrow, and within MDS marrow this ratio was higher in
`blasts than in the remainder of cells. This observation was
`
`Figure 6. Expression of FLIP protein in normal (n 5 3) and in MDS marrow
`(n 5 5). Shown is an original gel (top) and a Western blot for FLIP using marrow cells
`from 3 healthy donors (1,2,3), 4 marrow (JLP [RA], JAC [RA], GRD [tAML], and FWH
`[RA]), and one example of peripheral blood cells (PJC [RAEB]) from patients with
`MDS. MW indicates molecular weight.
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
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`BLOOD, 15 NOVEMBER 2001 z VOLUME 98, NUMBER 10
`
`TRAIL AND APOPTOSIS IN MDS
`
`3063
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`Table 1. Cytogenetically abnormal cells in MDS marrow before and after
`exposure to TRAIL as determined by FISH analysis
`
`Cytogenetically
`abnormal cells (%)
`
`Patient
`ID
`
`Diagnosis
`(FAB)
`
`Cytogenetic
`abnormality
`
`Before
`TRAIL
`
`After
`TRAIL
`
`255
`167
`221
`187
`256
`209
`236
`183
`218
`
`RA
`RA
`RA
`RA
`RA
`RAEB
`RAEB
`RAEB-T
`tAML
`
`5q2
`121
`5q2
`220
`27
`5q2
`27
`18
`18
`
`84
`14
`38
`51
`28
`73
`9
`3
`13
`
`0
`0
`0
`29
`15
`53
`4
`5
`32
`
`Fresh marrow cells from patients with known clonal cytogenetic abnormalities
`were cultured for 6 to 12 hours in the presence of TRAIL (300 ng/mL). After washing,
`FISH analysis with the appropriate probes was carried out on pre-exposure and
`postexposure samples, side by side.
`
`TRAIL for 6 hours. Results in 8 individuals are summarized in
`Table 1. After TRAIL exposure, the proportion of cytogenetically
`abnormal cells decreased in 6 of 9 MDS marrows studied,
`especially in marrow from patients with RA. While the results
`suggest that LZ-TRAIL eliminated predominantly abnormal cells
`in certain MDS marrows, results indicate considerable heterogeneity.
`
`In vitro hemopoiesis
`
`To determine whether the effects of human LZ-TRAIL on marrow
`cells were of functional relevance, we characterized in vitro
`hemopoiesis of marrow mononuclear cells from 21 patients with
`MDS (RA n 5 8, RARS n 5 3, RAEB n 5 3, CMML n 5 1,
`RAEBtn 5 4, and tAML from MDS n 5 2) and 6 healthy donors
`were propagated in LTMC in the presence or absence of human
`TRAIL (Figure 7). As postulated, numbers of colonies from normal
`marrow did not change in the presence of LZ-TRAIL (104% 6 5%
`of untreated group [100%]). In MDS marrow, colony numbers were
`slightly increased in the presence of LZ-TRAIL when results of all
`MDS marrows were combined (114% 6 14% of untreated group).
`However, results were strikingly dependent upon disease stage. In
`advanced MDS (RAEB-T and tAML) colony and cluster numbers
`decreased significantly in the presence of TRAIL (65% 6 7% of
`untreated marrow; P 5 .004 compared to normal marrow), whereas
`in patients with less advanced MDS (RA, RARS, and RAEB),
`colony numbers increased in the presence of TRAIL (157% 6 21%
`of untreated marrow, P 5 .037 compared to normal). These results
`suggest complex regulatory mechanisms and reflect
`the great
`heterogeneity of MDS. Nevertheless, the data are consistent with a
`model that proposes that hemopoiesis in less advanced MDS is
`suppressed by negative signals, presumably generated by clonal
`precursors,5 and elimination of those cells should result in im-
`proved hemopoiesis. Conversely, in advanced MDS, the abnormal
`precursor pool has expanded and is a major contributor to overall
`hemopoiesis.34 As a consequence, elimination of those cells should
`result in a decline in colony formation. An alternative consideration
`might be that cellular responses to TRAIL signaling differed for
`different disease stages.
`
`Discussion
`
`Increased cellular proliferation, along with exaggerated pro-
`grammed cell death (apoptosis), is one paradigm that has been used
`
`to explain the presence of a cellular or even hypercellular marrow
`concurrently with peripheral blood cytopenias in patients with
`MDS.35 Proliferation is likely to be due to dysregulated cytokine
`expression, while apoptosis could be mediated by either a lack of
`positive (survival) signals or an overproduction of negative (death)
`signals.5,8,27,36 Such a pattern could arise with an excess of
`bifunctional cytokines showing a posit