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Blood, 15 November 2006, Vol. 108, No. 10, pp. 3530-3537. Prepublished online as a Blood First Edition Paper on August 1, 2006; DOI 10.1182/blood-2006-04-013813.
NEOPLASIA Expression of interleukin-3 receptor subunits on defined subpopulations of acute myeloid leukemia blasts predicts the cytotoxicity of diphtheria toxin interleukin-3 fusion protein against malignant progenitors that engraft in immunodeficient miceFrom the Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada; and the Scott and White Memorial Hospital, Temple, TX.
The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3R  and  c subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT388IL3). Among 19 patient samples, the relative level of the IL-3R was higher than the IL-3Rc and highest in CD34+CD38-CD71- cells, enriched for candidate leukemia stem cells, compared with cell fractions depleted of such progenitors. Overall, the amount of IL-3Rc subunit did not vary among sorted subpopulations. However, expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rc expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34+CD38-CD71- cells from different samples; this value was correlated (r = .76, P = .05) with the ability of DT388IL3 to kill AML progenitors that engraft in 2-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus, quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors.
Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis characterized by the accumulation of large numbers of myeloid blast cells that fail to differentiate into functional blood cells.1 In spite of these obvious abnormalities in cell maturation there are now considerable data demonstrating a hierarchy of malignant progenitor cells in AML similar to that found in normal hematopoiesis and the existence of a small subpopulation of leukemia stem cells (LSCs) that possesses extensive proliferative capacity and the potential for self-renewal.2,3 Leukemic cells that initiate long-term hematopoiesis in stromal coculture (AML LTC-ICs) and suspension cultures and engraft in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (NOD/SCID leukemia-initiating cells [N/SL-ICs]) are typically present at a frequency of fewer than 1 in 10 000 malignant cells and express a cell-surface phenotype similar to that seen on normal multipotential long-term culture-initiating cells (LTC-ICs) and lymphomyeloid NOD/SCID mouse competitive repopulating units.4-7 Several recent studies have shown that N/SL-ICs can be phenotypically defined as CD34+, CD38-, human leukocyte antigen (HLA)-DR-, CD90-, CD71-, CD117-, and CD123+. This antigenic profile is intriguing in that it overlaps with normal stem cells (CD34+, CD38-, CD71-, HLA-DR-) but also has leukemia-specific features (CD90-, CD117-, and CD123++).4,8-16 The frequent relapses observed in patients with AML who achieve an initial remission of their leukemia suggest that LSCs might be relatively resistant to conventional chemotherapy and also might be responsible for re-establishing the malignant clone in patients with leukemia. These cells are thus obvious targets for novel therapeutic approaches in AML.
The human interleukin-3 receptor (IL-3R) is a heterodimeric structure.17 The
Fusion proteins in which the diphtheria toxin (DT) catalytic and translocation domains are genetically fused to a growth factor are a novel class of molecules which can selectively target and kill malignant cells expressing the relevant receptor.21-26 After ligand binding and receptor-mediated endocytosis, the toxin translocates to the cytosol, where it ADP-ribosylates elongation factor 2, leading to inactivation of protein synthesis and cell death.27-30 DT388IL3 consists of the truncated form of diphtheria toxin (DT388) linked to human IL-3. This fusion protein has been shown to target and kill cells which express the IL-3R, including AML blasts and progenitors.31 Previous studies have shown that this molecule will kill AML colony forming cells (AML-CFCs), long-term culture-initiating cells (AML LTC-ICs), and N/SL-ICs from many patient samples while showing little or no toxicity against analogous normal bone marrow progenitors.18 Although DT388IL3 is effective against many AML samples, only partial eradication of malignant progenitors was seen with some leukemias in spite of detectable expression of high-affinity IL-3R on target blasts. More recently, we determined that the level of IL-3R subunit expression (particularly the
The current study examined the possibility that expression of the IL-3R subunits might be heterogeneous among AML cells and progenitors. We were particularly interested in the possibility that IL-3R expression on the most primitive AML cells (ie, the candidate LSCs that initiate long-term engraftment and proliferation in immunodeficient mice), might be different than the expression detected on the majority population of leukemic cells that have limited proliferative capacity. The level of IL-3R subunit expression was measured by QRT-PCR in subpopulations of leukemic cells enriched for AML progenitors with a multiparameter fluorescence-activated cell sorter (FACS) strategy. These levels were then compared with the ability of DT388IL3 to kill N/SL-ICs from many of the same AML samples. The results demonstrate that the level of expression of the IL-3R
Blood and marrow samples Peripheral-blood (PB) samples from 19 newly diagnosed patients with AML and bone marrow (BM) from 3 healthy allogeneic transplant donors were obtained with the approval of the Clinical Research Ethics Board of the University of British Columbia and after obtaining informed consent from patients with AML and from donors in accordance with the Declaration of Helsinki. Light-density mononuclear cells were isolated and cryopreserved as previously described.5 Diagnosis and classification of AML were based on the criteria of the French-American-British (FAB) group.33 Cytogenetic analysis was performed on diagnostic bone marrow samples of patients with AML. Thawed cells were washed twice in Iscove modified Dulbecco medium (IMDM) containing 10% fetal calf serum (FCS) before use in the experiments described below. Normal BM was enriched for CD34+ cells using an immunomagnetic separation technique (EasySep; StemCell Technologies, Vancouver, BC, Canada) prior to the FACS isolation of subpopulations described under "FACS analysis and subpopulation isolation of AML and normal cells." DT388IL-3 fusion protein DT388IL3 was constructed by joining the human IL-3 cDNA to a truncated diphtheria toxin (DT388) sequence that lacks the native binding site via an intervening (G4S)2 linker. Recombinant protein was prepared, purified, stored, and tested as described previously.34 This material was found to kill IL-3R+ cell lines and AML-CFCs from primary AML samples expressing high-affinity IL-3Rs.19,31,34 The IL-3R binding affinity of this fusion toxin is similar to that of native human IL-3.22 In addition, the toxicity of DT388IL3 against IL-3R-bearing TF1/Bcl2 cells was reduced more than 120-fold by coincubation with excess human IL-3.31 Cultures of AML cells
AML cells were incubated for 24 hours at 37°C at a concentration of 1 x 106 cells/mL with or without DT388IL3. Equal fractions of the cells recovered from cultures with or without toxin were assayed in AML-CFC assay or in AML-CFC assays were performed by plating cells at 0.2 to 1.0 x 105 cells/mL in methylcellulose-based medium (StemCell Technologies) as previously described. Cultures were scored after 14 days at 37°C in a humidified atmosphere with 5% CO2 for the presence of clusters (4-20 cells) and colonies (more than 20 cells).5 FACS analysis and subpopulation isolation of AML and normal cells
Subpopulation analysis and isolation from AML PB and CD34+-enriched normal BM cells were performed with monoclonal antibodies directly coupled to the fluorochromes fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanin5-succinimidylester (Cy5). Anti-CD34-Cy5 and anti-CD71-FITC were kind gifts from Dr Peter Lansdorp (Terry Fox Laboratory, Vancouver, BC, Canada); Anti-CD38-PE was obtained from Becton Dickinson (San Jose, CA). Biotinylated antibodies recognizing CD123 (IL-3R Subpopulations of AML and normal BM cells labeled with anti-CD34, anti-CD38, and (for AML cells) anti-CD71 were sorted into IMDM (StemCell Technologies) with 20% FCS in microcentrifuge tubes at 4°C. Sort windows used to isolate the specific subfractions are shown in Figure 1. Sorted fractions were washed and used for RNA isolation and QRT-PCR analysis. QRT-PCR analysis
Total RNA was extracted from sorted cells using the Absolutely RNA, Microprep, or Nanoprep kits (Stratagene, La Jolla, CA). The RT reaction was performed in 20 µL with superscript II reverse transcriptase (Invitrogen, Burlington, ON, Canada) using random hexamer oligonucleotides (Amersham Pharmacia, Piscataway, NJ). Real-time PCR was performed using 12.5 µL 2 x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 1 µL of 20 pM-specific primers, 1 to 2 µL cDNA, and water to a final volume of 25 µL. Specific forward and reverse primers to produce approximately 100-bp amplicons for optimal amplification in real-time PCR of reverse-transcribed cDNA for human IL-3R
Transplantation and detection of AML cells in 2-microglobulin-deficient NOD/SCID mice
Mouse BM cells were prepared for FACS analysis as previously described7 and analyzed for the expression of CD45, a human-specific panleukocyte marker, using an antibody prepared in our center from American Type Culture Collection (ATCC) (clone no. HB10508; Rockville, MD). FACS analysis was performed on a Becton Dickinson FACScan or FACSort flow cytometer. The percentage of CD45+ cells was determined after excluding 99.9% of cells labeled with the isotype control and nonviable cells. Nonspecific binding of CD45 on mouse BM cells is reliably less than 0.1% to 0.3%.7 Thus, a difference between the immunoglobulin G1-negative (IgG1-) control and the CD45 expression of the treated mice of more than 0.1% was regarded as evidence for engraftment of human cells. Values shown for engraftment of AML cells in mouse BM are the mean values (± SD) of CD45+ cells (%) obtained for all mice in the cohort that survived to the time of analysis. Fluorescence in situ hybridization Cells from mouse BM were cytocentrifuged onto glass microscope slides and then fixed in methanol-glacial acetic acid. The human-specific centromeric repeat probe used to detect the +8 abnormality (plasmid D8Z2; ATCC) and the yeast artificial chromosome clone containing 550 kb human DNA encompassing the breakpoint on 16p13 (CEPHy904E02 854; Max Planck Institute, Berlin, Germany) to detect the inv(16) were labeled by nick translation with digoxigenin (DIG; Boehringer Mannheim, Mannheim, Germany). Chromosomes 8 and 16 probes and slides were treated as previously described.5 For probe detection, slides were incubated with a sheep anti-DIG-FITC antibody (Boehringer Mannheim) and counter-stained with PI as previously described.5 Slides were visualized on a Zeiss Axioplan (Oberkochen, Germany) fluorescence microscope using double or triple bandpass filters (Omega Optical, Battlebore, NC) to allow simultaneous visualization of FITC and PI signals. Statistical analysis Mean values of IL-3R subunit expression in subpopulations of AML and normal BM cells and correlation coefficients between variables were determined in Excel (Microsoft, Redmond, WA), and the significance of differences and correlations determined using a Student t test. A P value of less than .05 was considered significant.
Table 1 shows the clinical characteristics of the 19 patients with AML patients whose cells were tested in this study. Their ages at diagnosis ranged from 17 to 75 years and the FAB subtype of these leukemias was M4 (n = 10), M1 (n = 5), or M5 (n = 3). Cytogenetic analysis of the diagnostic BM specimens revealed abnormalities associated with a good prognosis [inv(16)] in 3 patients and a poor prognosis in 6 of these 19 patients according to the Medical Research Council (MRC, United Kingdom) or Southwest Oncology Group (SWOG) criteria.40,41
Comparison of QRT-PCR and FACS for detection of IL-3R subunit expression
In initial experiments the levels of expression of IL-3R subunits in AML blasts as detected by QRT-PCR was compared with detection by FACS analysis for 12 of the 19 patient samples shown on Table 1 using antibodies which detect IL-3R
IL-3R subunit expression on subpopulations of AML and normal BM cells enriched for progenitors Figure 1 shows a typical FACS profile of AML cells stained with antibodies against CD34, CD38, and CD71. Previous data had demonstrated that the CD34+CD38- phenotype includes N/SL-ICs from most patient samples while addition of CD71 negativity to this phenotype increases the enrichment of sorted cells for these primitive progenitors.15 These studies have also demonstrated that AML-CFCs are largely CD34+ in most patient samples but are more heterogeneous in their expression of CD388,15 (ie, most of these more mature progenitors express this antigen and thus are found in the CD34+CD38+ subpopulation, while N/SL-ICs are excluded). CD34- cells from most AML samples have little progenitor activity. Thus, PB cells from the 19 patients with AML (Table 1) were sorted into 4 subpopulations defined as CD34+CD38-CD71-, CD34+CD38-, CD34+CD38+, and CD34-. As shown in Table 3, the large proportion of AML cells from 16 of these patients were CD34+. Overall, mean frequencies of the CD34+CD38-CD71-, CD34+CD38-, and CD34+CD38+ subpopulations were 3.2% (range, 0.05%-25.3%), 5.3% (range, 0.08%-38.4%), and 40.2% (range, 0.7%-90.9%), respectively, while 27% (range, 2.1%-80.9%) of cells were CD34-.
QRT-PCR was used to detect expression of the IL-3R and c subunits in RNA isolated from the 4 sorted subpopulations of AML cells from these 19 patient samples. As shown in Figure 3, the median levels of expression of the subunit were higher than that of the c subunit for each isolated subpopulation (range, 4- to 15-fold). The level of c expression detected among the subpopulations of cells enriched for primitive progenitors (CD34+CD38- cells or such cells lacking CD71) was not significantly different from that detected in the subpopulations depleted of such cells (CD34+CD38+ or CD34- cells). However, expression of the IL-3R subunit was significantly higher among CD34+CD38-CD71- and CD34+CD38- cells than among CD34+CD38+ or CD34- cells (P < .002, paired t test).
RNA also was isolated from CD34+CD38- and CD34+CD38+ cells from 3 normal BM samples. QRT-PCR demonstrated that the mean (± SD) level of expression of the IL-3R and c subunits (relative to GAPDH set at 1000) was 21.6 (± 8.6) and 9.0 (± 5.1), respectively, for CD34+CD38- cells, and 8.2 (± 1.8) and 26.2 (± 3.8), respectively, for CD34+CD38+ cells. These values for the subunit were significantly (P  .002) lower than those obtained for the same subpopulations of AML cells. In contrast, the c subunit was expressed at similar (in CD34+CD38- cells) or higher (P = .003 in CD34+CD38+ cells) levels among normal than among malignant cells.
In our previous studies we had defined an expression of the IL-3R
AML blasts from these same samples were incubated for 24 hours with or without DT388IL3 at 50 or 250 ng/mL and then injected into cohorts of sublethally irradiated
DT388IL3 is a novel fusion protein which is currently undergoing clinical evaluation as a possible new therapeutic agent for the treatment of AML. Although data from tissue culture and mouse models had suggested that this drug would be active against many human AMLs, it also suggested that some leukemias would be much more sensitive than others.18,19,31 This was particularly true when DT388IL3 was tested against AML progenitors that engraft in NOD/SCID mice (N/SL-ICs).18 Thus, for some samples DT388IL3 was highly cytotoxic when tested against AML-CFCs but much less so against N/SL-ICs, while for other samples the drug had little effect against any class of AML progenitors. Evaluation of the expression of high- and low-affinity IL-3-binding sites on AML blasts demonstrated that the presence of IL-3Rs was necessary but not always sufficient to ensure leukemic progenitor kill by the fusion protein.19,34 Previous studies had also demonstrated that AML blast populations highly enriched for or depleted of progenitor activity could be obtained by isolating cells that varied in their expression of CD34, CD38, and CD71.8,15 N/SL-ICs are largely restricted to the CD34+CD38- subpopulation of AML blasts from most patient samples and can be further enriched by excluding CD71+ cells, while CD34- cells are typically depleted of all progenitor activity.8,15,16
Using FACS analysis other investigators have demonstrated that the IL-3R subunit is typically highly expressed on AML blasts, including the CD34+CD38- subpopulation.14,20 More recently this finding has been confirmed and extended by using MFI to quantify expression of both the IL-3R subunits on AML blasts labeled with anti-CD123 and anti-CD131.42 This measurement was also shown to correlate with the percentage of AML cells undergoing apoptosis when exposed to DT388IL3. As an alternative to flow cytometry we have used QRT-PCR to show that the level of IL-3R subunit expression in AML blasts predicted the killing of AML-CFCs by DT388IL3 and its variants.32 However, with both techniques some patient samples showed relative resistance to DT388IL3 cytotoxicity in spite of high-level IL-3R expression.
In initial experiments described here, flow cytometry was compared with QRT-PCR for its ability to detect and quantify the IL-3R subunits. Both techniques provided quantitative and reproducible data. However, there was no correlation between the FACS data (either MFI or percentage of positive cells) and QRT-PCR for the IL-3R
Since LSCs are the most relevant target population for novel antileukemic therapy such as DT388IL3 we used QRT-PCR to study the expression of IL-3R subunits on subpoulations of AML cells known to be enriched for or depleted of leukemic progenitors that engraft in immunodeficient (
Although N/SL-ICs are a small proportion of the total cells present in even the most enriched population studied here,15 and the number of AML samples studied in
In initial studies investigating the relative cytotoxicity of DT388IL3 against normal and leukemic progenitor cells, a striking lack of toxicity was seen against primitive normal multipotential progenitors, including those with lymphomyeloid potential, that are detected in NOD/SCID mice.18 This was somewhat surprising given the fact that expression of both IL-3R subunits has been demonstrated on CD34+CD38- cells from normal BM as well as cord blood and fetal liver using RT-PCR.43 We have confirmed the findings of this earlier study. However, using the QRT-PCR technique described in the current report the relative level of expression of the IL-3R A phase 1/2 clinical trial of DT388IL3 is currently under way in patients with relapsed or refractory AML.45 Data presented here suggest that it may be possible to use quantification of target molecules on AML blasts to predict which patients are likely to benefit from therapy with this fusion protein. The use of QRT-PCR for the IL-3R subunits is currently being tested in this context. Furthermore, these results suggest that detailed evaluation of subpopulations of malignant cells with different functional properties may reveal similarities and differences in gene expression that are important for the rational development of targeted antileukemic therapy.
Submitted April 3, 2006; accepted July 6, 2006.
Prepublished online as Blood First Edition Paper, August 1, 2006; DOI 10.1182/blood-2006-04-013813.
Supported by the Canadian Institute for Health Research and National Cancer Institute of Canada (D.E.H.), National Institutes of Health (NIH) grants RO1CA76178and RO1CA90263, and Leukemia and Lymphoma Society grant 6006-05 (A.E.F.).
The authors declare no competing financial interests.
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 USC section 1734.
Reprints: Donna E. Hogge, Terry Fox Laboratory, BC Cancer Agency, 675 West 10th Ave, Vancouver, BC V5Z 1L3 Canada; e-mail: dhogge{at}bccancer.bc.ca.
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Abstract
Introduction
indicates untreated cells; 
, cells treated with 50 ng/mL DT388IL-3; and 
, cells treated with 250 ng/mL DT388IL-3. Error bars indicate SEM level engraftment for all mice in the cohort. The level of engraftment was significantly (P < .05; Student t test) less than that obtained for the untreated control cells for the 4 patient samples treated with both doses of DT338IL3 shown in panel B.