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Journal of Clinical Oncology, Vol 18, Issue 19 (October), 2000: 3331-3338
© 2000 American Society for Clinical Oncology

Expression of Interferon Regulatory Factor 4 in Chronic Myeloid Leukemia: Correlation With Response to Interferon Alfa Therapy

By Manuel Schmidt, Andreas Hochhaus, Sven A. König-Merediz, Cornelia Brendel, Jutta Proba, Georg J. Hoppe, Burghardt Wittig, Gerhard Ehninger, Rüdiger Hehlmann, Andreas Neubauer

From the Zentrum für Innere Medizin, Abteilung Hämatologie/Onkologie/Immunologie, Klinikum der Philipps-Universität Marburg, Marburg; III Medizinische Universitätsklinik, Klinikum Mannheim der Universität Heidelberg, Heidelberg; Abteilung Molekularbiologie und Bioinformatik, Fachbereich Humanmedizin, Freie Universität Berlin, and Innere Medizin, Virchow Klinikum der Charité Berlin, Berlin; and Medizinische Klinik und Poliklinik I, Universitätsklinik Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Address reprint requests to Andreas Neubauer, MD, Zentrum für Innere Medizin, Abteilung Hämatologie/Onkologie/Immunologie, Klinikum der Philipps-Universität Marburg, Baldingerstraße, 35043 Marburg, Germany; email neubauer{at}mailer.uni-marburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Mice experiments have established an important role for interferon regulatory factor (IRF) family members in hematopoiesis. We wanted to study the expression of interferon regulatory factor 4 (IRF4) in various hematologic disorders, especially chronic myeloid leukemia (CML), and its association with response to interferon alfa (IFN-{alpha}) treatment in CML.

MATERIALS AND METHODS: Blood samples from various hematopoietic cell lines, different leukemia patients (70 CML, 29 acute myeloid leukemia [AML], 10 chronic myelomonocytic leukemia [CMMoL], 10 acute lymphoblastic leukemia, and 10 chronic lymphoid leukemia patients), and 33 healthy volunteers were monitored for IRF4 expression by reverse transcriptase polymerase chain reaction. Then, with a focus on CML, the IRF4 level was determined in sorted cell subpopulations from CML patients and healthy volunteers and in in vitro–stimulated CML cells. Furthermore, IRF4 expression was compared in the CML samples taken before IFN-{alpha} therapy and in 47 additional CML samples taken during IFN-{alpha} therapy. IRF4 expression was then correlated with cytogenetic response to IFN-{alpha}.

RESULTS: IRF4 expression was significantly impaired in CML, AML, and CMMoL samples. The downregulation of IRF4 in CML samples was predominantly found in T cells. In CML patients during IFN-{alpha} therapy, a significant increase in IRF4 levels was detected, and this was also observed in sorted T cells from CML patients. The increase seen during IFN-{alpha} therapy was not due to different blood counts. In regard to the cytogenetic response with IFN-{alpha}, a good response was associated with high IRF4 expression.

CONCLUSION: IRF4 expression is downregulated in T cells of CML patients, and its increase is associated with a good response to IFN-{alpha} therapy. These data suggest IRF4 expression as a useful marker to monitor, if not predict, response to IFN-{alpha} in CML.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTERFERONS (IFNs) ARE known to regulate immune response, cell growth, and antiviral activity in mammals. They act by binding their receptors, which leads to subsequent phosphorylation events and the association of activated transcription factors with different response elements in the promoter regions of IFN-regulated genes. Interferon alfa/beta (IFN-{alpha}/ß; type I IFNs) and interferon gamma (IFN-{gamma}; a type II IFN) mediate their action through distinct pathways and thus regulate various genes.1-6

Proteins of the interferon regulatory factor (IRF) family—such as IFN-stimulated gene factor 3-gamma, IFN consensus sequence binding protein (ICSBP), IRF-1, and IRF-2—are known to be regulated by IFNs and subsequently bind to IFN-stimulated response elements in IFN-dependent genes.7-11 In contrast, IRF4 (Pip/ICSAT/LSIRF/NF-EM5)—also a transcription factor of the IRF family—is not directly regulated by IFNs but by antigen receptor–mediated stimuli, such as CD3-crosslinking and anti-immunoglobulin M antibodies.12-15 In contrast to IRF-1 and IRF-2, which are widely expressed, ICSBP and IRF4 are tissue-restricted factors. ICSBP is expressed mainly in cells of hematopoietic origin, such as B cells and monocytes.10,16 IRF4 expression is highly restricted to lymphocytes of the B-cell type (pre-B, B, and plasma cells) and mature T cells.14

IRF-1-/- mice exhibit defects in T-cell differentiation and thymocyte development, and IRF-2–deficient mice have impaired bone marrow and B-cell development.17 Recently, it has been shown that ICSBP knockout mice reveal an even more striking anomaly: a granulocytic leukemia similar to chronic myeloid leukemia (CML) in humans.18 In ICSBP-/- homozygous mice, this loss of ICSBP more often leads to blastic transformation than in ICSBP+/- heterozygous mice, indicating a "dose-dependent" role of ICSBP in leukemogenesis. In keeping with these data, the absence of ICSBP mRNA has been described in myeloid leukemias, especially in CML, and stimulation experiments suggest a mechanism of downregulation.19 In contrast, IRF4-deficient mice develop an impaired immunoglobulin production and antibody response. Furthermore, these mice exhibit defective cytotoxic or antitumor responses, which suggests an essential role of IRF4 in the maturation of functionally intact B and T cells.20

The aim of this work was to investigate whether IRF4 plays a role in human leukemic transformation, especially in CML as it was observed with ICSBP. First, we analyzed the transcriptional level of IRF4 in different leukemic and normal hematopoietic tissues. Concentrating then on CML, we investigated cell subpopulations from healthy donors and CML patients for their IRF4 expression to specify the distribution in normal and malignant lymphocytes and monocytes. Furthermore, we studied the correlation of IRF4 expression with the cytogenetic response during IFN-{alpha} therapy in CML patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Samples
Patient samples were taken from three institutions (Virchow Klinikum, Berlin; Universitätsklinikum Carl Gustav Carus, Dresden; and Klinikum Mannheim der Universität Heidelberg, Mannheim, Germany). Most of the samples were from patients being treated in the ongoing German CML trials. Ten to 20 mL of heparinized peripheral blood (20 U/mL) were drawn after informed consent was given. Unless denoted otherwise, CML patients were untreated or treated with hydroxyurea and were Bcr-abl–positive. All acute myeloid leukemia (AML) patients had blast counts above 75%, and acute lymphoblastic leukemia (ALL) patients had blast counts above 50%.

Cell Lines
K-562, Jurkat, and U-937 were obtained from the American Type Culture Collection (Rockville, MD). EM-2, KU-812, BV-173, KASUMI-1, and NB-4 were from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). The Raji, Daudi, HL-60, MOLT-4, DHL-4, LAM-53, and EHEB cell lines were kindly provided by the laboratories of Prof. Dr. D. Huhn (Virchow Klinikum). All cell lines were maintained at 5% CO2 in RPMI 1640 medium with 1% glutamine (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco BRL) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany).

Cell Separation and Flow Cytometry
Mononuclear cells (MNCs) were separated from peripheral blood by centrifugation over a Ficoll (Biochrom) gradient and were stimulated with recombinant IFN-{alpha}2b 1,000 U/mL (Intron A; Essex Pharma GmbH, München, Germany) or IFN-{gamma} (Boehringer Mannheim, Mannheim, Germany).

CD19+ B cells were separated from peripheral blood using the MACS CD19 MultiSort Kit (Miltenyi Biotec GmbH, Sunnyvale, CA) according to the manufacturer’s instructions. Purity of some B-cell fractions were verified to approximately 90% by fluorescence-activated cell sorter (FACS) analysis. CD3+ T cells and CD14+ monocytes were separated from peripheral blood by using a cell-sorting procedure with a FACS Vantage (Becton Dickinson, Erembodegem-Aalst, Belgium), using CD3-phycoerythrin (Coulter-Immunotech, Hamburg, Germany) and CD14–fluorescein isothiocyanate antibodies (Becton Dickinson) as recommended by the manufacturer. The purity was also approximately 90%.

RNA Isolation and cDNA Synthesis
RNA was extracted from heparinized peripheral blood with using a commercial RNAzol kit (Paesel, Frankfurt, Germany). One microgram of total RNA was used for cDNA synthesis, as described previously.19

Expression Analysis by Reverse Transcriptase Polymerase Chain Reaction
Because of the restricted availability of RNA from leukemia patients, mRNA expression was analyzed by semiquantitative polymerase chain reaction (PCR). The PCR assay was performed as described previously19 under the following cycling conditions: 94°C/2 minutes for denaturation, then 94°C/1 minute, 61°C/1 minute, and 72°C/1 minute for 28 cycles, followed by 90°C/1 minute and 60°C/10 minutes. The primer sequences were as follows: IRF4 sense primer, 5'-TCCCCACAGAGCCAAGCATAAGGT-3'; and IRF4 reverse primer, 5'-AGGGAGCGGCCGTGGTGAGCA-3' (fragment of 436 base pairs). Beta-actin–mRNA expression was analyzed as described elsewhere.19 The PCR protocol was standardized such that the cycle number ensured that PCR amplification was in its exponential phase. The products were electrophoresed on a 3% agarose gel. Gels were stained with ethidium bromide and photographed. The gel photos were scanned, and integrated optical densities (IntOD) were calculated using ONE-Dscan 1.0 software (Scanalytics, Billerica, MA). The ratio of IntOD IRF4 to IntOD beta-actin was then calculated. Analysis of normal control samples suggested a value of 0.300 as the cutoff. Other reference genes, porphobilinogendeaminase (pbgd) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), revealed comparable results (data not shown). In addition, a dilution series of normal IRF4-positive MNCs with IRF4-negative K-562 cells was analyzed with the reverse transcriptase (RT)-PCR assay to test its accuracy. Also, the coefficient of variation for five positive controls was determined in at least five different experiments each, showing minor variations of 7% to 13% (data not shown). PCR products were verified by automated sequencing.

Determination of Cytogenetic Response to IFN-{alpha}
Cytogenetic response was assessed to analyze at least 10 metaphases and was categorized as good or poor. The good-response group included both complete responders (CRs; 0% Ph+ metaphases) and partial responders (PRs; 1% to 34% Ph+ metaphases). The poor-response group included patients with a minor response (MinR; 35% to 94% Ph+ metaphases) and no response (NR; 95% to 100% Ph+ metaphases).21

Statistical Analysis
Differences in the IRF4 expression of healthy individuals and of various leukemia samples were calculated by Fisher’s exact test. Differences in the IRF4 expression of sorted cells and samples from untreated CML and IFN-{alpha}–treated CML patients were calculated by Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of IRF4 in Cell Lines and Human Leukemia Samples
We first sought to analyze the expression patterns of IRF4 in different human leukemias. Therefore, cell lines and primary leukemia samples were analyzed.

To confirm the reliability of our RT-PCR assay results, we first investigated human cell lines, some of which have been analyzed previously.14,22 Various B-cell (DHL-4, Raji, Daudi) and T-cell lines (Jurkat, MOLT-4), the CML cell lines K-562, KU-812, and EM-2, the AML cell lines HL-60, KASUMI-1, and NB-4, and the monocytic cell line U-937 exhibited no or only weak IRF4 expression (data not shown). In contrast, only the pre–B-cell line BV-173 and the B-cell chronic lymphocytic leukemia cell lines EHEB and LAM-53 showed high IRF4 mRNA levels (data not shown).

We next determined the mRNA levels of IRF4 in peripheral-blood samples from healthy volunteers and compared them with the transcript numbers of samples obtained from patients with CML, AML, ALL, chronic lymphoid leukemia, and chronic myelomonocytic leukemia. Two (6%) of 33 healthy volunteers exhibited decreased IRF4 mRNA levels, whereas in samples of myeloid leukemias and myelodysplastic syndromes, the number of IRF4 transcripts was significantly reduced. A representative PCR gel is displayed in Fig 1. Sixty-two (93%) of 70 CML, 22 (76%) of 29 AML, and nine (90%) of 10 chronic myelomonocytic leukemia samples showed impaired IRF4 mRNA expression (P < .0001; Table 1; for healthy volunteers and CML patients, also see Fig 2). Because our CML samples included samples obtained at both diagnosis and during hydroxyurea administration, we subdivided these groups and detected no significant difference in IRF4 levels between those groups (data not shown). We then wanted to exclude the possibility that, in the case of CML, the lack of IRF4 transcripts was due to the absence of IRF4-expressing cells, such as lymphocytes. Thus, we analyzed the blood differentials from eight CML patients with low IRF4 mRNA levels and found no correlation between IRF4 level and percentage of lymphocytes (data not shown).



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  		Fig 1. IRF4 expression in peripheral blood from healthy volunteers and patients with various leukemias. IRF4 mRNA levels in CML (lanes 3 and 4) and AML patients (lanes 5 and 6) and healthy volunteers (NB; lanes 1 and 2) detected by semiquantitative RT-PCR.

 

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  		Table 1. IRF4 Transcript Numbers in Peripheral Blood From Healthy Volunteers and Patients With Different Kinds of Leukemias
 


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  		Fig 2. IRF4 expression in CML patients before and during IFN-{alpha} therapy. Samples from CML patients were taken at diagnosis or during hydroxyurea treatment (Dx; n = 70) (median, 0.027) and during IFN-{alpha} therapy (IFN; n = 47) (median, 0.436) (P < .0001). Samples were compared with those from healthy volunteers (NB; n = 33) (median, 0.547).

 
We also addressed the question of whether the expression of IRF4 was associated with risk groups in CML. Data for determination of the Sokal Index were available for samples of 21 CML patients at diagnosis using three risk groups: low (Sokal Index below 0.8), intermediate (0.8 to 1.2), and high risk (above 1.2). We found no significant correlation between IRF4 level and risk group, but the two samples with high IRF4 levels were in the low-risk CML group (Table 2).


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  		Table 2. IRF4 Transcript Numbers and Sokal Index in CML Samples
 
Unlike in myeloid leukemia samples, IRF4 expression in samples from lymphatic neoplasias was not significantly impaired (Table 1). Low IRF4 mRNA levels were detected in only three (30%) of 10 B-cell chronic lymphocytic leukemia samples, and six revealed relatively high IRF4 levels. An analysis of ALL samples showed that four (40%) of 10 had low IRF4 mRNA expression, with three of the four being T-cell ALL samples. The six IRF4-expressing ALL samples were subclassified; three could be identified as pre–B-cell ALL samples and two as undifferentiated ALL samples. All together, these data indicate a defect of IRF4 expression predominantly in T cells.

IRF4 Transcripts in Different Cell Subpopulations
Our above data in primary lymphatic leukemias point to IRF4 mRNA expression predominantly in samples originating from B-cell– but not from T-cell–derived diseases. With regard to CML, we wanted to study the expression patterns of IRF4 in CML cells in more detail. Thus, we isolated CD19+ B cells, CD3+ T cells, and CD14+ monocytes from CML patients (n = 5) and from healthy volunteers (n = 5) as controls using FACS analysis and magnetic beads cell sorting. In general, the purity of the analyzed fractions was at least 90% (data not shown). From these subpopulations, IRF4 mRNA expression was analyzed using RT-PCR. IRF4 mRNA expression was not detected in CD14+ monocytes from either healthy volunteers or CML patients (Fig 3). In contrast, in both groups CD19+ B cells showed IRF4 expression (Fig 3). However, CD3+ T cells revealed a significant difference between the healthy volunteers and the CML patients: Whereas normal T cells expressed IRF4 mRNA, no IRF4 was detected in T cells from CML patients (P = .032, Fig 3). These data point to a downregulation of IRF4 preferentially in T cells of CML patients. Thus, the observed lack of IRF4 in CML samples may be due to this phenomenon.



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  		Fig 3. IRF4 expression in cell subpopulations of healthy volunteers (NB; first bar) and CML patients (second bar). CD19+ B cells (left, P = .421), CD3+ T cells (middle, P = .032), and CD14+ monocytes (right, P = 1.000) were compared. The mean (± SE) of five samples is shown.

 
We investigated samples from CML patients in myeloid (n = 9) and lymphoid (n = 2) blast crisis. Except for a larger variation of IRF4 levels in myeloid samples, there was no detectable difference between these two small groups (median, 0.378 for myeloid and 0.235 for lymphoid), but more samples are needed for a more detailed analysis (data not shown).

Impact of In Vivo IFN-{alpha} Therapy on IRF4 Expression in CML Patients
IFN-{alpha} is frequently used to treat CML patients and has been shown to prolong survival.23,24 To investigate the effect of IFN-{alpha} on IRF4 mRNA levels in CML patients, we analyzed samples from 47 patients undergoing IFN-{alpha} therapy and compared them with the 70 previously analyzed CML samples taken at diagnosis or during hydroxyurea administration. We detected a significantly higher level of IRF4 transcripts in the samples from IFN-{alpha}–treated CML patients (P < .0001, Fig 2). The mean IRF4 expression was 0.103 (95% confidence interval, 0.062 to 0.143) for CML samples taken at diagnosis and 0.456 (95% confidence interval, 0.347 to 0.566) for samples from IFN-{alpha}–treated patients.

We then wanted to know whether the observed increase in IRF4 was due to an upregulation in T cells, which exhibited only a low level of IRF4 mRNA when taken from CML patients at diagnosis (see above). We analyzed CD3+ T cells from five patients undergoing IFN-{alpha} therapy and detected a significantly higher IRF4 level compared with T cells from untreated patients (Fig 4).



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  		Fig 4. IRF4 expression in T cells from untreated and IFN-{alpha}–treated CML patients. T cells from IFN-{alpha}–treated patients (IFN; gray box) and untreated patients (Dx; open box) were compared (P = .048). The mean (± SE) of five samples is shown.

 
Stimulation of CML Cells In Vitro
To investigate the effect of IFNs on IRF4 mRNA expression in CML cells in vitro, we isolated MNCs from a CML patient not treated with IFN-{alpha} and incubated them with IFN-{alpha} and IFN-{gamma}. No significant stimulation of IRF4 expression by IFN-{alpha} or IFN-{gamma} was detected (Fig 5 A). The observed increase in IRF4 mRNA levels even without treatment of MNCs (Fig 5A, lanes 2, 3, and 4) may due to stress during the experimental procedure and was not found in MNCs from two other CML patients (data not shown). In addition, we found no significant upregulation of IRF4 mRNA levels by IFN-{alpha} or IFN-{gamma} in stimulated CML and B-cell lines (data not shown).



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  		Fig 5. IRF4 expression in CML cells during in vitro stimulation. (A) Stimulation of MNCs for 6, 24, or 48 hours with IFN-{alpha} (lanes 5, 6, and 7) and IFN-{gamma} (lanes 8, 9, and 10); control samples in lanes 1 through 4. (B) Stimulation with phorbol myristate acetate (PMA; lane 2), cycloheximide (CHX; lane 3), tumor necrosis factor alfa (TNF-{alpha}; lane 4), and transforming growth factor beta (TGF-ß; lane 5) for 6 hours; control sample in lane 1.

 
Treatment with phorbol myristate acetate, a substance known to induce T-cell activation and differentiation, or cycloheximide, a protein synthesis inhibitor, increased the IRF4 levels, whereas tumor necrosis factor-alfa and transforming growth factor-beta did not increase IRF4 mRNA expression in MNCs of a CML patient (Fig 5B). MNCs of a blood donor showed the same reactions to the above substances (data not shown).

The observed upregulation of the IRF4 mRNA level both in vivo in CML patients (treated with IFN-{alpha}) and in vitro in CML cells (treated with phorbol myristate acetate or cycloheximide) indicates that the impaired IRF4 mRNA expression may due to a mechanism of downregulation rather than to genomic alteration.

Correlation of IRF4 Expression With Cytogenetic Response to IFN-{alpha} Therapy
To further investigate the impact of IFN-{alpha} treatment on IRF4 expression, we correlated the IRF4 expression to the cytogenetic response in CML patients receiving IFN-{alpha} therapy. It is well known that the response to IFN-{alpha} varies considerably: Approximately 80% of CML patients have a good hematologic response, but only 35% to 45% achieve a good cytogenetic response, with the rest cytogenetically resistant to IFN-{alpha}.25

Samples of 40 patients, which had been analyzed for IRF4 expression earlier (see above), were correlated with cytogenetic status after IFN-{alpha} therapy. There were 13 CR, 14 PR, two MinR, and 11 NR samples. Interestingly, with regard to IRF4 mRNA levels, a significant difference was found between the relatively high-expressing CR and PR groups and the low-expressing NR group (P = .021 or P = .001, respectively), whereas no difference was detected between CR and PR themselves (P = .396) (Table 3). The difference was even more convincing when good responders (CR/PR, 0% to 34% Ph+) and poor responders (MinR/NR, 35% to 100% Ph+) were compared, showing a significant correlation between high IRF4 expression and good response to IFN-{alpha} therapy (P = .0017) (Fig 6).


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  		Table 3. IRF4 Transcript Numbers in CML Samples From Different Responders to IFN-{alpha} Therapy
 


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  		Fig 6. Correlation of IRF4 expression with cytogenetic response to IFN-{alpha} therapy in CML patients. Twenty-seven good (0% to 34% Ph+: mean, 0.623; 95% confidence interval, 0.464 to 0.782; median, 0.577) and 13 poor responders (35% to 100% Ph+: mean, 0.249; 95% confidence interval, 0.145 to 0.354; median, 0.184) were compared (P = .0017).

 
For 20 CML patients, samples taken both at diagnosis and during IFN-{alpha} therapy were available, with 18 of them having low IRF4 levels before IFN-{alpha} therapy. In 11 cases, IRF4 levels increased during IFN-{alpha} therapy. The nine cases in which the IRF4 levels did not increase were analyzed for their cytogenetic response: there were five NRs, one PR, and three could not be determined (data not shown), correlating well with the above data.

In order to analyze whether the observed increase in IRF4 transcripts was due to differences in the distribution of cell compartments, we compared the IRF4 levels in CML samples to their blood differentials. For eight samples, all data were available; four of them were good responders and four were poor responders (Table 4). All good responders exhibited high IRF4 levels, whereas all poor responders showed low IRF4 levels (median, 0.652 and 0.121, respectively). Both groups had comparable leukocyte and lymphocyte counts (median, 33% and 29%, respectively), which indicates that the IRF4 upregulation was not due to variations in the blood differential.


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  		Table 4. IRF4 Transcript Numbers in CML Samples in Comparison to the Blood Differential
 
These data strongly suggest that IRF4 expression in CML patients during IFN-{alpha} therapy is dependent upon the response to the therapy, with normal mRNA levels only in good responders.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Altered signal transduction plays a major role in the oncogenesis of malignant human diseases. The fusion protein Bcr-abl, which is the hallmark of CML and has an activated part of the c-Abl nonreceptor tyrosine kinase, signals not only via the classical Ras-Raf cascade but also through proteins encountered in cytokine signal transduction pathways, such as STAT 1, 3, and 5.26-28 To prove that these genes are causally linked to leukemogenesis, knockout mice may be helpful. Recently, mice with germline deletion of the IRF ICSBP have been generated. Interestingly, their phenotype was not only immunocomprehension, as expected, but also that of CML.18 These data forced us to search for aberrant expression of IFN regulators in human leukemias.

IRF4 is a member of the growing family of genes enrolled in the control of IFN-dependent genes. In contrast to IRF-1 and IRF-2, expression of IRF4 is highly restricted to the lymphoid lineage.12,13 This may indicate a specific role of IRF4 in lymphoid development and control of its function. In line with this, IRF4 knockout mice harbor impaired immunoglobulin production and defective cytotoxic responses and finally develop lymphadenopathies, thereby supporting an essential role of IRF4 in lymphoid tissues. For the present study, we asked whether IRF4 may also be involved in human leukemogenesis. To this end, we used semiquantitative RT-PCR to analyze different human cell lines and primary human leukemias.

In keeping with others, we found the highest expression of IRF4 in lymphoid cell lines.14,22 In contrast, a complete lack of expression was detected in myeloid cell lines originating from CML and AML samples. We studied primary human leukemia samples, where the results in general were in line with the data obtained in the cell lines. We also asked whether sorted cells from various lineages of CML patients harbored perturbations of IRF4 transcript numbers. Therefore, T and B cells, as well as monocytic cells, were sorted. Healthy blood donors were used as controls. We observed a significant difference of expression in the T cell but not in the other investigated cellular compartments. Although CML derives from the uncontrolled proliferation of myeloid progenitor cells, many data actually point to an additional role of T cells in this disease. For instance, it has been discovered that the Bcr-abl peptide may be presented by HLA-A3, leading to induction of specific cytotoxic T lymphocytes.29,30 In keeping with this, CML has a significantly lower incidence in individuals with HLA-A3,31 which suggests that HLA-A3–positive individuals are somehow protected against expansion of Bcr-abl–presenting leukemia cells. Thus, from these data it seems that T cells may play an important role in CML.

Since our data pointed to a contribution of IRFs in the genetics of myeloid leukemias, we sought to study their role in treatment response. We had previously shown that in vivo IFN-{alpha} therapy leads to upregulation of another IRF family member, ICSBP.19 In vivo IFN-{alpha} therapy also resulted in a significant upregulation of IRF4, which takes place in the T-cell compartment. However, the expression level of IRF4 was not changed after in vitro incubation with IFN-{gamma} or IFN-{alpha}. This fits well with the fact that the promoter of IRF4 lacks an IFN-{gamma}–activated site and an IFN-stimulated response element, necessary for direct response to IFN-{gamma} and IFN-{alpha}, respectively. Thus, another different signal cascade may lead to the in vivo reaction observed in IFN-{alpha}–treated patients but not the direct signal cascade of IFN -> JAK -> STAT -> IRF.

The response to IFN-{alpha} is an important prognostic factor for survival in CML patients. Because approximately 60% of the patients do not respond cytogenetically to IFN-{alpha} therapy, it is useful to identify these resistant patients as early as possible. For this purpose, we wanted to investigate whether IRF4 expression may be correlated with cytogenetic response to IFN-{alpha} therapy. We found a highly significant correlation between a good response (CR, PR)/high IRF4 expression and a poor response (MinR, NR)/low IRF4 expression. The findings were not due to changes in the blood differential. These data may point to a role of IRF4 in the therapeutic effect of IFN-{alpha}. Since this was a retrospective analysis, a prospective study evaluating the role of IRF4 in reaching a cytogenetic response is mandatory and underway. Furthermore, IFN regulation through IRFs can be a possible target for gene therapy, ie, by upregulation of IRF4, which may be of benefit for CML patients.


   ACKNOWLEDGMENTS
 
Supported by grants from the Deutsche Forschungsgemeinschaft (to A.N. and B.W.), Bonn, Germany, the Deutsche José-Carreras-Leukämiestiftung e.V. (to A.N. and A.H.), Munich, Germany, and the Hector-Stiftung (to M.S. and A.N.), Mannheim, Germany. S.A.K.M. was funded by the Graduierten Kolleg Signaltransduktion from the Deutsche Forschungsgemeinschaft.

We are grateful to Prof. Dr. D. Huhn for help throughout this study. We thank Prof. Dr. I. Horak for his support of this study. We thank Dr. J. Mohm for providing clinical data on CML patients, Dr. M. Ritter for help collecting samples, and Dr. S. Nagel for critical discussion. In addition, we thank the clinicians and cytogeneticists participating in the German CML trials for sending samples and providing clinical and cytogenetic data.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Pestka S, Langer JA, Zoon KC, et al: Interferons and their actions. Annu Rev Biochem 56: 727-777, 1987[Medline]

2. Tanaka N, Taniguchi T: Cytokine gene regulation: Regulatory cis elements and DNA binding factors involved in the interferon system. Adv Immunol 52: 263-281, 1992[Medline]

3. Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415-1421, 1994[Abstract/Free Full Text]

4. Ihle JN: The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv Immunol 60: 1-35, 1995[Medline]

5. Schindler C, Darnell JE Jr: Transcriptional responses to polypeptide ligands: The Jak-STAT pathway. Annu Rev Biochem 64: 621-651, 1995[Medline]

6. Darnell JE Jr: STATs and gene regulation. Science 277: 1630-1635, 1997[Abstract/Free Full Text]

7. Miyamoto M, Fujita T, Kimura Y, et al: Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell 54: 903-913, 1988[Medline]

8. Harada H, Fujita T, Miyamoto M, et al: Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 58: 729-739, 1989[Medline]

9. Driggers PH, Ennist DL, Gleason SL, et al: An interferon gamma-regulated protein that binds the interferon-inducible enhancer-element of major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 87: 3743-3747, 1990[Abstract/Free Full Text]

10. Weisz A, Marx P, Sharf R, et al: Human interferon consensus sequence binding protein is a negative regulator of enhancer elements common to interferon-inducible genes. J Biol Chem 267: 25589-25596, 1992[Abstract/Free Full Text]

11. Fu X-Y, Kessler DS, Veals SA, et al: ISGF3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains. Proc Natl Acad Sci U S A 87: 8555-8559, 1990[Abstract/Free Full Text]

12. Matsuyama T, Grossman A, Mittrücker HW, et al: Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). Nucleic Acids Res 23: 2127-2136, 1995[Abstract/Free Full Text]

13. Eisenbeis CF, Singh H, Storb U: Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev 9: 1377-1387, 1995[Abstract/Free Full Text]

14. Yamagata T, Nishida J, Tanaka T, et al: A novel interferon regulatory factor family transcription factor, IRF4/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol Cell Biol 16: 1283-1294, 1996[Abstract]

15. Brass AL, Kehrli E, Eisenbeis CF, et al: Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU. 1. Genes Dev 10: 2335-2347, 1996[Abstract/Free Full Text]

16. Nelson N, Kanno Y, Hong C, et al: Expression of interferon regulatory factor family proteins in lymphocytes: Induction of Stat 1 and ICSBP expression by T cell activation. J Immunol 156: 3711-3720, 1996[Abstract]

17. Matsuyama T, Kimura T, Kitagawa M, et al: Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75: 83-97, 1993[Medline]

18. Holtschke T, Löhler J, Kanno Y, et al: Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 87: 307-317, 1996[Medline]

19. Schmidt M, Nagel S, Proba J, et al: Lack of interferon consensus sequence binding protein (ICSBP) transcripts in human myeloid leukemias. Blood 91: 22-29, 1998[Abstract/Free Full Text]

20. Mittrücker HW, Matsuyama T, Grossman A, et al: Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275: 540-543, 1997[Abstract/Free Full Text]

21. Hochhaus A, Yan XH, Willer A, et al: Expression of interferon regulatory factor (IRF) genes and response to interferon-alpha in chronic myeloid leukemia. Leukemia 11: 933-939, 1997[Medline]

22. Grossman A, Mittrücker HW, Nicholl J, et al: Cloning of human lymphocyte-specific interferon regulatory factor (hLSIRF/hIRF4) and mapping of the gene to 6p23–p25. Genomics 37: 229-233, 1996[Medline]

23. Tura S, Baccarani M, Zuffa E, et al: Interferon alpha-2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. N Engl J Med 330: 820-825, 1994[Abstract/Free Full Text]

24. Allan NC, Richards SM, Shepherd PCA: UK Medical Research Council randomized multicentre trial of interferon-alpha n1 for chronic myeloid leukemia: Improved survival irrespective of cytogenetic response. Lancet 345: 1392-1397, 1995[Medline]

25. Kantarjian H, O’Brien S, Anderlini P, et al: Treatment of myelogenous leukemia: Current status and investigational options. Blood 87: 3069-3081, 1996[Free Full Text]

26. Carlesso N, Frank DA, Griffin JD: Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med 183: 811-820, 1996[Abstract/Free Full Text]

27. Shuai K, Halpern J, ten Hoeve J, et al: Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 13: 247-254, 1996[Medline]

28. Ilaria RL Jr, Van Etten RA: P210 and P190 (BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J Biol Chem 271: 31704-31710, 1996[Abstract/Free Full Text]

29. Bocchia M, Wentworth PA, Southwood S, et al: Specific binding of leukemia oncogene fusion protein peptides to HLA class I molecules. Blood 85: 2680-2684, 1995[Abstract/Free Full Text]

30. Bocchia M, Korontsvit T, Xu Q, et al: Specific human cellular immunity to bcr-abl oncogene-derived peptides. Blood 87: 3587-3592, 1996[Abstract/Free Full Text]

31. Posthuma EF, Falkenburg JH, Apperley JF, et al: HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukemia: The Chronic Leukemia Working Party of the EBMT. Blood 93: 3863-3865, 1999[Abstract/Free Full Text]

Submitted September 29, 1999; accepted June 5, 2000.




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