Introduction

Acute leukemia (AL) is regarded the most common type of malignancy in children. Despite the good overall response of childhood patients to current chemotherapeutic treatments and the long-term event-free survival rates exceeding 80% [1], [2], a significant percentage of the population show resistance to therapy and/or relapse, often with devastating consequences. This has highlighted the importance of devising new therapies that will target the resistant clones and increase overall survival. Of significant interest are the genetic alterations that might affect the characteristics of hematopoietic stem cells (HSCs). For example, it has already been demonstrated that a subset of genes expressed in normal HSCs are highly reactivated in leukemic stem cells (LSCs), thus indicating that the LSCs express the self-renewal-associated programme normally characterising the HSCs [3]. Such findings suggest that leukemic transformation of progenitor cells is associated with ectopic reactivation of the genes responsible for self-renewal. Indeed, the presence of MLL rearrangements in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) seems to relate with high level expression of genes normally expressed in HSCs during the early stages of hematopoiesis, such as FLT3 and HOXA9 [4]–[10]. Consequently, the identification of specific genetic aberrations that characterise leukemic cells, such as abnormal gene expression, can lead to a better understanding of what constitutes their unlimited self-renewing properties and subsequently reveal potential molecular targets for successful therapy.

The most frequent targets of genetic alterations in human leukemias are transcription factor genes with essential functions in normal blood cell development. The Interferon Regulatory Factor 4 (IRF4) gene encodes a transcription factor important for hematopoietic development and immune responses. It is essentially involved in all developmental stages within the B-cell lineage (except during the germinal center (GC) reaction), with known critical functions in at least 3 key developmental processes: the termination of the GC B-cell transcriptional program, immunoglobulin class-switch recombination, and plasma cell development [11]–[13]. IRF4 is induced by mitogenic stimuli, such as antigen receptor engagement, lipopolysaccharides, and CD40 signaling [14], [15], which activate the Nuclear Factor-kappa B (NF-kB) pathway, ultimately leading to IRF4 promoter activation [16]–[18]. Despite functioning as a tumor suppressor gene in early B-cell development [11], [19], IRF4 is a well-established oncogene in multiple myeloma (MM) [20], with oncogenic implications extending to certain adult lymphomas and leukemias [13], [21]–[23]. High expression levels have been detected in chronic lymphocytic leukemia (CLL), where they have been linked to an unfavourable prognosis [24]–[27]. Very few studies have reported an association of IRF4 with childhood malignancy. In childhood ALL, IRF4 has been found significantly up-regulated in the more mature CD34-CD19⁺ LSC population [28], whereas others have reported that an intronic polymorphism of the gene relates to a greater risk association of males to the disease [29].

Considering the importance of IRF4 in key developmental stages of hematopoiesis, as well as its oncogenic implication in malignancies such as lymphomas and chronic leukemias, investigating its expression patterns in childhood leukemias could help in the understanding of how deregulation can contribute to leukemogenesis. In this study we investigate the transcript levels of IRF4 in the diagnostic samples of a cohort of 58 pediatric acute leukemia patients and 4 cell lines, including both ALL and AML, and compare them to those of healthy controls. Our aim is to determine whether aberrant IRF4 expression extends to childhood AML or to leukemic subtypes other than the more mature CD34-CD19⁺ ALL population. In addition, we examine whether there are any correlations between gene expression and specific cytogenetic characteristics, as well as other diagnostic variables, such as age, gender, and karyotype. Finally, we investigate the prognostic impact of high IRF4 expression in the clinical course of childhood leukemia, through possible correlations with minimal residual disease (MRD) and survival outcome.

Materials and Methods

Results

Quantitative Real Time RT-PCR analysis

A quantitative real-time RT-PCR assay was established for the gene of interest. IRF4 was found to be differentially over-expressed, as compared to the healthy controls, in 30 patients (51.7% of total cohort): 26 ALL (18 B-common, 3 pre-B, 4 T-cell and 1 infant leukemia of unknown immunophenotype), 4 AML (1 with M1 and 3 with M5 maturation), and 3 of the 4 cell-lines: REH (pre-B ALL), CCRF-CEM (T-cell ALL) and THP-1 (AML) (Figure 1). The patients over-expressing IRF4 are also highlighted in bold in Table S1. Five (5) of these 30 patients and the 3 cell lines mentioned above revealed a much higher expression ratio (R>11.6) than the rest (p = 0.000). In addition, the gene was found to be differentially down-regulated in 6 patients (∼10%) (Table 1). Therefore, patients were divided into 4 groups: subnormal ratio (ratio <2.4), normal (healthy) ratio (2.4≤ ratio <7), high ratio (7< ratio <11.6) and very high ratio (11.6< ratio). In the subnormal ratio group, the mean IRF4 expression was significantly lower (p = 0.000) than in the healthy controls group (File S3). Overall, the gene was found up-regulated in a broader spectrum of leukemic subtypes and maturation stages than previously thought, with the highest transcript levels appearing in the AML group (Figure 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. IRF4 expression levels in various immunophenotypic groups.

https://doi.org/10.1371/journal.pone.0072326.g001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. IRF4 expression levels in leukemia patients and cell lines.

https://doi.org/10.1371/journal.pone.0072326.t001

Correlation Analysis Between Gene Expression and Overall Survival

No significant correlation was found between abnormal IRF4 expression and overall survival (OS) (as measured in years of survival from the diagnosis) on comparing patients with normal and patients with high IRF4 expression (Figure 2, A–H). Please note that Patients 56 and 57 were diagnosed at a much earlier date (2003) and so were regarded as outliers and were not included in the analysis. Even though we have observed twice as many deaths (4 versus 2) in the patient group over-expressing IRF4 (Table S2), the Kaplan Meier curves have not produced a statistically significant difference (p>0.05). The results for estimated leukemia-free time (i.e. time to relapse) were marginally significant, as it can be seen in Figure 2 (E & G) (p = 0.08, CI = 90% and p = 0.05, CI = 95% respectively). Interestingly, when leukemia-free time was estimated within a specific time frame of 3-years (i.e. only the patients diagnosed in the years 2008, 2009 and 2010 were included in the analysis), a significant correlation appeared to exist between high IRF4 expression and relapse (p = 0.03) (Figure 2, I).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Overall survival (OS) and leukemia-free time analysis with respect to IRF4 expression.

Comparisons of OS included: normal levels of IRF4 vs. high levels of IRF4 (A), normal levels of IRF4 vs. very high levels of IRF4 (B), normal levels of IRF4 vs. both high and very high levels of IRF4 (C) and clinical remission vs. relapse irrespectively of IRF4 expression (D). Similarly, the same analysis was performed for leukemia-free time and comparisons included: normal levels of IRF4 vs. high levels of IRF4 (E), normal levels of IRF4 vs. very high levels of IRF4 (F), normal levels of IRF4 vs. both high and very high levels of IRF4 (G) and clinical remission vs. relapse irrespectively of IRF4 expression (H). Interestingly, when leukemia-free time was estimated within a certain time frame (3-year) high IRF4 expression appeared to correlate with leukemia-free survival (I).

https://doi.org/10.1371/journal.pone.0072326.g002

Discussion

In the present study we have found high IRF4 transcript levels in a significant number of patients (51.7%) and in 3 of the 4 cell lines, as compared to the healthy controls. This up-regulation seems to associate with a variety of leukemic subgroups, possibly reflecting the many roles of IRF4 in the immune system [12]. Considering that IRF4 is essential for several stages of normal B- and T-cell activation and differentiation, it is only logical to assume that aberrations in the IRF4 transcriptome would also reflect in lymphoid-cell associated malignancy.

A significant difference in IRF4 transcript levels was observed between the pre-B and B-common cohorts, as well as between the total B- and T-cell ALL groups (Figure 1), with higher transcript levels appearing in the B-common and T-cell subtypes, respectively. In the past, however, IRF4 mRNA expression has been found low in adult samples of T-cell ALL [34]. At this point it is difficult to validate the reasons for this difference, besides possible age-related dissimilarities in the biology of ALL, yet it points out that aberrant IRF4 transcriptional activity is developmental-stage-specific as well as cell-type-specific. It has already been demonstrated that IRF4 is expressed at varying levels throughout B-cell development, with expression peaking in plasma cells, therefore varying transcript levels in human malignancies could mirror IRF4 expression in normal lymphoid activation and differentiation [35], [36]. However, in contrast to the Gene Expression Omnibus (GEO) data profiles from healthy tissues [37] and a recent study from le Viseur et al [28], in our leukemia samples we have observed that the greater the distance from a differentiated B-cell, the higher the level of IRF4 expression. In other words, significantly higher transcript levels were observed in the B-common subgroup, than in the more mature pre-B subtype (CD34^dim/-CD19⁺). This suggests that, at least for the particular patient cohort studied here, ectopic expression of IRF4 does not exactly mirror the expression patterns encountered in normal B-cell development, but rather the situation seems to reverse. This observation could reflect differences in our experimental set-up, as opposed to the study from le Viseur et al [28], as we have not measured IRF4 expression in flow-sorted subpopulations from the same diagnostic sample but have rather based our analyses on the overall prevailing immunophenotype for every patient. On the other hand, the 3 cell lines, despite being phenotypically distant from differentiated cells, appeared to express even higher levels of the gene than the patients (all 3 fell into the very high ratio >11.6); this possibly implies that in childhood leukemia, IRF4 gene expression is not subject to the feedback regulation mechanisms that operate in normal tissues.

In the past, IRF4 expression was regarded an almost exclusive characteristic of the lymphoid lineage [15], but recent data have also demonstrated an involvement in myeloid cell differentiation [38]–[40]. The implication is still not entirely understood, but ectopic expression of IRF4 in myeloid progenitor cells in vitro was recently shown to inhibit myeloid cell growth and to tune the balance of lineage selection in myeloid progenitor cells by stimulating macrophage differentiation, whilst inhibiting neutrophil differentiation [41]. Subsequently, expression has been found down-regulated in chronic myeloid leukemias (CML), as well as in adult AML patient samples [34], [42], [43]. Here we report an association of IRF4 up-regulation with childhood AML, as it is in this patient cohort that we observe the highest transcript levels, including the AML cell line. Interestingly, 2 of the 4 AML patients over-expressing IRF4 fell into the very high ratio group (11.6< ratio) (Table 1) and were in fact positive for M5 maturation, whereas high expression was also observed in the M1 stage (without maturation). This could also highlight the role of IRF4 in early myeloid cell differentiation and, subsequently, in myeloid leukemia without maturation but, since the number of observed cases here is small, no such conclusions can be made at this point. Analysis of a larger cohort of patients and of myeloid cell populations at various maturation stages should be able to confirm or reject the validity of this notion. The opposing expression levels between adult and childhood AML (low versus high) are most probably the result of the different pathogenetic mechanism that discriminates between adult and childhood leukemias.

Significant differentially low IRF4 expression, as compared to the healthy controls (p = 0.000) was observed in 6 of the patient samples that we analysed (∼10% of total patients), all of which were B-cell ALL positive. This observation is consistent with the presence of a B-lineage-restricted repressor on the promoter of the IRF4 gene, which can become over-expressed in a cross-section of B-lineage leukemia [44], [45]. Since IRF4 binds to its own promoter region, creating the potential for positive auto-regulation, its expression can reach levels that activate the repressor [17]. Other leukemic subtypes seem to lack the repressor and therefore do not exhibit abnormally low IRF4 expression. Furthermore, at least one micro-RNA species has been identified as an IRF4 repressor. In the B-cell lineage, hsa-miR-125b (GC-enriched hsa-miR-125b) has been shown to directly down-regulate the expression of IRF4 and of the repressor PRDM1/BLIMP1 [46]. While IRF4 expression in non-B-cell leukemia can be deregulated toward higher levels, in B-lineage leukemia activation of the repressor may also facilitate a reduction in IRF4 transcript levels below the expected average.

Overall, as it can be seen in Figure 1, it seems that childhood leukemia, irrespective of subtype or cell maturation stage, is characterised by a minimum of approximately twice the amount of IRF4 gene expression encountered in healthy children (as measured in corrected expression levels (ΔCt)). This might suggest that there is a dose-dependency of childhood leukemia for ectopically expressed IRF4, a characteristic that could be explored further for its therapeutic potential. A therapy based on IRF4-knockout would most likely be hindered by the potentially detrimental side effects. IRF4-deficient mice show profound defects of the immune system, such as impaired lymphocyte function, inability to generate cytotoxic, antibody and antitumor responses, accompanied by a severe reduction in serum immunoglobulin concentrations [14]. Surprisingly, mice lacking one allele of IRF4 appear phenotypically normal, whereas a 50% knockdown of IRF4 mRNA and protein has proved effective in clearing MM cell lines [14], [47]. Perhaps the same should be applied to leukemic cells in order to evaluate whether modified or partial silencing of the gene appears to have an inhibitory or apoptotic effect, and thus whether it opens up a new opportunity of a therapy aimed at IRF4.

No significant correlation was found between high IRF4 expression and OS (Figure 2, A–H). This could be due to the small number of patients included in the analysis or perhaps to the fact that for most of these patients OS could not be calculated in terms of a 3-year or a 5-year survival. Whereas a marginally significant correlation appeared to exist between abnormal IRF4 expression and estimated leukemia-free time (p = 0.08 and p = 0.05) (Figure 2, E & G), a significant correlation appeared (p = 0.03) when leukemia-free time was estimated within a specific time frame of 3 years (Figure 2, I). This implies that high IRF4 expression might be used as an additional prognostic marker of relapse at the diagnosis of childhood acute leukemias. Further analyses on a larger number of patients over-expressing IRF4, over a longer period of time (i.e. 5-year survival), should be able to confirm this observation in the near future.

Additional efforts should be made in order to identify the putative targets of IRF4 and to determine the ones directly regulated by it. A number of proteins have already been identified as repressors in B-cells and found to interact with IRF4: PU.1, Spi-B, E47, NFAT, Stat6, Bcl-6 and Blimp-1 [14], [38], [48]–[50]. In addition, CD40 signaling has been found to directly induce the expression of IRF4 in GC B-cells which, in turn, binds to the promoter region of the BCL-6 gene to repress its expression [51]. We are currently investigating the expression patterns of other hematopoietic progenitor genes in a larger cohort of pediatric patients in an attempt to gain a better understanding of how deregulated transcription on key signaling pathways affects the malignant phenotype and disease progression in childhood leukemia.

Conclusions

In summary, our study shows that aberrant IRF4 gene expression is implicated in a variety of leukemic subtypes, possibly reflecting the many roles of the gene in the immune system. In contrast to other studies, we observe higher transcript levels in patients characterised by a more immature immunophenotype, and in T-cell leukemias than in leukemias of the B-lineage, implying that in childhood leukemia, IRF4 gene expression is not subject to the feedback regulation mechanisms that operate in normal tissues. In addition, ectopic expression of IRF4 does not seem to be a specific feature of lymphoid leukemia, but it also extends to myeloid leukemia, as it was in our AML group that the highest expression levels were observed. Again, our results are in contrast to other studies documenting a down-regulated IRF4 expression in adult myeloid leukemias [34], [42], [43], even though this is most probably due to age-related dissimilarities and a different pathogenetic leukemia mechanism. Interestingly, we show that childhood leukemia, irrespective of subtype or cell maturation stage, is characterised by a minimum of approximately twice the amount of IRF4 gene expression encountered in healthy children. This implies that there might be a dose-dependency of the disease for aberrantly expressed IRF4, a characteristic that could be explored therapeutically. In addition, a statistically significant correlation was found between high IRF4 expression and relapse, suggesting a prognostic value of IRF4 in the clinical course of childhood leukemias. Data from the past few years indicate that many important regulators of HSC development are also implicated in the abnormal self-renewal capacity of the LSCs. Only through the identification of these regulators, and of their downstream targets, will we gain a better understanding of what constitutes their unlimited self-renewing properties and subsequently reveal potential molecular targets for successful therapy.

Supporting Information

Acknowledgments

We thank Dr. M. Kanariou for providing access to the Flow Cytometry facility of the Immunology & Histocompatibility Department and Aspasia Divane at LIFE CODE Genetic Centre for Research and Diagnosis, for performing the karyotype and cytogenetic analyses.