IRF 4 as an Oncogenic Biomarker for Hematological Malignancies

The lymphocyte-specific transcription factor Interferon (IFN) Regulatory Factor 4 (IRF4) is crucial for lymphocyte development. Importantly, IRF4 has potent oncogenic and transforming properties, and its intimate interaction with lymphoid and myeloid malignancies has been increasingly recognized. In general, IRF4 exerts its function by transcriptionally regulating a pool of genes pivotal for cell development, oncogenesis and immune response. In clinical practice, IRF4 serves as an important prognosis and diagnosis marker for certain types of these malignancies. However, the oncogenic roles of IRF4 in most types of these malignancies and the molecular mechanisms underlying its interaction with them are largely not characterized. Accumulating evidence from cell culture shows that IRF4 regulates differential targets in distinct cancer contexts, depending on the“context-specific”co-regulator(s) associated with it in each setting. These complementary studies with in vitro cell culture systems are a necessary strategy which will provide molecular and mechanistic insights into the specific regulation and function of IRF4 in distinct cancer contexts, and may identify novel interventions specifically targeting IRF4 regulatory network for treatment. This review summarizes the evidence obtained from bench to bed showing the association of IRF4 with various types of hematological malignancies, with emphasis on molecular mechanisms underlying its regulation and its roles in these contexts.

IRF4 was cloned independently by different groups [4,14,15].Its closest family member is IRF8, and together, they both play critical roles for the development of immune cells (lymphocytes, myelocytes, and dendritic cells).Irf4 −/− mice are devoid of germinal centers (GCs) and plasma cells, with a severe reduction in the serum immunoglobulin (Ig) level and the failure of mounting protective antibody responses.
The expression of IRF4 is confined to immune cells including B cells, macrophages, and CD11b+ DCs, and is inducible by a variety of mitogenic stimuli including antigen receptor engagement, and TLR and CD40 signaling pathways [5].IRF4 is strongly up regulated upon co-stimulation of B cells with CD40 and IL4.Commonly, these stimuli all activate NFκB leading to IRF4 induction [16].Different from other IRF family members, expression of IRF4 is not induced by interferon.In B cell lineage, IRF4 expression is repressed by the transcription factor Mitf in mature B cells (native resting B cells), and is also particularly weak in GC B cells likely due to the absence of NFκB in these cells.The level of IRF4 protein is culminating in plasma cells, the terminally differentiated B cells, through a yet unknown mechanism.Among T cell lineages, its expression is the highest in activated T cells.
In this review, the association of IRF4 with multiple myeloma, DLBCL, and viral and other hematological malignancies is summarized and the known role and regulation of IRF4 in each cancer context are introduced.

IRF4 in multiple myeloma
Multiple myeloma(MM) is a malignancy of the terminally differentiated B lymphocytes, plasma cells, in which IRF4 is over expressed.Myc, which is over expressed owing to chromosomal translocation in this setting, induces expression of IRF4, and vice versa [29].Chromosomal translocation also contributes to IRF4 over expression in a small fraction of MM [33].Irf4 gene was identified as one of the six oncogenic chromosomal partners (the other five are Fgfr3, Ccnd3, Ccnd1, c-Maf, and Mafb) of IgH translocation in MM [33,34].
Transcriptome analysis has shown that IRF4 regulates a myeloma-specific gene expression pattern that combines IRF4 regulatory networks from activated B cells and plasma cells [29].In addition to Myc, other important IRF4 targets in multiple myeloma include Casp3, Cdk6, Ell2, and Tnfaip3 [29].Therefore IRF4 regulates multiple essential cellular processes such as cell cycle and apoptosis leading to myeloma cell proliferation and survival [27,29].
The Ets family members, PU.1 and its closely related factor SPIB, which are expressed in B cells, macrophages, and dendritic cells, are not expressed in MM [35].Thus, IRF4 transcriptional co-regulator which recruits IRF4 to its genomic targets in MM remains to be determined.

IRF4 in DLBCL
DLBCL, a type of aggressive lymphoma with poor prognosis and the most common subtype of non-Hodgkin lymphoma, accounts for approximately 40% of lymphomas among adults.Over expression of IRF4 is a hallmark of ABC (activated B-cell-like) subtype of DLBCL [5,23], owing to the constitutive activation of the prosurvival NFκB pathway [32], a process mediated by the adapter CARD11 in DLBCL.The constitutive activation of NFκB results from oncogenic mutations of BCR and My D88.ABCDLBCL is likely originated from late germinal center B cells which fail to terminally differentiate into plasma cells.IRF4 over expression may manifest the physiological attempt of these cells to activate the terminal plasma cell differentiation program [36].A recent report also shows that IRF4 is over expressed in a subtype of pediatric GCB-type DLBCL and follicular lymphoma grade 3 due to chromosomal translocation carrying Ig/Irf4 fusion loci [37].
In ABC DLBCL, IRF4 inhibits Irf7 gene transcription and therefore dampens type I IFN production, but stimulates NFκB signaling by transactivating Card11 gene promoter [35].Like its role in MM, about 50% knockdown of IRF4 is sufficient to kill ABC DLBCL cell lines, but not to other tested lymphoma and leukemia lines [35].Surprisingly, ChIP-Seq high throughput analysis has shown that, different from MM, IRF4 does not bind to Myc and Irf4 loci in ABC DLBCL lines, although MYC is highly expressed in ABC DLBCL, and that many other genes such as CD44 and CD40 are targeted by IRF4 in ABC DLBCL but not in MM [35].These observations suggest distinct IRF4 regulatory networks in MM and DLBCL.In addition, unlike most normal GC B cells which display mutually exclusive in the expression of MUM1 and Bcl-6, tumor cells in approximately half of IRF4-positive DLBCL are also Bcl-6 positive.The IRF4 coregulator, SPIB that is not expressed in MM, intimately interacts with IRF4 for its oncogenic function in ABC DLBCL [35].
Nevertheless, different clinical studies have shown conflicts in terms of the prognostic value of IRF4 with DLBCL; some report poor prognostic value, but others have failed to observe any significance [38].

IRF4 in Burkitt lymphoma
Burkitt lymphoma (BL) is a type of non-Hodgkin B-cell lymphoma which is highly aggressive.BL is subdivided in to EBVassociated endemic subtype (>99% are EBV+), the sporadic subtype in developed countries (15-25% are EBV+), and an AIDS-associated subtype (~30% are EBV+) clinical variants [39].BL is composed of monomorphic medium-sized B cells with a high proliferation rate, and c-Myc locus translocation as an oncogenic hallmark in all cases [40].Classically, like DLBCL, BL is considered to be germinal center in immunophenotype [41].In most reports, IRF4 is negative in BL cases.However, a study with 222 cases of well-characterized Burkitt lymphomas from Brazil with the classic phenotype and c-Myc translocation has revealed that 90 cases (40.5%) are IRF4-positive in the nucleus [41].

IRF4 and viral oncogenesis
Virus contributes to appropriately 20% of total malignancies in the globe [42,43].IRF4 is over expressed in EBV-transformed cells and associated lymphomas [20,[44][45][46], and in Human T-cell Leukemia Virus-1 (HTLV1)-transformed cell lines and associated Adult T-cell Lymphoma/Leukemia (ATLL) [31,[47][48][49][50].It is now understood that, in EBV-transformed cells, IRF4 is induced by the LMP1/NFκB signaling (Figure 1) [20,46].Consistent with this, IRF4 has been recently shown to be expressed in all EBV LMP1-driven tumors in mice [51].More recently, IRF4 has been shown to be stabilized by EBNA3 Cin EBV-transformed cells (Figure 1) [52].An over expression study also showed that IRF4 is a direct target for the EBV antigen EBNA2, as detected by microarray analysis (Figure 1) [53].Induction of IRF4 expression in the context of HTLV1 infection is complicated.It is induced in a Tax-independent manner in primary ATLL [54], but is induced in either Tax-dependent or Taxindependent manner in cell culture [47,49,54].Induction of IRF4  [20;44-46], and probably is also directly induced by EBNA2 [53].IRF4 protein is also stabilized by EBNA3C [52].Our recent study has disclosed that IRF4 is activated through c-Srcmediated tyrosine phosphorylation [91].We also have evidence showing that IRF4 is activated by the LMP1 signaling pathway, probably mediated by c-Src.c-Src protein is induced and activated in EBV latency [91].In addition to the LMP1 pathway, other lymphocyte-specific pathways may also contribute to IRF4 activation.Two transcriptional targets, BIC [21]and Irf5 [78], have been identified and confirmed so far for IRF4 in EBV latency.IRF4 induces BIC but represses Irf5 transcription.

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expression by Tax may be an important cellular event involved in HTLV1 leukemogenesis [50].IRF4 can also be induced by c-Rel or by other undefined cellular pathways in the absence of c-Rel or Tax [47,54].
LMP1 is the principal EBV oncogene, and is the only EBV product which transforms human and rodent fibroblasts in vitro [55,56].Up to 50% of LMP1 transgenic (Tg) mice had a threefold increase in lymphoma development in elderly period in comparison with LMP1negative mice [57].Moreover, on a T-cell ablated background, most LMP1 Tg mice developed DLBCL-like and plasmacytic tumors [51], As such, IRF4 can induce focus formation of mouse fibroblasts in soft agar, a typical sign of malignant transformation [20].The importance of IRF4 in LMP1 oncogenesis is underscored by the fact that IRF4 is expressed in all of these LMP1-driven tumors in mice [51].
Nevertheless, the role of IRF4 in EBV/LMP1 oncogenesis is unclear.Importantly, we have recently identified B-cell integration cluster (BIC) as the first miRNA-encoding gene induced by IRF4 in virus-transformed cells (Figure 1) [21].BIC encodes miR-155, which plays important roles in innate immunity [58,59], and is the first identified oncogenic miRNA (oncomiR) implicated in various types of cancers including lymphomas [60][61][62], breast cancer, leukemia, pancreatic cancer, and lung cancer [63,64].Targeted expression of miR-155 alone in B cells developed B cell malignancies in transgenic mice [65], and enforced expression in mouse bone marrow cells causes myeloid neoplasia [66].As an important miRNA in immunity and cancer, miR-155 preferentially targets SHIP1 [67], among many others [68][69][70].Like oncogenic IRFs, miR-155 is also associated with EBV latency [61,[71][72][73].BIC/miR-155 is induced by TLR signaling, TNFα, IFN-β, IFN-γ, EBV LMP1, LMP2A [74] and B cell receptor (BCR) engagement.However, little is known about the mechanism controlling its regulation [75].Therefore, our findings unveiled an intersection between two major and quite diverse models in the regulation of viral oncogenesis, and have provided valuable insights into the interaction between viral oncogenesis and immune mechanisms governed by them.For example, both factors are crucial regulators of germinal center reaction [36,76], which is implicated in lymphoma development and EBV latent infection [77].Also, IRF4 has been shown to repress IRF5 expression in EBV-transformed cells (Figure 1) [78].Furthermore, our microarray analysis shows that IRF4 regulates a pool of interesting genes in the context of EBV infection, including a subgroup of the genes such as Cdk6 and Ccnb1 which are also targeted by IRF4 in MM (data not published).Future pursuits on selected targets may unravel novel and specific roles for IRF4 in EBV oncogenesis, and broaden our knowledge in its interaction with viral oncogenesis and other associated cancers.

IRF4 and other hematological malignancies
In addition to MM, chromosomal translocation and genetic mutation of IRF4 have been found in peripheral T-cell lymphomas [79], and Chronic Lymphocytic Leukemia (CLL) [33,80].Also, IRF4 over-expression was found in various types of T cell lymphomas in addition to ATLL, in most Hodgkin lymphoma cases, and in follicular lymphoma, primary effusion lymphoma(PEL), primary central nervous system lymphoma, and anaplastic large cell lymphoma (ALCL) [16,53,81], as well as in the context of the acquired immunodeficiency syndrome (AIDS) and in post-transplant lymphoproliferative disorders (PTLD) [41].IRF4 may serve as one of the phenotypic markers of B-cell lymphoma histogenesis.In particular, IRF4 may be a marker for the transition from Bcl6+ germinal center B cell (GCB) to CD138+ immunoblasts and plasma cells [16].IRF4 may help in the discrimination of PEL versus other lymphomas involving the serous body cavities, which are usually IRF4 negative [16].
In addition to hematological malignancies, an interesting report has shown that the germline variant IRF4 rs12203592 T allele was associated with increased risks of several types of skin cancers including melanoma, squamous cell carcinoma and basal cell carcinoma [82].

IRF4 as a tumor suppressor
IRF4, when over-expressed, plays an important role in the pathogenesis of hematopoietic malignancies.However, in some certain cancer contexts such as B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and chronic myelomonocytic leukemia, IRF4 is down regulated.In these cases, IRF4 acts as a tumor suppressor [83][84][85].In Irf4 +/-heterozygous mice, c-Myc-induced leukemia was significantly accelerated [84].It is also notable that IRF4 plays a dual role for lymphocyte activation/development and death by summating distinct incoming signals [30,86], and thus plays a central role in integrating the life and death decisions for lymphocytes [87].

Activation of IRF4 in cancer
As a transcription factor, activation of IRF4 is prerequisite for its function.Serine phosphorylation of IRF4 by the kinase ROCK2 activates IRF4 leading to IL17/21 production in autoimmune response in mice [88].However, how IRF4 is activated in cancer is an open question which has never been mentioned.Many proteins involved in cancer signal transduction are tyrosine-phosphorylated.A few limited high throughput profiling studies have identified several tyrosine phosphorylation sites on IRF4 in different cancer contexts, including Y192 in MM [89], and Y37, Y122, Y125, Y428 and Y440 in Hodgkin lymphomas [90].Interestingly, our recent phospho-proteome analysis has shown that IRF4 is also tyrosine phosphorylated in EBV-transformed cells, and identified several phosphorylation sites including Y125, Y192, Y181 and Y428 on IRF4.We have concluded that Y125 is a promising phosphorylation site at least important for IRF4-associated lymphomas [91].We have further shown that the tyrosine kinase c-Src promotes IRF4 phosphorylation and activation, and identified Y62 and Y125 of IRF4 as two key sites responding to c-Src-mediated activation (Figure 1) [91].Moreover, we show that c-Src is constitutively expressed and activated in EBVtransformed cells [91].These findings indicate that IRF4 is activated through a c-Src-mediated pathway in EBV-transformed cells.However, our data suggests that c-Src is unlikely a direct kinase for IRF4 [91].Further study will be followed to confirm this claim and to identify the direct kinase(s).

Discussion and Prospectives
Increasing evidence has shown the implication of IRF4 deregulation in diverse hematological malignancies.IRF4 serves as a prognosis marker for MM and ABC DLBCL and was also proposed as a prognosis marker for classical Hodgkin lymphoma [92].IRF4 overexpression has been found in almost 100% cases of MM, classical Hodgkin lymphoma, primary central nervous system DLBCL, ISSN: 2325-2340 plasma cell myeloma and PEL.In many cases, IRF4 may be used as an important marker in combination with other markers for diagnosis.For example, for follicular lymphoma and DLBCL, IRF4 can be used with CD10 and Bcl6 for diagnosis [16].
However, the functional roles of IRF4 and the mechanisms underlying its interaction with these cancers remain to be elucidated.Study with cell culture systems is a necessary tool for this purpose.Targeted expression of IRF4 in mouse lymphocytes failed to develop any cancer [93], probably due to the fact that IRF4 requires co-factors for its function.Thus, gene-targeted mouse models are necessary to be established for the in vivo study of the interaction between IRF4 and other factors such as LMP1 in developing malignancies.
Currently, important work includes systematic and in-depth analyses of functional roles of IRF4 in distinct cancer contexts, the identification of "context-dependent" co-factors for IRF4, and the identification of potential lymphocyte-specific signaling pathways leading to IRF4 activation in each cancer context (Figure 1).These studies will highlight the importance of IRF4 in the pathogenesis of these cancers and will establish IRF4 as a unique therapeutic target for treating these hematological malignancies and other cancers [5].Since IRF4 is a lineage-dependent transcription factor, discriminating its specific roles in different cellular developmental stages and different cancer contexts may provide unique opportunities to target IRF4 regulatory network for treating these diverse proliferative diseases.Finally, we envision discovering novel signaling pathways and novel molecules such as the kinase(s) for IRF4 which may open up unique opportunities for therapeutic treatments of IRF4associated hematological malignancies.

Figure 1 :
Figure 1: A tentativescheme for IRF4 regulation in EBV latency.IRF4 expression is induced by the LMP1/NFκB axis[20;44-46], and probably is also directly induced by EBNA2[53].IRF4 protein is also stabilized by EBNA3C[52].Our recent study has disclosed that IRF4 is activated through c-Srcmediated tyrosine phosphorylation[91].We also have evidence showing that IRF4 is activated by the LMP1 signaling pathway, probably mediated by c-Src.c-Src protein is induced and activated in EBV latency[91].In addition to the LMP1 pathway, other lymphocyte-specific pathways may also contribute to IRF4 activation.Two transcriptional targets, BIC[21]and Irf5[78], have been identified and confirmed so far for IRF4 in EBV latency.IRF4 induces BIC but represses Irf5 transcription.