IRF-4 Suppresses BCR/ABL Transformation of Myeloid Cells in a DNA Binding-independent Manner*

Background: IRF-4 functions both as an oncoprotein and as a tumor suppressor in different cell context. Results: We found that IRF-4 inhibits BCR/ABL transformation of myeloid cells independent of its DNA binding and nuclear localization. Conclusion: The oncogenic and tumor suppressor functions of IRF-4 involve distinct pathways. Significance: The studies help to develop improved therapies for malignancies involving IRF-4. Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4-interacting protein found in the cytoplasm. However, IRF-4 tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain.

Interferon regulatory factor 4 (IRF-4) 2 plays a critical role in B, T, and dendritic cell development as well as in immune response regulation (1,2). IRF-4 was also originally identified as a proto-oncogene resulting from a t(6;14)(p25;q32) chromosomal translocation in multiple myeloma (3). In addition, IRF-4 expression has been shown to be an unfavorable prognostic factor in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (4). Recent studies have shown that expression of IRF-4 is essential for the maintenance of multiple myeloma cells (5), making IRF-4 an attractive target for the development of therapies for multiple myeloma.
In addition to its role as a transcription regulator, IRF-4 was recently found to function as a negative regulator of Toll-like receptor (TLR) signaling in the cytoplasm by interacting with the myeloid differentiation primary response protein 88 (MyD88), a common adapter protein for all TLRs except TLR3 (6,7). TLR signaling plays a central role in the innate immunity against microbial pathogenesis by inducing the expression of immunity and pro-inflammatory genes (8). In addition, TLRs are expressed in a wide variety of tumors and the activation of TLR signaling promotes tumor cell growth and survival (9 -11). IRF-4 binds to MyD88 on the same site bound by IRF-5, whose activation by interacting with MyD88 is essential for the induction of pro-inflammatory cytokines in response to TLR stimulation (12), thereby attenuating IRF-5 activation. Consistently, TLR-induced cytokine production is significantly increased in peritoneal macrophages from IRF-4-deficient mice (6,7). In addition, the activation of NF-B was also enhanced in response to TLR stimulation in IRF-4-deficient macrophages (6).
Though IRF-4 is also expressed in myeloid cells, its function in the myeloid lineage is not known. The closely related IRF family member IRF-8 plays an important role in myelopoiesis and in anti-viral immunity (13). IRF-8-deficient mice spontaneously develop a chronic myelogenous leukemia (CML)-like disease, indicating that IRF-8 functions as a tumor suppressor (14). Human CML is a myeloproliferative disease characterized by the underlying t(9;22)(q34;q11) reciprocal chromosome translocation resulting in what is known as the Philadelphia chromosome, which leads to the creation and expression of the fusion gene product BCR/ABL, a deregulated tyrosine kinase (15). We have shown that IRF-8 is down-regulated in a BCR/ABL-induced murine CML and that forced overexpression of IRF-8 in this model represses the resulting myeloproliferative disorder and prolongs survival (16). In addition, IRF-8 has overlapping functions with IRF-4 in early B cell development. Both proteins can interact with tran-scription factors PU.1 and E2A at the Ig and light chain enhancer regions (17) and both regulate Ikaros and Aiolos expression (18). Consistently, mice deficient in both IRF-4 and IRF-8 show a block in B cell development at the pre-B to immature B transition and display an accumulation of cycling pre B cells (19).
In contrast to its oncogenic function in mature lymphocytes, expression of IRF-4 is down-regulated in BCR/ABL positive CML and acute B-lymphoblastic leukemia (B-ALL) (20 -22). In CML, lower expression of IRF-4, like IRF-8, is correlated with a higher burden of pretreatment risk factors and a lower response rate to treatment with IFN-␣, the standard treatment for CML before the advent of tyrosine kinase inhibitors such as imatinib myselate (21,23). These data suggest that IRF-4 may have different functions in different cell contexts. Indeed, we have recently shown that IRF-4 and IRF-8 deficiencies can cooperate in the development of both myeloid and lymphoid tumors (24) and that IRF-4 deficiency facilitates the development of BCR/ ABL-induced B-ALL, while forced expression of IRF-4 potently suppresses the pathogenesis of BCR/ABL-induced B-ALL (25). These findings demonstrate that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development.
The finding of IRF-4 functioning as a tumor suppressor raises caution for developing therapies aiming to down-regulate IRF-4. Further studies on the mechanisms by which IRF-4 func-tions as an oncoprotein and as a tumor suppressor are necessary to develop improved therapies for malignancies involving IRF-4. IRF-4 contains multiple functional domains/motifs and localizes in both the nucleus and the cytoplasm. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 in suppressing BCR/ABL transformation in myeloid cells. We found that IRF-4 suppresses BCR/ABL transformation independent of its DNA binding and nuclear localization, and in a MyD88-independent manner. The IRF-association domain of IRF-4, on the other hand, is essential for its tumor suppressor function.

EXPERIMENTAL PROCEDURES
Mice-IRF-4 knock-out (KO) mice in C57BL/6 background were bred and genotyped as described previously (26). MyD88 KO mice in C57BL/6 background were kindly provided by Dr. Douglas Golenbock at the University of Massachusetts Medical School. Wild-type C57BL/6J mice were purchased from Taconic Farms (Germantown, NY). Mice used in this study were housed in the Association for Assessment and Accreditation of Laboratory Animal Care International-accredited Foster Animal Research Facility in Brandeis University and procedures were approved by the Institutional Animal Care and Use Committee of Brandeis University. DNA Constructs-Production of the murine stem cell virus (MSCV)-BCR/ABL-GFP-IRES-2xmyc-IRF-4, MSCV-BCR/ABL-GFP-IRES-2xmyc-Neo and MSCV-GFP-IRES-2xmyc-IRF-4 were described previously (25). Deletion mutants were prepared by PCR reaction using Pfu polymerase (Stratagene, La Jolla, CA). Construction of the IRF-4 DNA binding mutant (R98C99A) and the PU.1 interacting mutant (AS397) has been previously described (27). The cDNA of mouse MyD88 was amplified by PCR reaction using plasmid 13092 obtained from Addgene as a template. These PCR products were subcloned into the NotI and ClaI sites in-frame with an N-terminal Myc tag in MSCV-BCR/ABL-GFP-IRES-2xMyc tag plasmid or MSCV-GFP-IRES-2xmyc tag plasmid. All IRF-4 deletion mutants were confirmed by sequencing. The control vectors, MSCV-GFP-IRES and the MSCV-IRES-GFP, were made as described previously (25).
Cell Culture and Retrovirus Production-Bosc23 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 50 g/ml gentamicin (Invitrogen, Grand Island, NY). NIH3T3 cells were maintained in DMEM containing 10% donor bovine serum (DBS, Invitrogen), 50 g/ml gentamicin. Retroviruses were produced as described previously (28). Briefly, MSCV constructs were transfected into Bosc23 cells by the calcium phosphate precipitation method. Two days after transfection, retrovirus-containing supernatants were recovered and centrifuged. The viral titer was calculated based on the percentage of GFP-expressing NIH3T3 cells as described previously (29). NIH3T3 cell lines stably expressing both GFP-fused BCR/ABL and Myc-tagged IRF-4 mutant, or the control GFP were generated by retroviral infection as described (29). All GFP-positive cell lines were sorted by fluorescence-activated cell sorting (FACS) using a FACSAria (BD Biosciences, San Jose, CA). These cell lines were maintained and confirmed to express GFP by FACS analysis using a FACSCalibur TM Flow Cytometer (BD Biosciences, San Jose, CA), and data were analyzed with FlowJo software (TreeStar, San Carlos, CA).
Cytoplasmic and nuclear extracts were prepared from NIH3T3 cell lines expressing both GFP-fused BCR/ABL and Myc-tagged IRF-4 mutants by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) according to the manufacturer's instruction. The concentrations of cytoplasmic and nuclear extracts were measured by Bradford assay. The protein samples were separated by 6 -18% or 18% poly-acrylamide gradient gels, then transferred to nitrocellulose membranes in CAPS buffer. The membranes were probed with anti-IRF-4 (sc-6059, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Myc-Tag (9E10; Cell Signaling Technologies, Beverly, MA), and HRP-labeled rabbit anti-goat IgG or goat anti-mouse IgG (Pierce Biotechnology). Detection was carried out with Super Signal West Femto Chemiluminescence Reagents (Pierce Biotechnology). Blots were stripped and re-probed with nucleus-specific anti-Lamin B and cytoplasm-specific anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (sc-6216 and sc-47724; Santa Cruz Biotechnology).

IRF-4 Inhibits BCR/ABL-stimulated Bone Marrow Colony
Formation-It has been shown that IRF-4 is down-regulated in CML (20,21). We wondered whether IRF-4 could negatively regulate BCR/ABL transformation in myeloid cells. To address this question, we examined whether forced expression of IRF-4 could suppress BCR/ABL transformation. We first made retroviral constructs by inserting a BCR/ABL-GFP fusion with either IRF-4 or Neomycin resistance gene (Neo) into the murine stem cell virus (MSCV) vector as depicted in Fig. 1A. IRF-8 has been shown to inhibit BCR/ABL-induced bone marrow colony formation (16), and we thus included IRF-8 in this study as a control. Prior to conducting the transformation assay, we confirmed that BCR/ABL-GFP expression was similar for all these MSCV constructs in 32Dcl3 (32D) myeloid progenitor cells (Fig. 1B). In addition, phosphotyrosine levels were similar in BCR/ABL-GFPϩNeo, BCR/ABL-GFPϩIRF-4, and BCR/ABL-GFPϩIRF-8 expressing 32D cells (Fig. 1C), indicating that coexpression of IRF-4 or IRF-8 does not interfere with the kinase activity of BCR/ABL.
We then compared the abilities of the above retroviruses to stimulate cytokine independent bone marrow cell growth in soft agar. As expected, BCR/ABL-GFP, but not the GFP control, stimulated the formation of myeloid colonies under the condition used in the experiment (Fig. 2 and data not shown). Cultures of bone marrow cells infected with BCR/ABL-GFPϩIRF-4 or BCR/ABL-GFPϩIRF-8, on the other hand, had smaller and significantly fewer colonies after 10 days compared with BCR/ABL-GFPϩNeo infected cells (Fig. 2, A and B). Interestingly, BCR/ABL-GFPϩIRF-4-infected bone marrow cells formed significantly fewer colonies than BCR/ABL-GFPϩIRF-8-infected cells (Fig. 2B). These data show that IRF-4, like IRF-8, suppresses BCR/ABL transformation of myeloid cells, and suggest that IRF-4 may be an even more potent inhibitor of BCR/ABL transformation than IRF-8.
To rule out the possibility that the reduced transforming potential of BCR/ABL-GFPϩIRF-4 retrovirus is due to an artifact from the use of a bicistronic vector, we also performed bone marrow colony assay by co-transducing bone marrow cells with retroviral vectors expressing BCR/ABL and IRF-4 separately. In this experiment, the amount of BCR/ABL retroviruses was kept constant, while IRF-4 and GFP retroviruses with various ratios (0:6, 2:4, 4:2) were used to co-transduce the bone marrow cells with the BCR/ABL retroviruses (Fig. 2, C and D). At 10 days after plating, significantly fewer colonies were observed with the increased ratio of IRF-4 to GFP viruses. This result confirms the tumor suppressor activity of IRF-4 in BCR/ABL-induced transformation. The bicistronic vectors were used in the following experiments.

IRF-4 Inhibits BCR/ABL Transformation in a DNA Binding Domain-independent
Manner-IRF-4 contains multiple functional domains/motifs and has both nuclear and cytoplasmic functions. The DNA binding domain of IRF-4 has been shown to be essential for IRF-4 to regulate class switch recombination and immunoglobulin (Ig) secretion (30), but it is not required in the negative regulation of TLR signaling by IRF-4 (7). To identify the structure elements of IRF-4 that are involved in its function as an inhibitor of BCR/ABL transformation, we performed a structure/function analysis using a series of Myc-tagged IRF-4 mutants (Fig. 3A). To test whether the DNA binding ability is essential for IRF-4 to inhibit BCR/ABL-stimulated bone marrow colony formation, we generated two DNA binding domain (DBD) deletion mutants, as well as a mutant IRF-4 with R98/ C99A point mutations, which abolish the DNA binding ability of IRF-4 (30). Interestingly, all these mutants showed a similar tumor suppressor activity as the wild-type IRF-4 (Fig. 3, C and  D), indicating that the DNA binding ability of IRF-4 is not required for its tumor suppressor function.
It has been shown that the nuclear localization signal of IRF-4 is located in its DNA binding domain (7). Consistent with the previous finding, we found that the DBD deletion mutant (⌬DBD) was detected only in the cytoplasm, whereas the wild-type IRF-4 and the R98/C99A IRF-4 mutants were localized to both the nucleus and the cytoplasm. The small amount of 134 -410 mutant detected in the nucleus may be caused by cytoplasmic contamina- tion during fractionation. These results suggest that IRF-4 suppresses BCR/ABL transformation of myeloid cells in a DNA binding-independent manner and that the tumor suppressor function of IRF-4 primarily involves its cytoplasmic activities.

IRF-4 DNA Binding Domain Mutants Inhibit BCR/ABL Transformation in the Absence of the Endogenous IRF-4-
The transcription regulatory function of IRF-4 can be modulated by IRF-4-binding protein (IBP) and FKBP52, which were shown to interact with IRF-4 and inhibit IRF-4's DNA binding (31,32). It is possible that the DNA binding mutants of IRF-4 may sequestrate negative regulators, allowing the endogenous wild-type IRF-4 to suppress BCR/ABL transformation by regulating gene expression. To test this possibility, we performed a bone marrow colony formation assay using bone marrow cells from the IRF-4 KO mice. We found that the DBD deletion mutant of IRF-4 has the same ability as wild-type IRF-4 in suppressing BCR/ABL-stimulated bone marrow colony formation in the absence of endogenous IRF-4 (Fig. 4, A and B). Together, these data confirm that IRF-4 can inhibit BCR/ABL transformation in a DNA binding domain-independent manner.
The IAD Is Essential for IRF-4 to Suppress BCR/ABL Transformation-To further delineate which region of IRF-4 is involved in its tumor suppressor function, we analyzed more mutants of IRF-4. IRF-4 can regulate many genes by interacting with a transcription factor PU.1, which is important in normal myeloid and lymphoid development (1,33,34). To test the importance of the IRF-4/PU.1 interaction for IRF-4 tumor suppressor activity, we checked the tumor suppressor activities of PU.1 binding mutants of IRF-4, AS397 and 1-380 (27, 35). AS397 is an alanine substitution mutant that is unable to bind PU.1, and 1-380 has a C-terminal deletion including the PU.1 binding region (Fig. 5A). Interestingly, these two mutants showed similar levels of tumor suppressor activity as did wildtype IRF-4 (Fig. 5, C and D), suggesting that the IRF-4/PU.1 interaction is not essential for IRF-4 tumor suppressor activity. We next determined the importance of the IAD domain in IRF-4 tumor suppressor activity using four deletion mutants, 1-200, DBD, ⌬IAD, and ⌬ProϩIAD (Fig. 5A). All of the mutants were localized to both the nucleus and the cytoplasm (Fig. 5B). Bone marrow colony formation assays showed that these four mutants failed to inhibit BCR/ABL transformation (Fig. 5, C and D). Collectively, these data suggest that the IAD domain of IRF-4 is essential for its tumor suppressor function.
IRF-4 Inhibits BCR/ABL Transformation in a MyD88-independent Manner-One of the proteins that IRF-4 interacts with in the cytoplasm through its IAD domain is MyD88. To test whether MyD88 is required for the tumor suppressor function of IRF-4, we performed a bone marrow colony formation assay using bone marrow cells from MyD88 KO mice. The result showed that expression of the wild-type or DBD deletion mutant of IRF-4 significantly inhibits BCR/ABL-stimulated bone marrow colony formation (Fig. 6, A and B). These experiments demonstrate that IRF-4 functions as a tumor suppressor in a MyD88-independent manner.

DISCUSSION
In this study, we demonstrated that IRF-4 inhibits BCR/ABLstimulated cytokine-independent myeloid colony formation in a DNA binding-independent manner and that the IAD domain of IRF-4 is critical for its tumor suppressor activity. The tumor suppressor function of IRF-4 involves its cytoplasmic activities, but its cytoplasmic-interacting protein MyD88 is not required for IRF-4 to suppress BCR/ABL transformation.
IRF-4 not only regulates genes that play crucial roles in normal hematopoetic cell development but is also involved with many genes regulating cell proliferation and survival in multiple myeloma (2). In particular, IRF-4 directly regulates the expression of MYC, which is essential for multiple myeloma cell survival (5). As such, IRF-4 acts as an oncogene primarily through its transcription-regulation function. However, the function of IRF-4 in suppressing transformation of myeloid cells by BCR/ABL is independent of DNA binding and involves its cytoplasmic function. The results indicate that the oncogenic and tumor suppressor functions of IRF-4 involve distinct pathways.
IRF-4 interacts with a number of transcription factors. One possible mechanism by which the DNA binding domain mutants of IRF-4 inhibit BCR/ABL transformation is that mutant IRF-4 sequestrates transcription factors that are critical for cell growth/survival. However, the interactions between IRF-4 and its transcription partners are weak and their stable interaction relies on DNA binding (1,27). As such, the DNA binding domain mutants of IRF-4 are unlikely to function as dominant negative regulators.
IRF-4 localizes in both the nucleus and the cytoplasm. It is not clear how IRF-4 functions as a tumor suppressor in the cytoplasm. The general TLR signaling adapter, MyD88, has been shown to play critical roles in the development of several types of cancer (11). MyD88 deficiency leads to reduced growth of colon cancer in a APC min/ϩ mouse model (36), and MyD88dependent activation of ERK promotes tumorigenesis (37). Because MyD88 is one of the IRF-4 interacting proteins in the cytoplasm, we thought that the negative regulation of TLR signaling by IRF-4, through the IRF-4/MyD88 interaction, might contribute to its tumor suppressing activity. However, we found that IRF-4 and the DNA binding domain deletion mutant of IRF-4 still suppress BCR/ABL transformation in MyD88deficient bone marrow cells. These results suggest that IRF-4 suppresses BCR/ABL transformation by interacting with other proteins. Another IRF-4 binding protein is IBP (31). In addition to inhibiting IRF-4's DNA binding, IBP has been shown to function as an activator for Rac (38,39). It has been shown that Rac1 and Rac2 are key regulators of leukemogenesis induced by BCR/ABL (40). Thus, IRF-4 interaction with IBP may play a role in suppressing BCR/ABL transformation. This possibility will be tested in the future.
Our result showed that IRF-4 is a more potent inhibitor of BCR/ABL transformation than IRF-8 (Fig. 2). This seems puzzling at first, since IRF-4 KO mice, unlike IRF-8 KO mice, do not develop a myeloproliferative disease. A likely explanation, however, is the differential expression levels of IRF-4 and IRF-8 in myeloid cells. It has been shown that IRF-8 is more active than IRF-4 in myeloid cells due to its higher abundance (41). So even though IRF-4 is a more potent tumor suppressor in myeloid cells, its low expression in these cells makes it a weak tumor suppressor overall. However, our finding suggests that therapies up-regulating IRF-4 may be effective in treating CML.
IRF-4 has diverse functions in different cell contexts. It has been shown that IRF-4, as well as IRF-8, regulates Ikaros and Aiolos expression (18). These two genes belong to the Ikaros family of zinc-finger transcription factors and inhibit the expression of the surrogate light chain and promote cell cycle withdrawal in pre-B-cell development. IRF-4 also regulates the gene encoding the chemokine receptor Cxcr4 (42). The up-regulation of Cxcr4, the receptor for CXCL12, can promote migration of pre-B cells away from IL-7-expressing stroma cells, an event that is necessary for pre-B cell development. These studies show that IRF-4 and IRF-8 regulate genes important for the transition from large, cycling pre-B to small, resting pre-B cells. The transcriptional regulatory functions of IRF-4/8 may underlie, at least in part, the mechanism by which IRF-4/8 function as tumor suppressors in early B-cell development. IRF-4, therefore, may exert tumor suppressor function through different mechanisms in different cell contexts. The mechanisms by which IRF-4 functions as a tumor suppressor in other cell contexts will be studied in the future.