Molecular and Cellular Pathobiology The Transcription Factor IRF8 Counteracts BCR-ABL to Rescue Dendritic Cell Development in Chronic Myelogenous Leukemia

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8 / mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCRABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL–dysregulated genes and predicted that BCR-ABL has immunestimulatory potential. Indeed, IRF8-rescued BCR-ABL–expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML. Cancer Res; 73(22); 6642–53. 2013 AACR. Introduction Chronic myelogenous leukemia (CML) is a myeloproliferative disorder that accounts for approximately 15% of newly diagnosed cases of adult leukemia (1, 2). The causalmolecule of this disease is BCR-ABL, a 210 kDa oncoprotein encoded by the BCR-ABL fusion gene, which is generated by a t(9;22) chromosomal translocation (3). BCR-ABL possesses deregulated ABL tyrosine kinase activity that stimulates many downstream signals including the MYC, NF-kB, RAS, mitogen-activated protein kinase, phosphoinositide 3-kinase (PI3K), and STAT pathways. These signals lead to stimulation of cell proliferation, inhibition of apoptosis, and alteration of adhesion to stroma cells and extracellular matrix. CML begins with a chronic phase in which clonal BCR-ABLþ hematopoietic stem cells give rise to increased numbers of their progenies, particularly myeloid precursors and mature cells (mainly neutrophils) in the bone marrow, blood, and extramedullary tissues. After 3 to 5 years, if not treated, the disease progresses into a fatal blast crisis, characterized by accumulation of immature precursor cells with differentiation arrest. During the last decade, therapy with BCR-ABL–selective tyrosine kinase inhibitors (TKI), such as imatinib, nilotinib, and dasatinib, has greatly improved the prognosis of patients with CML (4). However, several problems still impede a complete cure of this disease. First, a minor but significant subset of patients fails to respond to or becomes resistant to TKIsmainly due to acquisition of point mutations in the ABL gene, such as T315I. Second, because leukemic stem cells are not efficiently eliminated by TKIs, discontinuing TKI therapy often results in CML recurrence (4). Third, while CML is highly sensitive to tumor immunity, TKIs may suppress functions of T cells and dendritic cells (5–7). Therefore, next-generation therapies for CML are required. Dendritic cells are the most potent professional antigenpresenting cells and are essential for the initiation and regulation of innate and adaptive immunity against pathogens and tumors (8). Dendritic cells are a heterogeneous population that can be divided into classical dendritic cells (cDC; further divided into CD8aþ dendritic cells, CD4þ dendritic cells, and double-negative dendritic cells in mice) and plasmacytoid dendritic cells (pDC) both in humans and mice (9). CD8aþ Authors' Affiliations: Departments of Immunology, Environmental Immuno-Dermatology, Internal Medicine and Clinical Immunology, and Experimental Animal Science, YokohamaCity UniversityGraduate School of Medicine; and Department of Hematology, Yokohama City University Medical Center, Yokohama, Japan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). T. Watanabe and C. Hotta contributed equally to this work. Corresponding Author: Tomohiko Tamura, Department of Immunology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 81-45-787-2612; Fax: 81-45-787-2615; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-0802 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(22) November 15, 2013 6642 on April 14, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from dendritic cells (BDCA3þ in humans) are a unique subset capable of priming CTLs by cross-presentation of dead cell materials and are thus critical for tumor immunity. pDCs produce a large amount of type I IFN, that is, IFN-a/b, upon Toll-like receptor (TLR)-7/8/9 signaling. Type I IFN elicits antiviral and antitumor responses. Of note, IFN-a was once used as a major therapeutic agent for CML (10, 11). CML has long been suggested to be highly sensitive to T cell– mediated tumor immunity (12). CML responds to immunemediated therapies, such as IFN-a, allogeneic stem cell transplantation (alloSCT), and donor lymphocyte infusion (13). IFN-a induces a specific T-cell response in CML (10, 11). In alloSCT, Tcell depletion from donor bone marrow cells results in a significant increase in relapse, especially in patients with CML. Yet, the fact that the disease develops in patients with CML suggests that tumor immunity is insufficient in patients with CML. Because T cells per se in patients with CML rarely express BCR-ABL, it is reasonable to suspect dendritic cells as a culprit. Indeed, several studies have shown that patients with CML have reduced numbers of cDCs and pDCs in the chronic phase, as compared with healthy individuals (14, 15). Other studies have reported that dendritic cells generated in vitro from monocytes or CD34þ cells of patientswith CMLare functionally defective in multiple aspects, such as actin organization, antigen processing, migration, maturation, and cytokine production (16, 17). In a murine CML model, defective homing and impaired induction of CTLs by BCR-ABL–expressing dendritic cells have been reported (18). However, the mechanism of impaired dendritic cell development in CML remains unknown. IFN regulatory factor-8 (IRF8) is a hematopoietic transcription factor that regulates the development of multiple immune cell types (19). IRF8 is required for differentiation of mouse cDCs (particularly CD8aþ dendritic cells; refs. 20, 21), pDCs (22), and monocytes (particularly Ly6Cþ monocytes; ref. 23), while inhibiting myeloid cell proliferation and neutrophil differentiation (24). IRF8 is also indispensable for the differentiation of CTLs (25). Thus, Irf8 / mice develop immunodeficiency and a CML-like syndrome (26). Importantly, mutations in the human IRF8 gene are associated with dendritic cell immunodeficiency (27). Furthermore, IRF8 expression is dramatically decreased in patients with CML (28, 29). IRF8 also overcomes themitogenic activity of BCR-ABL in differentiating myeloid progenitors in vitro (30). Coexpression of IRF8 in bone marrow progenitors can ameliorate BCR-ABL–induced myeloproliferative disorder in mice (31). Coexpression of IRF8 in a BCR-ABL–transformed mouse pro-B cell line induces a chemokine-dependent antileukemia immunity in mice (31–33). These findings imply that there is an antagonistic relationship between IRF8 and CML pathogenesis. However, this idea has not been tested in terms of dendritic cell biology yet. In this study, we have investigated how BCR-ABL and IRF8 are involved in the development and function of dendritic cells in CML using a murine model. Materials and Methods Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice and OT-I or OT-II T cell receptor transgenic mice (The Jackson Laboratory) were used at 8 to 12 weeks of age. All animal experiments were carried out in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan), and all protocols were approved by the Institutional Review Boards of Yokohama City University (Yokohama, Japan; Protocol #F11-85). Flow cytometry Flow cytometry was performed using FACSCanto II (BD Biosciences), and data were analyzed using the FlowJo software (TreeStar). For antibodies and their clone names, see Supplementary Materials and Methods. Separation of hematopoietic progenitors Murine lineage marker–negative (Lin ) cells were purified from bone marrow cells by the magnetic-activated cell sorting (MACS) system using the Lineage Cell Depletion Kit (Miltenyi Biotec) and anti-interleukin (IL)-7 receptor (IL-7R/CD127) antibody. For isolation of Lin Sca-1þc-Kitþ (LSK) cells, MACS-purified Lin cells were stained with antibodies against Sca-1 and c-Kit, and were then purified by the fluorescenceactivated cell sorting (FACS) system using a FACSAria II (BD Biosciences). Retroviral transduction and CML model mice The following murine stem cell virus (MSCV) retroviral vectors were used: MIG [MSCV-internal ribosome entry site (IRES)GFP], MIG-p210 (MSCV-p210-IRESGFP), MICD8 [MSCV-IRES-human truncated CD8 (hCD8t)], and MICD8-IRF8 (MSCV-IRF8-IRES-hCD8t). Lin cells were precultured for 24 hours with stem cell factor (SCF) and thrombopoietin (TPO) for transplantation, or SCF, IL-6, and IL-3 for in vitro experiments, and were then transduced with MSCVs by spinoculation for 2 consecutive days. All cytokines were purchased from PeproTech. For generating CML model mice, C57BL/6-Ly5.1 LSK cells transduced with MIG or MIGp210were injected intravenously into lethally irradiated syngenic C57BL/6-Ly5.2 recipients. qRT-PCR, ELISA, and immunoblot analysis Quantitative PCR (qPCR) with quantitative reverse transcription PCR (qRT-PCR) was performed using RNAiso Plus (Takara Bio), DNase I (Invitrogen), PrimeScript (Takara Bio), Thunderbird SYBR qPCR Mix (Toyobo), and an ABI PRISM 7900 sequence detection system (Applied Biosystems) according to the manufacturers' protocols. Primer sequences are described in SupplementaryMaterials andMethods. Datawere analyzed using the DDCT method and normalized against Gapdh levels. ELISA for IFN-a and IL-12p40 was performed using commercially available kits (PBL and BioLegend, respectively). For immunoblot analysis, anti-IRF8 (C-19; Santa Cruz Biotechnology), c-ABL (Cell Signaling Technology), phosphotyrosine (4G10; Millipore), and b-actin (AC-74; Sigma-Aldrich) antibodies were used. Dendritic cell culture BonemarrowLin cells transducedwithMSCVswerewashed 24 hours after the last spinoculation and cultured with human IRF8 Overrides BCR-ABL in Dendritic Cell Development www.aacrjournals.org Cancer Res; 73(22) November 15, 2013 6643 on April 14, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from fms-like tyrosine kinase-3 (Flt3)-ligand (Flt3L) for 7 days to induce dendritic cell differentiation. TKIs and a STAT5 inhibitor [N0-((4-oxo-4H-chromen-3-yl)methylene)nicotinohydrazide] were purchased from Santa Cruz Biotechnology. Trichostatin A (TSA) and 5-azacytidine (5-Aza) were purchased from Focus Biomolecules and Sigma-Aldrich, respectively. Microarray RNAs from two independent experiments were analyzed using a Whole Mouse Genome 8 60 K Microarray (Agilent) according to the manufacturer's protocol. Microarray data are available at the GEO/NCBI database (GSE44920). For details, see Supplementary Materials and Methods. T-cell response assays Antigen presentation capability of dendritic cells via MHC class II (MHC-II) was evaluated using OT-II T cells essentially as previously described (21). In vitro CTL assays were performed using OT-I T cells essentially by the method previously established (34). Results BCR-ABL inhibits dendritic cell development and Irf8 expression To investigate how dendritic cell development is affected in CML, we first used a murine CML model in which hematopoietic progenitors (LSK cells) were transduced with bicistronic MIG-p210 retrovirus (carrying cDNAs encoding BCR-ABL and GFP) in the presence of SCF and TPO, and then transplanted these cells into lethally irradiated mice. These mice (BCR-ABL mice), but not mice transplanted with empty MIG-transduced cells (MIG mice), exhibited splenomegaly and died by 4 to 8 weeks after the transplantation (Fig. 1A and Supplementary Fig. S1A). Wright–Giemsa staining and flow-cytometric analyses of splenic cells revealed that BCR-ABL mice had significant increases in the percentage and number of GFPþ (therefore BCR-ABLþ) neutrophils, a hallmark of CML, as compared with MIG mice (Fig. 1B and C). We found that both cDCs (CD11c cells) and pDCs (CD11clowPDCA-1þ cells) were dramatically diminished in BCR-ABL mice as compared with MIG mice; the percentage and number of GFPþ cDCs showed 16.7and 6.5-fold reductions, respectively, and those of pDCs showed 18.1and 8.1-fold reductions, respectively (Fig. 1D). More detailed analysis demonstrated that all cDC subsets, that is, CD8aþ dendritic cells, CD4þ dendritic cells, and CD8a CD4 (double-negative) dendritic cells, were significantly diminished (Supplementary Fig. S1B). In addition, we noted that the remaining GFPþ cDCs in BCR-ABL mice expressed lower levels of MHC-II compared with those inMIGmice (Supplementary Fig. S1C). In fact, these spared dendritic cells exhibited a reduced capability of presenting ovalbumin (OVA) antigen, as demonstrated by the proliferation of and IFN-g production by OT-II T cells (Supplementary Fig. S1D). These results indicate that BCR-ABL strongly and globally inhibits the generation of dendritic cells in vivo. BCR-ABL inhibits an early stage of dendritic cell differentiation Wenextanalyzedprogenitor populations in thebonemarrow. Dendritic cells originate frombonemarrow hematopoietic stem cells through monocyte-dendritic cell progenitors (MDP; Lin Sca-1 CD127 c-Kitint/þCD115þ) and then common dendritic cell progenitors (CDP; Lin Sca-1 CD127 Flt3þc-KitintCD115þ; refs. 9, 35). MDPs are a CD115þ population in myeloid progenitors (Lin CD127 Sca-1 c-Kitþ), and overlap with granulocytemonocyte progenitors (GMP; Lin CD127 Sca-1 c-KitþCD16/ 32þCD34þ) and to a lesser extent commonmyeloid progenitors (CMP; Lin CD127 Sca-1 c-KitþCD16/32lowCD34þ). Within myeloid progenitors, CMPs progress toward either GMPs or megakaryocyte-erythroid progenitors (MEP; Lin CD127 Sca1 c-KitþCD16/32 CD34 ). Interestingly, the bonemarrowGFPþ population in BCR-ABL mice lacked MDPs and CDPs (Fig. 1E). The percentages of total GFPþ myeloid progenitors were comparable between BCR-ABL mice and MIG mice (Supplementary Fig. S1E). GFPþ myeloid progenitors in BCR-ABL mice had relatively low percentages of CMPs and GMPs, which could be partially due to the lack of MDPs, while they contained amodestly increased percentage of MEPs and harbored an aberrant CD34 CD16/32þ population. Despite the sharp decrease in MDP counts, we noted that monocyte counts were not significantly affected in BCR-ABL mice (data not shown), consistent with the fact that monocyte counts are not reduced in patients with CML. The developmental route of these BCR-ABLþ monocytes is unknown, but one possibility is that they are derived from upstream myeloid progenitorswithout transitingMDPs.Overall, these data suggest that BCR-ABL affects the early stage of dendritic cell differentiation, particularly the generation of MDPs. We analyzed IRF8 expression in BCR-ABL and MIG mice. qRT-PCR analysis revealed that BCR-ABL suppressed Irf8 mRNA expression both in Lin bone marrow progenitors and splenic cells (Fig. 1F). Immunostaining for IRF8 in splenic dendritic cells demonstrated that the remaining few BCRABLþ dendritic cells expressed lower levels of IRF8 than control dendritic cells (Fig. 1G and Supplementary Fig. S1F). IRF8 overrides BCR-ABL to rescue dendritic cell differentiation To further investigate the mechanisms involved in this process, we examined whether BCR-ABL inhibits dendritic cell differentiation in vitro. We transduced MIG-p210 or emptyMIG retrovirus into bonemarrow Lin progenitors in the presence of SCF, IL-6, and IL-3, and the transduced cells were then cultured with Flt3L for 7 days to induce differentiation toward cDCs (CD11chighMHC-IIþ) and pDCs (CD11cþB220þCD11blow; ref. 21). In MIG-transduced control cultures, cDCs and pDCs were efficiently generated (Fig. 2A). In BCR-ABL–transduced cultures, however, the development of both cDCs and pDCs was significantly inhibited in terms of percentages and absolute cell counts, even though BCR-ABL– transduced cells showed a greater increase in cell number during the Flt3L culture than MIG-transduced cells. Wright– Giemsa staining revealed that while many of the MIGtransduced cells showed typical dendritic cell morphology, Watanabe et al. Cancer Res; 73(22) November 15, 2013 Cancer Research 6644 on April 14, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from BCR-ABL–transduced cells exhibited neutrophil-like, macrophage-like, or immature morphologies (Fig. 2B). Furthermore, the induction of Irf8mRNA expression during the Flt3L culture was almost completely abrogated by BCR-ABL (Fig. 2C). When a kinase-deadK1172Rmutant of BCR-ABLwas transduced into cells, neither dendritic cell development nor IRF8 expression was affected, suggesting that the kinase activity of BCR-ABL is required for the observed inhibitory effects (Fig. 2D). The expression levels of wild-type (WT) and K1172R BCR-ABL were comparable, and K1172R lacked autophosphorylation at tyrosine residues. We next asked whether restoration of IRF8 expression could improve dendritic cell differentiation impaired by BCR-ABL. To this end, we cotransduced Lin cells with MICD8-IRF8 (that expresses IRF8 and hCD8t) together with MIG-p210, and treated them with Flt3L. hCD8t, used as a transduction marker, does not transmit any signals because it contains no cytoplasmic domain. Flow-cytometric analysis of doubly transduced (i.e., GFPþhCD8tþ) cells revealed that the forced expression of IRF8 efficiently rescued differentiation of both cDCs and pDCs (Fig. 3A). The expression levels of Irf8 mRNA and IRF8 protein in A BCR-ABL MIG B BCR-ABL MIG