Dysregulation of c-Myb Pathway by Aberrant Expression of Proto-oncogene MYB Provides the Basis for Malignancy in Adult T-cell Leukemia/lymphoma Cells

The proto-oncogene MYB has been identified as the cellular counterpart of v-Myb; the latter oncogene is present in the avian myeloblastosis virus (AMV) and E26, the product of which induces myeloid and lymphoid leukemia in infected birds (1). In normal cells, c-Myb functions physiologically as a transcription factor and is involved in the regulation of hematopoietic cell development and proliferation. The primary structure and functional mechanisms of c-Myb have been well described (2–4).

The c-Myb protein has a molecular weight of approximately 75 kDa and consists of three major domains: an N-terminal DNA-binding domain (DBD), a central transactivation domain (TAD), and a C-terminal–negative regulatory domain (NRD). The TAD and DBD are essential for the function of c-Myb as a transcription factor, whereas the NRD is a critical regulator of c-Myb activity (2, 5). MYB mRNA contains several natural alternative splicing variants produced by the usage of alternative exons. Woo and colleagues first demonstrated that one MYB splicing variant encoded a c-Myb isoform bearing an additional 121 amino acids not present in wild-type (WT) c-Myb (6). Subsequently, Kumar and colleagues demonstrated that expression of the 89-kDa c-Myb isoform, encoded by a MYB transcript variant containing extra 363 base pairs between exons 9 and 10, induced higher cell viability than did WT c-Myb (7). Most recently, O'Rourke and Ness identified a series of MYB splicing variants formed using the alternative exons 8A, 9A, 9B, 10A, 13A, and 14A and demonstrated the expression patterns of these variants in hematopoietic cell lines (Jurkat, KG-1, and K562), as well as in primary acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (ALL) cells (8). The authors observed overall increases in total MYB RNA levels, as well as MYB-9A and -10A mRNA levels, in patients with AML and ALL. In particular, up to 180-fold increases in MYB-9A mRNA levels were observed in T-cell ALL samples compared with normal bone marrow samples. The authors also found that the MYB-9A and -10A mRNAs, which included premature termination codons (PTCs) within exons 9A and 10A, respectively, encoded c-Myb isoforms (c-Myb-9A and -10A, respectively) that lacked the C-terminal NRD region. Both isoforms, which lack NRD to a similar degree as that observed in v-Myb, exhibited transactivity levels similar to or even exceeding that of v-Myb.

Notably, given its roles in hematopoietic cell differentiation and proliferation, c-Myb is expressed only in immature myeloid and lymphoid T cells but not in normal mature T and B cells (9–13). Importantly, Maurice and colleagues reported that c-Myb plays a critical role in the determination of T-helper cell fate by directly targeting the GATA3 gene (14); another study subsequently investigated in detail the molecular regulatory mechanism of GATA3 gene expression by c-Myb (15). Conversely, c-Myb overexpression and dysregulation have been frequently observed in hematopoietic tumors, particularly AML, ALL, and lymphomas, and the implications of c-Myb overexpression/deregulation and leukemogenesis have been extensively investigated (4, 5). Despite the accumulation of knowledge about the physiologic roles of c-Myb in normal and tumor cells, the biologic significance of c-Myb overexpression in leukemic cells has not been completely clarified.

Adult T-cell leukemia/lymphoma (ATLL), one of the most aggressive T-cell malignancies, is caused by human T-lymphotrophic virus type 1 (HTLV-1) infection. More than a million HTLV-1 carriers have been reported in areas where HTLV-1 is endemic, such as Japan (∼1% of the national population; ref. 16). Although the lifetime incidence of ATL among HTLV-1 carriers is approximately 5%, estimations suggest that annually, approximately 1,000 deaths in Japan are attributable to ATL. HTLV-1 was discovered to be the causative agent of ATL in the early 1980s (17–19). HTLV-1 is mainly transmitted to infants from infected mothers through breastfeeding. HTLV-1 then leads to the immortalization of infected cells, and the accumulation of genetic disorders triggers malignant transformation in these cells, followed by a monoclonal expansion of ATL leukemic cells. Nevertheless, the underlying molecular mechanism of HTLV-1 pathogenesis and the genetic disorders responsible for the onset of ATL are poorly understood. Identification of the key molecules responsible for ATL leukemogenesis is necessary for the identification of new therapeutic targets for ATL.

Our laboratory previously conducted comparative analyses of the gene expression patterns in ATL cells (n = 52) and normal CD4⁺ T cells (n = 21); these revealed MYB mRNA overexpression and upregulation of the c-Myb pathway in ATL cells (GSE33615). As described previously, c-Myb expression is normally suppressed in mature T cells. Consequently, we hypothesized that the abnormal overexpression of c-Myb might allow this protein to function when it is not required, thus dysregulating cellular homeostasis in ATL cells. In the current study, we observed for the first time the overexpression of not only total c-Myb but also an oncogenic splicing variant, MYB-9A, in primary ATL cells. We demonstrated that c-Myb targets various genes that encode essential regulators of cell proliferation and that c-Myb-9A induces significantly higher levels of transactivation of these genes compared with WT-c-Myb. Our data suggest that c-Myb pathway dysregulation consequent to the unbalanced overexpression of c-Myb-9A, which overproduces and thus overactivates c-Myb target genes, might underlie the wide range of abnormalities and unlimited cell growth observed in ATL cells.

All cell lines used in the current study were obtained and maintained as previously reported (20, 21). Briefly, Jurkat (T-ALL patient-derived T-cell line), TL-Om1 (ATL patient-derived T-cell line), and MT-2 (HTLV-1 immortalized T-cell line) were maintained in RPMI (Gibco) containing 10% FBS (Gibco) at 37°C with 5% CO₂. HeLa (cervical cancer–derived epithelial cell line), 293T (human embryonic kidney–derived cell line containing the SV40 T-antigen), and 293FT (derivative of 293T) cells were cultured in DMEM (Nissui) containing 10% FBS (Gibco) at 37°C with 5% CO₂.

The cell lines used in this study were obtained and authenticated as follows. TL-Om1, derived from malignant T cells from a patient with ATL, was kindly provided by Dr. K. Sugamura in Tohoku University (Tokyo, Japan). We authenticated within 6 months of this study by FISH and HTLV-1 provirus–specific real-time PCR and confirmed that this cell line contained 1 copy/cell of HTLV-1 provirus at the site of 1p13 of chromosome 1 in the gDNA, maintaining the original characteristics of TL-Om1 (22). MT-2, HTLV-1 transformed T cell line, was kindly provided by Dr. H. Hoshino in Gunma University (Tokyo, Japan). We authenticated within 6 months of this study by the proviral integration site sequencing technique (23) and confirmed that this cell line contains 10 copies of HTLV-1 provirus/cell. We also confirmed that this cell line produced infectious HTLV-1 viral particles and expressed viral Gag-Tax fusion protein, which are major characteristics of MT-2 cells. Jurkat was obtained within 6 month of this study from ATCC that authenticated this cell line by short tandem repeat (STR) analysis. 293FT was obtained more than 6 months before this study from Riken Cell Bank that conducted a series of authentication analysis on this cell line including growth curve analysis, cell adhesion analysis, isozyme analysis, and STR analysis. Just after arrival to our laboratory, 293FT cells were cultured for several days in the conditioned medium and divided to 20 cryo-tubes as live cell stock, then stored in liquid N₂ until use. HeLa and 293T cells were gifted by the Japanese Foundation for Cancer Research (JFCR) more than 6 months prior to this study. We authenticated these cell lines by karyotype analysis within 6 months of this study.

Peripheral blood mononuclear cells (PBMCs) from patients with ATL and healthy volunteers were a part of those collected with an informed consent as a collaborative project of the Joint Study on Prognostic Factors of ATL Development (JSPFAD). The project was approved by the Human Genome Research Ethics Committee in the Institute of Medical Sciences, the University of Tokyo (IMSUT; Tokyo, Japan). Clinical information of patients with ATL and healthy volunteers enrolled in the current study is shown in Supplementary Table S6.

Total RNA was extracted using Isogen (Nippon Gene Co., Ltd.) following the manufacturer's protocol. Extracted total RNA samples were subjected to reverse transcription using SuperScript II (Life Technologies, Thermo Fisher Scientific), followed by quantitative real-time PCR using SYBR Premix Ex Taq and thermal cycler dice (both from Takara Bio Inc.) or by semiquantitative PCR. Primer sequences used for real-time PCR are shown in Supplementary Table S1.

c-Myb expression plasmids (c-Myb-8A, 9A, 9B, 10A, 13A, and 14A in pCDNA3 mammalian expression vector) were generous gifts from Dr. O'Rourke (8). FLAG-tagged c-Myb expression plasmids (WT-c-Myb, 9A, and 10A) were generated by inserting PCR-amplified cDNA fragments for each isoform into pME-FLAG vector at XhoI and SpeI sites.

For detection of proteins in Western blotting, the following primary antibodies were purchased from the indicated companies: c-Myb (#ab45150, Abcam), FoxM1 (#sc-500, Santa Cruz Biotechnology), AURKA (#121005, Cell Signaling Technology), EZH2 (#3147S, Cell Signaling Technology), NIK (#4994S, Cell Signaling Technology), NF-κB2-p52 (#sc-7386, Santa Cruz Biotechnology), NF-κB1-p50 (#sc-1190X, Santa Cruz Biotechnology), IκBα (#sc-371, Santa Cruz Biotechnology), P-IκBα (#9246S, Cell Signaling Technology), FLAG (F3165; Sigma-Aldrich Corporation), and β-actin (sc-69879; Santa Cruz Biotechnology, Inc.). For the secondary antibodies conjugated with alkaline phosphatase, anti-mouse IgG (S372B; Promega) and anti-rabbit IgG (S373B; Promega) were used depending on the host species of the primary antibody.

Chromatin immunoprecipitation (ChIP) against endogenous c-Myb was conducted in Jurkat and TL-Om1 cells, and ChIP against FLAG-tagged c-Mybs (WT, c-Myb-9A, and c-Myb-10A) was conducted in 293FT cells transiently overexpressing each FLAF-c-Myb. The ChIP procedures is described elsewhere (24) with minor modifications. For immunoprecipitation of c-Myb in Jurkat and TL-Om1, rabbit monoclonal c-Myb antibody (#ab45150, Abcam) was used. Normal rabbit IgG (#2729P, Cell Signaling Technology) was used as the negative control. For precipitation of FLAG-tagged c-Myb in 293FT cells, anti-FLAG M2 Affinity Gel (#A2220, Sigma) was used. For the negative control, the lysate was incubated with a normal mouse IgG (#I5381, Sigma), followed by precipitation with ProteinG 4 Fast Flow sepharose (#17-0618-01, GE Healthcare). Primer sequences for detection of c-Myb target promoters by real-time PCR are listed in Supplementary Table S2.

Activated NF-κB p65 was detected by immunocytochemistry in HeLa cells overexpressing c-Myb isoforms (WT, 8A, 9A, 9B, 10A, 13A, and 14A). Fixed HeLa cells, transiently overexpressing c-Myb isoforms (WT, c-Myb-8A, 9A, 9B, 10A, 13A, and 14A) or mock-transfected with the empty vector as the negative control, were fixed in 4% paraformaldehyde for 5 minutes, permeabilized in 0.1% Triton X-100 in PBS for 10 minutes, blocked in 3% BSA in PBS for 1 hour, and incubated with an mAb against activated subunit of NF-κB-p65 (#MAB3026, Millipore) followed by Alexa Fluor 546–conjugated goat anti-mouse IgG (A11030, Invitrogen). Cells were then observed by Zeiss710 laser scanning microscope.

Detection of NF-κB activity by electrophoretic mobility shift assay (EMSA) in nuclear extract of cells was conducted as described elsewhere (21). Briefly, nuclear extracts from Jurkat cells with c-Myb overexpression or knockdown and TL-Om1 cells with c-Myb knockdown were prepared and incubated with [γ-³²P]ATP-labeled oligo DNA probes for NF-κB (#E3291, Promega) and for Oct1 (#E3241, Promega). The samples were separated by electrophoresis with 4% acrylamide gels at 150 V for 1.5 hours and completely dried at 65°C for 3 hours. The band intensity of ³²P-labeled probe, binding to nuclear proteins, was detected by autoradiography.

Soft agar assays were conducted in NIH3T3 cells stably expressing WT-c-Myb, c-Myb-9A, or c-Myb-10A. NIH3T3 cells stably expressing WT-c-Myb, c-Myb-9A, or c-Myb-10A were established by transfecting c-Myb-pCDNA3 plasmid and selecting by addition of 0.8 mg/mL G418 for 10 days. HA-pCDNA3 plasmid was transfected as negative control. Selected c-Myb expressing NIH3T3 cells were seeded at a density of 1 × 10⁴ cells in 1 mL of 0.3% agarose in DMEM with 10% FBS over a cushion of 2-mL 0.5% agarose in DMEM with 10% FBS per well in 6-well plate. After the top agar was set, 500 μL/well of DMEM was gently overlaid as feeding medium. After culturing at 37°C with 5% CO₂ for 2 weeks, colonies were scored and photographed.

PCR-amplified promoter fragment of c-Myb target genes was subcloned into pGL4.10 plasmid (Promega). Primer sequences for PCR amplification of promoter region are shown in Supplementary Table S3. For FoxM1, the promoter reporter clone for human FoxM1 (HPRM13918) was purchased from GeneCopoeia, and the BglII/HindIII digested FoxM1 promoter fragment was inserted to pGL4.10.

In the current study, luciferase-based reporter assays were conducted for assessing (i) c-Myb target promoter activity, (ii) NF-κB activity, and (iii) HTLV-1 LTR activity. HEK293T cells were seeded at the concentration of 8 × 10⁴ cells/mL and 200 mL/well in 48-well culture plate 1 day before transfection. At 24 hours after seeding, 10 ng reporter plasmid (c-Myb target promoter-pGL4.10, 6× NF-κB–binding sites pGL4.10, or HTLV-1-LTR-pGL4.10), 5 ng RSV-Renilla-luciferase plasmid, and 200 ng c-Myb-pCDNA3 for a well were cotransfected by Lipofectamine 2000 (Invitrogen). At 24 hours after transfection, Renilla and firefly luciferase activities were measured using the Dual Luciferase Assay System (Promega) with the Centro LB 960 luminometer (Berthold Technologies). Detected firefly luciferase activity was divided by corresponding Renilla-luciferase activity to normalize transfection efficiency.

In the current study, two shRNAs targeting 3′ UTR and exon 8 of MYB mRNA were prepared. Two sets of sense- and antisense-DNA oligos targeting 3′ UTR and exon8 of MYB mRNA were annealed by boiling for 5 minutes followed by cooling down in room temperature for more than 1 hour. Annealed fragments were inserted at BglII/XbaI in pENT4-H1 vector (Invitrogen). The sh-c-Myb fragment in pENT4-H1 was further transferred to the destination vector, CS-RfA-EvBsd, by incubation with LR-clonaseII. For preparation of sh-Myb lentiviruses, 293FT cells were seeded at 1 × 10⁵ cells/mL and 10 mL/10-cm dish. At 24 hours after seeding, 17 ng sh-Myb-#1 or -#2- CS-RfA-EvBsd, 10 ng CAG-HIV-gp, and 10 ng pCMV-VSV-G/RSV-Rev were cotransfected by phosphate-Ca²⁺ method. At 72 hours after transfection, the culture medium was filtrated through 45-μm filter, and recombinant lentiviruses were precipitated by centrifugation at 10,000 rpm for 3 hours at 4°C. Precipitated viruses were resuspended in 100 μL of RPMI (FBS⁻) and stored at −80°C until use.

For infection, 2 × 10⁶ cells of Jurkat, TL-Om1, or primary ATL cells were resuspended in 100 μL sh-Myb-lentivirus stock and processed for centrifugation at 2000 rpm for 3 hours at 35°C to enhance infection. After centrifugation, cells were resuspended in 2 mL of RPMI and incubated at 37°C with 5% CO₂ for 72 hours before being used for experiments. Sh-c-Myb oligos used in the current study are shown in Supplementary Table S4.

Cells for FACS analyses were collected and resuspended in FACS buffer (PBS containing 2% FBS) prior to analysis. For apoptosis assays, cells were stained with PE-Annexin V in the binding buffer (all from PE AnnexinV Apoptosis Detection Kit I, #559763, BD Pharmingen) for 20 minutes at room temperature before analyses. All FACS analyses were conducted with FACSCalibur (BD Biosciences), with appropriate instrument settings and compensation, if necessary. Obtained data were analyzed by FlowJo ver.10 (TreeStar Inc.).

On the basis of the report by Marcos (25), which thoroughly examined and reviewed employment of morpholino oligos for regulation of alternative splicing, we designed FITC-conjugated 2 morpholino oligos, targeting the exon9A/intoron9 border (MYB-MO-#1) and exon9B/intron10 border (MYB-MO-#2) of MYB mRNA, respectively, with assistance from GENE TOOLS, LLC. The custom morpholino oligos, as well as the standard oligos (i.e., the control MO) were purchased from GENE TOOLS, LLC. The morpholino oligos were introduced at 10 μmol/L concentration to Jurkat, TL-Om1, MT-2 cells, and primary ATL cells at concentration of 1 × 10⁶ cells in 400 μL Cytomix buffer (25 mmol/L HEPES, 120 mmol/L KCl, 10 mmol/L KPO₄, 2 mmol/L EGTA, 0.15 mmol/L CaCl₂, 5 mmol/L MgCl, pH 7.6, by KOH) by electroporation (220∼250 V, 1,050 F, 720 Ω) using Electro Cell Manipulator 600 (BTX). Morpholino oligo sequences are shown in Supplementary Table S5.

Throughout the current study, two-tailed paired Student t test was performed to test the statistical difference between the experimental groups. Asterisks and sharps in the figures indicate a significant difference between the tested groups (*, #, P < 0.05; **, ##, P < 0.01; and ***, ###, P < 0.001; n > 3).

As shown in Fig. 1A, seven well-known transcript variants (NCBI: U22376.1, AJ606324.1) and isoforms of c-Myb have been reported [see the work of O'Rourke and Ness (ref. 8) for a detailed structure of each variant and encoded isoform]. First, the expression level of each MYB transcript variant was assessed using a qPCR analysis with variant-specific primers. All tested transcript variants, including MYB-9A and -10A, were overexpressed in ATL cells, particularly those isolated from patients with chronic and acute ATL (Fig. 1B; data not shown for MYB-8A, -9B, -13A, and -14A mRNAs). Moreover, Western blotting with a c-Myb–specific antibody revealed significantly increased c-Myb protein levels in ATL cells compared with normal CD4⁺ T cells (Fig. 1C).

Like the v-Myb oncoprotein, c-Myb-9A is expected to exhibit strong transforming activity. In soft agar assays using NIH3T3 cells engineered to overexpress various c-Myb isoforms (Fig. 2A), c-Myb-9A exhibited the strongest colony formation activity, whereas WT-c-Myb did not exhibit significant transforming activity (Fig. 2B).

The impact of c-Myb overexpression on cell growth was further evaluated by knocking down total c-Myb at the protein level. Jurkat and TL-Om1 cells were infected with recombinant lentiviruses to induce the overexpression of shRNAs against all MYB transcripts. The top panels of Fig. 3A show that both shRNAs effectively knocked down c-Myb at the protein level. Subsequently, Venus (+) cell populations (i.e., shRNA-expressing populations) were assessed for approximately 2 weeks after infection. We observed a specific reduction in Venus (+) cell populations, indicating selective cell death in c-Myb knockdown cells (Fig. 3A). Primary ATL cells were also subjected to c-Myb knockdown (n = 4). Again, both shRNAs effectively suppressed c-Myb protein expression (Fig. 3B, top left). The proportions of Annexin V (+) populations increased, particularly in response to sh-c-Myb-#2, indicating the induction of apoptosis following c-Myb knockdown in primary ATL cells (Fig. 3B). It may be noteworthy that the protein levels of both WT-c-Myb and c-Myb-9A were reduced following treatment with the shRNAs described herein (Fig. 3B).

Thus far, our results demonstrate that c-Myb is responsible for sustainable ATL cell growth. To assess the effects of the overexpression of c-Myb-9A, which harbors strong transforming activity, we attempted a c-Myb-9A–specific knockdown using morpholino oligomers. We tested 2 FITC-labeled morpholino oligomers (Supplementary Table S5) and found that MYB-MO-#2 effectively suppressed the expression of both c-Myb-9A and -9B (see Supplementary Fig. S1C). Consequently, we employed MYB-MO-#2 for the specific suppression of c-Myb-9A (Fig. 4A). First, we introduced MYB-MO-#2 to Jurkat, TL-Om1, and MT-2 cells. Exon-specific qPCR confirmed significant decreases in MYB-9A mRNA levels in all cell lines following MYB-MO-#2 treatment (Fig. 4B). FACS analyses of apoptotic cells demonstrated that a significantly higher proportion of FITC (+) cells (i.e., c-Myb-9A knockdown cells) were also Annexin V (+) compared with control Morpholino oligomer–transfected cells (Fig. 4B). Figure 4C demonstrates the influence of MYB-MO-#2–mediated c-Myb-9A–specific knockdown in primary ATL cells (n = 4). FACS analyses demonstrated that c-Myb-9A–specific knockdown also induced apoptosis in primary ATL cells. These results provide evidence for a major role of c-Myb-9A overexpression in ATL cell survival.

To evaluate the influence of c-Myb deregulation on transcriptional regulation, a series of ChIP assays was conducted to identify c-Myb target genes. Figure 5A presents the results of ChIP assays performed against endogenous c-Myb in Jurkat and TL-Om1 cells. Notably, c-Myb accumulated on the promoter regions of the tested genes. Consistent with the possibility that c-Myb may regulate those genes, the levels of the associated transcripts, except for IL6 mRNA (Supplementary Fig. S2), were upregulated in patients with ATL (n = 52), compared with healthy donors (n = 21), as determined in our laboratory using gene expression microarray analyses (GSE33615). These data indicate that c-Myb acts as a positive regulator of these target genes in ATL cells.

Among the well-known c-Myb isoforms, c-Myb-9A has the highest level of transactivity (8). We speculate that this increased transactivity might be attributable to the greater recruitment of c-Myb-9A to target regions compared with other isoforms. Therefore, we prepared plasmids that would express FLAG-tagged WT-c-Myb, c-Myb-9A, and c-Myb-10A and subjected 293FT cells overexpressing FLAG-c-Myb proteins to ChIP assays using a FLAG antibody. FLAG-c-Myb-9A was indeed detected at significantly higher levels on the tested target promoters compared with FLAG-WT-c-Myb (Fig. 5B). Furthermore, the accumulation of c-Myb on some target genes was enhanced under conditions of DNA hypomethylation (Supplementary Fig. S3).

Using a luciferase-based promoter reporter system for the indicated c-Myb target genes, we tested the activation of these reporters with the following c-Myb isoforms: WT, 9A, and 10A (Fig. 5C). Consistent with the ChIP assays (Fig. 5A and B), WT-c-Myb exhibited significantly higher reporter activities relative to the mock control on all tested promoters. Moreover, c-Myb-9A and -10A exhibited significantly higher transactivation of these promoters compared with WT-c-Myb. In addition, c-Myb-9A exhibited the highest transactivity for HTLV-1 LTR among the 7 c-Myb isoforms (Supplementary Fig. S4), which is known to be regulated by c-Myb (26).

Thus far, our results indicate that c-Myb upregulates several genes, the products of which play critical regulatory roles in cell proliferation. In addition, c-Myb-9A exhibits a hyperactive capacity for transactivation compared with WT-c-Myb. Among the tested c-Myb target genes, NIK, FoxM1, and AURKA are positive regulators of the NF-κB pathway. Thus, c-Myb might influence cellular NF-κB activity by regulating these components. Indeed, the activation of NF-κB by WT-c-Myb was confirmed using NF-κB reporter assays (Fig. 6A) and immunocytochemical analysis of activated p65 (Supplementary Fig. S5). Moreover, c-Myb-9A induced the highest level of NF-κB pathway activity among the tested c-Myb isoforms (Fig. 6A). In agreement with these results, both the protein and mRNA levels of NIK, FoxM1, and AURKA, together with the protein level of P-IκBα, were decreased following c-Myb knockdown in Jurkat and TL-Om1 cells (Fig. 6B and Supplementary Fig. S6). Conversely, the overexpression of c-Myb-9A in Jurkat cells increased the expression levels of those proteins, as well as of downstream NF-κB components such as NF-κB2-p52, NF-κB1-p50, and P-IκBα (Fig. 6B). Finally, we used an EMSA to test NF-κB activities in nuclear extracts from c-Myb overexpressing or knocked down Jurkat and TL-Om1 cells. In agreement with the data in Fig. 6B, NF-κB activity was increased most strongly in response to the overexpression of c-Myb-9A, followed by c-Myb-10A. In contrast, NF-κB activity was suppressed in Jurkat and TL-Om1 cells following c-Myb knockdown (Fig. 6C).

In this study, we have demonstrated for the first time the abnormal and unbalanced MYB transcript overexpression in ATL cells. We have demonstrated increases in the expression of not only overall c-Myb but also of c-Myb-9A, an isoform with a “gain -of-function oncogenic characteristics,” in ATL cells. In tumors, MYB gene overexpression and its influence on cellular homeostasis have been extensively studied (27–35). Conversely, limited information is available regarding the expression profiles of c-Myb isoforms, each of which possesses a considerably different transactivation capacity and transforming activity. In the current study, we explored the molecular impacts of c-Myb dysregulation consequent to unbalanced c-Myb isoform expression on the malignancy of ATL cells.

A variant-specific qPCR analysis of MYB mRNA revealed increased expression levels of all tested variants (data not shown for MYB-8A, 9B, 13A, and 14A) in patients with ATL compared with healthy volunteers; this tendency was most significant for MYB-9A mRNA (Fig. 1B). In addition, the magnitude of increased expression of this transcript variant was even greater in ATL cells from patients with chronic- and acute-type ATL compared with those from patients with smoldering-type ATL. In other words, c-Myb-9A levels might correlate with the risk of ATL progression. In contrast to WT-c-Myb, c-Myb-9A exhibited strong transforming activity in soft agar assays that used NIH-3T3 cells as an indicator of transformation (Fig. 2), thus confirming the “gain-of-function oncogenic characteristics” of this isoform.

The mechanism of MYB-9A mRNA overexpression has not yet been elucidated. We recently reported that abnormal splicing patterns of HELIOS mRNA, another hematopoietic transcription factor, were observed frequently in ATL cells (20). Moreover, we reported the drastic dysregulation of miRNA expression in ATL cells (21). Significant downregulation of miR-150, a well-known negative regulator of MYB mRNA expression (36), was observed in our previous miRNA microarray analysis of patients with ATL (GSE31629). Changes in transcriptional activity, mRNA stability, and splicing preferences might contribute to the overexpression of oncogenic MYB transcript variants.

The overall suppression of c-Myb–induced death in both ATL cell lines and primary ATL cells (Fig. 3). Moreover, the specific morpholino oligomer–induced knockdown of c-Myb-9A resulted in a significant increase in apoptotic cell rates in T-cell lines, regardless of HTLV-1 infection status, as well as primary ATL cells (Fig. 4). These data suggest that c-Myb-9A plays a crucial role in cell survival in c-Myb–overexpressing cells, including primary ATL cells.

Extensive attempts have been made to identify c-Myb target genes (14, 15, 37–40). A list of hundreds of putative c-Myb target genes was generated using a combination of c-Myb knockdown and gene expression microarray analyses. In the current study, we conducted ChIP analyses of nine putative c-Myb target genes that encode regulators of cell proliferation. Figure 5A demonstrates the enrichment of c-Myb on the promoters of all tested target genes. A gene expression microarray analysis (GSE33615) demonstrated that the expression levels of all tested c-Myb target genes, except for IL6 mRNA, were elevated in ATL cells (Supplementary Fig. S2), further supporting the notion that c-Myb is a positive regulator of these genes in ATL cells.

c-Myb-9A, which lacks a C-terminal NRD and is structurally similar to the v-Myb AMV oncoprotein, is known to exhibit a significantly higher transactivation capacity relative to that of WT-c-Myb (8). We wondered whether this higher transactivation capacity could be attributed to enhanced recruitment to target promoters. In a series of previous studies, C-terminal deletion mutants of c-Myb led to significant increases in DNA-binding activity, transactivation, and transformation activity, indicating the importance of the NRD in the negative self-regulation of c-Myb (41–45). Molvaersmyr and colleagues identified SUMOylation sites for degradation within the NRD (46). Our ChIP assays demonstrated significantly higher levels of FLAG-c-Myb-9A enrichment on the tested target promoters compared with FLAG-WT-c-Myb enrichment (Fig. 5B). In agreement with the ChIP assay results, luciferase-based promoter reporter assays revealed significantly higher levels of promotor transactivation with c-Myb-9A than with WT-c-Myb (Fig. 5C and Supplementary Fig. S4). Furthermore, c-Myb was generally capable of accessing the target regions more efficiently under conditions of Aza-dC treatment–induced DNA hypomethylation; under the same conditions, c-Myb-9A exhibited even more efficient recruitment, especially to GATA3, FOXM1, AURKA, EZH2, and IL-2Rα (Supplementary Fig. S3). In agreement with our results, Wang and colleagues demonstrated that WNT5A gene hypomethylation led to increased c-Myb recruitment (47). DNA methylation is a known marker of epigenetic silencing (48). Therefore, c-Myb-9A might have a stronger impact when the target gene is not silenced. Collectively, these data led us to speculate that the enhanced transactivation capacity of c-Myb-9A leads to the overexpression of c-Myb target genes in cells with elevated c-Myb-9A expression.

Finally, we revealed that c-Myb plays an important role in NF-κB pathway activation. Of the c-Myb target gene products evaluated in the current study, NIK is a key positive regulator of the noncanonical NF-κB pathway (49). AURKA is known to phosphorylate IκBα, leading to its degradation and activating NF-κB (50). FoxM1 transactivates AURKA gene expression to a similar extent as c-Myb and is also known as a strong positive regulator of cell cycle and cell proliferation (51). Recently, c-Myb and FoxM1 were found to share a considerable number of target genes and are expected to function synergistically (51). Constant activation of the NF-κB pathway, as well as its pro-oncogenic influences, has been well-documented and reviewed in ATL cells (52). Figure 6A shows that c-Myb indeed activates the NF-κB pathway and that c-Myb-9A induces the highest level of activation. In Fig. 6B and Supplementary Fig. S6, we demonstrate that NIK, FoxM1, and AURKA protein/mRNA levels, as well as the levels of downstream NF-κB components, were regulated in a manner dependent on the c-Myb expression level. In particular, c-Myb-9A overexpression enhanced the expression levels of the abovementioned proteins in Jurkat cells (Fig. 6B). In c-Myb-9A–overexpressing cells, the molecular impact of c-Myb-9A might be accelerated in a positive feedback manner by the activation of c-Myb itself, as well as synergistic activation between the c-Myb and FoxM1 pathways. This dysregulation of critical transcription factors at multiple stages might be responsible for the constitutive and drastic NF-κB activation observed in ATL cells. Using EMSA, we ultimately tested whether a direct relationship existed between the c-Myb expression level and NF-κB activity level in Jurkat and ATL-derived TL-Om1 cells. Figure 6C shows that the overexpression of c-Myb, especially c-Myb-9A, activates NF-κB in Jurkat cells, whereas shRNA-mediated c-Myb knockdown suppressed NF-κB activity in both Jurkat and TL-Om1 cells. Altogether, these data suggest that c-Myb plays a critical role in the regulation of NF-κB activity by controlling the expression of proteins involved in the NF-κB pathway, such as FoxM1, AURKA, and NIK. Moreover, it is highly possible that in the absence of a self-regulatory domain, c-Myb-9A can overproduce these gene products and thus hyperactivate downstream NF-κB activity compared with WT-c-Myb (Fig. 6D).

In summary, this study highlights for the first time the impact of c-Myb dysregulation on the malignant characteristics of ATL cells. Moreover, we have revealed that the aberrantly expressed oncogenic c-Myb isoform, c-Myb-9A, is responsible for extensive c-Myb pathway dysregulation. Our current study data suggest that the expression levels of c-Myb isoforms, including c-Myb-9A, are increased during the leukemogenesis and progression of ATL. This overexpression of c-Myb can trigger the dysregulation of cellular homeostasis; c-Myb-9A, which lacks self-regulatory machinery, further augments this disorder by inducing the undesirable overexpression of cellular proteins involved in cell proliferation, NF-κB activation, and transformation (Fig. 6D). Because the c-Myb pathway is profoundly involved in the regulation of T-cell function and composition, dysregulation of this pathway may be an effective therapeutic target for ATL. Further investigations to elucidate the molecular basis of c-Myb pathway dysregulation in ATL cells will be fundamental to the establishment of an effective therapeutic approach.

No potential conflicts of interest were disclosed.

Conception and design: K. Nakano, K. Yamaguchi, T. Watanabe

Development of methodology: K. Nakano, T. Watanabe

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Nakano, K. Uchimaru, A. Utsunomiya, T. Watanabe

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Nakano, T. Watanabe

Writing, review, and/or revision of the manuscript: K. Nakano, K. Uchimaru, A. Utsunomiya, T. Watanabe

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Nakano, T. Watanabe

Study supervision: T. Watanabe

The authors thank Dr. O'Rourke in University of New Mexico for his generous gifts of c-Myb expression plasmids. They also appreciate Dr. S. Firouzi in the University of Tokyo for the integration site analysis of MT-2 cells; Dr. H. Sato in the University of Tokyo for the karyotype analysis of HeLa and 293T cells; and Drs. H. Miyoshi and A. Miyawaki in RIKEN, Japan, for providing the Venus-encoding lentivirus vectors.

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, to T. Watanabe (No. 26293226 and No. 23659484) and to K. Nakano (No. 24501304).

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