BAALC Potentiates Oncogenic ERK Pathway Through Interactions with MEKK1 and KLF4

ORIGINAL ARTICLE BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4

K Morita1, Y Masamoto1, K Kataoka1, J Koya1, Y Kagoya1, H Yashiroda2, T Sato1,3, S Murata2 and M Kurokawa1,4

Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell- cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by upregulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Krüppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.

Leukemia (2015) 29, 2248–2256; doi:10.1038/leu.2015.137

INTRODUCTION Previous reports have suggested a fundamental role of MEKK1 in 11,12 Acute myeloid leukemia (AML) is a clonal disorder characterized anti-apoptotic function in myeloid cells and leukemogenesis. by differentiation arrest and accumulation of immature myeloid By contrast, ERK can trigger tumor suppressor pathways as well, such as cellular senescence, apoptosis and differentiation in a progenitors in the bone marrow, resulting in hematopoietic 13,14 failure. To date, several poor prognostic factors for AML have been context-dependent manner. Krüppel-like factor 4 (KLF4), reported, which include gene mutations in TET2, ASXL1 or a member of Krüppel-like family transcription factors, is induced 1 by ERK activation and mediates growth inhibition in human colon DNMT3A and overexpression of ERG, EVI1, MN1 or BAALC. In 15 general, overexpression of BAALC is observed in one-fourth of cancer cells. Although KLF4 has been shown to enhance stem-cell 2,3 like properties as one of four iPS factors, it has been drawing attention AML patients, and recent reports have shown its adverse impact 16 on the survival of AML patients across all karyotypes.4 BAALC in AML as an essential factor for monocytic differentiation. In this study, we aimed to investigate the molecular basis of encodes at least eight alternatively spliced transcripts in humans, BAALC in leukemia cell proliferation. We demonstrate here that and BAALC 1-6-8 is the major isoform expressed in hematopoietic through interactions with MEKK1 and KLF4, BAALC potentiates cells.5 In normal hematopoiesis, the expression of BAALC is limited oncogenic ERK pathway while inhibiting ERK-dependent differ- to the immature fraction, that is, CD34+ fraction rich in hemato- 6 entiation. This study could also determine whether concurrent poietic stem and progenitor cells. In a large cohort of patients with MEK inhibition and KLF4 induction would be a legitimate AML, high BAALC expression is associated with expressions of stem therapeutic modality for BAALC-high AML. cell markers.2 From these facts, it is suggested that BAALC is related to immaturity of hematopoietic cells, but the precise function of BAALCinleukemogenesisispoorlyunderstood. MATERIALS AND METHODS Aberrant activation of several oncogenic signaling cascades is Analysis of protein phosphorylation implicated in AML pathogenesis.7 Previous reports showed that AML cells transduced with BAALC or control vectors were stimulated by Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular phorbol myristate acetate (PMA) at 100 ng/ml for 6 h, and then were signal-regulated kinase (ERK) pathway is activated in a signiﬁcant harvested. After subsequent protein extraction, phosphorylation of proportion of AML patients and expected as a potential signaling molecules was examined by immunoblotting. therapeutic target.8 However, the anti-leukemic activity of MEK 9 inhibitor monotherapy in the clinical settings has been modest, Co-immunoprecipitation assay which necessitates further elucidation of the networks of signaling Co-immunoprecipitation (CoIP) was carried out as previously described.17 molecules in this pathway. Among them, mitogen-activated Immunoprecipitation was performed using anti-ERK, anti-MYC-tag, anti- protein kinase kinase kinase 1 (MEKK1) is a well-known kinase as HA-tag and anti-FLAG-tag antibodies. The precipitates were analyzed by well as a scaffold protein of key elements in ERK pathway.10 immunoblotting.

1Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; 3Department of Transfusion Medicine, The University of Tokyo Hospital, Tokyo, Japan and 4Department of Cell Therapy and Transplantation, The University of Tokyo Hospital, Tokyo, Japan. Correspondence: Dr M Kurokawa, Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: [email protected] Received 28 April 2015; accepted 21 May 2015; accepted article preview online 8 June 2015; advance online publication, 26 June 2015 BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2249 Time-lapse immunoprecipitation assay MEKK1 and KLF4 are novel interacting partners of BAALC AML cells transduced with BAALC or control vectors were starved for 24 h, To gain insight into molecular functions of BAALC, interacting then stimulated by PMA at 100 ng/ml for the indicated time periods. After partners of BAALC were explored with a yeast two-hybrid system subsequent cell lysis, extracted proteins were immunoprecipitated by anti- on a proteome-wide scale (Supplementary Figure S4A). Using MYC-tag antibody or anti-ERK antibody with sepharose G protein beads. BAALC as a bait and a cDNA library from adult human tissues as Protein–protein interaction was examined by immunoblotting. prey, we identified eight possible interacting molecules Immunoblotting (Figure 1e). Among them, we focused on MEKK1 and KLF4, both 17 of which are known to have substantial roles in Ras/Raf/MEK/ERK Immunoblotting was performed as previously described. See Supple- pathway, one of pivotal oncogenic signaling cascades in AML.10,18 mentary methods for details. To validate the interactions between BAALC and MEKK1 or KLF4, Yeast two-hybrid screening we performed CoIP assay in AML cells. Intriguingly, BAALC bound to MEKK1 more tightly in the presence of PMA, a potent activator Yeast two-hybrid screening was performed with the Matchmaker Gold of multiple cytoplasmic signal transduction cascades, while Yeast Two-Hybrid System (Clontech, Mountain View, CA, USA). Human – BAALC 1-6-8 cDNA was used as a bait and cloned into the pGBKT7 vector BAALC KLF4 complex was formed independently of PMA in frame with the GAL4 DNA-binding domain (pBD-BAALC). Yeast Y2H stimulation (Supplementary Figures S4B and E). Protein–protein GOLD cells were then co-transformed with pBD-BAALC, SmaI-linearized interactions between BAALC and MEKK1 or KLF4 were further prey vector (pGADT7) and a cDNA library generated from healthy human affirmed by in vitro pull-down assays using purified proteins from bone marrow. Interaction was confirmed as described in the Yeast Protocol mammalian cells (Figures 1f and g). These results illustrate that Handbook (Clontech). After extraction of plasmids from positive clones, BAALC directly interact with MEKK1 in a stimulation-dependent, inserted cDNA was sequenced by 3130 × L Genetic Analyzer (Applied and with KLF4 in a stimulation-independent manner. Biosystems, Waltham, MA, USA) and identical aliment was computationally searched by BLAST (Basic Local Alignment Search Tool, National Library of Medicine). N-terminal region of BAALC is necessary and sufficient for the interactions with MEKK1 and KLF4 Mice Next, we elucidated an essential binding region of each protein NOD/SCID/gamma (NSG) mice were purchased from the Jackson using a series of deletion mutants of BAALC, MEKK1 and KLF4. CoIP Laboratory (Bar Harbor, ME, USA). Littermates were used as controls in assay revealed that N-terminal region of BAALC (amino acids 1–35) all experiments. was an indispensable prerequisite for its binding to MEKK1 and KLF4 (Supplementary Figures S5A and B). Notably, this N-terminal region Xenograft mouse model of BAALC is conserved across all mammals expressing BAALC, Xenograft mouse model of human AML with high expression of BAALC indicating the functional importance of this region.5 HEL cells was developed using NSG mice. Before transplantation, NSG mouse was expressing deletion mutants of BAALC harboring N-terminal 1–35 treated with intraperitoneal injection of cyclophosphamide 100 mg/kg/day amino-acid sequence (Δ1, Δ2) showed comparable proliferation and for 2 days. At day 0 and day 1, 5 × 106 cells/body of Kasumi-1 cells transduced with indicated viruses were intravenously injected. Peripheral cell-cycle progression (Supplementary Figures S5C and E) to cells blood was collected twice a week and chimerism was checked by flow expressing full-length BAALC. By contrast, BAALC mutants lacking cytometer. Once peripheral blood chimerism of leukemia cells exceeded N-terminal domain (Δ3, Δ4) had no effect on proliferation and cell- 1%, treatment either by U0126 (25 μmol/kg/week intraperitoneally) or by cycle progression, underscoring a fundamental role of this region doxycycline (diluted in drinking water at 1 mg/ml+3% sucrose) was started. for proliferative capacity of BAALC. See Supplementary methods for more information. Furthermore, we found that C-terminal region of MEKK1 and KLF4 was necessary for their interactions with BAALC RESULTS (Supplementary Figures S6A and B). These findings indicate that BAALC promotes leukemia cell expansion the N-terminal region of BAALC is essential for cell-cycle fi progression of myeloid leukemia cells through interactions with We rst examined the expression levels of BAALC in primary C-terminal regions of MEKK1 and KLF4. human bone marrow samples from AML patients and control subjects as well as in seven AML cell lines. AML cells showed about 10-fold higher BAALC than control subjects (Figure 1a). BAALC was BAALC prolongs ERK activation by inhibiting reassociation of an − fi higher even in human primitive CD34+ CD38 AML cells compared ERK-speci c phosphatase MAP kinase phosphatase 3 with normal CD34+ CD38− cells rich in hematopoietic progenitor Considering that both MEKK1 and KLF4 are involved in ERK cells (Supplementary Figure S1A). Among seven AML cell lines, pathway, we postulated that BAALC induces leukemia cell Kasumi-1 and OCI-AML2 showed comparable BAALC levels as proliferation by potentiating MEK/ERK signaling. By BAALC over- BAALC-high AML patient samples, while BAALC expressions were expressions in BAALC-low AML cells, we found that BAALC low in HEL and MV4-11. From this finding, hereafter we used specifically upregulated the activities of ERK and its direct Kasumi-1 and OCI-AML2 as BAALC-high leukemia cells, and HEL downstream target ribosomal protein S6 kinase, 90 kDa and MV4-11 as BAALC-low cells (Figures 1a and b). When full- (p90RSK), without affecting the phosphorylation status of other length of BAALC (1-6-8 form) was lentivirally transduced to HEL upstream molecules of ERK (Figure 2a). The activities of other and MV4-11 cells, they showed increased proliferative capacity signaling pathways including PI3K-AKT, JAK-STAT and NF-κB and accelerated cell-cycle progression, while apoptotic status was pathways were not altered by BAALC (Supplementary Figure S7A), not altered (Figure 1c; Supplementary Figures S1C and F). On the suggesting that BAALC specifically affects the activity of ERK. other hand, BAALC-knocked down Kasumi-1 and OCI-AML2 cells Similarly, AML cells transduced with BAALC showed prolonged by retroviral short hairpin RNA (shRNA) showed opposite ERK activation compared with the control (Figure 2b; phenotype to BAALC overexpression. Moreover, phenotypes Supplementary Figures S7B and C). Consistent with these findings, associated with BAALC knockdown were completely rescued by knockdown of BAALC in AML cells had decreased ERK activation, additional expression of shRNA-resistant cDNA construct of BAALC which was reverted by additional expression of BAALC (Figure 1d; Supplementary Figures S2A and C and S3A and C). (Supplementary Figure S8A). These results indicate that BAALC promotes proliferation, cell- As shown above, BAALC potentiated ERK phosphorylation cycle progression of leukemia cells along with inhibition of without influencing upstream kinase activity of ERK, although leukemia cell differentiation. BAALC has no known kinase or phosphatase sequence motifs.6

© 2015 Macmillan Publishers Limited Leukemia (2015) 2248 – 2256 BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2250 We thus hypothesized that BAALC contributes to the sustained ERK analysis of CoIP assay, in which AML cells overexpressing BAALC phosphorylation by hindering the interaction between ERK and were harvested at the indicated time points after stimulation and mitogen-activated protein kinase phosphatase 3 (MKP3/DUSP6), an analyzed for the interaction between BAALC, ERK, MKP3 and ERK-speciﬁc phosphatase that mediates deactivation of ERK MEKK1. BAALC bound to MEKK1 immediately after PMA stimula- 19 signaling. To test this hypothesis, we conducted a time lapse tion, strongest at around 3–6 h after stimulation (Figure 2c;

Leukemia (2015) 2248 – 2256 © 2015 Macmillan Publishers Limited BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2251 Supplementary Figures S8B and C). As for the interaction between Nuclear KLF4 drives monocytic differentiation by constitutive ERK and MKP3, PMA treatment rapidly induced dissociation of activation of ERK pathway MKP3 from ERK, followed by MKP3 reassociation with ERK after 6 h To address whether the growth-promoting effect of BAALC over- of stimulation in control cells. On the contrary, in BAALC- expression on leukemia cells is dependent on the ERK pathway, the overexpressing cells, the recruitment of MKP3 to ERK at 6 h was constitutive active form of MEK1 (CA-MEK1, S218E/S222E) was hindered along with the stimulation-dependent BAALC-MEKK1 transduced into BAALC-low AML cells. In clear contrast to the case of interaction up to 9 h after stimulation (Figure 2d; Supplementary BAALC overexpression, CA-MEK1 decreased cell proliferation and Figures S8D and E). The extent of ERK phosphorylation was induced monocytic differentiation, despite the comparable phos- inversely correlated to the intensity of ERK–MKP3 interaction, phorylation of ERK to BAALC (Figures 3a and b; Supplementary underscoring the importance of MKP3 in ERK activity (Figure 2b; Figures S14A and C). To further investigate this phenotypic Supplementary Figures S7B and C). discrepancy, we focused on a biological function of KLF4, one of the other interacting partners of BAALC. The expression of KLF4 is 15,26 BAALC induces drug resistance in AML cells through ERK positively regulated by ERK in tumor cells from several origins, activation and it is considered to be involved in monocytic differentiation of 16 To further investigate the specificity and oncogenic potentiality of myeloid cells. When we compared the expression levels of KLF4 by BAALC in ERK pathway, we treated BAALC- or control vector- immunoblotting, nuclear expression of KLF4 was increased in CA- transduced AML cells by various signaling inhibitors and examined MEK1-transduced AML cells, accompanied by an increase of KLF4 in their proliferation. Both genetic and pharmacologic inhibition of whole-cell lysates. When KLF4 was knocked down by shRNA in this ERK pathway completely abrogated the growth advantage context, CA-MEK1-induced growth arrest was reversed along with conferred by BAALC, which were not observed by inhibiting other decreased KLF4 expression in the nucleus, although knock- signaling pathways (Supplementary Figures S9A, B and S10A). down of KLF4 had little effect on the proliferative capacity in the Growth advantage of BAALC-expressing cells was also reversed by absence of constitutive activation of ERK (Figure 3c; forced expression of MKP3 (Supplementary Figures S11A and C), Supplementary Figures S15A and B). CA-MEK1-induced differen- suggesting that BAALC affects proliferative capacity of AML cells in tiation was also counteracted by KLF4 depletion assessed by an ERK-dependent manner. These results collectively indicate that surface expression of monocyte differentiation markers and BAALC interacts directly with MEKK1, an essential scaffold protein microscopic morphology (Supplementary Figures S15C and D). for ERK activation, and prolongs ERK activation by inhibiting These data indicate that sustained activation of ERK pathway reassociation of MKP3 to ERK (Supplementary Figure S11D). induces monocytic differentiation in leukemia cells in a KLF4- High BAALC expression is clinically associated with overexpres- dependent manner. sion of genes involved in drug resistance, lower complete remission rate and refractoriness to chemotherapy in AML BAALC antagonizes KLF4 activity through ERK activation and cases.2,20 Given that ERK can exert its oncogenic activity through inhibition of nuclear KLF4 translocation increased resistance to chemotherapeutic drugs,21 we deduced We next explored the physiological significance of interplay that BAALC leads to drug resistance via ERK pathway. ATP-binding between BAALC and KLF4. When BAALC was transduced to AML cassette (ABC) superfamily represents a major mechanism of drug cells, KLF4 translocated from the nucleus to the cytoplasm resistance,22 and among them, ABCB1 (MDR1; multidrug resis- (Figure 3d; Supplementary Figures S16A and D). On the contrary, tance protein 1) and ABCG2 (BCRP1; breast cancer resistance knockdown of BAALC led to KLF4 shift from the cytoplasm to the protein 1) are well-known downstream targets of ERK nucleus (Figure 3e; Supplementary Figures S17A and D). When – pathway.23 25 We then treated AML cells with cytosine arabino- CD34+ CD38− bone marrow cells from AML patients were assessed side (AraC), an essential component of AML treatment, and for KLF4 localization, BAALC-high cases (the upper quartile of BAALC measured the mRNA expression of these ABC transporters. expression) mainly showed cytoplasmic KLF4, while KLF4 was Additive BAALC expression in AML cells potentiated the upregula- proportionally distributed both in the nucleus and in the cytoplasm tion of these genes in response to AraC treatment. This in BAALC-low cases (the lower quartile) (Figure 3f; Supplementary upregulation was selectively abrogated by ERK pathway inhibition Figure S17E). These data indicate that BAALC reduces the amount (Figure 2e; Supplementary Figure S12A), indicating that BAALC- of nuclear KLF4 by nucleo-cytoplasmic translocation. induced drug resistance is dependent on MEK/ERK pathway. To prove the hypothesis that BAALC inhibits ERK-mediated When the half-maximal inhibitory concentrations (IC50) were monocytic differentiation by relocating KLF4 from the nucleus to assessed, in line with the high expression of resistance-related the cytoplasm, we examined the effect of additional BAALC genes, BAALC-expressing cells were more resistant to AraC expression on proliferation and differentiation of CA-MEK1- treatment, of which were again specifically overcome by ERK expressing cells. BAALC overexpression in CA-MEK1-transduced pathway inhibition (Figure 2f; Supplementary Figure S13A). These AML cells restored proliferative capacity (Supplementary Figure S18A). results support the idea that BAALC induces drug resistance by CA-MEK1-expressing cells with simultaneous BAALC expression upregulating drug resistance genes in an ERK-dependent way, were resistant to CA-MEK1-induced differentiation (Supplementary which can be targeted by MEK inhibitor. Figures S18B and D). The amount of nuclear KLF4 was reduced by

Figure 1. BAALC potentiates proliferation of AML cells and interacts with MEKK1 and KLF4. (a) Relative BAALC expressions in primary human bone marrow cells from AML patients (n = 80), control subjects (n = 7) and AML cell lines (n = 7). Values are normalized to BAALC expression in control subjects. (b)RelativeBAALC expressions in AML cell lines (n = 7). Values are normalized to BAALC expression in primary human bone marrow cells from control subjects. (c) Growth curves of HEL and MV4-11 cells transduced with BAALC or control vector (mock) (n = 3). (d) Growth curves of Kasumi-1 and OCI-AML2 cells transduced with sh_Luc. or sh_BAALC. BAALC were overexpressed by lentivirus in sh_BAALC-transduced cells (n = 3). (e) Potential binding partners of BAALC detected by yeast two-hybrid screening. Coded sequences contained in prey vectors were shown for MEKK1 and KLF4. (f) Interaction between BAALC and MEKK1 was assessed by in vitro pull-down assay. To obtain soluble recombinant protein, shortened form of MEKK1 (MEKK1-Δ4) was utilized (see Supplementary Figure S6A). Puriﬁed MYC-tagged BAALC and HA-tagged MEKK1-Δ4 proteins were mixed and immunoprecipitated by anti-MYC tag antibody followed by blotting with anti-HA tag antibody. (g) Interaction between BAALC and KLF4 by in vitro pull-down assay. To obtain soluble recombinant protein, shortened form of KLF4 (KLF4-Δ2) was utilized (see Supplementary Figure S6B). Puriﬁed MYC-tagged BAALC and His-tagged KLF4-Δ2 proteins were mixed and immunoprecipitated by anti-MYC tag antibody followed by blotting with anti-His tag antibody. Data are mean ± s.e.m. values. *Po0.05, **Po0.01.

© 2015 Macmillan Publishers Limited Leukemia (2015) 2248 – 2256 BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2252 BAALC in CA-MEK1-expressing cells (Supplementary Figure S18E), To further elucidate the role of KLF4 in BAALC-derived supporting the hypothesis that BAALC blocks ERK-dependent proliferation and differentiation block with prolonged ERK differentiation by affecting the amount of KLF4 in the nucleus. activation, we assessed the effect of additional KLF4 expression

Figure 2. BAALC activates ERK pathway by inhibiting MKP3 reassociation and induces drug resistance in an ERK-dependent manner. (a) Immunoblotting of phosphorylated proteins of MAPK signaling cascade in HEL and MV4-11 cells transduced with MYC-tagged BAALC or control vector. Cells were stimulated with PMA (100 ng/ml) for 6 h. (b) Immunoblotting of phosphorylated ERK in HEL cells transduced with MYC- tagged BAALC or control vector. Cells were stimulated with PMA (100 ng/ml) for the indicated time periods. (c) Interaction between BAALC and MEKK1 in HEL cells transduced with MYC-tagged BAALC. Cells were stimulated with PMA (100 ng/ml) for the indicated time periods. Immune complexes were precipitated by anti-MYC tag antibody and blotted by anti MEKK1 antibody. (d) Interaction between ERK and MKP3 in HEL cells transduced with MYC-tagged BAALC. Cells were stimulated with PMA (100 ng/ml) for the indicated time periods. Immune complexes were precipitated by anti-ERK antibody and ERK-bound MKP3 was detected by anti-MKP3 antibody. (e) Expression levels of ABCB1 in HEL cells transduced with BAALC or control vector followed by treatment with AraC (1 μM) or DMSO for 48 h. Cells were conccurently transduced with dominant negative form of ERK (K71R) or treated by U0126 (5 μM), PD169316 (10 μM), SP600125 (10 μM), PI-103 (1 μM), WP1066 (2 μM) and SC-514 (10 μM). Values were normalized to that of mock-transduced HEL cells treated with DMSO (n = 3). (f) AraC sensitivity of HEL cells transduced with BAALC or control vector. Cells were treated with AraC at the indicated concentrations in the presence of transduction with dominant negative form of ERK (K71R) or treatment by U0126 (5 μM) or DMSO. Cell viabilities were determined by trypan blue exclusion assays (n = 3). IC50 values of BAALC- or control vector-transduced HEL cells are provided. Data are mean ± s.e.m. values. *Po0.05.

Figure 3. BAALC counteracts CA-MEK1-induced monocytic differentiation through cytoplasmic sequestration of KLF4. (a) Immunoblotting of phosphorylated ERK in HEL cells transduced with BAALC, CA-MEK1 or control vector. Cells were stimulated by PMA (100 ng/ml) for 6 h. (b) Growth curve of HEL cells transduced with BAALC, CA-MEK1 or control vector (n = 3). (c) Immunoblotting of total and nuclear KLF4 in HEL cells co-transduced with CA-MEK1 and sh_KLF4.(d) Immunofluorescence images of KLF4 in HEL cells transduced with MYC-tagged BAALC or control vector (scale bars, 10 μm). (e) Immunofluorescence images of KLF4 in Kasumi-1 cells transduced with sh_Luc. or sh_BAALC (scale bars, 10 μm). (f) Immunofluorescence images of KLF4 in CD34+ CD38− bone marrow cells from AML patients. Representative images from two AML patients with high and low BAALC expression were shown (scale bars, 10 μm). Data are mean ± s.e.m. values. *Po0.05. on BAALC-expressing cells. As expected, KLF4 overexpression overexpressing AML cells and decreased by BAALC knocking restored nuclear KLF4 and abrogated the differentiation block by down. Meanwhile, the expression of cyclin-dependent kinase BAALC (Supplementary Figures S19A and E). Furthermore, mRNA inhibitor 1 (CDKN1A, p21), an essential factor for KLF4-mediated expressions of key regulators in cell-cycle progression positively tumor suppression as a major transactivation target of KLF4,28 was regulated by ERK, such as cyclin-dependent kinase 6 (CDK6) decreased in BAALC-overexpressing AML cells and increased in and cyclin D1 (CCND1),27 were consistently increased in BAALC- BAALC-knocked down cells (Supplementary Figures S20A and B).

Figure 4. MEK inhibition combined with KLF4 induction is highly effective for BAALC-high AML in vitro and in vivo.(a) Correlation between BAALC expressions and sensitivities to MEK inhibitor of AML cell lines (n = 7). (b) Correlation between BAALC expressions and sensitivities to MEK inhibitor of CD34+CD38− fractions in bone marrow cells from AML patients (n = 30). (c) Schematic design of xenograft mouse models of human AML. Kasumi-1 cells or BAALC-transduced HEL cells were transplanted into NSG mice. (d) Overall survival of NSG mice transplanted with Kasumi-1 cells transduced with Tet-ON KLF4 followed by treatment with DMSO, U0126, Dox or U0126 plus Dox (n = 6). U0126 was administered intraperitoneally at 25 μmol/kg/week and Dox was given orally (diluted in drinking water at 1 mg/ml). (e) Overall survival of NSG mice transplanted with BAALC-overexpressing HEL cells transduced with Tet-ON KLF4 followed by treatment with DMSO, U0126, Dox or U0126 plus Dox (n = 6). (f) Schematic summary of this study. BAALC interacts with MEKK1 in the cytoplasm and upregulates ERK pathway as an adaptor protein. Constitutive activation of ERK pathway conventionally mediates monocytic differentiation via nuclear KLF4 accumulation. In contrast, in the presence of excess BAALC, KLF4 accumulation in the nucleus is blocked by cytoplasmic BAALC. Thus, BAALC promotes leukemia cell proliferation by activating ERK pathway while blocking differentiation of leukemia cells by preventing ERK-pathway mediated KLF4 accumulation in the nucleus. This unique dual function of BAALC might contribute to a formation of more aggressive leukemia.

Leukemia (2015) 2248 – 2256 © 2015 Macmillan Publishers Limited BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2255 These findings collectively indicate that BAALC promotes cell-cycle induction, is highly effective against BAALC-high leukemia cells progression by activating ERK pathway, and inhibits monocytic both in vitro and in vivo. differentiation by inhibiting nuclear translocation of KLF4. Our detailed experiments revealed that BAALC interacts with MEKK1, inhibits reassociation of ERK and MKP3, and MEK/ERK inhibition in combination with KLF4 induction is a consequently maintains ERK activity. Sustained activation potential therapeutic strategy against BAALC-high AML of ERK pathway promotes proliferation through cell-cycle progres- From the above-mentioned data, it is suggested that modulating sion in AML cells. In addition, ERK-dependent upregulation of ERK pathway and KLF4 expression would be effective for treating chemoresistance-associated transporters in response to cytotoxic BAALC-high leukemia. Thus, we established tetracycline-inducible agents was enhanced by BAALC overexpression, indicating that (Tet-ON) KLF4-expressing AML cell lines, in which KLF4 is induced BAALC mediates a variety of functions on AML cells by modulating by doxycycline. Given that BAALC exerts dual actions on leukemia ERK activity. Considering the association of higher BAALC cells through ERK and KLF4, we evaluated the combination expression among AML patients and paucity of promising drugs therapy by MEK inhibition and KLF4 induction in Kasumi-1 cells in clinical use targeting these transporters, the rationality of and BAALC-overexpressing HEL cells. Of note, there was an treating BAALC-high AML with MEK inhibitors to overcome ERK- inhibitory effect of MEK inhibition and KLF4 induction on cell mediated drug resistance should be emphasized. proliferation, possibly reflecting accelerated differentiation Although aberrant activation of ERK pathway is implicated in induced by KLF4 (Supplementary Figures S21A–C). the biology of AML,7 chronic activation of ERK induced by We next assessed the effect of MEK inhibition on the constitutive active form of MEK1 leads AML cells to monocytic proliferation of several AML cell lines. Interestingly, sensitivity to differentiation.29 In this report, we demonstrated that this MEK inhibitor was positively correlated to BAALC expression level differentiation of AML cells depends on KLF4 induction, which is (Figure 4a). The concentration of U0126 to inhibit the proliferation one of the other interacting partners of BAALC. In addition, we of AML cell lines with high BAALC (Kasumi-1, OCI-AML2) was 1/15 showed that BAALC inhibits the nuclear localization of KLF4 by to 1/25 of that, when compared to cells with low BAALC (MV4-11, interacting with KLF4 in cytoplasm. To our knowledge, this is the HEL) (Supplementary Figure S21D). BAALC expression was also first report to show that cytoplasmic sequestration of KLF4 confers positively correlated to in vitro sensitivity to U0126 among primary oncogenic potential. Our study also suggests that targeting KLF4 human AML cells (Figure 4b), highlighting MEK/ERK pathway as a would be a rational choice for overcoming treatment-resistant legitimate therapeutic target in AML with high BAALC expression. BAALC-high AML. Finally, we developed BAALC-high AML model mice using NSG Furthermore, our data indicated that MEK inhibition could mouse to investigate the combinational effect of MEK inhibition effectively treat BAALC-high AML. Although several MEK inhibitors and KLF4 induction in vivo (Figure 4c), by transplanting Kasumi-1 are already utilized in a clinical setting, the efficacy of MEK cells expressing either BAALC shRNA or control shRNA as well as fi by transplanting HEL cells expressing either BAALC or control inhibitor monotherapy is limited so far, rst because of the high probability of side effects due to its narrow therapeutic vector. These xenotransplants succumbed to AML characterized 30 by extensive infiltration of human CD45+ leukemia cells in the BM, concentration range. In our experiments using AML cell lines the liver and the spleen (Supplementary Figures S22A and B). and primary human samples, AML cells with higher expression of BAALC shRNA had a positive effect on mice transplanted with BAALC possessed higher sensitivity to MEK inhibitor U0126. In Kasumi-1 cells to prolong their survival, and exogenous BAALC addition, U0126 decreased the proliferation rate of leukemia cells had a negative effect on those transplanted with HEL cells, with high BAALC expression at significantly lower concentration recapitulating the adverse prognostic impact of high BAALC compared with that of BAALC-low cells. These results suggested expression in human AML (Supplementary Figures S23A and B). that AML patients with higher expression of BAALC might be Next, we transplanted these BAALC-high AML cells transduced effectively treated by MEK inhibitor at relatively low doses to with lentiviral Tet-ON vector encoding KLF4 into NSG mice. After reduce its toxicity. Moreover, MEK inhibitor can reverse the drug confirming the engraftment of AML cells in peripheral blood by resistance conferred by BAALC, which might be involved in flow cytometry, we divided engrafted mice into four groups; unfavorable prognosis of BAALC-high AML patients, and can control group, U0126 monotherapy group, doxycycline mono- potentiate the therapeutic efficacy of other antileukemic agents. therapy group, and U0126 plus doxycycline combination therapy Given that U0126 cannot reverse differentiation block caused by group. Compared with the modest effect of MEK inhibition or direct interaction of BAALC with KLF4 independently from ERK, KLF4 induction monotherapy on overall survival, MEK inhibition KLF4 induction therapy both in vitro and in vivo was combined fi plus KLF4 induction signi cantly prolonged the survival with MEK inhibition in this study. Our results clearly showed that (Figures 4d and e). By contrast, MEK inhibitor with or without concurrent MEK inhibition and KLF4 induction are highly effective KLF4 induction therapy exerted smaller impact on the survival for BAALC-high AML. Several drugs that can induce KLF4 have of NSG mice engrafted with low BAALC-expressing HEL cells, been evaluated in clinical and preclinical settings, making our compared with the cases of high BAALC-expressing AML cells approach feasible and highly promising.31 (Supplementary Figure S23C), underscoring the importance of To date, there have been few reports that provide a perspective these pathways in the maintenance of BAALC-high AML cells. 32–35 fi on the leukemogenic property of BAALC itself. Actually, our Overall, these ndings clearly indicate that MEK inhibition fi combined with KLF4 induction would be a promising therapeutic overexpression and knockdown study revealed the signi cance of strategy against BAALC-high AML (Figure 4f). BAALC as an unprecedented dual-acting protein, which functions as an adaptor protein in MAPK cascade and as a molecular trap against KLF4 in the cytoplasm, but this property of BAALC should DISCUSSION be elucidated by using antibodies that can detect endogenous In this study, we showed that BAALC promotes proliferation and BAALC in the future. Considering that MEK inhibition is effective drug resistance of AML cells by sustained activation of ERK for BAALC-high leukemia cells even at low doses and concurrent pathway through the interaction with MEKK1 and that BAALC KLF4 induction further enhances therapeutic efficacy, our results inhibits monocytic differentiation induced by ERK through nucleo- strongly indicate that combination therapy targeting this novel cytoplasmic translocation of KLF4. We also demonstrated that BAALC-ERK-KLF4 axis would become a promising strategy for inhibition of ERK pathway, especially combined with KLF4 refractory AML with high BAALC expression.

© 2015 Macmillan Publishers Limited Leukemia (2015) 2248 – 2256 BAALC as an oncogenic hub to drive ERK pathway K Morita et al 2256 CONFLICT OF INTEREST 14 Deschênes-Simard X, Gaumont-Leclerc MF, Bourdeau V, Lessard F, Moiseeva O, The authors declare no conﬂict of interest. Forest V et al. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation. Genes Dev 2013; 27:900–915. 15 Chen ZY, Tseng CC. 15-deoxy-Delta12,14 prostaglandin J2 up-regulates Kruppel- ACKNOWLEDGEMENTS like factor 4 expression independently of peroxisome proliferator-activated receptor gamma by activating the mitogen-activated protein kinase kinase/ We thank Dr T Kitamura for platinum-A (Plat-A) packaging cells, Dr H Miyoshi for extracellular signal-regulated kinase signal transduction pathway in HT-29 colon lentiviral vectors of CSII-EF-MCS-IRES2-Venus, CSII-EF-MCS-IRES2-hKO1, pENTR4- cancer cells. Mol Pharmacol 2005; 68: 1203–1213. H1tetOx1, CSIV-TRE-RfA-EF-KT, psPAX2 and pMD2.G, Dr E Takekawa for CA-MEK1 16 Feinberg MW, Wara AK, Cao Z, Lebedeva MA, Rosenbauer F, Iwasaki H et al. and MEKK1, Dr T Furukawa for MKP3, Dr S Yamanaka for KLF4, Dr M Ogata for ERK1 The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation. and Dr A de la Chapelle for BAALC. EMBO J 2007; 26: 4138–4148. 17 Yoshimi A, Goyama S, Watanabe-Okochi N, Yoshiki Y, Nannya Y, Nitta E et al. Evi1 AUTHOR CONTRIBUTIONS represses PTEN expression and activates PI3K/AKT/mTOR via interactions with polycomb proteins. Blood 2011; 117: 3617–3628. KM designed research, performed experiments, analyzed data and wrote the 18 Kim MO, Kim SH, Cho YY, Nadas J, Jeong CH, Yao K et al. ERK1 and ERK2 regulate manuscript. YM designed and supervised research, and wrote the manuscript. embryonic stem cell self-renewal through phosphorylation of Klf4. Nat Struct Mol 19 – KK designed research. HY and SM supervised two-hybrid screening. 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