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Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive

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Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive Published OnlineFirst June 23, 2017; DOI: 10.1158/0008-5472.CAN-16-2974 Cancer Therapeutics, Targets, and Chemical Biology Research Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive Acute Lymphoblastic Leukemia to TNFa Treatment Hayato Tamai1, Hiroki Yamaguchi1, Koichi Miyake2, Miyuki Takatori3, Tomoaki Kitano1, Satoshi Yamanaka1, Syunsuke Yui1, Keiko Fukunaga1, Kazutaka Nakayama1, and Koiti Inokuchi1 Abstract Acute lymphoblastic leukemias (ALL) positive for KMT2A/ to inhibit S100A6 expression in the presence of TNF-a.InKMT2A/ AFF1 (MLL/AF4) translocation, which constitute 60% of all infant AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor ALL cases, have a poor prognosis even after allogeneic hemato- immunity and lowered the penetrance of leukemia development. poietic stem cell transplantation (allo-HSCT). This poor progno- Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1- sis is due to one of two factors, either resistance to TNFa, which positive ALL, amlexanox broadened GVL responses and extended mediates a graft-versus-leukemia (GVL) response after allo-HSCT, survival. Our findings show how amlexanox degrades the resis- or immune resistance due to upregulated expression of the tance of KMT2A/AFF1-positive ALL to TNFa by downregulating immune escape factor S100A6. Here, we report an immune S100A6 expression, with immediate potential implications for stimulatory effect against KMT2A/AFF1-positive ALL cells by improving clinical management of KMT2A/AFF1-positive ALL. treatment with the anti-allergy drug amlexanox, which we found Cancer Res; 77(16); 1–8. Ó2017 AACR. Introduction involved in the graft-versus-leukemia (GVL) effect, or tumor immunity by upregulation of S100A6 expression followed The most prevalent mixed-lineage leukemia (MLL) rearrange- by interference with the p53–caspase pathway (3). S100A6 is a ment in acute lymphoblastic leukemia (ALL) generates the þ 10.5-kDa Ca2 -binding protein belonging to the S100 protein KMT2A/AFF1 (MLL/AF4) fusion gene due to the t(4;11)(q21; family, which has been reported to interact with and alter the q23) chromosomal translocation. ALL with t(4;11)(q21;q23) has conformation of p53 (4–7). Upregulation of S100A6 expression a bimodal age distribution with a major peak of incidence in early in KMT2A/AFF1-positive ALL inhibits p53 acetylation followed by infancy and accounts for over 50% of ALL cases in infants < 6 inhibition of caspase apoptotic pathway upregulation in the months old, 10% to 20% in older infants, 2% in children, and up presence of TNFa (8). to 7% in adults (1). Despite recent improvements in the overall Here, we focused on amlexanox (2-amino-7-isopropyl-5- treatment outcome for ALL patients, including allogeneic hemato- oxo-5H-chromeno[2,3-b] pyridine-3-carboxylic acid), a com- poietic stem cell transplantation (allo-HSCT), KMT2A/AFF1-pos- mon anti-allergic drug, which targets S100A6. Amlexanox was itive ALL is still associated with a poor prognosis (2). The complete reported to inhibit the translocation pathway of endogenous remission (CR) rate in children is as high as 88%, but the median S100A6 in endothelial cells (9). Amlexanox has also been overall survival (OS) is only 10 months, indicating an extremely reported to bind to S100A13, which is another member of poor prognosis. In adult patients with ALL, the CR rate is 75%, but the S100 protein family (10). S100A13 and acidic fibroblast the prognosis is also poor, with a median OS of 7 months (1). growth factor (FGF1) are involved in a wide range of important The poor prognosis of KMT2A/AFF1-positive ALL has been biological processes, including angiogenesis, cell differentia- suggested to be due to resistance to TNFa, which is the factor tion, neurogenesis, and tumor growth. Generally, the biolog- ical function of FGF1 is to recognize a specific tyrosine kinase on the cell surface and initiate the cell signal transduction 1Department of Hematology, Nippon Medical School, Tokyo, Japan. 2Depart- cascade. Amlexanox binds S100A13 and FGF1 and inhibits the ment of Biochemistry and Molecular Biology, Division of Gene Therapy Research heat shock–induced release of S100A13 and FGF1 (10). Amlex- Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan. anox has been used in a number of recent metabolic studies. 3Research Center for Life Science, Nippon Medical School, Tokyo, Japan. Reilly and colleagues reported that amlexanox acted as an H. Yamaguchi and K. Miyake contributed equally to this article. inhibitor of IKKe and TBK1 (11). They showed that treatment Corresponding Author: Hayato Tamai, Nippon Medical School, Sendagi 1-1-5, with amlexanox resulted in reduced inflammation, marked Bunkyo-Ku, Tokyo 113-8603, Japan. Phone: 81-3-3822-2131; Fax: 81-3-5685-1793; improvement in insulin sensitivity, and reduced hepatostea- E-mail: [email protected] tosis in mice with genetic or dietary obesity (11). The present doi: 10.1158/0008-5472.CAN-16-2974 study was performed to examine the effects of amlexanox in Ó2017 American Association for Cancer Research. KMT2A/AFF1-positive ALL. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 23, 2017; DOI: 10.1158/0008-5472.CAN-16-2974 Tamai et al. Materials and Methods ries) for Ag retrieval, rinsed twice for 10 minutes each time in PBS, and incubated for 15 minutes in 3% hydrogen peroxide in Cell culture methanol to quench endogenous peroxidase activity. For The KMT2A/AFF1-positive human ALL cell line RS4;11 was CD45R/B220 staining, the pretreated slides were incubated for purchased from ATCC. The KMT2A/AFF1-positive ALL cell line 60 minutes with anti-CD45R/B220 (BD Pharmingen) primary SEM was purchased from Deutsche Sammlung von Mikroorganis- Abs. To visualize anti-CD45R/B220 Ab binding, the slides were men und Zellkulturen GmbH (DSMZ). The cell lines were incubated for 30 minutes with FITC-conjugated anti-rat IgG obtained in 3 years and used within 6 months after receipt or (Jackson ImmunoResearch Laboratories). Nuclei were counter- resuscitation. To check mycoplasma we used a PCR-based meth- stained with Mayer's hematoxylin. The photomicrographs shown od, indirect staining and an agar and broth culture. SEM, SEM in the figures were taken with a SPOT Insight digital camera transduced with a lentiviral vector expressing luciferase controlled using SPOT Advanced Version 4.0.9 software (Diag- (SEM-Luc), and RS4;11 cells were cultivated in RPMI 1640 (Sig- nostic Instruments) with a Nikon Eclipse 80i microscope ma-Aldrich) supplemented with 10% FBS (PAN Biowest) at 37C equipped with Nikon Plan 2Â/0.08 NA and Plan 40Â/0.75 NA under 5% CO . 2 objectives. Comparison of CD45R/B220-positive tumor cells between the control group and amlexanox group was performed In vitro analysis of SEM and RS4;11 cell growth by counting the numbers of CD45R/B220-positive cells/mm2. SEM and RS4;11 were seeded in 6-well plates (2 Â 105 cells/ well) and incubated in vitro with TNFa (0 and 10 ng/mL; Wako) or Separation of human PBMCs Human peripheral blood mononuclear cells (PBMC; 2 Â 105 Human PBMCs were obtained by separation of heparinized cells/well) and amlexanox (Tokyo Chemical Industry; 0, 10, and blood from a healthy donor on a Ficoll–Histopaque gradient. The 100 mg/mL) for 48 hours before counting cells to examine the PBMCs were washed twice and resuspended in RPMI 1640 sup- effects of TNFa and amlexanox on leukemia cells. plemented with 25 mmol/L HEPES buffer, 2 mm glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin, and 10% heat- Western blotting analysis inactivated FCS (designated as FCS-RPMI). Western blotting analysis was performed as described previ- ously (3). SEM and RS4;11 cells were incubated for 48 hours and Animal experiments using PBMC-NOD/SCID mice collected for Western blotting analysis. Equal aliquots of lysates For in vivo analysis, 5 Â 105/body of SEM-Luc cells were injected from cell lines or homogenized mouse spleen were subjected to intraperitoneally (i.p.) into three groups of 10 nonobese diabetic/ fl 10% SDS-PAGE, transferred onto polyvinylidene di uoride severe combined immunodeficiency (NOD/SCID) mice. Each membranes, and immunoblotted with the following primary group of 10 mice was divided into two groups: the amlexanox antibodies (Abs): anti-S100A6 (calcyclin) Ab (Santa Cruz Bio- þ PBMC group (n ¼ 5) fed a diet containing amlexanox (0.02%), – technology), anti caspase-3, anti-cleaved caspase-3 Ab (Cell Sig- and the PBMC group (n ¼ 5) fed a diet without amlexanox. – naling Technology), anti-p53 (Santa Cruz Biotechnology), anti Feeding with each diet was started when SEM-Luc cells were –b acetyl-p53 (Millipore), and anti -actin Ab (Millipore). Can Get injected and supplied consistently until mice died. Mice in each Signal (Toyobo) was used to promote the reaction between group were injected i.p. with 4 Â 107/body of human PBMCs primary Ab and antigen (Ag). every 2 weeks. Non–PBMC-injected mice fed a diet without amlexanox were used as controls. In addition to overall survival KMT2A/AFF1 Animal experiments using the transgenic (OS) rate, tumor growth after injection of human PBMCs was mouse model assessed using an in vivo imaging system (IVIS) as described KMT2A/AFF1 þ transgenic (Tg) mice, which show CD45R/B220 previously (3). leukemia by 12 months of age, at which time lymphoma cells will fi have in ltrated the bone marrow (BM) and spleen, were estab- Statistical analysis KMT2A/AFF1 lished previously (12). This Tg mouse model has an The results of cell growth were analyzed by the Student t test, KMT2A/ essentially normal immune system. We divided 10 male assuming unequal variances and two-tailed distributions. Data AFF1 Tg mice at the age of 4 months into two groups: the are shown as the means Æ standard deviation of at least three n ¼ amlexanox group ( 5) fed a diet containing amlexanox samples.
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