Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive Acute Lymphoblastic Leukemia to Tnfα Treatment

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. 2017 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 biological 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 2017 American Association for Cancer Research. KMT2A/AFF1-positive ALL.

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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 2/0.08 NA and Plan 40/0.75 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 (2 105 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; 2 105 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 previously (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, 5 105/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 4 107/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 standard deviation of at least three n ¼ amlexanox group ( 5) fed a diet containing amlexanox samples. For survival analyses, event time distributions were n ¼ (0.02%) for 10 months, and the control group ( 5). All estimated using the method of Kaplan and Meier, and differences KMT2A/AFF1 Tg mice were killed at the age of 14 months for in survival rates were compared using the log-rank test. In all histopathological and Western blotting analysis. All animal analyses, P < 0.05 was taken to indicate statistical significance. experiments were performed in accordance with the regulations established by the Ethical Committee of Nippon Medical School and were approved by the Animal Care and Committee of Nippon Results Medical School (Approval number: 28-026). Both SEM and RS4;11 cells were sensitive to TNFa in the presence of 100 mg/mL of amlexanox, while both SEM and Histopathology and immunopathology RS4;11 cells showed resistance without amlexanox For histopathological analysis, mice were killed and tissues of First, to analyze the effects of amlexanox on KMT2A/AFF1- interest were fixed in 4% paraformaldehyde. Hematoxylin and positive ALL cell lines, SEM and RS4;11 were seeded in 6-well eosin (H&E) staining was performed using standard protocols. plates (2 105 cells/well) and incubated in vitro with TN-a (0 and For immunopathological analysis, the slides were fixed for 30 10 ng/mL; Wako) and amlexanox (Tokyo Chemical Industry; 0, minutes in 4% paraformaldehyde, microwaved for 5 minutes in 10, and 100 mg/mL). After 48 hours of incubation, viable cells Vector Antigen Unmasking Solution (pH 6.0; Vector Laborato- were counted using Trypan blue exclusion. KMT2A/AFF1-positive

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Amlexanox Suppresses KMT2A/AFF1 (MLL/AF4)-Positive ALL

Figure 1. Effects of amlexanox on KMT2A/AFF1-positive ALL cell lines with or without TNFa. The figure presents a summary of growth inhibition of KMT2A/AFF1-positive ALL cell lines by administration of amlexanox with or without TNFa (A, SEM; B, RS4;11). Although there was no significant inhibition by 10 ng/mL of TNFa without amlexanox, significant inhibition was observed with 10 ng/mL of TNFa in the presence of 100 mg/mL of amlexanox.

Figure 2. Effects of amlexanox on KMT2A/AFF1-positive ALL cell lines with or without PBMCs (2 105 cells/well). SEM and RS4;11 were incubated with PBMCs (2 105 cells/well) instead of 10 ng/mL of TNFa and viable cells were counted. SEM cells showed signiﬁcant growth inhibition by incubation with PBMCs in the presence of 10 or 100 mg/mL of amlexanox (P ¼ 0.0136 or 0.0019; A). RS4:11 cells also showed signiﬁcant growth inhibition by incubation with PBMCs in the presence of 100 mg/mL of amlexanox (P ¼ 0.0125; B).

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Figure 3. Mechanism underlying the effects of amlexanox on KMT2A/AFF1-positive ALL. A and B, Western blotting of lysates of KMT2A/AFF1-positive cell lines with amlexanox. Western blotting analysis showed that 100 mg/mL of amlexanox suppressed S100A6 activation and induced apoptosis by upregulation of acetyl p53/p53 and cleaved caspase-3/ caspase (A, SEM; B, RS4;11).

ALL cells (SEM or RS4;11) showed no significant growth inhibi- and RS4;11 cells were incubated with PBMCs (2 105 cells/well) tion by 10 ng/mL of TNFa in the absence or presence of 10 mg/mL instead of 10 ng/mL of TNFa and viable cells were counted. SEM of amlexanox (Fig. 1A and B). However, both cell lines showed cells showed significant growth inhibition on incubation with significant growth inhibition by 10 ng/mL of TNFa in the presence PBMCs in the presence of 10 or 100 mg/mL of amlexanox (P ¼ of 100 mg/mL of amlexanox (P ¼ 0.0085 for SEM, P ¼ 0.0196 for 0.0136 or 0.0019; Fig. 2A). RS4:11 cells also showed significant RS4;11; Fig. 1A and B). Next, to examine direct evidence that growth inhibition on incubation with PBMCs in the presence of amlexanox increases cell-mediated anti-leukemia effects, SEM 100 mg/mL of amlexanox (P ¼ 0.0125; Fig. 2B). These results

Figure 4. Results of amlexanox in KMT2A/AFF1 Tg mice. A, Comparison of the results of immunohistopathological analyses (H&E and CD45R/B220) between one mouse from the amlexanox group (amlexanox#1) and one mouse from the control group (control#1). Although CD45R/B220þ pro–B-cell leukemia infiltration (green fluorescence) was observed in the spleen and BM of control#1, no leukemia infiltration was observed in amlexanox#1. Although H&E staining in amlexanox#1 showed normocellular BM, control#1 showed hypercellular BM because of leukemia infiltration. Although H&E-stained spleen sections from amlexanox#1 showed normal structure of the spleen, control#1 showed destruction of the normal structure. B and C, Comparison of CD45/B220þ leukemia mass/mm2 between the control group (n ¼ 5) and amlexanox group (n ¼ 5). Both the spleen (B)andBM(C) of the amlexanox group had significantly less CD45/B220þ leukemia cell invasion in comparison with the control group (both P < 0.001).

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Amlexanox Suppresses KMT2A/AFF1 (MLL/AF4)-Positive ALL

Figure 5. Comparison of KMT2A/AFF1 Tg mice between the amlexanox group and control group. A, Western blotting analysis conﬁrmed in vivo that upregulation of S100A6 was inhibited and p53 acetylation and cleaved caspase-3 were activated in the amlexanox group. B and C, At the age of 14 months, KMT2A/AFF1 Tg mice in the amlexanox group had signiﬁcantly lower body weight than those in the control group (P < 0.001).

indicated that amlexanox increases both TNFa and cell-mediated of five mice in the amlexanox group at the age of 14 antileukemia effects. months. Figure 4A (top) shows a comparison of the results of H&E staining between one mouse from the amlexanox group Downregulation of S100A6 by amlexanox leads to growth (amlexanox#1) and one mouse from the control group (con- suppression of KMT2A/AFF1-positive human ALL cells through trol#1). Although the normal structure of the spleen was dis- the p53–caspase-3 pathway in the presence of TNFa rupted in control#1, no such effect was seen in the spleen of To analyze the molecular mechanism underlying the effect of amlexanox#1. Although BM of control#1 was hypercellular with amlexanox on the KMT2A/AFF1-positive human ALL cells, SEM tumor cells, that of amlexanox#1 was normocellular with no and RS4;11 were incubated for 48 hours, and collected for tumor invasion. Figure 4A (bottom) shows a comparison of the Western blotting analysis. As shown in Fig. 3, S100A6 was results of immunohistopathologic analysis between one mouse activated in the presence of 10 ng/mL TNFa, and activated from the amlexanox group (amlexanox#1) and one mouse from S100A6 was decreased and both acetyl-p53/p53 ratio and the control group (control#1). Although the spleen and BM of þ cleaved caspase-3/caspase-3 ratio were increased in cells treated control#1 showed CD45R/B220 leukemia infiltration, no leu- with 100 mg/mL of amlexanox in the presence of 10 ng/mL of kemia was observed in amlexanox#1. þ TNFa in the KMT2A/AFF1-positive human ALL cell line (Fig. 3A Figure 4B shows a comparison of CD45/B220 leukemia and B). mass/mm2 between the control group (n ¼ 5) and amlexanox group (n ¼ 5). Both the spleen and BM of the amlex- þ Amlexanox inhibited infiltration of pro–B-cell leukemia in the anox group had significantly less CD45/B220 leukemia cell KMT2A/AFF1 Tg mouse model invasion in comparison with the control group (both P < To examine whether amlexanox in combination with tumor 0.001). immunity suppressed the development of MLL/AF4-positive ALL, we used KMT2A/AFF1-positive Tg mice in which the immune Amlexanox inhibits S100A6 upregulation followed by the system is normal (12). In vivo analysis was performed using p53–caspase-3 apoptotic pathway in KMT2A/AFF1 Tg mice KMT2A/AFF1-positive Tg mice, which we established previously To examine the mechanism underlying the suppression of þ and show CD45R/B220 pro–B-cell leukemia by 12 months of leukemia by amlexanox in KMT2A/AFF1 Tg mice, we performed age (12). Although pro–B cell-leukemia infiltration was observed Western blotting analysis of the lysates from the spleens of in all five mice in the control group, no leukemia occurred in four KMT2A/AFF1 Tg mice in the amlexanox group and compared the

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Figure 6. Results of amlexanox in PBMC-NOD-SCID mice. A, OS curves of PBMC-NOD-SCID mice injected with 5 105 body of KMT2A/AFF1-positive ALL cells. The amlexanox þ PBMC group showed signiﬁcantly prolonged OS in PBMC-NOD-SCID mice injected with 5 105/body of KMT2A/AFF1-positive ALL cells. B and C, Figure 5B and C show comparisons of SEM-Luc volumes between the PBMC þ amlexanox group and control group. On day 84 after transplantation, the SEM-Luc volume was signiﬁcantly lower in the amlexanox þ PBMC group than in the control group (P ¼ 0.003).

results with those for KMT2A/AFF1 Tg mice in the control group. Figure 6B and C show comparisons of the volumes of SEM-Luc Western blotting analysis indicated the inhibition of S100A6 and between the amlexanox þ PBMC group and control group. On upregulation of p53 acetylation as well as cleaved caspase-3 levels day 84 following transplantation, the volume of SEM-Luc was in the amlexanox group in comparison with the control group significantly lower in the amlexanox group than the control group (Fig. 5A). (P ¼ 0.003). There was no significant difference in SEM-Luc between the PBMC group and control group (P ¼ 0.120). Amlexanox-treated KMT2A/AFF1Tg mice were significantly smaller than those in the control group Discussion Reilly and colleagues reported that amlexanox reduced The results presented here suggested that amlexanox inhibits obesity in mice by inhibition of IKKe andTBK1(11),andthis the resistance of KMT2A/AFF1 (MLL/AF4)-positive ALL to TNFa may be a problem when considering use of amlexanox in by downregulating S100A6 expression. The results of this study human infants. Therefore, we monitored body weight of mice indicated that amlexanox blocks upregulation of S100A6 expres- treated with amlexanox. As shown in Fig. 5B, KMT2A/AFF1 Tg sion followed by interference with the p53–caspase pathway in mice in the amlexanox group had significantly lower weight at KMT2A/AFF1-positive ALL both in vitro and in vivo. The hypo- the age of 14 months than those in the control group (Fig. 5C; thetical mechanism underlying these effects is shown in Fig. 7. P < 0.001). With respect to the genomic mechanism of KMT2A/AFF1, KMT2A is an H3K4 histone methyltransferase, and leukemia transforma- Comparison of PBMC-NOD/SCID mice transplanted with tion by KMT2A fusion requires the H3 lysine 79 (H3K79) methyl- SEM-Luc transferase DOT1L, which is recruited to the KMT2A fusion To examine whether amlexanox in combination with GVL transcriptional complex. Suppression of DOT1L inhibited expres- effect could suppress the development of KMT2A/AFF1-positive sion of AFF1 target genes, indicating that H3K79 methylation is a ALL, we used PBMC-NOD/SCID mice transplanted with KMT2A/ distinguishing feature of KMT2A/AFF1-positive ALL and plays a AFF1-positive ALL. As shown in Fig. 6A, PBMC-NOD/SCID mice key role in maintenance of AFF1-driven gene expression (13). transplanted with SEM-Luc in the amlexanox þ PBMC group However, our therapeutic target of KMT2A/AFF1-positive ALL is showed significantly longer survival than those in the control not this genomic mechanism but the resistance of KMT2A/AFF1 group (P ¼ 0.011). The PBMC group did not show significantly (MLL/AF4)-positive ALL to TNFa by downregulating S100A6 longer survival than those in the control group (P ¼ 0.93). expression.

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Amlexanox Suppresses KMT2A/AFF1 (MLL/AF4)-Positive ALL

another member of the S100 protein family, S100A13 (10). Although there have been no reports regarding the relations between amlexanox and S100A8 and S100A9, it is possible that this drug affects these proteins. Another major advantage of the use of amlexanox is that it is already a commonly used anti-allergy drug and its cost is not high in contrast to almost all other molecular target drugs. However, there is a problem when considering use of amlexanox as treatment for human infants. As reported by Reilly and colleagues, amlexanox prevents weight gain by inhibition of IKK-e and TBK1 (11). Our KMT2A/AFF1 Tg mice fed amlexanox were also significantly smaller than those fed the control diet (P < 0.001; Fig. 5B and C). This is an important problem for the treatment of infants, although it would not be an issue in adults. Despite these problems, treatment targeting S100A6 by amlexanox may increase tumor immunity and the effects of hematopoietic stem cell transplantation against KMT2A/AFF1-positive leukemia, thus Figure 7. making it useful for treatment of KMT2A/AFF1-positive leukemia. Working hypothesis for the effect of amlexanox on KMT2A/AFF1-positive ALL. Amlexanox induces the caspase-3 apoptotic pathways in KMT2A/AFF1-positive ALL through downregulation of S100A6, followed by p53 acetylation. Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed.

Authors' Contributions We previously reported that the poor prognosis of KMT2A/ AFF1 a Conception and design: H. Tamai, H. Yamaguchi K. Inokuchi -positive ALL may be caused by resistance to TNF , which is Development of methodology: H. Tamai, H. Yamaguchi the factor involved in the GVL effect, or tumor immunity by Acquisition of data (provided animals, acquired and managed patients, upregulation of S100A6 expression followed by interference with provided facilities, etc.): H. Tamai, H. Yamaguchi, K. Inokuchi the p53–caspase apoptotic pathway (3). Amlexanox, known as an Analysis and interpretation of data (e.g., statistical analysis, biostatistics, anti-allergic drug, may exploit the GVL effect or tumor immunity computational analysis): H. Tamai, H. Yamaguchi, M. Takatori, T. Kitano in KMT2A/AFF1-positive ALL because it inhibits the resistance of Writing, review, and/or revision of the manuscript: H. Tamai, H. Yamaguchi, KMT2A/AFF1 a In vivo KMT2A/ K. Miyake, S. Yamanaka, K. Fukunaga, K. Nakayama, K. Inokuchi -positive ALL to TNF . analysis using Administrative, technical, or material support (i.e., reporting or organizing AFF1 -positive Tg mice showed that amlexanox in combination data, constructing databases): H. Tamai, H. Yamaguchi with tumor immunity may suppress the development of MLL/ Study supervision: H. Tamai, H. Yamaguchi, K. Miyake, K. Inokuchi AF4-positive ALL. Therefore, using amlexanox after CR may be effective to suppress relapse of KMT2A/AFF1-positive ALL. Acknowledgments The GVL effect on KMT2A/AFF1-positive ALL may also be The authors thank Dr. R. Van Etten of Harvard Medical School for the kind induced by amlexanox in PBMC-NOD/SCID mice. Specifically, gift of the pMSCVneop230 BCR/ABL plasmid for establishment of our KMT2A/ allogeneic hematopoietic stem cell transplantation (allo-HSCT) is AFF1 Tg mice. expected to show beneficial effects in combination with amlexanox despite its lack of efficacy in KMT2A/AFF1-positive ALL Grant Support patients reported to date (2). Interestingly, Spijkers-Hagelstein H. Tamai was supported in part by grant 40465349 from the Ministry of and colleagues reported that overexpression of the S100 protein Health and Welfare of Japan and the Ministry of Education, Science, and Culture family members S100A8 and S100A9 in KMT2A/AFF1-positive of Japan. H. Tamai was also supported by grants from SENSHIN Medical þ ALL was associated with failure to induce free-cytosolic Ca2 and Research Foundation. The costs of publication of this article were defrayed in part by the payment of prednisolone resistance (14). These observations taken together page charges. This article must therefore be hereby marked advertisement in with our previous study suggest that high levels of S100 protein accordance with 18 U.S.C. Section 1734 solely to indicate this fact. expression by KMT2A/AFF1-positive ALL may be the main factors involved in therapy resistance and poor prognosis of KMT2A/ Received November 1, 2016; revised March 11, 2017; accepted June 6, 2017; AFF1-positive ALL. Amlexanox was also reported to bind to published OnlineFirst June 23, 2017.

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Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1 -Positive Acute Lymphoblastic Leukemia to TNFα Treatment

Hayato Tamai, Hiroki Yamaguchi, Koichi Miyake, et al.

Cancer Res Published OnlineFirst June 23, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2974

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