Cabozantinib is selectively cytotoxic in acute myeloid leukemia cells with FLT3-internal tandem duplication (FLT3-ITD)

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

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Q2 Original Articles

Cabozantinib is selectively cytotoxic in acute myeloid leukemia cells with FLT3-internal tandem duplication (FLT3-ITD) Q1 Jeng-Wei Lu a, An-Ni Wang a, Heng-An Liao a, Chien-Yuan Chen b, Hsin-An Hou b,

Chung-Yi Hu a,c, Hwei-Fan Tien b, Da-Liang Ou d,*, Liang-In Lin a,c,** a

Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan c Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan d Department of Oncology, National Taiwan University, Taipei, Taiwan b

A R T I C L E

I N F O

Article history: Received 20 October 2015 Received in revised form 30 March 2016 Accepted 1 April 2016 Key words: Acute myeloid leukemia FLT3-ITD Cabozantinib

A B S T R A C T

Cabozantinib is an oral multikinase inhibitor that exhibits anti-tumor activity in several cancers. We found that cabozantinib was significantly cytotoxic to MV4-11 and Molm-13 cells that harbored FLT3-ITD, resulting in IC50 values of 2.4 nM and 2.0 nM, respectively. However, K562, OCI-AML3 and THP-1 (leukemia cell lines lacking FLT3-ITD) were resistant to cabozantinib, showing IC50 values in the micromolar range. Cabozantinib arrested MV4-11 cell growth at the G0/G1 phase within 24 h, which was associated with decreased phosphorylation of FLT3, STAT5, AKT and ERK. Additionally, cabozantinib induced MV4-11 cell apoptosis in a dose-dependent manner (as indicated by annexin V staining and high levels of cleaved caspase 3 and PARP-1), down-regulated the anti-apoptotic protein survivin and up-regulated the proapoptotic protein Bak. Thus, cabozantinib is selectively cytotoxic to leukemia cells with FLT3-ITD, causing cell-cycle arrest and apoptosis. In mouse xenograft model, cabozantinib significantly inhibited MV4-11 and Molm-13 tumor growth at a dosage of 10 mg/kg and showed longer survival rate. Clinical trials evaluating the efficacy of cabozantinib in acute myeloid leukemia (AML) with FLT3-ITD are warranted. © 2016 Published by Elsevier Ireland Ltd.

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Introduction Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by acquired genetic changes in hematopoietic progenitor cells. Abnormal proliferation and differentiation frequently result in increased numbers of immature myeloid cells that are unable to function properly [1–3]. FMS-like tyrosine kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase (RTK) family and is mainly expressed in hematopoietic stem cells and progenitor cells. Upon binding to the FLT3 ligand (FL), the FLT3 receptor undergoes dimerization and autophosphorylation, inducing the activation of downstream signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and Ras/Raf/mitogenactivated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways. FLT3 normally regulates the survival and proliferation of hematopoietic progenitor cells [4–6].

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J.-W. Lu, A.-N. Wang and H.-A. Liao contributed equally to this work. * Corresponding author. Tel.: +886 2 23123456 ext 88662; fax: +886 2 33936523. E-mail address: [email protected] (D.-L. Ou). ** Corresponding author. Tel.: +886 2 23123456 ext 67342; fax: +886 2 23711574. E-mail address: [email protected] (L.-I. Lin).

FLT3 is also expressed at high levels in the leukemic blasts of 70–100% of patients with AML. Internal tandem duplication (ITD) mutations in the juxtamembrane region of FLT3 (FLT3-ITD) result in ligand-independent autophosphorylation, constitutive activation of FLT3 and additional activation of the STAT5 signaling pathway [7]. Constitutive activation of FLT3 confers survival and proliferative advantages to leukemic cells [7]. FLT3-ITD, one of the most common mutations in AML, occurs in approximately 30% of AML patients and is associated with a poor prognosis [8]. FLT3-ITD has been suggested as a potential therapeutic target. Several small-molecule FLT3 inhibitors have been developed and evaluated in clinical trials, including midostaurin (PKC-412) [9], lestaurtinib (CEP-701) [10], tandutinib (MLN-518) [11], sorafenib (BAY-43-9006) [12], and quizartinib (AC220) [6]. Although these tyrosine kinase inhibitors (TKIs) have shown efficacy in preclinical studies and encouraging initial responses in clinical trials, the clinical experience of these agents demonstrated minimal clinical efficacy, presumably due to dose-limiting toxicities that preclude potent FLT3 inhibition or short-term responses [13,14]. Therefore, next-generation FLT3 inhibitors need to be discovered or to be generated. Cabozantinib (XL-184, N-(4-((6,7-Dimethoxyquinolin-4yl)oxy)phenyl)-N-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide) is an oral multikinase inhibitor that targets MET, vascular endothelial growth factor receptor 2 (VEGFR2), rearranged during

http://dx.doi.org/10.1016/j.canlet.2016.04.004 0304-3835/© 2016 Published by Elsevier Ireland Ltd.

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transfection (RET), KIT, tyrosine kinase with immunoglobulin-like and extracellular growth factor-like domain 2 (TIE-2), and FLT3, all of which have been implicated in tumor pathogenesis [15]. Previous studies have shown that cabozantinib potently inhibits multiple receptor tyrosine kinases, reduces cell viability in various cancer cell lines and exhibits effective anti-tumor activity in animal xenograft models [15,16]. The US Food and Drug Administration (FDA) approved cabozantinib for the treatment of progressive metastatic medullary thyroid cancer (MTC) in 2012 [17]. Clinical trials for the treatment of prostate cancer, hepatocellular carcinoma, metastatic renal cell carcinoma and non-small cell lung cancer are ongoing. However, there is very little information about the application of cabozantinib for the treatment of leukemia. We aimed to elucidate the efficacy and mechanism of action of cabozantinib in acute myeloid leukemia.

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Materials and methods

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Cell lines and chemicals

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The human leukemia MV4-11, THP-1, RS4;11, K562, HL60, and U937 (ATCC, Manassas, VA, USA) cell lines and MOLM-13 and OCI-AML3 (DSMZ, Brunswick, Germany) cell lines were maintained in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel). All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The origin and characteristics of MV4-11, MOLM13, OCI/AML3 and U937 cell lines were reported previously [18]. These cell lines were authenticated (16-Markers STR) by Food Industry Research and Development Institute, Hsinchu, Taiwan. The genetic profiles of these cell lines were identical to reported genetic profiles. Human peripheral blood mononuclear cells (PBMC) from one healthy donor without other detailed information were isolated from EDTA-stabilized blood by using a Histopaque-1077 density gradient (Sigma-Aldrich, St. Louis, MO). This study was approved by the Institutional Review Board of National Taiwan University Hospital (serial no. 201012144RC), and written informed consent was obtained from this donor. Cabozantinib (XL-184) was purchased from Selleck Chemicals for in vitro experiments and Cabozantinib-malate was kindly provided by Exelixis, Inc. (San Francisco, CA, USA) for in vivo experiments. These chemicals were dissolved in DMSO (Sigma) and stored at −20 °C.

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Cell viability assays

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Following treatment with cabozantinib or DMSO for 72 h, the effects of these drug treatments on the viability of the leukemia cell lines were assessed using a Colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI). Leukemic cells were seeded in 96-well plates at 2500 cells per well and were exposed to various concentrations of drugs for the indicated times. At the end of each set of experiments, 20 μl of CellTiter 96 AQueous One Solution Reagent was added to each well, and the cells were incubated at 37 °C for 2 h. Finally, the absorbance of the contents in each well was measured at 490 nm using a SpectraMax M5 (Molecular Devices, CA). The half-maximal inhibitory concentration (IC50) was determined by an MTS assay and calculated with the CompuSyn software (ComboSyn, Inc., Paramus, NJ). Viable cells were also determined by trypan blue exclusion. Each experiment was performed in triplicate.

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Flow cytometric analysis

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For cell cycle analysis, cabozantinib-treated cells were harvested, washed in icecold phosphate-buffered saline (PBS), fixed in 70% cold ethanol, re-suspended in 0.25 ml of PBS and 1 ml of DNA extraction buffer (192 ml of 0.2 M Na2HPO4 and 8 ml of 0.1 M citric acid, pH 7.8), incubated at room temperature for 5 min, and then stained with a DNA staining solution (PBS containing 20 μg/ml propidium iodide [PI] and 200 μg/ml DNase-free RNase) for at least 30 min at room temperature in the dark. For apoptosis assays, the cells were stained with a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ) and analyzed with a Coulter Epics XL-MCL flow cytometer (Beckman Counter, Inc., Brea, CA, USA) to determine the phase of cell apoptosis (early versus late).

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Western blot analysis

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After cabozantinib treatment, cells were collected, washed with PBS, and lysed with 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 1% protease inhibitor and 1% phosphatase inhibitor cocktail. Protein concentrations were determined using a Quick Start Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved by electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Darmstadt, Germany). The membrane was blocked with Pierce Protein-Free Blocking Buffers (Thermo Scientific, MA,

USA). The following antibodies were used: anti-FLT3, anti-PARP-1, anti-survivin, and anti-p27 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho-FLT3 (pY589/591), anti-Erk1/2, anti-phospho-Erk1/2 (pT202/pY204), anti-Akt, antiphospho-Akt (pS473), and anti-cyclin E from Cell Signaling Technology (Beverly, MA, USA); anti-phospho-STAT5 (pY694), anti-STAT5, anti-caspase3, anti-Mcl-1, antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH), and anti-tubulin from Genetex (San Antonio, TX, USA); and anti-Bak from Calbiochem (Darmstadt, Germany). The bound antibodies were detected using an Immobilon Western Chemiluminescent Horseradish Peroxidase (HRP) substrate (Millipore Corporation, Billerica, MA, USA) and imaged with a Fujifilm LAS-4000 (FUJIFILM, Tokyo, Japan).

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Quantitative real-time PCR

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Total RNA was isolated by using RNAzol B Reagent (Tel-Test; Friendswood, TX, USA). The RNAs were then reverse transcribed to cDNA by using Moloney murine leukemia virus reverse transcriptase (Epicentre, Robbinsville, NJ, USA), coupled with oligodTs and random hexamers under standard conditions. Quantitative real-time PCR (QRT-PCR) was carried out in an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR green as the detection dye (Power SYBR®Green PCR Master Mix, Applied Biosystems). PCR conditions consisted of 1 cycle at 50 °C for 2 min and 95 °C for 10 min, followed by up to 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (annealing/extension). Primer specificity was confirmed by dissociation curves following the reaction. The sequences of primer pairs were as follows: Survivin–forward, 5′-CATCTCTACATTCAAGAACTGG-3′ and reverse, 5′-GGTTAATTCTTCAAACTGCTTC-3′; hypoxanthine phosphoribosyltransferase (HPRT)– forward, 5′-TGACACTGGCAAAACAATGCA-3′ and reverse, 5′-GGTCCTTTTCACCAGCAAGCT3′. Survivin transcript levels were normalized to those of HPRT and were calculated using the ΔCT method as follows: relative expression = 2−ΔCT, where ΔCT = CT(Survivin) − CT(HPRT).

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Tests of in vivo anti-tumor efficacy and its molecular mechanism

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To assess the efficacy of cabozantinib in vivo, we used a mouse xenograft experiment. Female BALB/c athymic (nu+/nu+) mice aged 6–8 weeks were purchased from the National Laboratory of Animal Breeding and Research Centre, Taipei, Taiwan. The mice were subcutaneously inoculated in the flank with 6 × 106 MV4-11, 4 × 106 OCI-AML3 or 2 × 106 U937 cells in a 1:1 homogeneous mixture of PBS and Basement Membrane Matrigel (BD Biosciences; final volume 0.1 ml). The tumor volume was calculated using the following formula: volume (mm3) = (width) 2 × length × 0.5. When tumor size reached 100–200 mm3, mice were randomly divided into several groups and orally administered various dose of cabozantinib-malate or vehicle. Cabozantinib-malate, formulated in sterile water/5 mmol/l HCL, was administered at 10 or 30 mg/kg via oral gavage. When tumor volume reached 2000 mm3, animals were euthanized and the tumor was collected and fixed overnight in neutral pHbuffered formalin. The protocol for the xenograft experiments was approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University, and conformed to the criteria outlines in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. In addition, the tumors were also collected at the indicated time points of drug treatment for further immunoblotting and immunohistochemical analysis [19,20].

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Immunohistochemical staining

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Collected tumor tissues were fixed in 10% neutral buffered formalin, paraffinembedded, and the immunohistochemical staining was carried out as previous reports [19]. Briefly, tissues were deparaffinized using xylene and rehydrated with series dilutions of ethanol in a stepwise fashion. Endogenous peroxidase activity was then blocked using 1X tri-EDTA (pH 9.0) buffer at 100 °C for 10 min, and tissue sections were treated with 3% H2O2 for antigen retrieval. Slides were incubated at 4 °C overnight using rabbit anti-ki-67 (1:100 dilutions, Abcam, Cambridge, MA, USA) or rabbit anti-active caspase 3 (1:100 dilutions, BD Biosciences, San Jose, CA, USA). After washing with 1x PBS, this was followed by development using the EnVision™ + Dual Link System (Dako, Carpinteria, CA, USA). The slides were counterstained with hematoxylin for 1–2 minutes and examined by light microscopy after being dehydrated, cleared, and mounted on slides. Ki-67 positive cells were counted under high power (400×). Three tumor samples in each group were analyzed, and the numbers were the average of counting 4 HPF in each sample.

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Statistical analysis

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All data were representative of at least three independent triplicate experiments. Quantitative data are expressed as means ± SDs. Differences between the treatment groups and the DMSO control group were compared by Student’s t test. Survival rates were analyzed by Kaplan–Meier and comparisons of survival curves and median survival were analyzed by log-rank test. P values of less than 0.05 were considered to be significant.

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Fig. 1. Cabozantinib (XL-184) selectively decreased the viabilities of FLT3-ITD-positive MV4-11 and MOLM-13 cells, resulting in IC50 values at the nanomolar level. Leukemia cell lines were treated with DMSO or increasing concentrations of cabozantinib for 72 h, and cell viability was measured by MTS assay. Data are the mean ± SD from 3 independent experiments.

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Results Cabozantinib is a potent inhibitor of leukemia cell proliferation with FLT3-ITD We compared the sensitivity of a panel of leukemia cell lines (MV4-11, MOLM-13, OCI-AML3, THP-1, RS4;11, K562, U937 and HL60) to cabozantinib using an MTS assay. Cabozantinib inhibited FLT3-ITD-positive MV4-11 and MOLM-13 cell proliferation in a dosedependent manner with IC50 values of 2.4 ± 0.3 nM and 2.0 ± 0.8 nM, respectively (Fig. 1). By contrast, in normal PBMCs and FLT3-ITDnegative leukemia cells, cabozantinib treatment resulted in IC50 values of greater than 2 μM, suggesting drug potency with specificity. Cabozantinib inhibits FLT3-ITD-dependent signaling Given that the constitutive ligand-independent activation of FLT3ITD leads to the activation of downstream signaling pathways, including the STAT5, PI3K/Akt, Ras/Raf/MEK/ERK pathways, we first examined the inhibitory effects of cabozantinib on the phosphorylation of FLT3 and downstream signaling pathways. We found that FLT3 phosphorylation was strongly down-regulated following treatment with as low as 1 nM cabozantinib for 2 h (Fig. 2A). Furthermore, we found that the phosphorylation of STAT5, Akt and ERK1/2 was also inhibited by cabozantinib in a dose-dependent manner in MV411 cells (Fig. 2B). By contrast, the phosphorylation of Akt and ERK1/2 was unaffected by cabozantinib in FLT3 wild-type OCI-AML3 cells (Fig. 2C). To exclude that the inhibitory activity against c-MET, RET and VEGFR2 might contribute to the growth arrest induced by cabozantinib, we also analyzed the effect of these molecules in MV411 and OCI-AML3 cells. As shown in supplementary Fig. S1, there were no c-MET, RET and VEGFR2 expressions in MV4-11 cells. Although some RET and MET expressions were shown in OCI-AML3 cells, no p-RET was shown and cabozantinib only had a minimum effect on p-MET.

Cabozantinib induces cell-cycle arrest and apoptosis in leukemia cells with FLT3-ITD To elucidate the molecular mechanism of the anti-proliferative effects of cabozantinib, cell cycle and apoptosis analyses were performed. We found that treatment of MV4-11 and MOLM-13 cells, but not of OCI-AML3, with as low as 5 nM cabozantinib induced significant G0/G1 cell cycle arrest within 24 h in a dosedependent manner (supplementary Fig. S2; Fig. 3A–C). Cabozantinib down-regulated the key G1/S transition regulator cyclin E and up-regulated the cyclin-dependent kinase (CDK) inhibitor p27, thereby leading to G0/G1 cell cycle arrest (Fig. 3D), suggesting that the dysregulation of cyclin E and p27 was involved in the effects of cabozantinib. For apoptosis analysis, increased percentages of sub-G1 cells (19.7 ± 3.7%) and annexin V-positive cells (37.1 ± 6.7%) were observed following treatment of MV4-11 cells with 50 nM cabozantinib (Fig. 4A and B). Consistent with the effects of cabozantinib on sub-G1 and annexin V, significant amounts of cleaved caspase 3 and PARP-1 were observed (Fig. 3E). Similar results were demonstrated in MOLM-13 cells, with sub-G1 cells and annexin V-positive cells amounting to 40.9 ± 5.2% and 88.3 ± 2.2%, respectively (Fig. 4D and E). Following 72 h of treatment with cabozantinib, 57.7 ± 12.5% and 84.5 ± 7.4% of sub-G1 MV4-11 cells and MOLM-13 cells, respectively, underwent apoptosis (Fig. 4A and B), indicating that cabozantinib induced apoptosis of FLT3-ITD harboring MV4-11 and MOLM-13 cells, but not OCI/AML3 cells with FLT3-WT (Fig. 4C and F), in a dose and time-dependent manner. Further analysis of apoptosis-associated proteins revealed that cabozantinib was able to down-regulate the anti-apoptotic protein survivin and up-regulate the pro-apoptotic protein Bak in the MV4-11 cells (Fig. 5A). Exposure of the MV4-11 cells to cabozantinib significantly inhibited survivin mRNA expression (Fig. 5B), indicating that cabozantinib suppressed the expression of survivin at the transcriptional level.

Please cite this article in press as: Jeng-Wei Lu, et al., Cabozantinib is selectively cytotoxic in acute myeloid leukemia cells with FLT3-internal tandem duplication (FLT3-ITD), Cancer Letters (2016), doi: 10.1016/j.canlet.2016.04.004

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Fig. 2. Cabozantinib specifically inhibited the phosphorylation of FLT3 (A) and downstream signaling molecules such as STAT5, Akt and ERK1/2 in FLT3-ITD MV4-11 cells (B). By contrast, the phosphorylation of downstream FLT3 signaling molecules was unaffected by cabozantinib in FLT3 wild-type OCI-AML3 cells (C). Cells were treated with cabozantinib for 2 h, and the expression of pFLT3, FLT3, pSTAT5, STAT5, pAkt, Akt, pErk1/2, and Erk1/2 was measured by Western blotting.

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Cabozantinib inhibits growth of subcutaneous MV4-11 xenograft tumors In order to characterize the in vivo biological activity of cabozantinib, MV4-11 tumors were harvested, homogenized and analyzed by western blot analysis for phospho-FLT3, phospho-Stat5, phospho-Akt and phospho-Erk. We found that phosphorylation of

FLT3 and its downstream STAT5 were completely abolished in MV4-11 tumors after 4 h treatment and persisted up to 24 h. However, phosphorylation of Akt showed a similar pattern following the 30 mg/kg dose with inhibition at 4 h, but rebounded at 24 h. Inhibition of phosphorylated Erk at 4 h showed variant results among individuals (Fig. 6A). In addition, positive active caspase-3 immunostain on MV4- Q5 11 tumor treated with 10 mg/kg and 30 mg/kg cabozantinib or

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Fig. 3. Treatment with cabozantinib for 24 h induced prominent G0/G1 cell-cycle arrest in MV4-11 (A) and MOLM-13 (B), but not in OCI/AML3(C), cells in a dosedependent manner. MV4-11 cells treated with cabozantinib for 24 h demonstrated down-regulation of the key G1/S transition regulator cyclin E and up-regulation of the CDK inhibitor p27 (D) as well as activation of caspase 3 and cleavage of PARP-1 (E). Data are the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 4. Apoptosis was observed as shown by increased percentages of sub-G1 cells following treatment of MV4-11 (A) and MOLM-13 (B), but not of OCI/AML3 (C), cells with cabozantinib. Annexin V and PI analyses also demonstrated that cabozantinib treatment for 72 h triggered apoptosis in MV4-11 (D) and MOLM-13 (E), but not in OCI/ AML3 (F), cells in a dose-dependent manner. Annexin V-positive cells represent cell populations at the early and late stages of apoptosis. Data are the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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vehicle (Supplementary Fig. S3) demonstrated that apoptosis was induced by cabozantinib in vivo. Therefore, daily oral administration was suggested in the following anti-tumor and survival analysis. To determine the antitumor efficiency of cabozantinib, mice bearing FLT3-ITD cell lines (MV4-11, Molm-13) and FLT3-WT cell lines (U937, OCI/AML3) were orally dosed (dosage of 10 mg/kg for 5 days followed by one-day rest for a total of 4 cycles). With well tolerance of the drug in mice, no effects on body weight were observed during dosing period. On the other hand, as shown in Fig. 6B, tumor growth in MV4-11 and Molm-13, but not in U937 and OCI/ AML3, was well inhibited in group of 10 mg/kg post dosage with p

343 values of <0.0001, 0.0024, 0.5977 and 0.1063, respectively. Intrigu344 ingly, further observation revealed that the repression of MV4-11 345 tumors was sustained for approximately 1 month in 10 mg/kg group 346 while no tumor growth was observed in 30 mg/kg group for ap347 proximately 2 months after treatment (supplementary Fig. S4). A second-round treatment was performed during relapse of tumor Q6 348 349 growth with an average volume over 1000 mm3 in the MV4-11 tumor 350 xenograft mice after first-round administration. The cabozantinib 351 was still effective for repression of MV4-11 tumor growth (supple352 mentary Fig. S4). The tumor re-grew again immediately after 353 stopping second-round cabozantinib administration.

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Fig. 5. (A) Down-regulation of survivin and up-regulation of Bak were observed in MV4-11 cells treated with cabozantinib for 24 h. (B) Cabozantinib inhibited the mRNA expression of survivin in MV4-11 cells treated for 24 h. Relative mRNA expression was quantified by real-time PCR. Data are the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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354 Fig. 6. Mouse xenograft of AML tumor growth after treatment with cabozantinib. Mice bearing MV4-11 tumor were administrated with a single dose of 30 mg/kg cabozantinib and tumors were harvested at 4 h and 24 h after administration. Tumor lysates were analyzed for FLT3, p-FLT3, STAT5, p-STAT5, Akt, p-Akt, Erk and p-Erk by western blot. 355 Expression level of phosphorylated protein was normalized with GAPDH by using ImageJ software (A). Cabozantinib induces significant regression of subcutaneous FLT3356 ITD tumors, but not FLT3-WT tumors in athymic mice (B). Kaplan–Meier survival curves of different groups of mice were plotted against days after injection treated with 357 358 cabozantinib or vehicle. (C). H&E and Ki-67 immunostaining was performed on MV4-11 tumor treated with cabozantinib or vehicle (D). Quantification of the Ki-67 positive 359 cell number (E) and comparison of tumor weight with or without cabozantinib (F). Data are the mean ± SD from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. 360 Q10 Scale bars = 200 μm. 361 362 After tumor inoculation, mice treated with vehicle or cabozantinib 363 with a dosage of 10 mg/kg were monitored for survival as well. As 364 shown in Fig. 6C, treatment with 10 mg/kg cabozantinib had sur365 vival advantage in mice with Molm-13 and MV4-11 cells, but not 366 Q7 with U937 or OCI/AML3 cells. A trend toward decrease in the pro367 liferation marker Ki-67 was also noted in cabozantinib-treated groups 368 in a dose-dependent manner (p < 0.0001) (Fig. 6D and E). In addi369 tion, tumor weight significantly decreased in the cabozantinib370 treated groups (p < 0.0001) (Fig. 6F). These data suggested that 371 cabozantinib selectively inhibited FLT3-ITD tumor growth in vivo. 372 In addition to mouse subcutaneous model, xenotransplantation 373 in zebrafish was used as a second animal model [21]. In the 374 xenotransplantation zebrafish model, we first verified that there was 375 no discernible toxicity to zebrafish with dosage of 100 nM 376 cabozantinib (supplementary Fig. S5A and B), and then we in377 jected MV4-11 cells labeled with CM-Dil into the yolk sac of a 2-day 378 old zebrafish larva, and subsequently treated with cabozantinib or 379 DMSO at another day. We found that treatment with up to 100 nM 380 cabozantinib for 48 h induced a 5-fold decrease of fluorescence in381 tensity in the fish (supplementary Fig. S5C and D), demonstrating 382 the ability of cabozantinib to decrease the leukemic burden in vivo.

Discussion In this study, we demonstrated that cabozantinib acts selectively on AML with FLT3-ITD. Its activity was probably based on the inhibition of the phosphorylation of FLT3 and its downstream targets, STAT5, ERK and AKT, and thereby leads to cell-cycle arrest. In addition, cabozantinib induced cell apoptosis through both downregulation of the anti-apoptotic protein survivin and up-regulation of Bak. In this study, we also demonstrated that cabozantinib decreased survivin transcription and decreased the phosphorylation of Akt and STAT5 in leukemic cells with FLT3-ITD, which is in accordance with previous reports stating FLT3-ITD may up-regulate survivin via the PI3K/Akt [22] and STAT pathways [23] at the transcriptional level. Although Mcl-1, a Bcl-2 family member, plays an important role in controlling apoptosis [24,25], in this study, treatment with cabozantinib did not down-regulate Mcl-1 expression in MV4-11 cells. Taken together, we suggest that the inhibition of cell proliferation and the induction of apoptosis by cabozantinib are, at least in part, related to the inhibition of FLT3-ITD and the subsequent PI3K/Akt signaling pathway activity. In mouse xenograft model, cabozantinib strongly inhibits the growth of FLT3-ITD-bearing

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MV4-11 and Molm-13 tumors. This effect of cabozantinib, at a dose of 30 mg/kg, lasted over 2 months, indicating that this drug would be an interesting alternative of the treatment of AML patients harboring FLT3-ITD mutations. Compared with various solid tumor cell lines, such as stomach (SNU-1, SNU-16), brain (U87MG), lung (H441, H69), prostate (PC3) and breast (MDA-MB-231) carcinoma cell lines, which all show IC50 values in the micromolar range, medullary thyroid carcinoma (TT) and myeloid leukemia (MV4-11) appear much more sensitive to cabozantinib, with IC50 values of 94 nM [26] and 2.4 nM (measured in this study), respectively. The US FDA approved cabozantinib for the treatment of patients with advanced/progressive metastatic medullary thyroid cancer (MTC) in 2012. The recommended daily dose of cabozantinib for these patients is 140 mg daily [27]. Drug-related toxicities were observed, including grade 3 palmarplantar erythrodysesthesia and elevations in AST/ALT and lipase. Although following a single 175-mg dose of cabozantinib, steadystate plasma concentrations were at the micromolar level [28], our data appear that a much lower dose of this drug may be sufficient for AML patients with FLT3-ITD. A phase I trial evaluating the use of cabozantinib to treat patients with relapsed or refractory AML (NCT01961765) is currently ongoing. It is known that FLT3-ITD mutations are associated with adverse outcomes [29,30] with conventional standard treatment, but the significance of the less common FLT3-TKD mutations remains controversial [31,32]. Several tyrosine kinase inhibitors, including sorafenib, AC220, MLN518 and PKC412, were able to effectively and selectively inhibit signaling pathways driven by FLT3-ITD. However, compelling clinical and laboratory evidence has demonstrated that FLT3-ITD with additional tyrosine kinase domain (TKD) mutations, mainly in the “gatekeeper” residue F691 (F691L) and the Q8 activation loop (AL) residue D835 (D835V/Y/F) and residue Y842 (Y842C/H), confer drug resistance [14,33–35]. Cabozantinib has been reported to be a potent inhibitor of RET and demonstrates different effects in the presence of various RET mutations. In contrast to its effects on cells with the M918T mutation (IC 50 = 27 nM), cabozantinib had minimal effects on cells with the Y791F mutation (IC50 = 1173 nM) and was not active in the presence of cells with the V804L mutation (IC50 > 5000 nM) in any of the biochemical assays performed. On the other hand, cellular assays showed that cabozantinib could inhibit a human MTC cell line harboring an activating C634W mutation in RET, with an IC50 value of 85 nM [26]. In addition, an in vitro kinase assay demonstrated that cabozantinib had similar effects on wild-type and mutant MET (Y1248H, D1246N, and K1262R) [15]. Therefore, cabozantinib may probably be effective in certain kind of resistant mutants. However, the efficacy needs further clarification. This study is the first report to demonstrate the potent activity of cabozantinib against human myeloid leukemia cells with FLT3ITD rather than with wild-type FLT3 both in vitro and in vivo. Recently, several FLT3-targeting agents, including midostaurin, lestaurtinib, sorafenib, quizartinib, and tandutinib, have been explored for the treatment of AML [6]. Some of these FLT3-targeting agents are now undergoing phase III studies for the treatment of AML patients with FLT3-ITD [36]. Whether cabozantinib is an optimal drug remains to be explored in future trials.

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Authors’ contributions J-W L and A-N W performed in vitro experiments, H-A L and D-L O performed animal experiments. C-Y C, H-A H and D-L O performed statistical analysis. J-W L, A-N W, H-A L, C-Y H, H-F T and L-I L analyzed and interpreted the data. J-W L, A-N W, H-A L, D-L O and L-I L wrote the paper. L-I L designed and coordinated the research study.

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