The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in Cutaneous T-Cell Lymphoma via ITK-mediated signaling pathway

Journal Pre-proof The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in Cutaneous T-Cell Lymphoma via ITK-mediated signaling pathway Hongyang Li, Cheng Wang, Pengcheng Ma, Mengli Zhang, Hua Yang, Shengtao Yuan, Jun Wei, Lei Tao, Kun Qian, Man Xu, Lingjun Li

PII:

S0923-1811(20)30043-8

DOI:

https://doi.org/10.1016/j.jdermsci.2020.01.013

Reference:

DESC 3567

To appear in:

Journal of Dermatological Science

Received Date:

14 October 2019

Revised Date:

10 January 2020

Accepted Date:

21 January 2020

Please cite this article as: Li H, Wang C, Ma P, Zhang M, Yang H, Yuan S, Wei J, Tao L, Qian K, Xu M, Li L, The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in Cutaneous T-Cell Lymphoma via ITK-mediated signaling pathway, Journal of Dermatological Science (2020), doi: https://doi.org/10.1016/j.jdermsci.2020.01.013

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The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in Cutaneous T-Cell Lymphoma via ITK-mediated signaling pathway Hongyang Lia, Cheng Wanga, Pengcheng Maa, Mengli Zhanga, Hua Yangb, Shengtao Yuanc, Jun Weia, Lei Taoa, Kun Qiana, Man Xud, Lingjun Lia,*. a

The Hospital for Skin Disease, Institute of Dermatology, Chinese Academy of

Medical Science, Peking Union Medical College, Nanjing 210042, PR China. Department of dermatology, Chongqing Hospital of Traditional Chinese Medicine,

Chongqing 400011, PR China. c

Jiangsu Center for Pharmacodynamics Research and Evaluation, China

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Pharmaceutical University, Nanjing 210009, China.

The Department of Clinical Laboratory, The Fourth Affiliated Hospital of Nanjing

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b

Medical University, Nanjing 210031, PR China.

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*Corresponding author: Lingjun Li, The Hospital for Skin Disease, Institute of

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Dermatology, Chinese Academy of Medical Science, Peking Union Medical College, Add: 12 jiangwangmiao street, Nanjing 210042, China; Tel: +86-25-85478929; Fax:

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+86-25-85471862; E-mail: [email protected].

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Hightlights 

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The retinoid derivant ECPIRM selectively inhibited the proliferation of Hut78 and MJ cells rather than Myla cells. Targeted inhibition of Interleukin-2-inducible T-cell kinase (ITK) induced cell apoptosis. ECPIRM efficiently combined ITK and inhibited ITK-mediated signaling pathway. ECPIRM suppressed tumor growth in Hut78-xenografted nude mice model via ITK-mediated signaling pathway. 1

ABSTRACT Background: The treatment of Cutaneous T-cell lymphoma (CTCL) met huge challenges because of the heterogeneity and the scarcity of targeted drugs. ECPIRM derived from isotretinoin exhibited strong anti-proliferation effects in Hut78 and MJ cells rather than Myla cells. However, there was no data regarding the potential target of ECPIRM for its selective activity.

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Objectives: To investigate the potential target of ECPIRM for its selective anti-proliferation activity.

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Methods: We evaluated the cell viability of CTCL cells after ECPIRM treatment, and detected the effects of ECPIRM on the biomarker genes of CTCL. Subsequently, the

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mRNA and protein level of Interleukin-2-inducible T-cell kinase (ITK) was

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determined. Then the induction of apoptosis triggered by ITK inhibitor BMS-509744 and ITK siRNAs were detected, and the docking of ECPIRM interacted with ITK and

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the effects of ECPIRM on ITK-mediated signaling pathway were analyzed. Finally, we evaluated the anti-growth activity of ECPIRM in Hut78-xenografted nude mice,

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and the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt were

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determined.

Results: ITK was highly expressed in Hut78 and MJ cells rather than Myla cells, and targeted inhibition of ITK triggered cell apoptosis. ECPIRM efficiently bound the hydrophobic active pocket of ITK, and significantly inhibited ITK-mediated signaling pathway. In addition, ECPIRM suppressed tumor growth in Hut78-xenografted model, and upregulated the expression of cleaved caspase 3 and inhibited the expression of 2

ITK, p-ERK and p-Akt in tumor tissues, which was consistent with in vitro study. Conclusion: ECPIRM might provide a novel strategy for CTCL by inhibiting ITK-mediated signaling pathway. Keywords: Cutaneous T-cell lymphomas; retinoids; ECPIRM; ITK; apoptosis.

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1. Introduction Cutaneous T-cell lymphoma (CTCL) was a highly heterogeneous non-Hodgkin lymphomas characterized by the residing of malignant T lymphocytes in the lesions of

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skin [1]. The incidence of CTCL was 6.4 per million, which made it the second most

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common extra-nodal non-Hodgkin lymphomas [2]. Mycosis fungoides (MF) and Sézary syndrome (SS) were the most common form of CTCL, both of them accounted

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for more than 80% of CTCL, and MF was the main form of CTCL [2-4]. Owning to

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the heterogeneity and the scarcity of special targets, the precise treatment of CTCL also met huge challenges.

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Current medicinal therapeutic approaches of CTCL in clinic mainly included IFN-α, histone deacetylase (HDAC) inhibitor and retinoids. As immune adjusting

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reagent, retinoids played important roles in the clinical treatment of CTCL, and the main biochemical function depended on the activation of retinoic acid receptor (RAR) or retinoid X receptor (RXR) to inhibit the proliferation of malignant cells [5]. The RXR agonist bexarotene exhibited pro-apoptosis acticity and inhibitory effects on angiogenesis and migration and infiltration of malignant T lymphocytes into the skin, 3

which was approved by FDA for the treatment of CTCL in 1999 [6]. Furthermore, isotretinoin and acitretin were also reported to exert therapeutic effects in CTCL [7]. However, the side effects triggered by retinoids seriously affected the continuous application of retinoids in clinic [8, 9]. Hence, the development of new retinoid was also needed for the efficient treatment of CTCL. ECPIRM (the N-(4-ethoxycarbonylphenyl) isoretinamide, C29H33O3N) was a

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compound that derived from isotretinoin, which exhibited anti-proliferation effects on cutaneous squamous carcinoma cell and CTCL cells via RAR/RXR-independent manner [10, 11]. As the previous study reported, ECPIRM induced dramatic cell

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apoptosis in Hut78 cells rather than human lymphocytes via JAK/STAT signaling

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pathway [10]. However, the potential target of ECPIRM that indicated the selective effects was not determined by the previous work. Here, our recent study revealed that

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the high level of Interleukin-2-inducible T-cell kinase (ITK) might be the main target

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for the special activity in CTCL cells, and targeted inhibition of ITK might provide a new strategy for the treatment of CTCL. Our study would lay a strong foundation for

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the development of new ITK-targeted drugs and the personalized treatment of CTCL.

2. Materials and methods 2.1. Chemicals and reagents The retinoid derivant ECPIRM was synthesized in our lab (Fig.1A) [11]. ITK inhibitor BMS-509744 and Cell Counting Kit-8 (CCK8) were purchased from

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MedChem Express (Princeton, NJ, USA). TRIzol® Reagent, HiScript® II Reverse Transcriptase SuperMix, SYBR® qPCR Master Mix and apoptosis detection kit (Annexin V-PI Staining) was purchased from Vazyme Biotech Co., Ltd (Nanjing, China). Lipofectamine™ RNAiMAX Transfection Reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibody against ITK was obtained from Abcam (Cambridge, MA, USA), and antibodies against ERK, p-ERK, Akt,

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p-Akt, cleaved caspase 3 and GAPDH were commercially available from Cell Signaling Technology (Beverly, MA, USA).

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2.2. Cell culture

The human CTCL cell line Hut78 was purchased from CoBioer Biotechnology

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(Nanjing, China), and cell line Myla was purchased from BeNa Culture Collection

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(Beijing, China). The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin (Gibco, Grand

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Island, NY, USA) using in BB150 incubator (Thermo Fisher Scientific Inc.,

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Langenselbold, Germany) at 37 ℃ with 5% CO2. 2.3. CCK8 assay

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CCK8 assay was used to detect the proliferation inhibition of ECPIRM or

BMS-509744. Hut78 cells were seeded into each 96-well plate with 8,000 cells per well, and Myla cells were seeded with 4,000 cells per well. Subsequently, cells were treated with different concentrations of the drugs for 72 h. Then 20 μl CCK8 solution were added into each wells. After 2 to 4 hours, the plates were detected at the 5

absorbance of 450 nm using Multiskan Spectrum (Thermo Scientific, Waltham, MA, USA). The inhibition rate or cell viability was caculated as the percentage of negative controls. 2.4 Quantitative real-time PCR (RT-qPCR) Cells were treated with indicated concentrations of ECPIRM for 72 h, and the

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sample were then collected using TRIzol® Reagent. The RNA were reversely transcribed into cDNA using HiScript® II Reverse Transcriptase SuperMix. RTq-PCR was run in an ABI 7300 PCR instrument by 1 cycle of 95 ℃ for 5 minutes, 40 cycles

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of 95 ℃ for 10 seconds and 60 ℃ for 30 seconds, and 1 cycle of 95 ℃ for 15

seconds, 60 ℃ for 60 seconds and 95 ℃ for 15 seconds. The sequences of primers

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2.5 Western blotting

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used in the research were shown in Supplementary Table S1.

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After the treatment of drugs, cell samples were collected using cell lysis buffer with phosphatase inhibitor and protease inhibitor inside. Enhanced BCA protein assay

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was determined to analyze the concentrations of each sample. Subsequently, 40 μg total protein was used in the SDS-PAGE electrophoresis system, and the protein in

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gels were then transfected into 0.45 μm PVDF membrane. After blocking with 2.5% bovine serum albumin (BSA) for 1 hour, the membrane was then incubated with primary antibody overnight at 4 ℃, the dilution ratio of the antibody was based on the instruction manuals. After washing with TBST for 3 times every 10 minutes, the membrane was then incubated with secondary antibody for another 1 hour at room 6

temperature. Finally, the membrane was exposed using enhanced chemiluminescence reagents (Millipore) by Molecular Imager@ ChemiDocTM XRS+ Image System (Bio-Rad Laboratories, Inc., Richmond, CA, USA). 2.6 Apoptosis detection CTCL cells were seeded into 6-well plate at the density of 300,000 cells per well,

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and cells were then directly treated with drugs for 72 h, or transfected with siRNAs for 48 h, the samples were then collected. After washing with cold PBS for 2 times,

the cells were suspended with 500 μl binding buffer and stained with 5 μl Annexin V

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and 5 μl PI for 15 minutes. The induction of apoptosis was detected by BD FACS

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Calibur flow cytometry. 2.7 RNA interference

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We transfected the cells using RNAiMAX Transfection Reagent, and processed

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all the operation according to the manufacturer’s protocol. The negative control, ITK siRNA oligos were purchased from GenePharma (Shanghai, China). The sequences

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were as follows: ITK siRNA #1, sense 5'- GGACAGGAAUGGGCAUGAATT -3' and antisense 5'-UUCAUGCCCAUUCCUGUCCTT-3'; ITK siRNA #2, sense 5'-

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CCGGAUUUCGGUUGUACAATT UUGUACAACCGAAAUCCGGTT

-3' -3'.

UUCUCCGAACGUGUCACGUTT

and Scrambled

-3'

and

antisense siRNA

sense

antisense

5'5'5'-

ACGUGACACGUUCGGAGAATT -3' were used as negative control. The cells were incubated with the complex of RNAiMAX reagent and siRNAs for 48 h, and the 7

samples were then collected for western blotting and apoptosis detection. 2.8 Nude mice xenograft study Female NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NCG) nude mice (Animal certificate number 201812933 and Permit number SCXK (Su) 2015-0001) at the old of 5 weeks with body weight from 18 to 22 g were obtained from the Model Animal Research

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Center of Nanjing University. 2×106 Hut78 cells were injected into the subcutaneous tissue of mice armpit. When the tumor tissue volume was grown with

about 100 mm3, the mice with analogous tumor volume were randomly divided into

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four groups, which contained six mice inside. In Hut78 xenograft model, ECPIRM were dissolved in 0.5% CMC-Na and intragastrically administrated with dosages of

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10 mg/Kg, 50 mg/Kg and 100 mg/Kg, respectively. Furthermore, an equal amount of

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0.5% CMC-Na was given in the negative control. After the administration for 10 days, mice were euthanized and the tumor tissues were then resected and collected by

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formalin fixation. Relative tumor volume (RTV) were calculated by the following formula：RTV = Vt/V0, where Vt represented the tumor volume at day t, and V0

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represented the volume at day 0. The animals were all housed and cared under

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standard conditions according to the protocols that approved by China Pharmaceutical University.

2.9 Immunohistochemical analysis The expression of Cleaved caspase 3, ITK, p-ERK and p-Akt in tumor tissues of NCG nude mice was detected using immunohistochemical analysis. The tissue sections 8

embedded by paraffin were deparaffnized using xylene, and treated with gradient concentration of alcohol (100%, 95%, 85% and 75%). Subsequently, the sections were soaked in 0.01 mol/l sodium citrate and heated to boil for 2 times. 3% hydrogen peroxide was dropped on the sections at room temperature to remove the interference from endogenous peroxidase. The samples were then washed thrice with double-distilled water and incubated with 1% normal goat serum at room temperature

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for 20 minutes. Primary antibodies against cleaved caspase 3, ITK, p-ERK and p-Akt were incubated for 12 hours at 4 ℃，and the dilution ratio of these antibodies was based on the instruction manuals. After washing with PBS for 3 times every 5 minutes, the

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samples were incubated with secondary antidody for another 1 hours at room

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temperature. Finally, the slides were observed by converted microscopy, and the

plus 6.0 software.

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2.10 Statistical analysis

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integrated optical density of the positive staining was analyzed using by Image-pro

The data in our study were calculated and expressed as mean ± S.D. using Student’s

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t-test (two-tailed), and the statistical significant differences was calculated by

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GraphPad Prism 5.0 software. We used *P < 0.05, **P < 0.01 or ***P < 0.001 to represent significant differences in our study. 3. Results

3.1 ECPIRM selectively inhibited the proliferation of Hut78 cells. As our previous study reported, ECPIRM exhibited stronger anti-proliferation 9

effects in Hut78 cells than other retinoids, such as all-trans retinoic acid, 13-cis-retinoic acid and bexarotene [10]. To determine the effects of ECPIRM on the growth of other CTCL cells, we compared the anti-proliferation activity of ECPIRM between Hut78, MJ and Myla cells. As shown in Fig.1B, the anti-proliferation effects emerged significant difference in the above cells, and the IC50 of ECPIRM in Hut78 and MJ cells was 10.33±0.64 μM and 16.86±0.48μM, respectively, while the IC50

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in Myla cells was 63.93±6.64 μM. Moreover, ECPIRM dramatically inhibited the cell viability in a dose-dependent manner in both of Hut78 and MJ cells rather than

Myla cells (Fig.1C). Furthermore, we evaluated the effects of bexarotene in the above

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cells, and found that the IC50 of ECPIRM was lower than bexarotene in both Hut78

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and MJ cells (Supplementary Fig.S1). Hence, the results demonstrated that ECPIRM exhibited selective inhibitory effects in Hut78 and MJ cells.

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As reported, 17 genes (CCL18, CCL26, FYB, T3JAM, MMP12, LEF1, LCK, ITK,

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GNLY, IL2RA, IL-26, IL-22, CCR4, GTSF1, SYCP1, STAT5A, TOX) were determined to distinguish MF/SS from benign inflammatory dermatoses, which were

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important biomarkers for the diagnosis of CTCL [12]. Here, we detected the effects of ECPIRM on the mRNA expression of the above genes. As shown in Fig.1D, we found

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that ECPIRM exhibited effects on the expression of 14 genes, and 3 genes (MMP12, IL-22 and SYCP1) could not be detected in Hut78 cells because of excessive Ct value in RT-qPCR assay. Importantly, the mRNA level of ITK was maximally decreased compared with other genes, and the inhibitory effects were exerted in dose-dependent manner in both Hut 78 and MJ cells (Fig.1D-E). Considering the great difference of 10

ECPIRM activity on CTCL cells, we compared the basic expression of ITK, and found that both mRNA and protein level in Hut78 and MJ cells were greatly higher than that in Myla cells (Fig.1F-G). Above all, these results suggested that high expression of ITK might be the important biomarkers for the selective anti-cancer activity of ECPIRM in Hut78 and MJ cells.

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3.2 Targeted inhibition of ITK induced cell apoptosis in Hut78 cells. ITK, a member of the Tec family, was a non-receptor protein tyrosine kinase,

which played important roles in the proliferation, maturation and differentiation of T

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cells[13]. ITK was highly expressed in T-cell acute lymphoblastic leukemia (ALL) and T cell lymphoma compared to other cancer types (Fig.2A). However, there was

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no data regarding targeted inhibition of ITK for the inhibition of Hut78 cells. Here,

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we firstly used the selective and ATP-competitive ITK kinase inhibitor BMS-509744 to evaluate its anti-proliferation effects in Hut78 cells, and found that BMS-509744

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exhibited a low IC50 at 4.83±0.67μM, and induced cell apoptosis in Hut78 cells in a dose-dependent manner (Fig.2B-C). Subsequently, we then interfered the expression

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of ITK by siRNAs, and similarly found that targeted inhibition of ITK expression also

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triggered obvious apoptosis in Hut78 cells (Fig.2D-E). Taken together, ITK was an important target for the proliferation inhibition in CTCL cells. 3.3 ECPIRM efficiently combined ITK and inhibited ITK-mediated signaling pathway. The previous report and our present study prompted that ITK might be the important target for ECPIRM in Hut78 cells. Here, we used Autodock Vina and Free 11

Maestro of Schrodinger software to evaluate the combination between ECPIRM and ITK protein. As shown in Fig.3A-B, we found that ECPIRM tightly combined with the hydrophobic active pocket of ITK protein with the lowest binding position at -9.0 kcal/mol, and the hydrogen bonds formed by ITK interacted with ECPIRM were located at the amino acid sites ILE 369, GLY 441 and CYS442 of ITK protein. These results proved that ECPIRM exhibited strong binding activity with three-dimensional

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structure of ITK. As reported, the roles of ITK inside cells were mediated by the downstream ERK and Akt signaling pathway[14, 15]. Here, we detected the effects of

ECPIRM on the relative expression of ITK, p-ERK and p-Akt using western blotting

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analysis. In Fig.3C-D, we found that ECPIRM treatment significantly decreased the

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protein level of ITK, and simultaneously suppressed the relative expression of p-ERK and p-Akt in Hut78 cells in dose-dependent manner. Although bexarotene exhibited

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significant inhibitory effects in CTCL cells, it showed no obvious changes on the

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relative level of ITK protein (Fig.3E-F). Overall, our results demonstrated that ECPIRM efficiently combined with ITK protein and inhibited ITK-mediated

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signaling pathway in Hut78 cells.

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3.4 ECPIRM suppressed tumor growth in Hut78-xenografted nude mice model via ITK-mediated signaling pathway. The above results revealed that ECPIRM exhibited strong anti-proliferation

effects on Hut78 cells with high level of ITK expression in vitro. Here, we performed in vivo experiment to evaluate the effects of ECPIRM on the growth of the tumor in Hut78-xenografted model. As shown in Fig.4A, we found that ECPIRM treatment 12

exhibited weak growth inhibition at the early stage of the administration, and exerted obvious inhibitory effects on Hut78-xenografted tumor after 10 days treatment in dose-dependent manner according to the relative tumor volume. The resected xenograft tumor size furtherly determined the inhibitory activity of ECPIRM in Hut78-xenografted model (Fig.4B). Furthermore, ECPIRM treatment did not trigger obvious toxicity in vivo according to the similar nude mice weight of each group

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(Fig.4C). Combined the activity against tumor growth in Hut78-xenografted model,

we detected the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt in resected xenograft tumor tissues using immunohistochemical analysis. As shown in

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Fig.4D-H, we found that ECPIRM treatment significantly induced the expression of

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cleaved caspase-3 in a dose-dependent manner, and the expression of ITK, p-ERK and p-Akt were also greatly decreased after the administration of ECPIRM.

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Collectively, this set of experiments revealed that ECPIRM exhibited obvious

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anti-growth effects via induction of apoptosis and inhibition of ITK-mediated signaling pathway in Hut78-xenografted model, which furtherly demonstrated the

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similar molecular mechanisms in vitro.

4. Discussion CTCL was a heterogeneous group of skin-homing T-cell cancer, which represent about 75% of all the primary cutaneous lymphomas, the majority of which were MF and SS in clinic. The diagnosis of CTCL required the integration of clinical 13

observation and histopathologic verification, and the systemic therapies included biologic-response modifiers, HDAC inhibitors and antibody-based methods [16]. However, the scarcity of biomarkers resulted in complexity of diagnosis and shortage of precise treatment of CTCL. The latest study reported several genes (CCL18, CCL26, FYB, T3JAM, MMP12, LEF1, LCK, ITK, GNLY, IL2RA, IL-26, IL-22, CCR4, GTSF1, SYCP1, STAT5A, TOX) that were able to distinguish patients of

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MF/SS from benign inflammatory dermatoses, which provided important basis for the identification of vital biomarkers in CTCL [12]. Combined our previous work and the

recent study, we demonstrated that ECPIRM exhibited selective anti-proliferation

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activity in Hut78 cells rather than Myla cells and human lymphocytes. Subsequent

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study revealed that the high level of ITK in Hut78 might be the crucial reason for the pharmaceutical mechanism of ECPIRM.

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ITK played crucial roles in the proliferation, maturation and differentiation of T

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cells [13], which was highly expressed in T-cell ALL and T cell lymphoma compared to other cancer types (Fig.2A). The researchers identified a compound CTA191

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through the drug docking screening, and found that CTA191 induced cell death by inhibition of the phosphorylation of ITK and its downstream kinase IkB in CTCL cell

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line CRL2105 and HTB176 cells [17]. Here, our study revealed that targeted inhibition of ITK resulted in cell apoptosis. Importantly, ECPIRM efficiently binded the hydrophobic active pocket of ITK protein and inhibited the mRNA and protein level of ITK, which might be the important reason for the selective activity of ECPIRM in Hut78 cells. Furthermore, the activation of T-cell receptor signaling in 14

T-cell lymphomas facilitated the resistance to chemotherapy via ITK/NF-κB/GATA-3 axis [18], which prompted that ECPIRM might be an ideal agent for the reversal of drug resistance by the inhibition of ITK-mediated signaling pathway. ECPIRM was chemically synthesized from isotretinoin, and exhibited anti-proliferation effects on cutaneous squamous carcinoma cells and CTCL cells, and exerted no significant skin stimulation compared with all-trans retinoid acid, which

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attributed to the independence of RAR/RXR activation [10, 11]. As reported, JAK/STAT3 signaling pathway was inhibited by ECPIRM treatment in Hut78 cells

[10]. Coincidentally, the study revealed that ITK fused with protein tyrosine kinase

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(PTK) triggered the activation of tyrosine phosphorylation events in Peripheral T Cell

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Lymphoma (PTCL)，and the downstream target was explored and STAT3 was found to be highly phosphorylated [19]. Hence, these clues suggested that targeted inhibition

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of ITK by ECPIRM treatment might be the upstream factor for the inhibition of

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JAK/STAT3 signaling pathway.

In clinic, the side effects triggered by retinoids seriously affected the continuous

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application of retinoids [8, 9]. In our previous study, we found that ATRA cream induced obvious skin irritation reactions in BALB/c mice after the topical treatment

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for 2 days, such as erythema and desquamation, and the degree peaked at the fifth day after treatment．However，neither erythema nor desquamation was observed in BALB/c mice during 21 days treatment with 0.075%ECPIRM gel [20]. Furthermore, as shown in Fig.4C, we found that ECPIRM treatment did not trigger obvious toxicity in vivo according to the similar nude mice weight of each group. 15

In summary, our present work demonstrated that ECPIRM exhibited selective anti-proliferation activity in Hut78 cells in vitro via the targeted inhibition of ITK-mediated signaling pathway. In addition, ECPIRM efficiently combined with the protein structure of ITK by virtual docking using Autodock Vina and Free Maestro of Schrodinger software. Furthermore, in vivo study revealed that ECPIRM suppressed tumor growth in Hut78-xenografted model, and induced apoptosis and inhibited

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ITK-mediated signaling pathway in tumor tissues, which was consistent with in vitro

study. In conclusion, ECPIRM might provide a novel strategy for the precision

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treatment of CTCL by the targeted inhibition of ITK.

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Conflicts of interest

Acknowledgements

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The authors declare no conflict of interest.

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Our research was financially supported by the Natural Science Foundation of Jiangsu Province (BK20180157) and supported by the Fundamental Research Funds

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for the Central Universities (3332019105) and the National Natural Science

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Foundation of China (no. 81602788). We are very grateful for the funding from the above organizations.

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[17] I. Bustos-Villalobos, J.W. Bergstrom, N.E. Haigh, J.I. Luna, A. Mitra, A.I. Marusina, A.A. Merleev, E.A. Wang, A. Sukhov, H. Sultani, R. Liu, G. Bhardwaj,

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W. Guo, H.J. Kung, K.S. Lam, E. Maverakis, ITK inhibition for the targeted treatment of CTCL, J Dermatol Sci 87(1) (2017) 88-91. [18] T. Wang, Y. Lu, A. Polk, P. Chowdhury, C. Murga-Zamalloa, H. Fujiwara, K.

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Suemori, N. Beyersdorf, A.C. Hristov, M.S. Lim, N.G. Bailey, R.A. Wilcox, T-cell Receptor Signaling Activates an ITK/NF-kappaB/GATA-3 axis in T-cell

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Lymphomas Facilitating Resistance to Chemotherapy, Clin Cancer Res 23(10) (2017) 2506-2515.

[19] N.N. Fathi, D.K. Mohammad, A. Gorgens, S.E. Andaloussi, R. Zain, B.F. Nore, C.I.E. Smith, Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression, Biochem Biophys Res Commun 504(4) (2018) 749-752. [20] M. Zhang, J. Wei, P. Ma, L. Li, K. Qian, L. Tao, Effect of a tretinoin derivative 18

ECPIRM on retinoic acid receptors and skin irritation response to it in mice,

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Chin J Dermatol 49(6) (2016) 420-424.

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Figure Legends Fig.1. ECPIRM selectively suppressed cell proliferation and inhibited ITK expression in Hut78 cells. (A) The chemical structure of ECPIRM (C29H33O3N, molecular weight 443.59). (B) CCK-8 proliferation assay was used to detect the proliferation of Hut78, MJ and Myla cells after treatment with different concentrations of ECPIRM for 72 h. (C) The cell viability of Hut78, MJ and Myla

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cells after the treatment with indicated concentrations of ECPIRM were shown. (D)

Hut78 cells were treated with 10 μM ECPIRM for 72 h, and the mRNA levels of CTCL-specific genes in Hut78 cells were detected by RT-qPCR analysis. (E). The

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fold change of ITK mRNA expression after ECPIRM treatment were shown. The

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comparison of the relative mRNA level (F) and protein level (G) of ITK were shown in these cells. The data shown were expressed as mean ± S.D. using Student's t-test

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(two-tailed). *indicates the significant difference compared with control. *P＜0.05,

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**P＜0.01 and *** P＜0.001.

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Fig.2. Targeted inhibition of ITK triggered cell apoptosis in Hut78 cells. (A) ITK was highly expressed in T-cell ALL and T cell lymphoma compared to other cancer types. Data was obtained through the Cancer Cell Line Encyclopedia (CCLE) project (https://portals.broadinstitute.org/ccle/home). (B). CCK-8 assay was used to detect the 21

proliferation of Hut78 cells after treatment with different concentrations of ITK inhibitor BMS-509744 for 72 h. (C). The apoptosis detection of BMS-509744 in Hut78 cells using Annexin V and PI staining. (D). Hut78 cells were transfected by ITK siRNAs using Lipofectamine RNAiMAX Reagent for 48 h, and the expression of ITK was confirmed by western blotting analysis. (E). Deletion of ITK by siRNAs

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triggered cell apoptosis in Hut78 cells.

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Fig.3. ECPIRM efficiently combined ITK and inhibited ITK-mediated signaling pathway. ITK (PDB: 4PQN) was docked with ECPIRM using Autodock vina software, and the lowest binding position (-9.0 kcal/mol) was selected. (A). The surface figure of ECPIRM filled in the hydrophobic active pocket of ITK protein was shown. (B). The amino acid of ITK interacted with ECPIRM formed hydrogen bond interaction. (C). Hut78 cells were treated with indicated concentrations of ECPIRM

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for 72 h, and cell samples were collected for western blotting analysis for ITK, ERK, p-ERK, Akt and p-Akt. (D). The histograms showed the relative protein level of each protein. (E). Hut78 cells were treated with indicated concentrations of bexarotene for

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72 h, and cell samples were collected for western blotting analysis for ITK. (F). The

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histograms showed the relative protein level of ITK. The data shown were expressed as mean ± S.D. using Student's t-test (two-tailed). *indicates the significant difference

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and *** P＜0.001.

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compared with control. N.S. showed no significant difference, *P＜0.05, **P＜0.01

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Fig.4. ECPIRM suppressed the tumor growth in Hut78-xenografted nude mice via ITK-mediated signaling pathway. (A). The changes of relative tumor volume was shown during drug administration. (B). The Image of resected xenograft tumor after ECPIRM treatment was shown. (C). The mice weight of each group were shown in the line chart. The tissues from Hut78-xenografted models were analyzed by HE staining (D) and immunohistochemistry analysis of Cleaved caspase 3 (E), ITK (F),

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p-ERK (G) and p-Akt (H), and the histograms showed the integrated optical density of positive staining of each antibody. The data shown were expressed as mean±S.D.

using Student's t-test (two-tailed). *indicates the significant difference compared with

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control. N.S. showed no significant difference, *P＜0.05, **P＜0.01 and *** P＜

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0.001.

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