Ethacrynic Oxadiazole 6u Induces Apoptosis by Targeting mTOR/c-Flip Axis and Noxa in Lymphoma Cells

Abstract

Background

Evasion of apoptosis is a major cause of therapy failure in lymphoma. We studied a covalent compound 6u with an α, β-saturated carbonyl group derived from the diuretic drug ethacrynic acid to induce apoptosis of lymphoma cells by targeting antiapoptotic proteins.

Results

We found that 6u induces apoptosis in 4 lymphoma cell lines, Daudi, Romas, Jeko-1 and Jurkat, accompanied by down-regulation of c-Flip and Mcl-1, as well as upregulation of Noxa. The down-regulation of Mcl-1 relies on Noxa induction and that the down-regulation of c-Flip is mediated by inhibiting protein synthesis pathway through covalent binding to mTOR complexes. Overexpression of c-Flip and silencing Noxa attenuate 6u-induced apoptosis. 6u modified with the saturated carbonyl structure loses the mTOR inhibition and apoptosis induction abilities. 6u exhibits potent anti-lymphoma effects in mantle cell lymphoma Jeko-1 xenografts without causing toxicity.

Conclusion

6u functions as an apoptosis inducer through the α, β-saturated carbonyl group to decrease c-Flip and to induce Noxa. 6u represents a new type of covalent agent for targeting lymphoma by inducing apoptosis.

Introduction

Ethacrynic acid (EA) is a clinical used diuretic drug with glutathione s-transferase P1-1 (GSTP1-1) inhibition ability, and is reproposing as an anticancer agent [1]. The α, β-unsaturated carbonyl moiety is the functional group of EA for inhibiting GSTP1-1 activity and cell growth [2]. To improve its antitumor activity, we have done structural modifications through esterification and incorporation of a heterocyclic oxadiazole or a thiazol group [3–5]. These compounds contain the α, β-unsaturated carbonyl moiety with increased cell growth inhibition and apoptosis induction abilities.

Previously mechanistic studies revealed that these compounds inhibited cell growth through GSTP1-1 inhibition-independent pathways. By focusing on one of the EA esters, ethacrynic acid butyl-ester (EABE), we found that it induces apoptosis through reactive oxygen species (ROS) production [2]. However, we found that EABE enhances arsenic trioxide (ATO)-induced apoptosis in leukemia and lymphoma cells through inhibiting the GSTP1-1 activity [6]. The EA oxadiazole analogues are more effective than EABE and EA to induce apoptosis [4]. By focusing on two ethacrynic oxadiazole analogues, 6s and 6u, we found that both compounds exhibited similar apoptosis induction abilities in HL-60 cells. Although both compounds contain the α-, β-unsaturated carbonyl moiety, 6u does not inhibit GSTP1-1 activity [4]. However the apoptosis induction abilities of both compounds are attenuated by the overexpression expression of GSTP1-1 [7]. The cell lines without GSTP1-1 expression are much more responsive to both compounds. Further mechanistic studies revealed that the apoptosis induction was correlated with the downregulation of antiapoptotic proteins c-Flip, Mcl-1, and XIAP [7]. Our results supported that EA and its analogues function as antitumor agents through the α, β-unsaturated carbonyl group.

The α, β-unsaturated carbonyl group can form covalent bonds with cysteine proteins through mannish reaction [8]. Although covalent agents through this type of reaction have limitation for drug development, FDA has approved several covalent tyrosine kinase inhibitors with acrylamide group for cancer therapy [9]. The proteins bond by the α, β-unsaturated carbonyl moiety of EA have not be identified before. Previously we have found that EA analogues decreased the protein levels of c-Flip, Mcl-1, and XIAP with a short-life [7]. These protein levels are controlled by mTOR translation signaling pathways. The mTOR regulates cell proliferation and survival through forming two complexes with Raptor (mTOR complex 1; mTORC1) or Rictor (mTOR complex2; mTORC2), to activate p-P70S6K and p-S6 and to increase protein translation [10], which could be the potential EA analogue’s targets. In this study we focus on 6u to investigate its abilities of apoptosis induction in lymphoma cell lines without GSTP1-1 and to explore the potential binding proteins. We found that 6u binds to mTOR/Raptor/Rictor through the β-unsaturated carbonyl group and exhibits in vivo anti-lymphoma activity with accepted toxicity. 6u represents a new type of covalent compounds for lymphoma therapy.

Materials and Methods

Reagents

6u, E2 and 6u-Bio were synthesized and the chemical structures were confirmed with magnetic resonance (MR) spectroscopy. Ibrutinib (IBN), Rapamycin and AZD8055 were obtained from MedChemExpress (MCE). The general caspase inhibitor Q-VD-Oph and cycloheximide (CHX) were obtained from Selleckchem Co. (Houston, TX). Antibodies to PARP were obtained from BD Bioscience; to c-Flip from ENZO Life Sciences; and to Mcl-1, GSTP1-1, Bcl-2 and β-actin were from Santa Cruz Biotechnology, Inc. The antibodies to Puma, Bim, Bax, Bak, mTOR and p-mTOR (Ser2448) were from Cell Signaling Technology, Inc. Noxa was from Abcam, Inc (Cambridge, MA). p-P70S6K (Thr389), p-S6, Rictor and Raptor were from Proteintech Group, Inc. Streptavidin-HRP were obtained from Cell Signaling Technology. Noxa siRNA were obtained from Thermo Fisher Scientific.

Cell Lines

Human lymphoma leukemia Daudi, Jurkat and Romas cells obtained from ATCC (Rockville, MD) and Jeko-1 cells from National collection of Authenticated Cell Culture, Shanghai were cultured in RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mmol/L L-glutamine, and 10% (v/v) heat-inactivated heat inactivated fetal bovine serum (FBS).

Establishment of c-Flip Overexpression Cells

Jeko-1 cells were transfected using electroporation in 100 μL of nucleofector solution (Lonza Nucleofector Kit) with 2.0 μg pcDNA3.1-flag-Vector or 2.0 μg pcDNA3.1-flag-FLIP (HonorGene) and plated in 6-well plates with 2 mL supplemented RPMI-1640 medium. After the initial transfection for 48 h, the cells were treated with 1.0 mg/mL G418 to select the c-Flip expression cells. The expression of the transfected c-Flip was confirmed using Western blot probed with the anti-Flag and anti-Flip antibodies.

Biotinylated Compound Binding Assay

Daudi cells were lysed in NP40 lysis buffer, and the lysates were incubated with 20, 40, or 80 μM Biotin-labeled 6u (6u-Bio) for 1 h at room temperature. Proteins were separated by SDS-PAGE, transferred onto a membrane, and then probed with Streptavidin-HRP.

NeutrAvidin Magnetic Beads Pull-down Assay

Daudi cells lysed in NP40 lysis buffer was prepared and incubated with 80 μM 6u-Bio for 1 h at room temperature, respectively. Then 20 μL of NeutrAvidin magnetic beads were added and incubated at 4 ℃ overnight. The mixture was washed by 1% NP40 buffer and added loading buffer then heated at 98 ℃. SDS-PAGE electrophoresis was performed and probed with the relative antibody.

Proteomic Analysis

Proteins pulled-down with NeutrAvidin magnetic beads from lysate of Daudi cells treated with 6u-Bio were enzymatically digested into peptide segments. The peptide segments were separated using reversed-phase high performance liquid chromatography (RP-HPLC). Identification and quantification of proteins were analyzed through MR spectrometry. KEGG analysis was performed using BMKCloud (www.biocloud.net).

Quantitation of Apoptotic Cells, Western Blot Analysis, Immunoprecipitation (IP), and siRNA Knock-down were Done as we Reported Before [6]

Xenografts

These experiments were conducted in accordance with the requirement of animal experiment ethics committee of Shenyang Pharmaceutical University. NOD/SCID mice (8 weeks, Beijing Weitonglihua) were injected with 5 × 10⁶ Jeko-1 cells subcutaneously in the right flank. Mice with tumors of about 100 mm³ were randomized to four groups of five mice each. Animals were treated with 25 mg/kg IBN, 3 mg/kg 6u, and 6 mg/kg 6u once daily for 12 days. Tumor growth and body weight of mice were monitored daily. Tumor volumes were measured every other day using digital calipers.

Statistical Analysis

The student t-test was used to determine the significance of differences between two groups. One-way ANOVA was used for multiple groups. p < 0.05 was considered to be statistically significant.

Results

6u Inhibits Cell Growth with Arrest in Sub-G1 Phase Independent of GSTP1-1 Expression

6u is one of the EA oxadiazole analogues with the α, β-unsaturated carbonyl group (Fig. 1A). To compare the growth inhibition of 6u, four lymphoma cell lines were used. Among the four cell lines, Daudi does not express GSTP1-1 while other three lines with the GSTP1-1 expression (Fig. 1B). The growth inhibition rates of 6u were tested among the four cell lines after treatment of 72 h. The IG₅₀ of 6u in Daudi, Romas, Jeko-1 and Jurkat cell lines are 0.46 μM, 0.72 μM, 0.87 μM, and 1.40 μM, respectively. The Jurkat cells with the highest GSTP1-1 expression were less sensitive to 6u. The other three lines with or without GSTP1-1 are responsive to 6u at the same levels. Fluorescence-activated cell sorting (FACS) analysis with PI staining revealed that 6u induced Sub-G1 arrest at Daudi, Romas, Jeko-1 and Jurkat cells with the rates of 38.25%, 30.95%, 35.32% and 15.00% at 2 μM treatment for 12 h, respectively (Fig. 1D). These data suggest that 6u inhibits cell growth through cytotoxic effect independent of GSTP1-1 expression.

Fig. 1.

6u inhibits lymphoma cell growth and induces Sub-G1 arrest independent of GSTP1-1 expression. A The structure of 6u. B Basel levels of GSTP1-1 protein of Daudi, Romas, Jurkat and Jeko-1 cells tested by Western blot analysis. C The growth inhibition curves of four cell lines treated by 6u for 72 h. Data shown are the mean ± SD of three independent experiments. D The four cell lines were treated with 6u at the indicated concentrations for 12 h. The Sub-G1 cells were detected by FACS with PI staining. * p <0.05; ** p <0.01; *** p <0.001 compared to the control group by t-test

6u Induces Apoptosis Through Down-regulation of c-Flip and Mcl-1 as well as Upregulation of Noxa

By focusing on two cell lines, Daudi (without GSTP1-1) and Romas (with GSTP1-1), we tested the apoptosis induction. FACS analysis with Annexin V/PI staining assay showed 24.54% and 37.84% of apoptotic cells in Daudi and Romas, respectively, after treatment at 2 μM for 12 h (Fig. 2A). Daudi cells were more sensitive than Romas to 6u at 0.5 and 1 μM. Western blot analysis was used to detect the apoptosis-related protein changes regulated by 6u at 12 h treatment. Among the tested proteins, 6u decreased the levels of Mcl-1 and c-Flip and increased the levels of Noxa, these changes were associated with the PARP cleavage in both cell lines. The levels of Puma, Bim, Bax, Bak and Bcl-2 were not changed after 6u treatment (Fig. 2B). The levels of GSTP1-1 in Romas were not changed. The abilities of 6u decreasing c-Flip and Mcl-1 were attenuated in Romas cells compared with Daudi cells at 1 μM treatment, but there is no difference between the two lines at 2 μM treatment. These data suggest GSTP1-1 has a protective role which can be neglected at the adjusted concentration.

Fig. 2.

6u induces apoptosis with down-regulation of c-Flip and up-regulation of Noxa. A Daudi and Romas cells were treated with 6u at the indicated concentrations for 12 h. Apoptosis cells were measured by FACS with Annexin V/PI staining. B Daudi and Romas were treated with 0.5 μM, 1 μM and 2 μM of 6u for 12 h. The levels of apoptosis related protein were determined with Western Blotting. C Daudi cells were treated with 6u at 2 μM for the indicated time, the levels of PARP cleavage, c-Flip, Mcl-1 and Noxa were measured by Western Blotting. D Daudi cells were pretreated with 25 μM of the caspase pan-inhibitor Q-VD-Oph for 4 h, and then following with 2 μM 6u for 12 h. The protein levels were determined by Western Blotting

To identify the key proteins involved in 6u-induced apoptosis and to exclude the influence of GSTP1-1, we tested the time-dependent changes of proteins in Daudi cells treated with 2 μM 6u for 3 h, 6 h, 9 h and 12 h. PARP was cleaved at 6 h with c-Flip and Mcl-1 down-regulation and Noxa induction (Fig. 2C). We then tested whether the c-Flip and Mcl-1 reduction and Noxa increase are due to activated caspase in apoptotic cells. The general caspase inhibitor Q-VD-OPh blocked the PARP cleavage, but not the down-regulation of c-Flip and Mcl-1 nor the upregulation of Noxa (Fig. 2D). These data suggest that these proteins are regulated by 6u ahead of apoptosis induction.

We then tested the roles of Noxa and c-Flip in triggering apoptosis of 6u treatment. Jeko-1 cells were used to silence Noxa to test its effect on 6u-induced apoptosis. Silencing Noxa attenuated the effects of 6u-induced PARP cleavage and Mcl-1 reduction, but not c-Flip reduction (Fig. 3A). The proportion of apoptotic cells following 6u treatment were decreased from 59.13% to 9.26% after treatment with 2 μM 6u (Fig. 3B). To test the role of c-Flip, we generated Jeko-1 cells transfected c-Flip with an expression vector. Western blot showed that the c-Flip levels were significantly increased compared with the empty vector transfected cells. The PARP cleavage and Mcl-1 reduction, but not Noxa induction, were blocked in Jeko-1/c-Flip cells treated with 6u (Fig. 3C). The proportion of apoptotic cells following 6u treatment were attenuated from 40.85% to 10.51% with overexpression of c-Flip (Fig. 3D). These findings support that both c-Flip and Noxa are important factors for 6u-induced apoptosis.

Fig. 3.

c-Flip and Noxa are the key factors to control 6u-induced apoptosis. A Jeko-1 cells were transfected with control siRNA and Noxa siRNA for 18 h and then treated with 2 μM 6u for 12 h. The protein levels were detected by Western blot. B Apoptotic cells of Jeko-1 cells transfected with siRNA/Con or siRNA/Noxa determined by FACS analysis with Annexin V/PI staining. C Jeko-1 cells with overexpression of c-Flip were established. Jeko-1/Vector and Jeko-1/c-Flip cells were treated with 6u for 12 h and the protein levels were measured with Western blot analysis. D Apoptotic cells of Jeko-1/Vector and Jeko-1/c-Flip cells treated with 6u at 2 μM for 12 h were determined by FACS with Annexin V/PI staining

6u Decreases c-Flip Through Inhibiting mTOR-mediated Protein Synthetic Pathways

To determine the mechanism of c-Flip reduction, we first examined the protein degradation rate after treatment with CHX, a protein synthesis inhibitor, in Daudi cells. 6u decreased the levels of c-Flip at the same rates in presence with or without CHX (Fig. 4A, B). The proteasome inhibitor MG132 did not block the down-regulation of c-Flip in Jeko-1 cells treated with 6u (Fig. 4C). These data indicate that 6u does not accelerate the c-Flip protein degradation. The c-Flip is a short-life protein and its protein level is maintained by mTOR signaling pathway [11, 12]. To investigate the role of mTOR signaling in 6u treatment, we compared the inhibition of 6u on the key factors of mTOR signaling with the mTORC1 inhibitor Rapamycin and the mTORC1/mTORC2 inhibitor AZAD8055 [13]. Western blot analysis showed that the levels of p-mTOR, p-P70S6K, p-S6 and c-Flip were reduced by all the three compounds. However, RARP cleavage was observed in 6u and AZAD8055 treated cells, but not in Rapamycin treated cells. Meanwhile the Noxa was induced by 6u, but not by Rapamycin and AZD8055 (Fig. 4D). FACS analysis with Annexin V/PI staining showed 24.84%, 9.27% and 15.58% of apoptotic cells after treatment with 1 μM 6u, Rapamycin and AZD8055, respectively (Fig. 4E). These data suggest that 6u functions as an mTOR inhibitor to inhibit c-Flip protein synthesis. The ability of Noxa induction by 6u makes it as a more effective apoptosis inducer than the selective mTOR inhibitors, Rapamycin and AZD8055. To confirm the effects of 6u on mTOR and Noxa are caused by the α-, β-unsaturated carbonyl moiety, we synthesized an analogue E2 with the α, β-saturated carbonyl structure (Fig. 4F). Western blot analysis showed that E2 lost the abilities of inhibiting mTOR signaling members without inducing PARP cleavage and c-Flip downregulation (Fig. 4G).

Fig. 4.

6u decreases c-Flip though inhibiting the mTOR signaling pathway. A Daudi cells were treated with CHX in the presence or absence of 2 μM 6u for the indicated times. The protein levels were analyzed with Western blot analysis. B The density of the protein bands were quantified by Image J software and the protein decrease curves were drawn. C Jeko-1 cells were pretreated with 10 μM MG132 for 2 h and then following 1 μM 6u treatment for 6 h. The c-Flip levels were determined with Western blot analysis. D Daudi cells were treated with 1 μM 6u, Rapamycin and AZD8055 for 12 h. The protein levels were measured by Western blot analysis. E Apoptosis cells were measured by FACS with Annexin Ⅴ/PI staining. F The structure of E2. G Daudi cells were treated with E2 for 12 h. The related protein levels were measured by Western blot

6u Inhibits mTOR Activation by Interrupting the Complexes with Raptor and Rictor

Although we found that 6u inhibits the downstream signaling pathway, it does not inhibit the mTOR enzyme activity through the pure enzyme inhibition assay (data not shown). Since E2 with the α, β-saturated carbonyl structure lost the mTOR inhibition ability, we thought the inhibition of mTOR resulting from the covalent binding. We then synthesized the biotin tagged 6u (6u-Bio, Fig. 5A). To determine if 6u-Bio binds to proteins, Daudi cell lysates were mixed with 6u-Bio at concentrations of 0, 20, 40 and 80 μM. The mixture was separated with Western blot analysis and probed with Streptavidin-HRP. The Western blot results showed that 6u-Bio binds to various proteins (Fig. 5B). The proteins bond to 6u-Bio were then pulled with NeutrAvidin beads and subjected with Proteomics analysis. We identified mTOR, MAPK3 and other proteins bond to 6u-Bio. KEGG pathway enrichment analysis revealed the enriched signaling including oxidative phosphorylation, PI3K, mTOR signaling pathways and apoptosis pathway. The highly enriched signalings are oxidative phosphorylation and mTOR pathways (Fig. 5C). The binding of 6u-Bio to mTOR, Rictor and Raptor was confirmed using NeutrAvidin pulled down proteins by Western blot analysis (Fig. 5D). We then used 6u to treat Daudi cells at the 2 μM and did IP with mTOR antibody. 6u interrupted the interaction of mTOR with Raptor and Rictor (Fig. 5E). These data support that α, β-unsaturated carbonyl structure of 6u bind to the mTORC1/2 complex and separated Raptor and Rictor to inhibit mTORC1/2 activities to inhibit protein synthesis and proliferation (Fig. 5F).

Fig. 5.

6u binds to mTOR and interrupts the complex. A The structure of biotin-tagged 6u (6u-Bio). B 6u-Bio was incubated with Daudi cell lysate for Western blot analysis and probed with Streptavidin-HRP. C Proteins pulled-down with NeutrAvidin beads were analyzed using MR. The KEGG analysis showing the protein pathways enriched by 6u-Bio. D Proteins pulled-down with NeutrAvidin beads were analyzed with Western blot and probed with mTOR, Rictor and Raptor antibodies. E Daudi cells were treated with 2 μM 6u for 12 h. Cell lysates were prepared and immunoprecipitated with mTOR antibody, and probed with mTOR, Rictor and Raptor antibodies. F The potential mechanism of 6u targeting mTOR complex and protein synthesis

6u Inhibits Lymphoma Growth in Xenografts

To explore the in vivo anti-tumor effects of 6u, we inoculated Jeko-1 cells into NOD-SCID mice subcutaneously. The mice were treated with 6u (3 mg/kg), 6u (6 mg/kg) and IBN (25 mg/kg) daily for 12 days. Ibrutinib (IBN), an BTK inhibitor used to treat B-cell lymphoma, was used as a positive control. The tumor images and weights at the end of experiment were shown in Fig. 6A & B. The suppression rates of tumor treated with 6u (6 mg/kg), 6u (3 mg/kg) and IBN (25 mg/kg) were 73.63%, 34.75%, 29.88%, respectively. The tumor growth rates in mice treated with different drugs showed that 6u is more active than IBN (Fig. 6C). The body weights of mice did not change during the treatment (Fig. 6D). There is no toxic sign observed in mouse organs treated with 6u based on Hematoxylin–Eosin (HE) staining analysis (Fig. 6E).

Fig. 6.

6u inhibits the growth of lymphoma xenografts. A Tumor image dissected from mice treated with 25 mg/kg IBN, 3 mg/kg 6u, and 6 mg/kg 6u for 12 days. B The tumor weights. C The tumor growth rates of Jeko-1 cells in mice during the treatments. D Body weights of mice during treatment procedure. E The tissue H&E staining of organs from mice treated with 6u and IBN. Scale bars, 500 μm. * p < 0.05; ** p < 0.01 compared to the control group by t-test. # p < 0.05; ## p < 0.01 by two-way ANOVA test

Discussion

Lymphoma is a heterogenic disease with different genetic alterations. Chemotherapy is still the main therapy for the most types of lymphomas. Evasion of the apoptosis is the mechanism of therapy resistance of lymphoma [14]. The apoptosis process is mainly controlled by the antiapoptotic Bcl-2 family proteins Bcl-2, Mcl-1 and Bcl-xL in mitochondrial-mediated pathway and the c-Flip protein in death-receptor-mediated pathway [15, 16]. These proteins have been used as targets for developing therapeutic agents. So far only the Bcl-2 inhibitor venetoclax approved for Chronic Lymphocytic Leukemia (CLL) therapy with limited effects in lymphoma patients probably due to expression of other proteins such as Mcl-1 and/or Bcl-xL [17]. Targeting Mcl-1 is not successful due to the toxicity [18]. Inhibitors targeting c-Flip have not been reported before. Here we reported a new compound 6u to target c-Flip through inhibiting its protein synthesis and Mcl-1 through inducing Noxa.

6u is an analogue derived from EA, a covalent compound used as a diuretic drug in clinic. EA contains an α, β-unsaturated ketone structure with GSTP1-1 inhibition ability and has been used to overcome resistance of chemotherapeutic agents by inhibiting GSTP1-1 [19]. EA also exhibited weak antiproliferative activity in tumor cells. The GSTP1-1 and antitumor inhibition abilities can be separated by glutathione conjugation. The EA-glutathione conjugation inhibits GSTP1-1 activity and loses the antitumor effects [20]. Therefore the α, β-unsaturated ketone is the functional group of EA to mediate the cell growth inhibition [3]. To improve antitumor activity of EA, we did structure modification remaining the α, β-unsaturated ketone moiety and obtained EA oxadiazol analogue 6u. 6u is tenfold more active to inhibit HL-60 cell growth than EA without GSTP1-1 activity inhibition [4]. However, the antitumor effect of 6u could be attenuated by overexpressed GSTP1-1 [7]. Here we found that Jurkat line with the highest GSTP1-1 expression was the least responsive and that the Daudi line without GSTP1-1 was the most responsive to 6u. We also found that 6u with the saturated ketone structure loses cell growth inhibition ability in Daudi cells. Our data support that the α, β-unsaturated ketone moiety is the functional group of 6u to inhibit lymphoma growth independent of GSTP1-1 inhibition.

We focused on Daudi cells without GSTP1-1 expression to study the mechanism of 6u to inhibit cell growth. 6u exhibited cytotoxic effects with Sub-G1 arrest and apoptosis induction. We found three factors correlated with 6u-induced apoptosis, downregulation of c-Flip and Mcl-1, as well as induction of Noxa. Further study revealed that the down-regulation of Mcl-1 is mediated through Noxa induction, which leads to Mcl-1 degradation. Noxa can be induced through endoplasmic reticulum and c-Jun N-terminal kinase (JNK) activation [21]. Previously we reported that EA derivatives induced apoptosis through ROS-mediated JNK [2], which could lead to Noxa induction and Mcl-1 degradation reduction.

The c-Flip is a key factor to block death receptor-mediated apoptosis pathway [22]. It has been reported that high expression levels of c-Flip predict poor prognosis in lymphoma cells [23]. We found that overexpression of c-Flip could completely block 6u-induced apoptosis. These data support that the down-regulation of c-Flip plays an important role in 6u-induced apoptosis. We found that 6u did not promote c-Flip protein degradation, rather inhibited its protein synthesis. Further studies revealed that 6u like mTOR inhibitor Rapamycin and AZD8055 to inhibit down-stream factors of p-mTOR, p-70S6K and p-S6. However, 6u works differently from Rapamycin and AZD8055 to inhibit mTOR. It works through binding to mTOR, Raptor and Rictor and interrupts the complexes of mTORC1/mTORC2. 6u, but not Rapamycin and AZD8055, induced Noxa with potent apoptosis induction. The dual effects of 6u to inhibit mTOR and to induce Noxa make it more effects than single mTOR inhibitors for lymphoma therapy.

6u contains a special α, β-unsaturated carbonyl moiety which is different from those existed from natural products [24]. Although this structure is thought to be difficult for drug development, the EA with this moiety is already used for diuretic therapy with acceptable toxicity in clinic [9]. We found that 6u significantly inhibits tumor growth of mantle cell lymphoma Jeko-1 cells in vivo without observed toxicity and exhibits more potent effective than BTK inhibitor Ibrutinib. Ibrutinib is also a covalent inhibitor with an acrylamide to bind to BTK cysteine 481 and approved for mantle cell lymphoma [25]. 6u represents a new type of covalent agents to target antiapoptotic proteins for lymphoma therapy.

Authors’ Contributions

Y.K., S.F., J.Z. and Y.J. prepared Figs. 1, 2, 3, 4, 5 and 6; Z.T. and X.B prepared for Fig. 3; Y.W. and P.G prepared for Fig. 5; J.S., Y.H., and G.Z. synthesized the compounds; Y.K., J.Z., S.W. and Y.J. wrote the manuscript; All authors reviewed the manuscript. Y.J. received the funding.

Funding

This work was supported by the National Science Foundation of China (No.82173844), General Project of the Education Department of Liaoning Province (No. JYTMS20231374) and The Samuel Waxman Cancer Research Foundation.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

All animal experiments were approval by the Ethics Committee of Shenyang Pharmaceutical University. (Permit Number: YPU-IACUC-S2023-1129–111).

Consent for Publication

No applicable.

Competing Interests

The authors declare no competing interests.