eIF4A1 Inhibitor Suppresses Hyperactive mTOR-Associated Tumors by Inducing Necroptosis and G2/M Arrest

Aberrantly activated mechanistic target of rapamycin (mTOR) signaling pathway stimulates translation initiation/protein synthesis and eventually causes tumors. Targeting these processes thus holds potential for treating mTOR-associated diseases. We tested the potential of eFT226, a sequence-selective inhibitor of eIF4A-mediated translation, in the treatment of mTOR hyperactive cells caused by the deletion of tuberous sclerosis complex 1/2 (TSC1/2) or phosphatase and TENsin homology (PTEN). eFT226 preferentially inhibited the proliferation of Tsc2- and Pten-deficient cells by inducing necroptosis and G2/M phase arrest. In addition, eFT226 blocked the development of TSC2-deficient tumors. The translation initiation inhibitor is thus a promising regimen for the treatment of hyperactive mTOR-mediated tumors.


Introduction
Hamartoma tumor syndrome can be caused by inactivating mutations of either PTEN, TSC1, or TSC2 [1,2]. As the second most frequently altered tumor suppressor in cancer, the loss of PTEN causes Cowden syndrome and endometrial carcinoma [2,3]. Loss-offunction mutations of TSC1 or TSC2 triggers the tuberous sclerosis complex, a benign tumor syndrome affecting heart, brain, lungs, kidneys, and skin [4]. Deficient TSC1 or TSC2 is also observed in hepatocellular carcinoma (HCC) and bladder cancer [5][6][7]. PTEN and TSC1/2 are major negative regulators of the AKT-mTOR pathway [8,9]. mTOR promotes translation initiation and elongation through the regulation of the eIF4E-binding proteins (4E-BPs), ribosomal protein S6 kinases (S6Ks), and eIF4F (which comprises the cap-binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A) [10]. These events in return control essential cellular processes including cell growth, proliferation, and survival. Inactivating mutations of TSC1/2 or PTEN causes the activation of the mTOR signaling pathway [11,12]. The mTOR inhibitor rapamycin displayed laboratory and clinical benefits for various tumors, such as TSC, bladder cancer, HCC, and endometrial carcinoma [6,[12][13][14][15]. However, rapamycin exerts, primarily, a cytostatic effect. The recurrence of tumors occurred after therapy was discontinued, rendering rapamycin a lifelong therapy [16]. Besides, there are rapamycin-associated cumulative toxicities and adverse effects, including stomatitis, wound healing complications, glucose intolerance, and hyperlipidemia [17][18][19]. Given that hyperactive mTOR often leads to the dysregulation of protein synthesis, targeting cap-dependent translation is therefore an attractive cancer therapy [20][21][22]. eFT226 (Zotatifin) is the first eIF4A inhibitor to enter human clinical trials [23]. It promotes eIF4A binding to specific mRNA sequences with recognition motifs in the 5 -UTRs and interferes with the assembly of the eIF4F complex downstream of mTOR [24]. Its sensitivity correlates with the mTOR-mediated activation of eIF4A [25]. The main purpose of this study is to investigate the potential therapeutic effects and underlying mechanisms of eFT226 on Tsc2and Pten-deficient tumors. We showed that Tsc2and Pten-deficient mouse embryo fibroblasts (MEFs) are more sensitive to eFT226. eFT226 suppressed their proliferation by inducing necroptosis and G2/M phase arrest, and blocked growth of Tsc2-deficient tumors. The specific inhibition of RIPK1 signaling with necrostatin-1(Nec-1) partially reversed the suppressive effect of eFT226. These data suggest that eFT226 holds promise as a new strategy for tumors with deficient TSC1/2 or PTEN.

Flow Cytometry
AnnexinV-647 and PI detection kit (Yeasen, Shanghai, China) was used to detect cell death. Cells were collected and washed with PBS and then resuspended in 1× binding buffer. Cells suspension (100 µL) was then transferred to a 5-mL culture tube and stained with 5 µL Annexin V-647 and 10 µL propidium iodide (PI) for 15 min at room temperature in dark. After addition of 200 µL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Cell Cycle Analysis Kit (Yeasen, China) was used to analyze DNA content and cell cycle profile. The cells were collected and washed with PBS and then placed in 70% ethanol overnight. Cell suspension (100 µL) was then transferred to a 5-mL culture tube and stained with 10 µL PI and 10 µL RNase for 30 min at room temperature in dark. After addition of 200 µL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Intracellular ROS generation was estimated using DCFH-DA (Yeasen, China). After cells were incubated with 10 µM DCFH-DA for 30 min and washed with PBS, measurements were performed using Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The ROS level is proportional to the mean fluorescence intensity (MFI) of a fluorescent probe DCFH-DA.

Real-Time Quantitative PCR
Total RNA was extracted from cells using Trizol (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. RNA was reversely transcribed using PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) in a total volume of 20 µL reaction, a total of 1 µg mRNA was converted into complementary DNA (cDNA). β-actin was used as internal control. Amplification was using BlastTaq TM 2 × qPCR MasterMix (Applied Biological Materials, Canada. The conditions for qRT-PCR were as follows: pre-denaturation at 95 • C for 5 min, then denaturation at 95 • C for 15 s and annealing at 60 • C for 30 s. The primers used were as follows: β-Actin forward 5 -AGAGGGAAATCGTGCGTGAC-3 reverse 5 -CAATAGTGATGACCTGGCCGT-3 ; RIPK3 forward 5 -GAGATGGAAGACACGGCACT-3 reverse 5 -GGTGGTGCTACCAAGGAGTT-3 ;

Cell Viability Assay
Cell viability was determined using CCK8 cell counting kit (Yeasen, China). Cells were seeded into 96-well plate at a density of 2 × 10 4 cells/well and were treated with various concentrations of eFT226 for dedicated time, cells were then incubated with 10 µL of CCK8 per well for 2 h, and the absorbance at 450 nm was measured with a microplate reader (Multiskan MK3; Thermo Fisher, Waltham, MA, USA).

Colony Formation Assay
Cells (500 cells/well) were seeded into 6-well plates and then treated with eFT226, DMSO was used as the control. After 2 weeks, the numbers of colonies were counted under a light microscope. Images of the colonies were captured using a camera.

Animal Experiments
A total of 26 healthy female BALB/c nude mice aged 5-6 weeks were purchased from Beijing HFK Bioscience (Beijing, China), and all animals were housed at the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (22-24 • C, 40-60% relative humidity), food and water were freely available, and the light/dark-cycle was maintained for 12/12 h, with the light turned on at 6:00 am. For the Tsc2-deficient tumors, one million NTC/T2 deficient (Tsc2 −/− , Tp53 −/− ) cells [29] and two million PLC/PRF/5 cells were inoculated into the right flanks of the mice, respectively. When the tumor size reached 80-100 mm 3 , the mice were randomly divided into four groups using random number table: control group (5% dextrose in water) (n = 8 for Tsc2 −/− , Tp53 −/− MEFs, n = 5 for PLC/PRF/5 cells) and eFT226 group (1 mg/kg) (n = 8 for Tsc2 −/− , Tp53 −/− MEFs, n = 5 for PLC/PRF/5 cells), respectively, once weekly for three weeks. The mice were weighed every other day. Then the mice were sacrificed, and the tumors were collected.

Histology Study
Tumor samples harvested from tumor-bearing nude mice were fixed in formalin, sectioned, and stained with hematoxylin and eosin (H&E) following the standard protocols. Immunohistochemistry for detection of Ki67 was performed according to standard protocols.

Statistical Analysis
GraphPad Prism V.8 was used for statistical analysis (San Diego, CA, USA). Data are presented as the means ± SD. The unpaired Student's t-test was used for the comparison between two groups. One-way ANOVA was used for comparing multiple groups of quantitative data. (* p < 0.05, ** p < 0.01, *** p < 0.001). All data are representative of at least three independent experiments.

Tsc2-and Pten-Deficient Cells Are More Sensitive to eFT226 Treatment
Since the hyperactivation of mTOR and translation initiation factors were presented in Tsc2and Pten-deficient MEFs ( Figure 1A), we examined the effect of eFT226 on the viability of mTOR-activated cells. Tsc2-deficient MEFs were more sensitive to rapamycin as previously reported [30,31] (Figure 1B). Biochemical analysis shows that eFT226 ( Figure 1C) [23], a selective eIF4A1 inhibitor, preferentially attenuated the proliferation and colony formation assay of Tsc2and Pten-deficient MEFs ( Figure 1D-G). These findings suggested that Tsc2and Pten-deficient cells are more sensitive to eFT226 treatment.

eFT226 Suppresses Tsc2-Deficient Tumor Growth
The tumor-bearing nude mouse model is a widely used pre-clinical model for the screening of drugs in vivo [29]. The NTC/T2 deficient cell line was reported to induce Tsc2 −/− tumors in nude mice [29]. To check the efficacy of eFT226 in hyperactive mTORassociated tumors, we first treated nude mice bearing Tsc2-deficient tumors with 1 mg/kg eFT226 once per week. eFT226 blocked tumor growth, as indicated by a reduced tumor volume and weight in the treatment group ( Figure 8A-C), without an effect on the body weight of mice ( Figure 8D). Besides, H&E staining showed that the eFT226 group displayed necrotic areas infiltrated with inflammatory cells, and immunohistochemistry staining displayed that the Ki-67 expression level, a cell-proliferation marker, was down-regulated in the eFT226 group ( Figure 8E). In addition, a PLC/PRF/5 xenograft mouse model was also established. As shown in Figure 5, eFT226 dosing for three weeks suppressed tumor volume and weight in PLC/PRF/5 cell-bearing mice ( Figure 9A-C). There was a slight decrease in the body weight of the eFT226-treated group compared to the vehicletreated group ( Figure 9D). Overall, these results indicate that eFT226 hinders Tsc2-deficient tumor growth.

Discussion
Activating mutations of proto-oncogenes such as EGFR, PI3K, and AKT as well as inactivating mutations of tumor suppressor PTEN or TSC1/2 causes the activation of the mTOR signaling pathway in a wide range of cancers [11,12]. However, the mTOR inhibitor sirolimus (rapamycin) is cytostatic but not cytotoxic. Although rapamycin achieved limited success in a few tumor syndromes such as TSC [36], most of the tumors are refractory to rapamycin treatment. Better or alternative regimens are therefore sought after. mTORmediated translation initiation is augmented in many cancers. The antineoplastic activity of eFT226, an inhibitor of the translation initiation factor eIF4A1, has been reported in a variety of cancer cells [25], eFT226 recently became the first rocaglate to enter clinical evaluation for advanced solid tumors in humans (ClinicalTrials.gov: NCT04092673) [23]. In the present study, Tsc2and Pten-deficient cells were more sensitive than WT cells to eFT226 treatment. eFT226 significantly blocked tumor growth with limited drug toxicity in nude mice bearing Tsc2-deficient MEFs and the HCC cells PLC/PRF/5. eFT226 inhibits translation initiation through forming a ternary complex with eIF4A and AGAGAG polypurine RNA oligonucleotides, preventing eIF4A1 releasing from the polypurine RNA motif [23,37,38]. In our study, eFT226 inhibits the eIF4F complex of Tsc2and Pten-deficient MEFs without affecting the expression of eIF4A.
Cell death and the cell cycle function in a coordinated manner. Cell cycle arrest induces RIP3 phosphorylation and enhances necroptosis [39] The cyclinB1/CDK1 complex is the essential player in G2/M transition. eFT226 induced G2/M arrest, presumably by downregulating Cdc25c, CDK1, and cyclinB1 in our study. The G2/M phase arrest caused by eFT226 in Tsc2and Pten-deficient MEFs could be partially attenuated by Nec-1. eFT226 exerted broad anti-tumor activity across different cancer cell lines via the induction of apoptosis [23,40]. In this study, eFT226 induced necroptosis by upregulating the expressions of RIPK1, RIPK3, and MLKL in Tsc2and Pten-deficient MEFs. Nec-1 blunted eFT226induced necroptosis and the elevation of RIPK3 and p-MLK. Even though we do not know the reason contributed to the discrepancy between previous reports and our study, eFT226mediated apoptosis or necroptosis may be cell type-dependent. Nevertheless, eFT226 may be a novel regimen for the treatment of common cancers such as endometrial cancer and glioblastoma and rare diseases such as TSC and Cowden Syndrome. mTOR inhibition by rapamycin and rapalogs mainly accumulates cells in the G1 phase of the cell cycle [41], while eFT226 induces G2/M phase arrest [23]. CDKs are master regulators of cell division and their inhibitors block cells at different stages of the cell cycle [42]. It is of interest to test whether combinations of mTOR inhibitors, CDK inhibitors, and translation initiation factor inhibitors trigger a durable cell cycle arrest for the treatment of cancer cells.
In conclusion, eFT226 preferentially inhibited Tsc2and Pten-deficient cells by inducing necroptosis and G2/M arrest. In addition, eFT226 blocked Tsc2-deficient tumor growth. Inhibiting the activity of the eIF4F complex may represent a cancer vulnerability that could be clinically exploited to overcome chemoresistance and tumor heterogeneity. The present study provides preclinical evidence for the potential clinical application of eFT226 in hyperactive mTOR-associated tumors.