Breast cancer cells were reported to up-regulate human prolactin receptor (PRLR) to assist their growth through the utilization of prolactin (PRL) as the growth factor, which makes PRLR a potential therapeutic target for breast cancer. On the other hand, advanced cancer cells tend to down-regulate or shed off stress signal proteins to evade immune surveillance and elimination. In this report, we created a fusion protein consisting of the extracellular domain of MHC class I chain-related protein (MICA), a stress signal protein and ligand of the activating receptor NKG2D of natural killer (NK) cells, and G129R, an antagonistic variant of PRL. We hypothesize that the MICA portion of the fusion protein binds to NKG2D to activate NK cells and the G129R portion binds to PRLR on breast cancer cells, so that the activated NK cells will kill the PRLR-positive breast cancer cells. We demonstrated that the MICA-G129R fusion protein not only binds to human natural killer NK-92 cells and PRLR-positive human breast cancer T-47D cells, but also promotes NK cells to release granzyme B and IFN-γ and enhances the cytotoxicity of NK cells specifically on PRLR-positive cells. The fusion protein, therefore, represents a new approach for the development of breast cancer specific immunotherapy. Breast cancer is the most common diagnosed and cause-of-death cancer in women all over the world . Most brNatural killer (NK) cells are a subset of innate cytotoxic lymphocytes serving a critical role in tumor immunosurveillance. NK cells integrate the signals from its activating and inhibitory receptors to target malignant and virus infected cells . The natMany strategies have been developed to target NKG2D receptor or NKG2D ligands in cancer immunotherapy, including up-regulation of NKG2D or NKG2D ligands, grafting NKG2D or NKG2D ligands with antibodies, cytokines, death receptor or signaling domains of activating receptors . For exaIn this study, we created a novel fusion protein consisting the extracellular domain of MICA and the PRL variant G129R. We hypothesized that the fusion protein MICA-G129R will bridge NK cells and PRLR-positive breast cancer cells. When binding to PRLR, the fusion protein labels the breast cancer cells with MICA, which attracts and activates NK cells to kill the breast cancer cells.CCCaagcttGAGAGGGTGGCGACGTCGGGG and the 3’ primer TCTggatccAGAACCACCACCAGAACCACCACCAGAACCACCCCCAGAGGGCACAGGGTG that contained a (GGGS)×3 linker and cloned into vector pcDNA3.1/Zeo(+) . The G129R sequence (c.85 to c.681) without the signal peptide (p.1-28) and stop codon, was amplified by PCR from a pCR3.1_G129R plasmid provided by Dr. Wen Y. Chen as a gift with 5’ primer CCggatccTTGCCCATCTGTCCCGG and the 3’ primer CGCctcgagGCAGTTGTTGTTGTGGATGATT and cloned into vector pLenti7.3 . Then the sequences of G129R with the V5 tag and the 6×His tag in the pLenti7.3 vector were amplified by PCR with the 5’ primer CCggatccTTGCCCATCTGTCCCGG and the 3’ primer TGAgcggccgcTCAATGATGATGATGATGATGACCGGTACGCGTAGAATC containing a stop codon in the 3’ primer and cloned into the vector pcDNA3.1/Zeo(+) at the 3’ end of MICA. The sequence of the fusion gene MICA-G129R with the V5-tag and 6×His tag was confirmed by DNA sequencing.The MICA extracellular domain sequence (c.-23 to c.894) was amplified by PCR from a pcDNA3.1_MICA/IL-12 vector constructed in our lab  with theCCCaagcttGAGAGGGTGGCGACGTCGGGG and the 3’ primer TGAgcggccgcTCACCCAGAGGGCACAGGGTG containing a stop codon; G129R sequence (c.1 to c.684) with signal peptide (p.1-28) and stop codon was directly cut from the pCR3.1_G129R plasmid and inserted into pcDNA3.1/Zeo(+) vector. To make a PRLR expression vector, the full length cDNA sequence of PRLR long isoform was amplified by PCR from a PRLR cDNA plasmid  with 5’ primer tggCTTAAGccaccATGGAGGAAAATGTGGCATCTGC and 3’ primer AAGActcgagTCAGTGAAAGGAGTGTGTAAAACATGC, and cloned into pcDNA3.1(+) . All these inserted sequences in the vectors were confirmed with DNA sequencing.To make control vectors, MICA extracellular domain (c.-23 to c.894) was cloned into the pcDNA3.1/Zeo(+) vector using 5’ primer 2 humidified incubator. All the media, fetal bovine serum and donor horse serum were purchased from Corning ; other supplements were from ThermoFisher Scientific . Two hundred units per ml of recombinant human IL-2 from PeproTech  was used in NK-92 culture.T-47D, NK-92, 293 and HeLa cell lines were obtained from the American Type Culture Collection  and cultured following the methods recommended by ATCC at 37°C in a 5% COAll the cell transfections were carried out using Lipofectamine 2000 (ThermoFisher Scientific) following the instruction of the manufacturer. To obtain stable clones, the transfected cells were cultured in selecting media containing 400 μg/ml of zeocin  for pcDNA3.1/Zeo(+) vector transfection or 500 μg/ml of Geneticin  for pcDNA3.1(+) vector transfection. After selection, 3 to 5 cell clones in each transfection were picked and respectively cultured. The protein expression of the transfected gene was verified with Western blot for each clone. The clone expressing the most protein of the transfected gene was used in the study.6 cells of 293/MICA-G129R stable clone were cultured in a T75 flask with 12 ml of culture media without zeocin. A 50 μL sample of the conditioned media was collected from the culture every 24 hours from day 1 to day 5. The media were centrifuged at 1000 g for 15 minutes, then 40 μl of supernatant was collected and froze at -80°C until examination. Forty microliters of fresh medium were used as the conditioned medium of day 0. For preparation of conditioned media, 4 × 106 of 293 cells without transfection (for control conditioned media), 293/MICA-G129R cells (for MICA-G129R conditioned media), 293/MICA cells (for MICA conditioned media) or 293/G129R cells (for G129R conditioned media) were respectively cultured in 12 ml culture media without zeocin for 72 hours. The supernatants were collected, centrifuged at 1000 g for 15 minutes, passed through 0.22 μm filters and stored at -80°C until use.To test the production of MICA-G129R fusion protein by the transfected cells, 4 × 10The conditioned media or the conditioned media diluted with fresh completed media to the desired concentrations were mixed with protease inhibitor cocktail  with a final concentration of 1% and loading buffer  with a final concentration of 20%, and then boiled for 10 minutes. All the prepared loading samples was loaded to the gel with the same volume if not specified. The proteins in the samples were separated by SDS-PAGE (10% or 12% polyacrylamide gels) and then transferred onto 0.45 μm pore-size nitrocellulose membranes (Bio-Rad). The proteins on the membrane were stained with ponceau S to confirm that the samples were loaded equally across all lanes. The membranes were then blocked with 5% non-fat dry milk in TBST  overnight. The proteins were detected with primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP). Blots were developed using enhanced chemiluminescence (ECL) detection reagents  and exposed to X‑ray films. The films were scanned and quantified using ImageJ . The following antibodies were used in Western blot: monoclonal mouse anti-human MICA (sc-137242), PRL (sc-46698), PRLR (sc-377098) antibodies, mouse IgG light chain binding protein conjugated to HRP (mIgG BP-HRP) (sc-516102) as secondary antibody  and monoclonal mouse anti-V5 antibody (#80076) .Two hundred thousand T-47D cells were seeded on two glass coverslips in two 35 mm dishes with culture media and cultured overnight. After the media were removed, one coverslip was covered with control conditioned media, and the other one was covered with MICA-G129R conditioned media. After incubation for two hours, the slides were washed with PBS, fixed with 4% formaldehyde, blocked with 1% BSA for 30 minutes, incubated with mouse anti-human MICA  primary antibody for one hour, and then chicken anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488  for one hour. The slides were mounted using mounting buffer with DAPI and observed with a fluorescence microscope (LEICA DMi8).One million NK-92 cells were collected and equally separated into two groups. They were respectively incubated with control conditioned media or MICA-G129R conditioned media for two hours. The cells were then harvested, washed with PBS on ice, blocked with 1% BSA for 10 minutes, incubated with mouse anti-human PRL  primary antibody for 20 minutes, and incubated with chicken anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488  for 15 minutes. After three times PBS wash, the cells were pelleted and resuspended in PBS, and then analyzed using flow cytometer (BD Accuri C6).2 humidified incubator for 24 hours. After centrifugation at 500 g for 5 min, 50 μl of supernatant was collected from each well for determining the cytotoxicity using the CytoTox 96 nonradioactive cytotoxicity assay  following the manufacturer’s protocol. The assay determines the cytotoxicity by measuring the lactate dehydrogenase (LDH), a stable cytosolic enzyme, released by the lysed cells. For each conditioned media treatment, many control wells were set at the same time for the calculation of the cytotoxicity: NK cells only (for effector cell spontaneous LDH release), target cells only (target cell spontaneous LDH release), target cells with lysis solution (for target cell maximum LDH release) and medium only (background).The target cells including T-47D cells, HeLa cells, 293 cells, 293/PRLR cells were seeded in 96-well plates and incubated overnight. Then the media was removed and replaced with 50% Alpha Minimum Essential Medium and 50% of conditioned media with NK-92 cells at the effector/target ratios of 5:1, 2:1, 1:1 and 1:2. The co-cultures were incubated in a 37°C, 5% CO2HPO4 pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% IGEPAL and 1 mM DTT) containing 1 M KCl and 25 mM imidazole followed by a wash with 10 ml low salt Buffer A containing 150 mM KCl and 25 mM imidazole. The bound MICA-G129R protein was eluted by adding 0.5 ml of Buffer A containing 150 mM KCl and 500 mM imidazole to the beads, incubated for 5 min, then the column was drained and the elution buffer containing MICA-G129R protein was collected. This elution step was repeated for two more times. The three elution fractions containing the purified MICA-G129R were pooled, passed through a 0.22 μm filter and stored at -80°C until use.Fusion protein MICA-G129R in supernatant of 293/MICA-129R stable clone was purified from the conditioned media using nickel sepharose beads . Five hundred microliters of beads were mixed with 90 ml MICA-G129R conditioned media and rotated overnight at 4°C. The slurry was applied to a column to allow the beads to pack. The MICA-G129R conditioned media was collected after passing through the column twice. Then the column was washed with 10 ml high salt Buffer A  fibers were packed as the HPLC column as previously described . The sepThe proteins in the purified MICA-G129R solution were quantified using Pierce BCA Protein Assay  according to the manufacture’s introduction. The provided albumin (BSA) in the assay was made serial dilutions as the protein standards.To examine the release of granzyme B and IFN-γ of NK-92 cells, NK-92 cells were cultured with or without T-47D cells at the ratio of 1:1 in the presence of the purified MICA-G129R protein at a final concentration of 125 nM  or the elution buffer in the protein purification as control. After 6 hours incubation and a quick spin, the supernatant was collected for ELISA. Human granzyme B ELISA kit  and Human IFN-γ uncoated ELISA kit  were used for the ELISA measurements according to the manufacture’s protocols.T-47D cells alone or with NK-92 cells at the ratio of 1:1 were cultured with the purified MICA-G129R protein at the final concentration of 125 nM or the elution buffer from the protein purification as control for 6 hours. After removal of the media or media with the floating NK-92 cells, the T-47D cells of each treatment were washed with PBS and lysed. The total proteins in the cell lysate of each sample were measured using BCA assay. The same amount of total proteins from each sample was used for detecting the caspase-3 activity using the Capspase-3 fluorometric kit  according to the manufacture’s instruction.t-test (for comparisons between two groups) or one-way analysis of variance (ANOVA) (for comparisons between three or more groups) and then Tukey’s HSD test. P < 0.05 was considered to indicate a statistically significant difference.In all the experiments, each treatment was performed in three replicates unless otherwise indicated and the experiments were repeated at least twice. The results were expressed as the mean ± standard deviation (SD). The significant differences were evaluated using either two-tailed Student’s The gene sequences encoding the extracellular domain of MICA and the PRL mutant, G129R were cloned and inserted into the mammalian expression vector pcDNA3.1/Zeo(+). A (GGGS)×3 linker sequence was added to hinge the MICA and G129R and provide flexibility. A V5 tag and a His tag were aligned downstream of the MICA and G129R segments in the open reading frame in the vector . The genThe MICA-G129R expression vector was transfected into 293 cells that do not express detectable MICA or PRL proteins. Clones resistant to the antibiotic zeocin were selected and expanded. The MICA-G129R fusion protein in the conditioned media from the clones was confirmed using anti-MICA, anti-PRL and anti-V5 antibodies . The cloExpression vectors for MICA alone and G129R alone were respectively transfected into 293 cells. Stable clones were established and the expression of the MICA protein or G129R protein was confirmed by Western blot analysis .To investigate if fusion protein MICA-G129R can bind to breast cancer cells, PRLR-positive breast cancer cell line T-47D cells were incubated with control or MICA-G129R conditioned media and then stained with mouse anti-MICA primary antibody followed by an anti-mouse secondary antibody conjugated with green fluorescent dye Alexa Fluor 488. The T-47D cells incubated with MICA-G129R conditioned media showed strong green fluorescence, while the cells with control conditioned media did not , demonstA human NK cell line, NK-92, was also incubated with the control and MICA-G129R conditioned media and stained with mouse anti-PRL primary antibody followed by an anti-mouse secondary antibody conjugated with Alexa Fluor 488. The NK-92 cells were then analyzed by flow cytometry. The result shows that the curve of NK-92 cells incubated with the MICA-G129R conditioned media shifted to the right compared to NK-92 cells incubated with the control conditioned media, demonstrating that the MICA-G129R protein also binds to NK-92 cells .To investigate if fusion protein MICA-G129R could enhance the cytotoxicity of NK cells on PRLR-positive breast cancer cells, the control and MICA-G129R conditioned media were added to the co-culture of NK-92 cells and T-47D cells at different effector/target ratios  for 24 hours and the cytotoxicity on T-47D cells was measured using a lactate dehydrogenase (LDH) cytotoxicity assay. Compared to control conditioned media, the MICA-G129R conditioned media induced 21%, 21%, 15% and 13% more T-47D cell death at the different effector/target ratios of 5:1, 2:1, 1:1 and 2:1 . To confTo validate that PRLR on the target cells is necessary for fusion protein MICA-G129R’s enhancement of cytotoxicity, PRLR-negative cell lines HeLa cells, 293 cells , and PRLhttps://www.bioinformatics.org/sms/prot_mw.html). Therefore, the molar concentration of MICA-G129R protein in the purified solution was 11.9 μM.Fusion protein MICA-G129R was designed with a His tag to assists its purification and concentration. Using the nickel resin chromatography, fusion protein MICA-G129R was successfully purified from the conditioned media. HPLC analysis of the purified MICA-G129R using a C-CP fiber column ,35 reveaTo further quantify fusion protein MICA-G129R in the purified solution, the purified solution was serially diluted. The MICA-G129R in the dilutions was detected using Western blot, quantified and analyzed . Result To determine if purified fusion protein MICA-G129R activates NK-92 cells, granzyme B and IFN-γ released by NK-92 were measured in NK-92 alone or NK-92 cells co-cultured with T-47D cells with or without the purified MICA-G129R fusion protein. The elution buffer in the protein purification was used as control. It was found that purified MICA-G129R significantly stimulated NK-92 cells to release granzyme B and IFN-γ in both NK-92 cells alone and co-culture of NK-92 cells with T-47D cells . While cTo investigate if apoptosis was induced in T-47D cells by the granzyme B and other contents released from the granules of NK cells, the activity of caspase-3 in the T-47D cells was measured using a fluorometric assay after carefully removing the NK-92 cells. The caspase-3 activity in the T-47D cells co-cultured with NK-92 cells and MICA-G129R protein was found significantly higher than that observed in the T-47D cells co-cultured with NK-92 cells but without MICA-G129R. Without NK-92 cells, the activity of caspase-3 in T-47D cells was very low, and MICA-G129R protein could not induce any remarkable difference .When the same level of purified MICA-G129R as the conditioned media was added to the co-culture of NK-92 cells and T-47D cells, it induced a significantly increased target cells death .Fusion protein MICA-G129R was confirmed not only able to bind to PRLR-positive breast cancer cells and NK cells , but alsTo quickly confirm the concept that fusion protein MICA-G129R induces cytotoxicity of NK cells on PRLR-positive breast cancer cells, conditioned media containing MICA-G129R were initially used in the study. Fusion protein MICA-G129R was then purified and quantified . Very feDifferent from most of bispecific proteins that typically use antibodies for targeting, fusion protein MICA-G129R uses the antagonistic G129R to specifically target PRLR-positive cells. The PRL/PRLR pathway has been studied for decades because of its etiological role in breast cancer . While PG129R as an antagonist has the great potential to target the PRL/PRLR pathway since its binding to PRLR does not promote growth of breast cancer cells, instead, induces autophagy-related cell death . The G12In vivo study has to be done to answer these questions.MICA has been reported to be shed from advanced cancer cells to evade NKG2D-mediated immune detection and elimination. The shedding of MICA results in its release into the circulation. Soluble MICA was found significantly elevated in the sera of patients with leukemia ,44, colo+ T cells, γδ T cells, and some activated CD4+ T cells [in vivo study with a particular focus on γδ T cells because of their ability to infiltrate solid tumors [Since NKG2D is also expressed on NKT cells, CD8 T cells , fusion d tumors . The infd tumors ,60. The Direct cell-cell contact between NK cell and the target cell to form an interface structure, immunological synapse, is required in the cytotoxicity of NK cells ,62. The In vivo mice models will also be included in the study of MICA-G129R for its circulation, tumor-targeting, NK cell-infiltration, efficacy and safety in inducing cytotoxicity and combination with other therapeutics. The in vivo interaction of human NK cells and MICA-G129R can be investigated in immunodeficiency mice xenografted with human PRLR-positive breast cancer cells. MICA also binds to mouse NKG2D and activates mouse NK cells [in vitro studies. This is a big challenge for all the solid tumor immunotherapies. Maybe combination of MICA-G129R with NK cell transfer is a possible way in the in vivo studies.One of the limitations of this study is that T47D was the only cell lines used, so more breast cancer cell lines with different PRLR expression levels will be included in future study. NK cells , so the NK cells , which iHeterogeneity of tumor-targeted antigens is one of the hallmarks of cancer and one of the obstacles in cancer immunotherapy, especially in solid tumors. Drug-resistance often develops when only one antigen is targeted. The strategy of fusing with MICA can be extended to become a universal strategy. Different antibodies, like Trastuzumab, Pertuzumab (anti-HER2), Matuzumab (anti-EGFR) can all be respectively fused with MICA and used together as a cocktail, so that multiple antigens in cancer can be targeted at the same time. NK cells can be derived from health donors, umbilical cord blood or induced pluripotent stem cells (iPSCs) and engineered to minimize the antigenicity and expand to be allogenic and off-the-shelf, and used together with the MICA fusion proteins. MICA and NKG2D can be engineered to bind more specifically with higher affinity to avoid the interaction with the soluble MICA and the intrinsic NK cells. This platform is like a “multi-bit screwdriver”. The handle of the screwdriver is the engineered allogenous off-the-shelf NK cell, while the MICA fusion proteins are the bits targeting different “screws” on cancer cells.In conclusion, a novel fusion protein MICA-G129R was created and demonstrated able to bind to PRLR-positive breast cancer cells and NK cells, promote the release of granzyme B and IFN-γ by NK cells and enhance the cytotoxicity to PRLR-positive breast cancer cells.S1 FigA. Western blot of PRLR with cell lysates of HeLa and T-47D cells. B. Western blot of PRLR with cell lysates of PRLR transfected 293 cells. The PRLR protein in three stable clones of PRLR transfected 293 cells were detected. The untransfected 293 served as the control. The clone 2 was used for the flowing study as the PRLR ectopically expressed 293 cells.(TIF)Click here for additional data file.S2 FigThe white arrow indicates the bands of MICA-G129R. CM indicates the lane with the MICA-G129R conditioned media. M indicates the lanes with the protein standard marker. P indicates the lanes with the purified MICA-G129R protein solution. The loading volume of each sample was indicated above each line.(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


T790M ratio significantly correlated with osimertinib response and was similar between the 1st/2nd-G EGFR-TKIs in 1st/2nd-G EGFR-TKI-refractory tumors.The most frequent mechanism of resistance after 1st/2nd-generation (G) epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is secondary point mutation Thr790Met (T790M) in EGFR. Afatinib followed by osimertinib (Afa group) may provide better outcomes for T790M-positive non-small cell lung cancer (NSCLC) than 1st-G EGFR-TKI followed by osimertinib (1st-G group). We studied 111 consecutive NSCLC patients with T790M mutation treated with osimertinib after progression following 1st/2nd-G EGFR-TKI between March 28, 2016 and March 31, 2018. We analyzed the ratio of T790M to EGFR-activating mutation (T790M ratio) in post EGFR-TKI resistance re-biopsy tissue using droplet digital polymerase chain reaction. And investigated whether afatinib purified the T790M mutation more than 1st-G EGFR-TKI. Among 60 patients with preserved re-biopsy tissue, we analyzed 38 having adequate DNA content. The response rate in Afa group was 81.8% (n = 11) and 1st-G group was 85.2% (n = 27). The mean T790M ratio in total population was 0.3643. The ratio in those with response to osimertinib was significantly higher than in the non-responders (0.395, 0.202;  However, cancer cells inevitably develop acquired resistance (AR) to EGFR-TKIs. Although EGFR-TKI treatment shows a durable response against NSCLC harboring EGFR mutations, most patients experience cancer relapse within 1–1.5 years following treatment with first-line 1st- and 2nd-generation (G) EGFR-TKIs. The most common resistance mechanism involves a secondary point mutation Thr790Met (T790M) in EGFR. It impairs the binding of TKIs to EGFR and is detected in approximately 50–60% of patients with 1st and 2nd-G EGFR-TKI-refractory tumors9. Osimertinib was developed for its activity against T790M by covalently binding to T790M-muted EGFR10, and it has been shown to be effective against AR of T790M-positive NSCLC after 1st- or 2nd-G EGFR-TKI treatment11. The RR in the trial was about 70% and use of osimertinib in 1st- and 2nd-G EGFR-TKI-refractory tumors was established as a standard treatment worldwide.Based on randomized trials showing superior progression-free survival (PFS), response rate (RR), and more favorable safety profiles when compared with standard first-line platinum-based doublet chemotherapy in non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutation12. However, in the Asian subset  analysis of OS in the FLAURA study, osimertinib was not superior to 1st-G EGFR-TKIs13. There may be no molecular targets for therapy due to the heterogeneity of resistance mechanisms, which are not well understood15. As a result, during clinical care for most patients following cancer progression after osimertinib treatment, chemotherapy is the only remaining option for second-line treatment.Moreover, osimertinib is associated with a longer PFS and overall survival (OS) than 1st-G EGFR-TKIs against advanced NSCLC harboring EGFR mutation (exon-19 deletion and L858R) as a first-line treatment16. A study showed that afatinib treatment followed by osimertinib indicated an extremely long treatment time to failure (TTF) and OS, even after removal of selection bias17. Furthermore, we reported that treatment with afatinib followed by osimertinib (Afa group) may provide better outcomes for T790M-positive NSCLC than that with 1st-G EGFR-TKIs (1st-G group)18. These results may be explained by the possibility that afatinib exhibits increased efficacy by targeting co-occurring EGFR mutations that enrich T790M cells.In contrast, afatinib demonstrated superior RR, PFS, and a trend of longer OS compared to gefitinib in the LUX-Lung 7Therefore, to investigate whether afatinib purifies T790M mutation more effectively than the 1st-G EGFR-TKIs, we analyzed the difference in the ratio of T790M mutation to EGFR-activating mutation ratio (T790M ratio) between the Afa and 1st-G group by using droplet digital polymerase chain reaction (ddPCR).We conducted a multicenter-retrospective study across three medical centers  in Japan. The study design and methodology were approved by the Institutional Review Board of each participating institution , and it was conducted in accordance with the Declaration of Helsinki and the World Health Organization’s Guidelines for Good Clinical Practice. We obtained the informed consent from participants and used an opt-out method so that patients and their families could refuse to participate in the study.EGFR mutation (Exon-19: deletion19 or Exon-21: L858R) before initial EGFR-TKI treatment to reduce bias towards patient conditions. Furthermore, we excluded cases where we detected T790M mutation in the pre-treatment tissue by EGFR test in general practice.Between March 28, 2016 (the date osimertinib was approved in Japan) and March 31, 2018, study participants were consecutively enrolled according to the following criteria: patients with T790M mutation who were treated with osimertinib after AR to EGFR-TKIs at any time for advanced NSCLC, had good Eastern Cooperative Oncology Group (ECOG) performance status (PS): 0–2, and had major We used RIKEN GENESIS CO., LTD.  to measure exon-19 deletion, L858R, and T790M by droplet digital polymerase chain reaction (ddPCR). The ddPCR analysis was performed using QX200 AutoDG Droplet Digital PCR System (BioRad). In this study, we excluded samples that had less than 1000 total copies/20 uL well or less than 10 copies with EGFR-activating mutation/20 uL well. T790M ratio was calculated by the following method. First, the fractional abundance  of activating mutation and T790M were measured with droplet count by ddPCR. Next, T790M ratio defined the ratio of T790M to activating mutation.19. We used Fisher’s exact tests for categorical comparisons of data and compared differences in continuous data using the Wilcoxon test. Kaplan–Meier curves were used to evaluate PFS, which were compared using the log-rank test. Median values and 95.0% confidence intervals (CIs) were also reported. All statistical analyses were conducted using R software, version 2.8.1 (http://R-project.org) . p < 0.05 was considered a statistically significant difference.We evaluated the systemic response to osimeritinib using the Response Evaluation Criteria in Solid Tumors (RECIST) ver1.1Of the 111 enrolled patients, the re-biopsy tissue obtained from each patient before osimertinib treatment was preserved for 60 patients. Among them, 38 samples yielded enough DNA for ddPCR analysis, and the T790M ratio was assessed Fig. .Figure 1p = 0.0231 . The T790M ratio in patients who obtained complete response (CR) or partial response (PR) to osimertinib in the osimertininb group (mean: 0.395) was significantly higher than in patients with stable disease (SD) or progressive disease (PD) mean: 0.202); 02; p = 0p < 0.0001  was significantly longer than that in the SD or PD subgroup (77 days [95%CIs 52-not available (NA)]); 47–710) wp = 0.03691. The median PFS in patients with T790M ratio ≥ 0.35 was longer when compared with patients with T790M ratio < 0.35 , however this was not statistically significant   was higher than that in patients with T790M ratio < 0.35 (70% 14/20]); /20); p =p = 0.0231. However, we did not observe a difference in the T790M ratio between the 1st- and 2nd-G EGFR-TKIs. The T790M ratio in the Afa group (mean: 0.3711) was similar to the 1st-G group (mean: 03616); p = 0.9693.This is the first analysis to reveal a potential implication that afatinib purifies the T790M mutation more than the 1st-G EGFR-TKI. This study suggested that the T790M ratio in 1st/2nd-G EGFR-TKI-refractory tumors significantly correlated with response to osimertinib. The T790M ratio in CR or PR subgroup of the osimertinib group (mean: 0.395) was significantly higher than in the SD or PD subgroup (mean: 0.202); 10. Therefore, although osimertinib is also effective against sensitive EGFR mutations, the most effective targets are T790M-positive EGFR mutations. In contrast, afatinib irreversibly binds to EGFR, ERBB2, and ERBB4 and blocks transphosphorylation of ERBB3 to inhibit all ERBB family signaling20. Two hypotheses were proposed: the first is that the broader inhibition of the EGFR family may improve the efficacy of EGFR-TKIs for NSCLC with EGFR mutation, and the second is that the broader inhibition of the EGFR family may attenuate T790M in cancer cells during relapse after EGFR-TKI treatment. Osimertinib could also be more effective against the AR of T790M after EGFR-TKI. The results of LUX-Lung7 and ARCHER1050 supported this theory in the first-line treatment for NSCLC with EGFR mutation21. Furthermore, afatinib provided the efficacy for uncommon EGFR mutations in preclinical and clinical studies23. In the analysis of GIO-TAG trials, the duration of response to osimertinib in patients receiving afatinib and subsequent osimertinib was highly encouraging25.Osimertinib was developed as a drug against T790M that works by covalently binding to T790M-muted EGFR28.In our study, the concentration of T790M correlated with the response to osimertinib, and our results suggested that the degree of attenuation of T790M is important in eliciting the effects of osimertinib. Some prior studies have also shown similar results: the ratio of T790M to EGFR-activating mutation in cell samples or plasma samples may predict the response to osimertinib or rociletinib18. We intended to collect all the samples in our previous study; however, this was not possible. Another reason is that later lines of therapy may have advanced clonal evolution and may not reflect the effect of osimertinib after 1st-G EGFR-TKI and afatinib treatment.However, we did not observe differences in the clonality of T790M after AR between afatinib and 1st-G EGFR-TKI. One reason was that the response of osimertinib after AR was the same in Afa group and 1st-G group when we collected the available tissue, although our previous study revealed that the objective RR and disease control rate were significantly higher in the Afa group than in the 1st-G groupp = 0.715). We believed that the PFS of osimertinib after AR in NSCLC with T790M mutation was influenced by co-mutation.Next, Pearson correlation analysis showed no significant relationship between T790M ratio and PFS of osimertinib after AR of EGFR-TKIs (r = 0.06123, 29, lung adenocarcinoma seems to follow a multistep progression from atypical adenomatous hyperplasia to adenocarcinoma in situ, and finally invasive adenocarcinoma30. EGFR driver alterations are acquired in the early step of cancer progression and can be identified in most neoplastic cancer cells31. Furthermore, by layering exacerbations of disease after treatment, especially EGFR-TKI use, cell progression will show different genomic patterns of selection through different lines of therapy32. In this setting, EGFR-sensitive mutant tumor cells may coexist with sub-clonal tumor cells harboring other gene mutations, and co-mutations such as TP53 affect the efficacy of osimertinib34. Furthermore, histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes35.Based on clinal evolution, which was reported for the first time as early as 1976We must consider some limitations of the present study. First, this was a retrospective study, and the participants were selected based on obtainment of adequate samples. Second, the line of treatment for EGFR-TKIs was not determined. Since the lines of EGFR-TKIs varied from case to case, the effectiveness of osimertinib may also vary accordingly. Although osimertinib response was not different between the two groups, we focused on the attenuation of tumor cells by EGFR-TKIs immediately before the administration of osimertinib to clarify the correlation between T790M ratio and osimertinib response. Third, in this study, we examined only exon-19 deletion, L858R and T790M by ddPCR and did not perform next generation sequencing. Therefore, we cannot know about the presence of other EGFR mutations, amplifications, alternative bypass pathways or co-mutations that may provide a mechanism of resistance to osimerutinib.In our analysis, the T790M ratio in 1st/2nd-G EGFR-TKI-refractory tumors significantly correlated with osimertinib response. Furthermore, the T790M ratio between the 1st- and 2nd-G EGFR-TKIs was almost similar in patients with EGFR-TKI-refractory NSCLC who showed response to osimertinib. Further analysis in a larger number of prospective studies is warranted to confirm our results.


The Editor-in-Chief has retracted this article. After publication, two of the panels in Figure 1C were found to overlap with figures in another published article [1], specifically: Figure 1C left panel overlaps with Figure 2A of [1]. Figure 1C middle panel overlaps with Figure 6 of [1]. The Editor-in-Chief therefore no longer has confidence in the integrity of the data in this article. The authors have agreed to this retraction but not to the wording of this retraction notice. [1] Manno D, Serra A, Buccolieri A, Panzarini E, Carata E, Tenuzzo B, Izzo D, Vergallo C, Rossi M, Dini L (2013) Silver and carbon nanoparticles toxicity in sea urchin Paracentrotus lividus embryos. BioNanoMat 14(3–4):229–238.Former article version (PDF 1612 KB)Below is the link to the electronic supplementary material.


EGFR gene. Major EGFR mutations (>90% of EGFR-mutated lung cancer) are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs). Many uncommon EGFR mutations have been identified, but little is known regarding their characteristics, activation, and sensitivity to various EGFR-TKIs, including allosteric inhibitors. We encountered a case harboring an EGFR-L747P mutation, originally misdiagnosed with EGFR-del19 mutation using a routine diagnostic EGFR mutation test, which was resistant to EGFR-TKI gefitinib. Using this minor mutation and common EGFR-activating mutations, we performed the binding free energy calculations and microsecond-timescale molecular dynamic (MD) simulations, revealing that the L747P mutation considerably stabilizes the active conformation through a salt-bridge formation between K745 and E762. We further revealed why several EGFR inhibitors, including the allosteric inhibitor, were ineffective. Our computational structural analysis strategy would be beneficial for future drug development targeting the EGFR minor mutations.Approximately 15–30% of patients with lung cancer harbor mutations in the  To treat cancers harboring driver oncogenes, which play an important role in the formation and growth of tumors, molecularly targeted drugs have been developed and have shown remarkable antitumor effects by specifically blocking the driver oncogene-oriented signaling pathways in lung cancer2. Epidermal growth factor receptor (EGFR) is the major driver oncogene in lung cancer, especially in Asian population, and a number of molecular target drugs have been developed and approved.Lung cancer is a major malignancy with a high mortality rate. Non-small-cell lung cancer (NSCLC) accounts for approximately 75–85% of all lung cancersEGFR gene are mainly concentrated in exons 18–21. Mutations in these regions change the spatial structure of the enzyme’s functional domain and lead to constitutive activation of EGFR and its downstream signaling activation3, in particular, a five to six amino acid deletion in exon 19 (del19) and a point mutation in exon 21 (L858R) account for >85% of all EGFR mutant lung cancers4. These activating mutation harboring NSCLCs are sensitive to the first-, second-, and third-generation EGFR tyrosine kinase inhibitors (TKI), including gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib6. The first-generation EGFR-TKIs, namely, gefitinib and erlotinib, have been shown to be effective in NSCLC with EGFR-activating mutations (del19 or L858R). The second-generation EGFR-TKIs, namely, afatinib and dacomitinib, irreversibly target the pan-ERBB family proteins. The third-generation EGFR-TKI, osimertinib, is effective against EGFR major activating mutations and T790M mutated EGFR, the most common acquired resistance mutation to first- and second-generation EGFR-TKIs8. On the other hand, a number of uncommon EGFR mutations have been identified, with each mutant demonstrating a different sensitivity to EGFR-TKIs11. Primary resistance has also been reported in uncommon EGFR mutation-positive NSCLCs such as EGFR exon 20 insertion mutation or L747P mutation. Walsh et al. reported a case diagnosed with exon 19 deletion that was resistant to EGFR-TKI using commercial diagnostic kits Terascreen® and Cobas®. However, using next-generation sequencing (NGS), they determined that the patient harbored the L747P mutation, but not del1912. L747P results from codon 747 of exon 19 with a 2-bp mutation (c.2239_2240TT > CC). This mutation is believed to promote carcinogenesis in the same manner as other common EGFR mutations. A limited number of case reports have indicated that lung adenocarcinoma patients with an EGFR-L747P mutation have different sensitivities depending on the type of EGFR-TKI19. From three-dimensional structure modeling of rare EGFR mutant kinases with an exon 19 insertion mutation with Leu to Pro substitution at the 747 residue, the 6 amino acids insertion with L747P mutation is predicted to prevent the stabilization of the inactive conformation of EGFR kinase20. Clinical case reports have shown the EGFR-L747P mutant is resistant to gefitinib or erlotinib, but the detailed structural mechanisms underlying the L747P induction of resistance to gefitinib or erlotinib have not been clarified because no detailed structural analysis for an L747P mutation has been conducted.The oncogenic mutations in the Because the clinical use of NGS is increasing, it is expected that the identification of L747P mutation-positive cases with a diagnosis of EGFR-del19 will also increase.50 of EGFR-TKIs to EGFR-L858R or del19 mutant but not to EGFR-L747P mutant cells. Microsecond-timescale molecular dynamics (MD) simulation analysis revealed structural insights into how the EGFR-L747P mutation induces the constitutive activation of EGFR as well as the different drug sensitivities, including that of the EGFR allosteric inhibitor.In this study, we encountered an EGFR-L747P mutant case diagnosed as EGFR-del19. The EGFR-L747P mutation was introduced into Ba/F3 cells, and the oncogenicity and EGFR-TKI sensitivities were examined by comparing EGFR-del19- or L858R-expressing Ba/F3 cells. The L747P mutant was confirmed to be resistant to gefitinib and erlotinib but sensitive to afatinib and dacomitinib. In addition, we found that the EGFR-L747P mutant was resistant to the EGFR allosteric inhibitor EAI-045 in combination with anti-EGFR antibody cetuximab, although the combination therapy was effective for EGFR-L858R mutants. Interestingly, the anti-EGFR antibody combination treatment decreased the ICEGFR  combined with an anti-angiogenic agent containing combination therapy for 4 months until progression in the context of a clinical trial, but the patient’s condition progressed within four months. We performed rebiopsy with bronchoscopy, and the EGFR gene mutation was confirmed to be del19 using the Cobas® kit. Later, cytotoxic chemotherapy was provided as a second-line treatment. However, the tumor grew rapidly and progressed within a course. Afatinib was subsequently introduced as a third-line therapy in June 201X + 2. The treatment was successful, and the tumor reduced rapidly. However, the tumor relapsed after about four months and was diagnosed as a progressive disease Fig. . Later, FR Table .Fig. 1ClBecause the characteristics of EGFR-L747P have not been well studied, we introduced the EGFR-L747P mutant and two other major active mutations L858R and del19 into Ba/F3 cells to evaluate their characteristics.50 as compared with the L858R or del19 mutant EGFR expressing cells. As previously reported, EGFR-TKI resistant T790M mutants showed a marked resistance to gefitinib, erlotinib, afatinib, and dacomitinib, but not to osimertinib  or EGFR-TKI resistant mutants (L858R/T790M and del19/T790M) introduced into Ba/F3 cells. We determined that the EGFR-L747P mutant, but not the L858R and del19 mutant expressing cells, is resistant to gefitinib and erlotinib but similarly sensitive to second-generation EGFR-TKIs afatinib or dacomitinib. In comparison with osimertinib, the L747P mutant showed a slightly higher ICnib Fig. . Immunobnib Fig. .Fig. 2Dr22, suggesting that the free energy estimation using MP-CAFEE can correctly predict how each resistant mutation will affect drug-binding. The same strategy was applied to EGFR exon 20 insertion mutations and successfully quantified the drug sensitivity of mutant EGFR kinases23. Thus, in this study, we attempted to predict the binding affinity of the EGFR-L747P mutant with gefitinib. Because the crystal structure data of the EGFR-L747P mutant have not been reported previously, the L747P mutation was modeled based on the structure of WT EGFR with gefitinib (PDB code: 2ITY). Then, the binding free energy of gefitinib-EGFR-L747P or L858R was calculated by MP-CAFEE. As a result, the calculated binding affinity for L747P significantly decreased compared with that for L858R, which was attributed to the loss of the van der Waals interactions  free energy (ΔG) simulation called MP-CAFEE to calculate the binding affinities of ALK-TKIs to multiple ALK-TKI resistant mutants, such as ALK-L1196M, G1202R, or I1171N. Our simulation demonstrated a clear linear correlation between the experimental IC50 and ΔG of each ALK-TKI to ALK mutantsons Fig.  while thons Fig. , as wellons Fig. , in whicThe L747P mutation also decreased the binding affinity of osimertinib by inducing a positional displacement of the bound drug  how the L747P mutation affects stabilities of the active and inactive conformations, and (2) why the allosteric inhibitor was ineffective for the L747P mutant. The mean structures of 1 μs × 3 MD simulations showed that the orientation of the αC-helix in the L747P mutant is distinct from that in the WT or L858R mutant Fig. . A salt 29. However, real-world data are unknown because of the misidentification of L747P by multiple diagnostic kits. In clinical practice, the response rate of EGFR-TKI is approximately 60–80%, and there is a certain proportion of patients with primary resistance. Several mechanisms have been reported as the primary resistance mechanisms. For example, BIM polymorphism, with a certain deletion polymorphism in the intron of the BIM gene, was shown to be related to the reduced expression of the proapoptotic form of BIM protein-containing BH3 domain that results in apoptosis resistance induced by EGFR-TKI treatment30. In addition, in high AXL protein-expressing cells, the inhibition of mutant EGFR by EGFR-TKI activates AXL through the inhibition of SPRY4 and induces resistance by promoting survival signaling through AXL31. Among the primary resistant cases, a certain number of EGFR-L747P mutations might also be included, at least in the first-generation EGFR-TKI-treated patients, and second EGFR-TKI might be effective for those cases. Thus, from the viewpoint of cancer precision medicine, unveiling the drug sensitivity profile for each minor mutation may be important in increasing effectiveness and reducing primary resistance.In this study, we report our experience with a lung cancer patient with an EGFR-L747P mutation diagnosed as EGFR-del19 by routine PCR-based diagnostic tests. We confirmed that the clinical diagnostic tests (cobas® or Scorpion ARMS®) provide positive results as del19 against the L747P mutation. Although the Cq value seemed to be slightly higher than del19, this could likely be attributed to the fact that the L747P mutation occurs in the middle of the deleted nucleotides in EGFR exon 19 deletion (ELREA 746–750). There are many types of EGFR exon 19 deletion mutations, in which 9 (three amino acids: L747–E749) to 24 bp (six amino acids: S752–I759) near the 746–750 amino acids are deleted, and in the diagnostic PCR tests, the multiplex primers are set to detect those multiple deletion mutants. The detailed frequency of each exon 19 deletion mutation and the sensitivities to each EGFR-TKI have not been fully studied at present. In addition, there have been only a few reports describing the frequency of the L747P mutation in lung cancer. In one report, the frequency of EGFR-L747P or L747S mutations was shown to exit in 0.59% of EGFR mutant NSCLCs (12 of 2031 cases of EGFR mutant cancer)As previously reported, we confirmed that the EGFR-L747P mutant showed a different sensitivity to EGFR-TKIs: it was resistant to gefitinib, erlotinib, and brigatinib; intermediately sensitive to osimertinib; and highly sensitive to afatinib or dacomitinib. Interestingly, we found that the L747P mutant was not further sensitized to afatinib by combining it with anti-EGFR antibody cetuximab, although the EGFR-L858R mutant, del19 mutant, L858R/T790M, or del19/T790M became more sensitive to afatinib in combination with cetuximab. In addition, the EGFR-L747P mutant but not the EGFR-L858R or L858R/T790M mutant was highly resistant to the EGFR allosteric inhibitor EAI-045 with cetuximab. In addition, we newly found that IKK inhibitor LY2409881 specifically showed inhibitory activity against EGFR-L747P but not against L858R or del19.Since the tyrosine kinase shared similar structure and conserved multiple amino acid sequence, we checked the conservation of EGFR L747 residue in other tyrosine kinase. EGFR-L747 corresponds to ERBB2-L755, ALK-L1152, ROS1-L1981, and NTRK1-L564. ERBB2-L755S is the most frequently altered codon and well-known driver oncogenic mutation observed in breast or colorectal cancer. Thus, our current analysis might contribute to understand the structure and possible drug sensitivity to the ERBB2-L755S mutation. On the other hand, ALK-L1152R was reported as ALK-TKI crizotinib resistant mutation, and ROS1-L1981 and NTRK1-L564 mutations were not well characterized.32. For example, we clearly demonstrated that our calculated binding free energies between ALK mutants with each ALK-TKI showed a linear correlation with the experimental IC50 data22. Currently, it is still challenging to find a new drug candidate with high accuracy using only computational simulation. However, it is likely that drug screening driven by in silico screening with a much higher accuracy will become more common in the near future.From the computational simulation, we noted the following three aspects: (1) the L747P mutation markedly decreased the van der Waals interaction between EGFR tyrosine kinase and gefitinib, a first-generation EGFR-TKI, resulting in the resistance to gefitinib; (2) the L747P mutation induces a structural change in the αC-helix orientation toward P-loop and helps in forming the salt bridge between K745 and E762 residues to fix the active conformation; and (3) this conformational change via the L747P mutation prevents binding of the allosteric inhibitor EAI-045. Although our suggested molecular mechanism of drug resistant acquired by the L747P mutation needs to be experimentally validated -drug complex), MD simulations of drug target proteins and protein-drug binding free energy prediction using MP-CAFEE have been practically applied to genomic medicineIn this study, we report our experience of a case with an EGFR-L747P mutation first diagnosed as an EGFR-del19 mutant. We confirmed that this mutant is resistant to gefitinib and erlotinib, less sensitive to osimertinib than EGFR-del19, and sensitive to afatinib and dacomitinib.An NSCLC patient with an EGFR mutation was administered EGFR-TKI treatment in the Department of Thoracic Medical Oncology at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research (JFCR). Patient provided written informed consent and agreed to the use of their residual biopsy samples after disease progression. This study was performed as an approved clinical observation study by the Institutional Review Board of the Cancer Institute Hospital at JFCR.33. Paired-end sequencing (2 × 150 bp) was performed on the MiSeq platform. Next, raw reads were preprocessed by removing the Illumina adapter sequences and low-quality bases using Trimmomatic-0.3934, with the LEADING:15 TRAILING:15 SLIDINGWINDOW:4:30 option. After trimming, < 40-bp-long reads were discarded. The quality-controlled reads were aligned onto the human genome sequence (UCSC hg38) by HISAT235, and SAMtools v1.8 was used to convert the obtained SAM file to a BAM file36. Nucleotide variants and indels were detected using Mutect2 implemented in GATK v4.0.7.038. During this process, we recalibrated the quality scores and filtered ambiguous mutations following GATK best practices recommendations40. Detected mutations were annotated by VEP41.Genomic DNA was extracted using an RNeasy Mini Kit (Qiagen). For targeted amplicon sequencing, the library was prepared using a Haloplex custom panel (Agilent), which is designed to detect well-known cancer-associated somatic mutationsA list of the kinase inhibitors used in the experiments and the companies from which they were purchased is shown below. Afatinib (from Chemietek) was used at 0.03 nM–1 μM, Brigatinib , Cetuximab , Dacomitinib , EAI-045 , Erlotinib , Gefitinib , Osimertinib .Ba/F3 murine bone marrow-derived pro-B cells (purchased from RIKEN BRC) harboring EGFR mutations were cultured in low-glucose Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (FBS), kanamycin, and 0.5 ng/mL of interleukin-3 (IL-3). 293FT human embryonic kidney cells (purchased from Invitrogen) were cultured in high-glucose DMEM supplemented with 10% FBS and kanamycin (Meiji Seika Pharma). EGFR-L747P, del19, L858R, del19/T790M, and L858R/T790M mutant Ba/F3 cells were generated by lentiviral infection produced by a pLenti6.3-EGFR-L747P del19, L858R, del19/T790M, or L858R/T790M plasmid and packaging plasmids as indicated below. All cells were regularly tested to ensure that they were free of mycoplasma contamination.PrimersMutagenesis primer for creating EGFR-L747P- EGFR-L747P-F: CGTCGCTATCAAGGAACCAAGAGAAGCAACATCTCC- EGFR-L747P-R: GGAGATGTTGCTTCTCTTGGTTCCTTGATAGCGACGSequencing primers for EGFR- EGFR ORF F1: GTGGCGGGACATAGTCAGCAGTG- EGFR ORF F2: CCTCAGGCCATGAACATCACCTG- EGFR ORF F3: CCGTGAGTTGATCATCGAATTC- EGFR ORF R1: GAATTTGCGGCAGACCAGGCAG- EGFR ORF R2: CTTCCGAACGATGTGGCGCCTTC- EGFR ORF R3: CAGCTTTGCAGCCCATTTCTATCSequencing primers for EGFR exon19- hEGFR-Ex19-F: GGCAGCATGTGGCACCATC- hEGFR-Ex19-R: GCCTGAGGTTCAGAGCCATG- hEGFR-Ex19-Fseq: GATCCCAGAAGGTGAGAAAG- hEGFR-Ex19-Rseq: GAGGATTTCCTTGTTGGC.The EGFR-L747P, del19, L858R, del19/T790M, or L858R/T790M mutant was created by Quikchange mutagenesis using pENTR-EGFR, and lentiviral plasmid pLenti6.3-EGFR-L747P or other mutants were cloned by LR clonase II using pENTR-EGFR (L747P or other mutants) and pLenti6.3-DEST (Invitrogen). Lentivirus was prepared by transfecting pLenti6.3-EGFR-L747P or other mutants with a helper plasmid (ViraPower) in 293FT cells. Ba/F3 cells were infected using lentivirus-containing medium supplemented with polybrene (8 µg/mL), and after an incubation of 24 h, the infected cells were selected using 7 µM blasticidin (Invitrogen) for one week. After the selection, cells were cultured in a culture medium without IL-3.50 was determined using a nonlinear regression model with a sigmoidal dose response in GraphPad.The 72-h cell viability assay was carried out by seeding 2000 cells/well of Ba/F3 cells into black, clear-bottom, 96-well plates. On the same day, serially diluted drugs were added to the cells and incubated for 72 h. After drug treatment, we measured cell viability using the CellTiter-Glo assay (Promega) according to the manufacturer’s protocol. GraphPad Prism version 8.0 (GraphPad software) was used to analyze the data. The ICg for 10 min. Equal amounts of protein from cell lysates were loaded on SDS-polyacrylamide gels for electrophoresis separation. Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with antibodies against phosphor-EGFR , total EGFR , phospho-Akt , total Akt , phospho-ERK , total ERK1/2 , phospoh-S6 , total S6 , or β-actin . ECL Prime Western Blotting Detection Reagent  and Amersham Imager 600  were used for signal detection.Cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer  and boiled at 100 °C for 5 min, or lysed in TNE lysis buffer , 0.5 M NaCl, 1 mM EDTA, Phostop, and Complete mini). Protein concentrations were measured using BCA Protein Assay Reagent (Thermo Fisher Scientific) after centrifugation at 20,000 The initial structures of wildtype EGFR in complex with gefitinib, afatinib, dacomitinib, and osimertinib were obtained from the Protein Data Bank . The structures of disordered loops and flexible side chains were modeled using the Structure Preparation module in the MOE (Molecular Operating Environment) program v. 2016.08 . The N- and C- termini were capped with acetyl and N-methyl groups, respectively. Titratable residues remained in their dominant protonation state at pH 7.0. Each mutation was introduced into the structure of wildtype EGFR using the Structure Preparation module in MOE. Gefitinib, afatinib, dacomitinib, and osimertinib were protonated to exist in an ionized state in the solution .42 As we had reported previously27, computational systems of the EGFR-drug complexes were prepared and their MD simulations were performed. For each EGFR mutant–drug pair, five sets of 50 ns production runs were executed with different velocities, and an additional 950 ns simulation was completed for each of the three trajectories. Three sets of 20 ns production runs were implemented for the solvated drug system. The binding free energy (ΔG) of each EGFR-TKI toward EGFR-WT, L747P, or L858R was calculated using MP-CAFEE , which constitutes one of the alchemical free energy perturbation methods43. ΔG for each EGFR mutant was computed as previously described44.All MD simulations were performed using the GROMACS 2016 programt-tests as appropriate. Significant probability (P) values are indicated as ***P < 0.001, **P < 0.01, and *P < 0.05.Data are presented as mean ± SD unless otherwise specified. Pairwise comparisons between groups were made using paired or unpaired Student’s Further information on research design is available in the Reporting SummarySupplementary InformationSupplementary Data 2Supplementary Data 1


Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of pulmonary vessel walls caused by excessive pulmonary arterial smooth muscle cell (PASMC) proliferation. Our previous clinical studies have demonstrated the importance of the downregulated circRNA in PAH. However, the role of upregulated circRNAs is still elusive. Here, we identified the upregulated circRNA in PAH patients, hsa_circWDR37_016 (circWDR37), as a key regulator of hypoxic proliferative disorder of pulmonary arterial smooth muscle cells (PASMCs). Quantitative real-time PCR (qRT-PCR) analysis validated that exposure to hypoxia markedly increased the circWDR37 level in cultured human PASMCs. As evidenced by flow cytometry, 5-ethynyl-2′-deoxyuridine (EdU) incorporation, wound healing, and Tunel assay, silencing of endogenous circWDR37 attenuated proliferation and cell-cycle progression in hypoxia-exposed human PASMCs in vitro. Furthermore, bioinformatics and Luciferase assay showed that circWDR37 directly sponged hsa-miR-138-5p (miR-138) and was involved in the immunoregulatory and inflammatory processes of PAH. Together, these studies suggested new insights into circRNA regulated the pathology of PAH, providing a new potential therapeutic target for PAH treatment. Pulmonary arterial hypertension (PAH) is a progressive disorder defined by a mean pulmonary artery pressure (mPAP) ≥ 25 mmHg. Resulting from the restricted blood flow through pulmonary artery (PA) circulation, the pathological increases in pulmonary vascular resistance (PVR) initiate the right ventricular (RV) compensatory responses . However, RV adaptation is not enough to overcome the hemodynamic compromises in pH, leading to RV hypertrophy, dilatation, and ultimate right heart failure . AlthougRecently, noncoding RNA and their processing machinery have been shown as the common hallmark of pH, particularly microRNA (miRNA) and long noncoding RNA (lncRNA) which regulate pH progressions –6. Circu2 (5%) atmosphere at 37°C. hPASMCs were characterized by immunofluorescence with antibodies specific to α-smooth muscle actin. To ensure reliable positive results, two to four generations of each primary hPASMCs cell line were used for experiments. For hypoxic culture, hPAMSMCs were exposed to CO2 (5%)/O2 (3%)/balance N2 for 24-48 h.Human Pulmonary Artery Smooth Muscle Cells (hPASMCs) from ScienCell Research Laboratories  were cultured in Smooth Muscle Cell Medium  in a COβ-Actin was used as the internal control. Primers specific for circWDR37  and β-actin  were purchased from Sangon Biotech . qRT-PCR was performed on a thermocycler ABI Prism 7300 fast  for 35 cycles. The relative expression of each group was analyzed using the 2^ΔΔCt- method [Total RNA from cultured hPASMCs were isolated with TRIzol extraction. Using high-capacity cDNA Reverse Transcription Kit  and Fast SYBRGreen Master Mix , the expression of circWDR37 was determined by qRT-PCR analysis. t method .To regulate the expression of circWDR37 in hPASMCs, one specific 2-O-methyl antisense oligoribonucleotide complementary to the mature circWDR37 (TACGTAGAGAAATCGACACTCTTAATGAACGTTTAGCTGCTGAAGGACAAGCGATTGATGGAGCAGAGCTGAGTAAGGGCCAACTCAAAACAAAAGCCAGTCACAGCACCAGCCAGCTCTCCCAGAAACTGAAGACCACTTACAAGGCTTCCACCAGCAAG) was synthesized by GenePhama Co., Ltd. . A scrambled RNA  labeled with 5′-carboxyfluorescein (FAM) was used to evaluate the transfection efficiency and served as the negative control (NC). The efficiency of siRNA of circWDR37 (si-circWDR37) was evaluated by qRT-PCR with divergent primers as described above.After serum deprivation, the hPASMCs were transfected with NC and si-circWDR37, respectively, using X-treme Gene siRNA Transfection Reagent  followed by manufactural instructions. After 4-6 hours, the cells were switched to SMCM supplied with FBS (10%) and cultured for another 24-48 hours under normal or hypoxic conditions.The hPASMCs were cultured in 96-well and transfected with different groups as described above. The cells transfected with NC were served as controls. After 24 h culture, cell proliferation capacity was evaluated by EdU Cell Proliferation Kit with TMB . EdU, an analog of thymidine, was incorporated into the DNA synthesis of cells. Under the catalysis of copper ion, EdU was labeled with biotin, and then, HRP streptavidin was added to combine with biotin, and the color was determined by TMB chromogenic solution. The plates were read using a spectrophotometer microplate reader  with a single wavelength of 370 nm.μL cell suspension was inoculated and transfected after cell adhesion. In addition, 10 μL CCK-8 solution  was added to each well, and the absorbance was measured at 450 nm after 2 hours of culture.WST-8 is a compound similar to MTT. In the presence of electron coupling reagent, WST-8 can be reduced to orange-yellow formazan by some dehydrogenase in mitochondria. The color is linear with the number of cells. In each well of the 96-well plate, 100 μL RNase A, and 25 μL propidium iodide (PI) at 37° C for 30 min. The cell cycle was detected at 488 nm by flow cytometry.The cell cycle was measured using the cell cycle and apoptosis analysis kit according to the manufacturer's instructions . Briefly, 48 hours after transfection, cells were digested with trypsin, collected, and washed with precooled PBS. Cells were then fixed with precooled 70% ethanol for 24 h. Subsequently, the cells were centrifuged at 1000 × g for 5 min and incubated with 500 staining buffer, 10 The hPASMCs were transfected with different groups and cultured on 24-well plates. The cell motility and migration were compared by wound-healing assay. The cells transfected with NC were served as controls. Briefly, the “wound” of each group was created by a pipette and captured by microscopy. After 24 h culture, the “wounds” were recaptured, and the proliferation and migration rates were analyzed and calculated.μL TUNEL detection solution  was added into each well and incubated at 37°C for 1 hour. Fluorescence microscopy  was used to image FITC-labeled cells under 488 nm excitation and 530 nm emission. The green fluorescent cells were calculated as apoptotic cells.The hPASMCs were transfected with different groups as described above. The cells transfected with NC were served as controls. The apoptotic hPASMCs were detected using a TdT-mediated one-step TUNEL apoptosis Assay Kit . After transfection, the hPASMCs were fixed with 4% paraformaldehyde for 30 minutes and washed with PBS. PBS containing 0.3% Triton X-100 was added for 5 minutes at room temperature. After cell penetration, 50 The wild-type sequence of circWDR37 was subcloned downstream of the Renilla luciferase reporter gene of the pSI-check2 expression vector  named as WT-WDR37. The sequence of circWDR37 with 4 mutation sites in positions 76, 78, 80, and 82 was synthesized and subcloned into the downstream of pSI-check2 named as (M1-WDR37). The sequence of circWDR37 with 3 mutation sites in positions 108, 110, and 113 was synthesized and subcloned into the same site as WT-WDR37 in pSI-check2 (M2-WDR37). Double-stranded nucleotides were designed to mimic endogenous mature hsa-miR-328-3p  and hsa-miR-138-5p  synthesized by GenePhama Co., Ltd. . A scrambled RNA was used as a negative control (NC). WT-WDR37/M1-WDR37/M2-WDR37 mixed with miR-328/miR-138/NC was cotransfected into HEK-293 cells. Luciferase activities were measured with a double-luciferase reporter assay kit  on a luminometer  after one-day culture.With the ceRNA hypothesis, the potential targets/miRNAs of circRNAs were predicted using homemade prediction software based on Miranda and TargetScan . Throughhttp://www.geneontology.org), which includes biological process (BP), cellular component (CC), and molecular function (MF), three domains. The P value produced by topGO denoted the significance of GO terms enrichment in the circRNA-miRNA-targeted genes. Pathway analysis was identified by KyotoEncyclopedia of Genes and Genomes . The P value cut-off is 0.05 , which denoted the significance of the pathway relevant to PAH. The target genes of circWDR37 were further annotated in terms of the diseases using the KEGG orthology-based annotation system (KOBAS) 3.0.The molecular functional roles of the circRNA target genes profiles were analyzed using Gene Ontology . Mean values and standard deviations and statistical differences were examined with Excel 2013 (Microsoft Corporation). Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post hoc. When WDR37 gene , with a G/T splice junction site. As hypoxia is a well-known factor for PAH, driving abnormal pulmonary arterial smooth muscle cell (PASMC) functions and the consequent hypoxic pulmonary arterial remodeling, we first invested the expression of circWDR37 in response to hypoxia in cultured human PASMCs (hPASMCs). As shown in In our previous study, we have screened the circRNA expression in PAH-COPD patients and found circWDR37 is the most significantly upregulated circRNA in PAH patients with the higher predictive power; therefore, we conducted this study on investigating its roles in PAH pathogenesis. As shown in Using si-circWDR37 transfection, we next assessed its effects on the proliferation of PASMCs under hypoxic conditions. Compared to normal groups transfected with scrambled RNA (NC), hypoxia markedly increased cell viability, which was attenuated by si-circWDR37 transfection . Indeed,It is well known that PAH-PASMCs exhibit an unbalanced apoptosis phenotype in vitro. We next assessed the role of circWDR37 in cell migration and apoptosis. Similarly, hypoxia increased the cell migration rate and antiapoptotic ability, which can be greatly attenuated by si-circWDR37 transfection under hypoxic conditions . Taken tAs competing endogenous RNA (ceRNA) circRNAs compete with messenger RNAs (mRNAs) for microRNAs (miRNAs). By sharing common miRNA response elements (MREs), circRNAs can act as miRNA “sponges” to regulate mRNA activity targeted by normal miRNA , 28. To In MRE predictions, there were two 7 seed-nucleotide regions at the positions 76-83 and 107-113 of circWDR37 matching the miR-328 and miR-138, respectively . The seeSustained pulmonary vasoconstriction leads to pulmonary vascular remodeling (PVR) such as pulmonary arterial intimal thickening and medial hypertrophy with different PA vasculature cell dysfunction . SeveralAbnormal proliferation and apoptosis of PASMCs is one major pathological process in hypoxic PVR. Extending from our previous studies, current findings further proved dysregulated circular RNAs contribute to the pathogenesis of PAH by regulating the proliferative disorder of PASMCs . Differehttp://mirdb.org/) (data not shown). The unidentified targets for miR-138 may contribute to the pathogenesis of PAH, and the underlying mechanisms were still unclear. Therefore, the effects of circWDR37/miR-138/gene (pathway) axis in hypoxic PASMCs and PAH models are still needed to be studied further. With continuous progress in the field of circRNAs, the ceRNA network for circWDR37 should be revealed in the near future. Still, the upregulated hsa_circWDR_37 in COPD-PAH patients regulated hypoxic proliferation, cell cycle progression, migration, and apoptosis of hPASMCs indicated its role in the pathogenesis of hypoxic PAH.Recently, increasing studies have studied the functional role of circRNA in the experimental PAH model. In mice with hypoxia-induced PAH, circ-calm4 has been proved to be upregulated by hypoxia and regulate the proliferation of PASMCs via sponging miR-337-3p/Myo10 signal transduction axis . Among tThe function pathway and KEGG disease analyses for the target genes of the circWDR37 in our study further provided new insight into the circRNA-regulated mechanisms of PAH. Among the enriched GO terms in BP, the cytokine-mediated biological process was identified. Many studies have proved that the levels of inflammatory cytokines, like interleukins, monocyte chemotactic protein 1, and tumor necrosis factor are elevated in patients with PAH . ProinflBesides, there were some limitations in the present study. Firstly, the function studies were performed only on the human PASMC cell line for the limited information of rat/mouse circRNAs. In the commonly used circRNA database, there are more than 80 thousand human circRNAs while there are only 27 thousand mouse circRNAs. The unknown circRNAs of other species still need to be identified in the future. Secondary, we mainly reported the function of circWDR37_016 in regulating cell proliferation and cell-cycle progression of hypoxic PASMC in the present study, the miR-138 regulatory cascade underlies hypoxia-induced cell proliferation, and migration by circWDR37_016 should be studied in the near future. Lastly, the function of circWDR37 should be confirmed in PAH animal models and other possible functions such as transcriptional or translation regulation still need to be considered in further functional studies.In conclusion, our present study provides evidence for the role of circWDR37 in hypoxic hPASMCs. Increased levels of circWDR37 might drive the excessive proliferation, cell cycle progression, antiapoptosis, and migration of PASMCs by sponging miR-138, contributing to hypoxic remodeling of pulmonary vessels. Thus, inhibition of circWDR37 may reverse the progression of PAH, suggesting a new therapeutic target.


Epigenetic modifications in RNA are known to play critical roles in cell differentiation through regulating expressions of some key genes including members of the suppressor of cytokine signaling (SOCS) family. The present study aimed to unveil the relationship of SOCS mRNA methylation induced by methyltransferase like 3 (METTL3) with Graves’ disease (GD).+ T cell microarray of GD tissues and isolated peripheral blood mononuclear cells (PBMCs). Furthermore, expressions of METTL3 targeted genes were detected using METTL3 knock-down experiment in RAW264.7 cells.Differently expressed genes (DEG) in GD tissues were identified using microarray analysis and further validated using CD4+T cells of GD compared to the controls. Bioinformatic analysis was undertaken by searching databases of found genes of the SOCS family that possessed many mRNA m6A modification loci. METTL3 knock-down experiment revealed that expressions of SOCS family members SOCS1, SOCS2, SOCS4, SOCS5, and SOCS6 were increased after METTL3 knock-down.High throughput microarrays revealed that METTL3 and SOCS molecules were aberrantly expressed in thyroid tissues and CD4For the first time, the present study revealed the relationship between m6A modification and GD and indicated that METTL3 may be involved in the development of GD by inducing mRNA m6A methylation modification of SOCS family members. Graves’ Disease (GD) is the main clinical sub-type of autoimmune thyroid diseases (AITDs), leading to a decline in quality of life and increasing the risk of death, and thus threatening public health. It is the cause of 90% hyperthyroidism, annually affecting 20-50 in 100,000 people, mainly aged between 30-50 years old, among whom, 5% were females and only 0.5% were males –6. Simil6-methyladenosine (m6A) is the most abundant and stable form of mRNA modification in most species and is also an important epigenetic marker involved in all aspects of the life process. mRNA m6A modification has been reported to influence mRNA alternative splicing, translation, and stability, thereby regulating the expression and function of targeted genes (6A modification is associated with the differentiation and immune response of immune cells (6A modification including m6A methyltransferase (writer), m6A demethylase (eraser), and m6A RNA binding protein (reader) has dramatically deepened our understanding of RNA epigenesis  (mRNA ms (IEGs) . Among ts (IEGs) . Studiess (IEGs) . Howeverhttp://david.abcc.ncifcrf.gov/) (https://www.ncbi.nlm.nih.gov/geo/) and found a genome-wide gene expression dataset, GSE9340, which includes 18 GD thyroid tissues. Adopting the same analysis, we screened the DEGs from GSE9340. We combined DEGs from our microarray data and GSE9340, and carried out the gene co-expression network bioinformatics analysis by using the WGCNA R software package as described previously  in our previously published GD microarray, which included 4 GD cases and 3 controls . In addirf.gov/) , 24, we eviously . Finally+ T cells from 15 GD patients and 10 controls. Furthermore, we isolated peripheral blood mononuclear cells (PBMCs) from whole blood samples of 26 newly recruited GD patients and 26 age- and sex-matched healthy volunteers and extracted total RNA from these PBMCs samples. The TRAb were positive for all the GD patients. For the healthy controls, we chose those who had normal thyroid function and negative TRAb, anti-thyroglobulin antibody (TgAb), and anti-thyroid peroxidase antibody (TPOAb). Those with thyroid diseases or other autoimmune diseases were excluded from the control group. After qualification and quantification by Nano Drop 2.0, we examined mRNA levels of METTL3, METTL14, FTO, and ALKBH5 using real-time qPCR with primers GTGATCGTAGCTGAGGTTCGT and GGGTTGCACATTGTGTGGTC for METTL3, TGGACCTTGGAAGAGTGTGTTT, and CATGAGGCAGTGTTCCTTTGTT for METTL14, TTGCCCGAACATTACCTGCT, and TGTGAGGTCAAAAAAGCGCAGAG for FTO, TCAAGCCTATTCGGGTGTCG, and AGCAGCATATCCACTGAGCA for ALKBH5, respectively.To further validate the expression of METTL3 and SOCS family members, we first explored Dataset GSE71956, which provides a genome-wide gene expression profile in CD4To explore the function of METTL3, we knocked down METTL3 in a mouse monocyte macrophage leukemic cell line RAW264.7 by transfecting METTL3 shRNA with METTL3 shRNA. The METTL3 shRNA sequences were designed as follows: upper-GATCCGGTTCGTTCCACCAGTCATAATTCAAGAGATTATGACTGGTGGAACGAACCTTTTTTG, lower-AATTCAAAAAAGGTTCGTTCCACCAGTCATAATCTCTTGAATTATGACTGGTGGAACGAACCG. Using the real-time PCR method, we identified that METTL3 gene expression was largely down-expressed after the interfering. Then we detected the gene expression of SOCS family members, such as SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7, by real-time PCR. The primers of these genes are shown below:SOCS1 former: GACGCCTGCGGCTTCTATT,reverse: CAGCTCGAAAAGGCAGTCG;SOCS2 former: ACGGAATGGGACTGTTCACC,reverse: AAGGCAGTCCCCAGATCGTA;SOCS3 former: TGGTCACCCACAGCAAGTTT,reverse: TCGCTTTTGGAGCTGAAGGT;SOCS4 former: CGGAGTCGAAGTGCTGACAG,reverse: ACTCAATGGACGAACAGCTAAG;SOCS5 former: TTCCCATGAGAACTTACAGCAAG,reverse: TTTTGTGCTAAATCCGAGCCA;SOCS6 former: AAGCAAAGACGAAACTGAGTTCA,reverse: CAGCTCCCGAATAAAGAGTCATC;SOCS7 former: GAAACCCAGGTTGACAAGAACT,reverse: TCCACAAGCGATACTGTCTCA.SPSS 20.0 software  was used to carry out the statistical analysis. A T-test was performed to analyze the differences in gene expression between two groups. WGCNA R software package was used to find functional pathways including DEGs. p value smaller than 0.05 was considered statistically significant.6A methylation, such as METTL3. GO function enrichment analysis indicated that the genes in the blue module are mainly involved in immune response pathways such as innate immune response (Bonferoni P = 3.18E-23) and adapted immune response (Bonferoni P = 2.92E-20) pathways , which targets mRNA m6A modification to screen m6A modification loci. The results revealed numerous m6A modification loci on mRNA of SOCS family members. We used bioinformatic database RMBase v2.0 , while SOCS3 mRNA level was not significantly changed after METTL3 knockdown .6A modification as a form of epigenetic modification has become a new research hotspot in various kinds of diseases including autoimmune diseases . The patients/participants provided their written informed consent to participate in this study.R-hS and J-aZ designed this study. C-qG, PD, and X-rL collected the samples. C-qG extracted RNA from samples. PD performed the real time qPCR. X-rL performed cell culture experiments. R-hS analyzed data and wrote the draft. J-aZ reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant No. 81800696 and 81873636), Science and Technology Development Fund of Pudong New District Minsheng Scientific Research  Project (No. PKJ2018-Y39), Talent Youth Cultivation Plan of Pudong New District (No. PWRq2020-11), Shanghai Medical Key Specialty (No. ZK2019C09) and “Top-100 Talent Cultivation Plan” of Shanghai University of Medicine and Health Sciences (No. B3-0200-20-311008-30).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South America has caused severe human congenital and neurologic disorders. Thus, it is vitally important to determine the inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to the ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost the ability to inhibit infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection. Zika virus (ZIKV) was first isolated from rhesus monkeys in the Zika forest of Uganda, Africa, in 1947 . An epidUbiquitin-specific peptidase 38 (USP38) is a member of ubiquitin-specific processing enzymes family, and it has been reported that it inhibits type I IFN signaling during viral infection , which iThe envelope of mature ZIKV is composed of the E protein and M protein, and the main function of ZIKV E protein is receptor binding, membrane fusion, and host cell entry ,21,22,23In this study, our results revealed that overexpression of USP38 in Hela cells inhibited ZIKV invasion, and that Hela cells stably expressing USP38 shRNA significantly promoted ZIKV invasion. ZIKV infection had no effect on USP38 expression in Hela cells. Furthermore, USP38 could interact with the ZIKV E protein and co-localize with ZIKV E protein in the cytoplasm. Meanwhile, USP38 decreased E protein polyubiquitination. At the same time, the deubiquitinase activity of USP38 was indispensable for inhibiting the replication of ZIKV. In conclusion, we revealed a new function of USP38 in viral infection and this may provide a new potential therapeutic target for ZIKV-associated diseases.2. C6/36 cells were maintained in an incubator at 30 °C with 5% CO2. HEK293T, Hela , A549  and C6/36  cells were purchased from the American Type Culture Collection . HEK293T cells, A549 cells, and Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. C6/36 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. HEK293T and Hela cells were maintained in an incubator at 37 °C with 5% COAntibody against Flag (F3165) (1:1000), HA (H6908) (1:2000), and GAPDH (G9295) (1:5000) were purchased from Sigma . Antibody against Myc (2276S) (1:2000) was purchased from Cell Signaling Technology . Antibodies against ZIKV NS5 protein (GTX133312) (1:1000) and Envelope protein (GTX133314) (1:1000) were purchased from GeneTex . Antibody targeting USP38(ab72244) (1:1000) was purchased from Abcam . PEI was purchased from Sigma . RPMI-1640 and DMEM were purchased from Gibco . Protein ladder (26616) was purchased from Thermo Scientific . Complete, EDTA-free Protease Inhibitor Cocktail was purchased from Roche . Normal rabbit immunoglobulin G (IgG), and normal mouse immunoglobulin G were purchased from Invitrogen Corporation .Cells were lysed at 4 °C with lysis buffer in the presence of cocktail (Roche), centrifuged at 4 °C at 12,000 rpm for 10 min to collect the supernatant, mixed with the loading buffer, and then subjected to 8% or 10% SDS-PAGE and Western blotting. For Co-IP analysis, 50 µL of supernatant after centrifugation was used for Western blotting. The left lysate was incubated with the indicated primary antibody overnight. The next day, the mixture was incubated with A/G-Sepharose for 2 h and then added to the loading buffer for Western blot analysis. The formula of the lysis buffer used in this study is as follows: 50 mM Tris-HCl, pH7.4–7.6, 300 mM NaCl, 1% Triton-X, 5 mM EDTA, and 10% glycerol. After being transfected with plasmids or infected with the virus as indicated, Hela cells were washed three times with PBS, then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% BSA, and finally the cells were incubated with the primary antibody overnight at 4 °C. The next day, the secondary antibodies and DAPI were added to the cells after washing off the primary antibodies. The cells were analyzed using a confocal laser scanning microscope .RNA is extracted by Ultrapure RNA Kit (CWBIO), cDNA is reverse transcribed by HiScript II Reverse Transcriptase (Vazyme). All specific primers for testing were designed by Primer Bank or Primer Premier 5.0. The primer sequences were as follows: ZIKV forward: 5′- GGTCAGCGTCCTCTCTAATAAACG-3′; and ZIKV reverse: 5′-GCACCCTAGTGTCCACTTTTTCC-3′, GAPDH forward: 5′-ATGACATCAAGAAGGTGGTG-3′; GAPDH reverse: 5′-CATACCAGGAAATGAGCTTG-3′. USP38 forward: 5′-TCATCAGGAGCCTAACCACC-3′; USP38 reverse: 5′-TCAGGAGAGCAATTACCCACG-3′. The sequence of shRNA targets human USP38 gene (sh-USP38-1) used in this study was as follow: 5′-CGTCTAATACTATGACTGTTA-3′; (sh-USP38-2): CCAGAGATTCTTACTGGTGAT. The shRNA was cloned into pLKO.1 vector (Sigma-Aldrich) and then transfected with psPAX2 and pMD2.G into HEK293T cells. After 36–48 h of transfection, the cell supernatants were collected and filtered through a 0.45-µm filter. Hela cells were infected by collected supernatants for 24 h and 4 μg/mL polybrene (Sigma) was used to assist this infection, after that infected Hela cells were selected by puromycin (Sigma) for 5 days and detected by immunoblot analysis.ZIKV strain z16006  isolated by the Institute of Pathogenic Microbiology, Center for Disease Control and Prevention of Guangdong  was used in this study. Myc-UB, Myc-K48, Myc-K48R, Myc-K63, and Myc-K63R plasmids were kindly gifted by Dr. Ying Zhu of Wuhan University. The cDNA of human USP38 was provided from Jiahuai Han laboratory of Xiamen University and then cloned into pcDNA3.1 (+)-3× Flag and pCAGGS-HA vectors. The N-terminal domain (1–400aa), C-terminal domain (401–1004aa) of USP38, USP38 (C454A/H857A/D918N) mutant and ZIKV Envelope protein of the corresponding fragment of ZIKV cDNA were cloned into pcDNA3.1 (+)-3 × Flag vector and pCAGGS-HA vector. The expression plasmids Envelope (1–193aa), Envelope (1–296aa), Envelope (1–406aa), Envelope (52–505aa), Envelope (132–505aa), and Envelope (280–505aa) were constructed into pcDNA3.1 (+)-3× Flag vectors.t-test was used for analysis between two groups of samples, and one-way ANOVA was used for analysis between multiple groups of samples. p < 0.05 was considered statistically significant .All experiments are repeated three times independently and the statistical analysis of these experimental data was completed by GraphPad Prism 8 software. Student’s Previous studies determined that, during viral infection, USP38 inhibits type I interferon pathway by degrading TBK1, thereby inhibiting the synthesis of type I IFN . Here, wNext, the mechanism by which USP38 represses ZIKV infection was explored. HEK293T cells were co-transfected with HA-USP38 and Flag-C, Flag-prM, and Flag-E, respectively. The results of co-immunoprecipitation (Co-IP) showed that USP38 specifically interacted with ZIKV E protein, but failed to interact with ZIKV C protein or prM protein A. ReciprUSP38 contains two domains, the N-terminal USP domain and the C-terminal domain containing Ubiquitin carboxyl-terminal hydrolase (UCH). To determine which domain is responsible for the interaction with E protein, we constructed three plasmids, pFlag-USP38 expressing the full-length of USP38, pFlag-USP38-N expressing the N-terminal domain of USP38, and pFlag-USP38-C expressing the C-terminal domain of USP38. Co-IP analysis results showed that E protein interacted with the full-length of USP38 and the C-terminal domain of USP38, but could not interacted with the N-terminal domain of USP38 E. ZIKV E protein contains three domains, a central b-barrel (domain I), an elongated finger-like structure (domain II), and a C-terminal immunoglobulin-like module (domain III) . Based oPrevious studies have reported that ZIKV E protein promotes the attachment and entry of the virus into host cells , and theThe type of E ubiquitination removed by USP38 was then explored. Interestingly, we noticed that USP38 could remove both K48-linked polyubiquitin and K63-linked polyubiquitin of E protein C. These We speculated that deubiquitinase activity of USP38 may play an important role in the restriction of ZIKV infection. To verify this speculation, we constructed a mutant of USP38-MUT (C454A/H857A/D918N) in which the deubiquitinase enzyme activity of USP38 was eliminated. HEK293T cells co-transfected with Flag-E, Myc-UB and HA-USP38 or HA-USP38-MUT. The results showed that ZIKV E protein polyubiquitination catalyzed by Myc-UB were significantly removed by USP38, but relatively unaffected by USP38-MUT A. In addSince the outbreak of Zika virus in South America in 2015, it has aroused public attention . NumerouZIKV belongs to the genus flavivirus within the family Flaviviridae, and the flavivirus envelope protein is responsible for virus entry and represents a major target of neutralizing antibodies . The posBased on our data, we proposed the molecular mechanism of USP38 against ZIKV infection. USP38 combines with the central b-barrel (domain I) and elongated finger-like structure (domain II) of E through its C-terminal domain. USP38 was capable to attenuate both K48- and K63-linked ubiquitination of E protein to prevent ZIKV infection. Overall, our study provided new insight into the regulatory mechanisms of Anti-Zika responses and offers a potential target for ZIKV infection.