TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify  MUSASHI-2 (MSI2) expression are biomarkers of resistance to PRMT5-targeted therapy in B-cell lymphoma. Moreover, combining PRMT5 inhibition with MSI2 or BCL-2 inhibitors blocks the translation of key drivers of lymphoma, c-MYC and BCL-2, inhibiting cell growth.Inhibition of the protein arginine methyltransferase PRMT5 has been suggested as a promising therapy for lymphoma. Here, the authors show that TP53 loss of function and  Protein arginine methyltransferase-5 (PRMT5) is the major enzyme responsible for the symmetric dimethylation of arginine on a variety of cytoplasmic and nuclear substrates, including histones, most notably at H3R2, H3R8, H4R3, and H2AR3, and these can result in both repression or transcription of genes2. PRMT5 knockout mice are embryonically lethal and conditional knockout studies in mice demonstrate major defects in the function of hematopoietic stem cells and B cells in various stages of development4.The protein arginine methyltransferases (PRMTs) family is a group of nine enzymes that transfer a methyl group from a cofactor, S-adenosyl methionine, to arginine residues on their target protein. PRMTs are classified into three groups based on their methylation pattern: monomethylation, symmetric dimethylation, or asymmetric dimethylation8. Among others, PRMT5 methylates P53 thereby modifying its DNA-binding activity and suppressing the expression of proapoptotic and antiproliferative genes, which contributes to lymphomagenesis9. Furthermore, PRMT5 increases de novo syntheses of key lymphoma-drivers, MYC and CYCLIN D1 by repressing miR-33b, miR-96, and miR-50310. Moreover, PRMT5 cooperates with MYC to sustain splicing fidelity, which is key to ensure tumor maintenance of MYC-driven lymphomas11, and promotes the upregulation of BCL-6 repressive activity in DLBCL12. Collectively, these data suggest selective targeting of PRMT5 may have a therapeutic value in lymphoma. This led to the development of the first selective inhibitors of PRMT5, of which three have progressed into clinical trials.Non-Hodgkin’s lymphoma (NHL) is the 7th most common cancer in adults and remains largely incurable with current treatment modalities. PRMT5 is overexpressed in germinal center-derived lymphomas and mantle cell lymphoma and plays an important role in lymphomagenesis by regulating several oncogenic pathways13. Currently, its close analogue, GSK3326595, was the first to be approved for Phase I clinical trials in patients with NHL and advanced solid tumors (NCT02783300). Due to the increased interest of PRMT5-targeted therapy application in the clinical setting and the potential development of drug resistance, we investigated the mechanisms of resistance to PRMT5 inhibition. To this end, we performed a genome-wide CRISPR/Cas9 screen in the mantle cell lymphoma line, Z-138 and a validation CRISPR/Cas9 screen in the DLBCL cell line, OCI-LY19. The top sensitizing gene was TP53, which validated our screens, as it is a well characterized target of PRMT59. Furthermore, we found that TP53 deletion and the hot spot TP53R248W mutation are biomarkers of resistance to PRMT5-targeted therapy.Here, we study the mechanisms of resistance and sensitivity to the selective PRMT5 inhibitor, GSK3203591 (GSK-591). This compound belongs to a series of non-SAM analogs that inhibit PRMT5 by occupying the substrate binding pocket in the presence of the PRMT5’s cofactors, MEP50 and SAM19. We found that c-MYC and BCL-2 are key targets of the MSI2/PRMT5 axis, which drives resistance to PRMT5 inhibition in B-cell lymphomas.We identified the RNA-binding protein MUSASHI-2 (MSI2), as the top driver of resistance to PRMT5 inhibition. MSI2 controls protein translation by binding primarily to the 3′UTRs of mRNAs. MSI2 plays a key role in hematopoietic stem cell activation, myeloid leukemia, and chronic lymphocytic leukemia (CLL) and its expression correlates with poor prognosis in multiple hematological malignancies, but its role in B-cell lymphoma is not currently knownMoreover, our study provides mechanism-based combination strategies to overcome resistance to PRMT5 inhibitors that can define therapeutic interventions to be applied in further clinical trials. First, the combination of GSK-591 with Ro 08-2750, which inhibits MSI2 activity, results in loss of c-MYC and BCL-2 protein expression. The second strategy utilizes dual targeting of PRMT5 and BCL-2, using GSK-591 and venetoclax, that exhibits potent anti-tumor efficacy and induction of apoptosis across a wide range of lymphomas.21, and to expand on these studies, we induced genetic depletion of PRMT5 with a short hairpin RNA (shRNA). PRMT5 knockdown reduced proliferation in 6 cell lines across different subtypes of B-cell lymphoma  rescues Z-138 cells from PRMT5 knockdown-mediated toxicity  and from DMSO and GSK591-treated cells, then the sgRNA abundance was measured by deep sequencing Fig. . Normalip-value < 0.1 and log2FC < −2 for depletion and log2FC > 2 for enrichment), which yielded 316 sensitizing genes and 89 genes involved in resistance to GSK-591  as essential genes for lymphoma cells survival in our unbiased CRISPR screen   is found in this pathway and was identified as the top driver of resistance to GSK-591  analysis. We found that 591 Fig. . ConsequTP53 may be a target of GSK-591. Accordingly, we investigated whether P53 wild type expression was essential for GSK-591 activity. Here, we used Z-138 and OCI-LY19 cells with wild type TP53 and then generated isogenic, TP53 stable knockout cells using CRISPR/Cas9 using sgRNAs targeting TP53. In the parental cells, GSK-591 treatment increased P53, PUMA, and BAX protein expression and induced PARP cleavage, but failed to do so in P53-deficient cells  were biallelic mutations in R248 of TP53  or carrying TP53R248W , . We found that MSI2 expression is significantly higher in GCB-DLBCL , higher expression of MSI2 exhibited significantly shorter overall survival times compared to those patients with low expression of MSI2 , , Fig. . We examBCL Fig. . Moreove0), Fig. . Further0), Fig. . To exam591 Fig. . These d25 , a small molecule that inhibits MSI RNA-binding activity and has anti-proliferative activity in myeloid leukemia cells and chronic lymphocytic leukemiaone Fig. . Both gep-value < 0.05 and log2FC ≥2). However, the combination of Ro and GSK-591 synergistically induced changes in the transcriptome of Z-138 cells with 1531 transcripts upregulated and 1069 downregulated upon combination treatment   Fig. .Fig. 4CoCDK4, CDK2, CHK1, CDC25A, and related transcription factors, including E2F1   Fig. . Intereses) Fig. , a key rion Fig. . We furtion Fig. . Consistion Fig.  and reduion Fig. . Taken t29. Moreover, we found previously that MSI2 binds and regulates c-MYC translation in myeloid leukemia cells30. We tested if PRMT5 and MSI2 inhibitors alone or in combination affected c-MYC mRNA in lymphoma cells. RNA-Seq analysis showed that treatment with GSK-591 and Ro alone did not affect c-MYC or MSI2 at the mRNA level, however, the combination of these drugs subtly reduced MSI2 mRNA  but did not induce significant changes to c-MYC mRNA  with Z-138 cells treated with either vehicle, GSK-591, Ro, and the combination. We confirmed that MSI2 binds c-MYC mRNA in the control cells and GSK591-treated cells, but the treatment with Ro or the combination significantly impaired MSI2 binding to c-MYC RNA . In this fusion, ADAR marks MSI2 binding sites that can be identified by RNA-sequencing   Fig. . MSI2-Hy01) Fig. .Fig. 6Rop-value < 0.05), while PRMT5 inhibition did not yield significant changes to MSI2 edit sites. The combination of Ro and GSK-591 did not induce significant changes in MSI2 binding when compared with Ro alone ,  differentially expressed genes in Z-138 cells treated with Ro, GSK-591 and the combination (DeSeq data), 2) the Ro-specific MSI2 targets (HyperTRIBE) and 3) genes that confer resistance to PRMT5 inhibition (genome-wide CRISPR screen). The analysis revealedt the anti-apoptotic protein BCL-2, hexamethylene-bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) and the spindle and kinetochore-associated protein 2 (SKA2) as candidates that could be part of the PRMT5-MSI2 axis Fig. .Fig. 7MSBCL-2 mRNA and treatment with Ro or with the combination significantly blocked MSI2 binding to BCL-2  and did not affect the binding to HEXIM1  and HEXIM1  mRNAs, while SKA2 was modestly upregulated upon combination treatment   to pharmacological inhibition of the methyltransferase. Additionally, PRMT5 inhibition in vivo exhibited modest anti-tumor activity. To help understand which pathways or genes contribute resistance and sensitivity to PRMT5 inhibition, we performed a genome-wide CRISPR/Cas9 screen.37. MEP50 is a known cofactor of PRMT5, and enhances PRMT5 binding affinity to its substrates38, hence it is not surprising that MEP50 was identified as a top hit in this screen. Likewise, PRMT1 shares common substrates with PRMT5, thus the dual inhibition of PRMT1 and PRMT5 results in potent anti-tumor activity in different cancer models40. However, our unbiased screen did not identify MEP50 nor PRMT1 as top targets, suggesting the existence of cell context dependencies for GSK-591 action.Previously, Gao et al., performed a CRISPR/Cas9 screen in the presence of a PRMT5 inhibitor using a small library targeting epigenetic regulators  in non-small cell lung cancer cells. They identified MEP50 and PRMT1 as key molecular targets of GSK-591TP53 as one of the most sensitizing genes to GSK-591 in lymphomas. PRMT5 regulates P53 through maintaining the splicing fidelity of MDM4 and thus negatively regulates P53 function42. Additionally, PRMT5 may directly methylate P53 on R333, R335 and R337, preventing its oligomerization with MDM2, which prevents TP53 binding to its target genes42. Our data support a link between PRMT5 and P53 with several experiments. First, we observed that most cell lines with wild-type TP53 are sensitive to GSK-591. Second, we demonstrated that P53 is required for GSK-591 activity because its loss conferred resistance to PRMT5 inhibition. Finally, reintroduction of wild-type TP53 in cells harboring TP53 missense mutations was sufficient to restore lymphoma cells’ sensitivity to GSK-591. These data indicate that GSK-591 inhibits PRMT5 and the functional consequences are mediated through P53.Our screens identified TP53 genetic alterations are found throughout the gene structure, but the functional relevance for each mutation has not been fully characterized and could be dependent on cell context. Some mutations are suggested to have either a loss-or gain-of-function45. However, mutations localized in the DNA-binding domain are likely to act like loss-of-function mutation as they abrogate TP53 transcriptional activity47. We found the R248W mutation of TP53 is associated with resistance to GSK-591 in vitro and in vivo. The hotspot TP53R248W mutation induces dominant negative activity preventing TP53 binding to its target promoters and correlates with shorter survival in patients across different types of cancer50. GSK-591 failed to induce TP53 targets, PUMA, P21 and BAX in the TP53R248W isogenic cell lines, even though P53 was upregulated. In agreement with our results, the chemotherapeutic agent, daunorubicin, induces P53 protein expression in TP53R248Q cells, however, this protein is not able to recruit targets, such as P2148. Thus, we hypothesize that PRMT5 might still be able to bind and methylate TP53R248W, therefore, GSK-591 treatment results in the upregulation of a dominant negative form of TP53 that mimics its deletion. Our findings have clinical implications as TP53R248W/Q is the most common site-specific mutation in all cancers, thus it could be used for patient selection in the ongoing and future clinical trials with PRMT5 inhibitors.MSI2 is highly expressed in DLBCL patients and strongly correlates with PRMT5 expression. Elevated MSI2 expression is considered a biomarker of poor outcome in multiple hematological diseases, such as chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL)53. We observed that MSI2 depletion reduced about 50% tumor growth and resistant tumor cells escaped due to inefficient MSI2 knockdown in a lymphoma xenograft model. Considering this, it would be interesting to conduct studies on B-cell lymphoma mouse models to determine whether MSI2 is required for both lymphoma initiation and progression.Our CRISPR/Cas9 screens identified the RNA-binding protein MSI2 as the top driver of resistance to PRMT5 inhibition. We found that 26. We demonstrate that the combination of Ro with GSK-591 induces synergistic antiproliferative activity and proves to be a viable therapeutic strategy to target B-cell lymphomas. Previously, we found Ro reduced binding of MSI2 to selected targets using RNA-IP 26. Our HyperTRIBE analysis found that Ro inhibited MSI2 binding to its targets regardless of the frequency of binding. It is also not clear why some targets were inhibited more than others. Over 1500 mRNAs were identified as Ro-specific targets in B-cell lymphoma. These Ro-targets were enriched for pathways previously linked to MSI2 in hematopoiesis and leukemogenesis. Thus, our data supports the need to develop more potent and clinically relevant molecules targeting MSI2 in multiple hematological malignancies.Previously, it has been shown that inhibition of MSI2 with Ro reduced disease burden in AML and CLL modelsMLL-leukemia self-renewal programs by retaining the translation of HOXA9, c-MYC, MYB, and IKZF231. MSI2 directly binds and maintains TETRASPANIN 3 (TSPAN3), branched-chain amino acid aminotransferase 1 (BCAT1) and FMS-like tyrosine kinase 3 (FLT3)55. Besides promoting translation, MSI2 mediates repression of its targets, including NUMB and P2126.MSI2 drives leukemia progression and stem cell renewal through a variety of mechanisms involving multiple pathways. We previously found that MSI2 maintains MYB and IKZF2, and Ro treatment resulted in their loss of protein abundance.Using HyperTRIBE, we identify direct targets of MSI2 that cooperate with the resistance mechanism to GSK-591 in B-cell lymphoma. Consistent with previous studies, in B-cell lymphoma, MSI2 binds to canonical targets such as 57. PRMT5 depletion and genetic deletion block the binding of splicing factors, such as SRSF1 and SRSF2, to their mRNA targets59. Moreover, another study demonstrated that the combination of PRMT5 and PRMT1 inhibitors is synergistic in AML due to the inhibition of the arginine methylation of a large compendium of RBPs40. However, MSI2 was not identified among PRMT5’s methylation targets in these studies, which is consistent  with the fact that PRMT5 inhibition alone did not affect MSI2 RNA-binding activity in our study. Nevertheless, regulation of multiple RBPs could lead to a combinatorial effect when both MSI2 and these targets are affected.PRMT5-mediated arginine methylation of RBPs is required for optimal binding activityThe differential expression analysis revealed the combination of GSK-591 and Ro is synergistic, inducing global changes in the transcriptome of B-cell lymphoma cell lines. Thus, the combination resulted in greater downregulation of signatures associated with cell cycle, c-MYC targets and upregulation of the P53 pathway. Previous studies have demonstrated that both PRMT5 and MSI2 maintain c-MYC expression in cancer cells through independent mechanisms, but we found MSI2 and PRMT5 cooperate by retaining c-MYC translation. Both pharmacological inhibition and genetic depletion of PRMT5 and MSI2 resulted in downregulation of BCL-2. Additionally, we found c-MYC and BCL-2 are  critical components of the PRMT5/MSI2 axis. It would be interesting to investigate whether the mechanism of resistance to PRMT5-targeted therapies mediated by the oncogenic axis, MSI2/c-MYC/BCL-2, could be extrapolated to the other cancers. These results suggest that PRMT5 and MSI2 cooperate in sustaining the shared translation of common oncogenic signaling pathways, which underscores the relevance of the dual targeting to prevent feedback mechanisms of resistance induced by the single agent.62. Combination of GSK-591 and venetoclax resulted in potent anti-proliferative activity across different lymphoma subtypes and even in the most resistant lymphoma cells, lower doses of both drugs were required in combination, which could help to prevent potential off-target toxicity.Venetoclax is the only selective BCL-2 inhibitor approved for treatment of patients with chronic lymphocytic leukemia and small lymphocytic leukemia. However, we and others have described multiple mechanisms of resistance to BCL-2 inhibition in different types of cancer, including B-cell lymphomasTP53 LOF and MSI2 expression may be useful for patient stratification in clinical studies with PRMT5 inhibitors. Furthermore, we provide a strong mechanism-based rationale for the therapeutic use of PRMT5 inhibitors in combination with MSI2 or BCL-2 inhibitors.In summary, our study provides data for the role of the PRMT5/MSI2 axis in regulating the key lymphoma drivers, c-MYC and BCL-2. Of major clinical relevance, we demonstrated that Primary patient samples were collected and approved by the Institutional Review Boards with the Biospecimen collection/banking study 06-107 and 12-245. The use of the samples for research purposes is covered under the Biospecimen research protocol 16–266.Human MCL-derived cell lines Z-138, Rec-1, Jeko-1, Mino, JVM-2, JVM-13, and BL-derived cell lines Raji, EB-1, Daudi, Ramos and CA-46 were obtained from ATCC (American Type Culture Collection). Human DLBCL-derived cell lines SUDHL-4, SUDHL-6, SUDHL-8, SUDHL-10 OCI-LY19, DB, NUDHL-1, U-2973, OCI-LY3, U-2932, Ri-1 and OCI-LY10, Hodgkin’s cell lines L-428, HDLM-2 and the MCL cell line, Maver-1, were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures . Cell lines HBL-1, TMD-8, SUP-M2, SUDHL-1, Karpas-299 and BJAB are not commercially available, and were kindly provided by Dr. R.E. Davis . P-4936 cells  were provided by Dr. J. Zhang . Cell lines were fingerprinted by STR analysis at the Integrated Genomic Operation Core, IGO . All cell lines, PDX samples and primary samples were authenticated using a targeted deep sequencing assay of 585 cancer genes (HemePACT) at the IGO (MSKCC). Cell lines were cultured in RPMI 1640 medium supplemented with 10%–20% heat-inactivated fetal bovine serum , 1% L-glutamine, and penicillin-streptomycin in a humid environment of 5% CO2 at 37 °C.+ Naive B cells were obtained from healthy donors. PBMC were isolated by Ficoll  density centrifugation and labeled with CD19 Microbeads (MACS Cat. 130-050-301).CD19Lymphoma patient cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum , 1% L-glutamine, and penicillin-streptomycin in a humid environment of 5% CO2 at 37 °C.GSK3235025 (GSK-025) used for the in vivo studies and GSK3203591 (GSK-591) for the in vitro experiments were provided by GlaxoSmithKline through MTA. Venetoclax was purchased from Selleckchem . Ro 08-2750 was purchased from Tocris.For the genome-wide CRISPR screen, lentiviral particles were made from the Brunello library (Addgene 73179) that contains 76,441 sgRNAs, targeting each gene with 4 different guides, and 1000 non-targeting control sgRNAs.For the validation CRISPR-Cas9 screen, a CRISPR KO library was designed containing 2020 sgRNAs targeting the top-ranked 316 enriched hits and 89 depleted hits from the genome-wide CRISPR screen, for a total of 405 genes (using the 4 sgRNAs per gene from the Brunello library), plus 200 non-targeting and 200 safe-harbor sgRNAs.6 cells (estimated library coverage of about 1000 cells per guide) were selected for 3 days with 4 μg/mL puromycin to enrich for cells carrying sgRNAs. Post-selection, 80 ×106 cells were sequenced to identify genes critical for survival, T0 control. Remaining cells were expanded for 3 days. Then, cells were treated with DMSO or GSK-591 (IC80) for 8 days and survived cells were harvested.Z-138 (for the genome-wide CRISPR screen) or OCI-LY19  cells expressing Cas9 were infected with the library for 48 h at a multiplicity of infection (MOI) of 0.3. Two infection replicates were performed. Two days after the infection, a total of 100 ×10p-values and FDR) at the gene level. This function implements a correlation-adjusted version of the Wilcoxon–Mann–Whitney test63. The sensitizing and resistant hits rank was obtained by applying two criteria: differential expression  and the abundance of sgRNAs in the same direction depletion or enrichment (number of sgRNAs per gene > = 3).Genomic DNA was isolated from all samples, and the sgRNA sequences were amplified by PCR . 48 h after transduction cells were selected using 10 and 12 μg/ml blasticidin, respectively, for 5 days. Puromycin resistance cassette from LentiGuide-Puro vector backbone (Addgene 52963) was swapped for GFP and the following guide RNA sequences: TP53 KO1: 5′-GATCCACTCACAGTTTCCAT-3′; TP53 KO2: 5′-GAGCGCTGCTCAGATAGCGA-3′; BCL-2 KO: 5′-GGCCTTCTTTGAGTTCGGTG-3′ were cloned into it. Z-138 and OCI-LY19 cells were transduced with lentiviral sgRNAs particles and 3 days post-transduction GFP-positive cells were sorted. Successful knockout was confirmed by immunoblotting.TP53 R248 genomic region (hg38 chr17:7674171-7674270) were designed by GuideScan. gRNA sequences most proximal to R248 were cloned into lenti-guide-dsRed vector backbone  and Sanger sequencing at the IGO (MSKCC).Sequences of target region from potential 60. The following antibodies for western blotting were purchased from Abcam: PRMT5 , MSI2 , H3R2me2 , H4R3me2 , HEXIM1  and NOXA  were purchased from Abcam. P53 , c-MYC , BCL-2 , BAX , MEP50 , SmD3me2 , PARP , Cleaved caspase-3 , P21 , PUMA , MDM2  CYCLIN B1 , CDK4 , CHK1 , RAD51 , GFP  and α-TUBULIN  were purchased from Cell Signaling Technology. β-ACTIN  and anti-FLAG  were purchased from Sigma-Aldrich. SKA2  was purchased from Invitrogen.Immunoblot analyses were performed following standard procedures as described5 cells/well with either vehicle (DMSO 0.1%) or increasing concentrations of drugs. Viable cell number was determined at 4- and 8-days drug incubation. On days of cell counts, the media and GSK-591 were replaced and cells split back to a density of 2.5 ×105 cells/well. Cell viability was assessed using CellTiter-Glo Luminescent assay (Promega). 80 μL of the cultured cells were transferred to opaque, white bottom 96-well plates and mixed with 20 μL of CellTiter-Glo reagent. The mixture was incubated for 30 min at RT and read using EnSight Multimode plate reader (PerkinElmer) for luminescence. Data was normalized as percent viability and graphed by non-linear regression curves in Graph Pad PRISM 7.0.Cells were seeded in 24-well plates at 2.5 ×10log . A log-odds value of zero indicates that the combination treatments are additive; a negative value indicates synergy between the drugs and a positive value indicates antagonism.To establish the interaction between two drugs in combination assays, we calculated the log-odds; the value: G367A/R368A and PRMT5 wild type were a gift from Dr. Joae Wu . GFP-P53 and FLAG-BCL-2 were from Addgene . pLKO_TRC005 vector was used for silencing PRMT5 (sequence: 5′-AGGGACTGGAATACGCTAATT-3′). pLKO_TRCN0000062809 Sequence of MSI2 shRNA (sequence:5′CCGGGTGGAAGATGTAAAGCAATATCTCGAGATATTGCTTTACATCTTCCACTTTTTG-3′).pBabe-puro-Flag enconding PRMT5ad libitum. Temperature and relative humidity were maintained at 20 ± 2 °C and 65%, respectively. Female NSG mice (NOD scid gamma mice) were obtained from the Jackson laboratory. 6-week old NSG female mice were injected subcutaneously with 10 million cells together with matrigel. Once tumors reached a volume of 100mm3, mice were randomized to receive either vehicle control (0.5% methylcellulose) or GSK-025, 100 mg/kg orally twice/day for 3 weeks. For the drug combination studies, mice were randomized to receive either vehicle, GSK-025 50 mg/kg twice daily and/or venetoclax 100 mg/kg/daily by oral gavage administrated. Mice were observed daily throughout the treatment period for signs of morbidity/mortality. Tumors were measured three times weekly using calipers, and volume was calculated using the formula: length x width 2 × 0.52. Body weight was also assessed three times weekly. After 3 weeks of treatment tumor samples were collected for immunoblotting. All studies were conducted in accordance with the MSKCC and GSK policies on the Care, Welfare and Treatment of Laboratory Animals and were reviewed according to the Institutional Animal Care and Use Committee either at MSKCC or GSK. The tumor burden was not exceeded in any of the studies performed.The animals were housed in individually ventilated cages  with 12 h dark, 12 h light conditions. The animals were fed food and water 29). Z-138 cells were transduced with virus expressing MSI2-ADAR or MIG empty vector. 48 h after transduction GFP-positive cells were sorted. 24 h after treatment with 5uM of GSK-591 and Ro-0812 cells were harvested, and RNA was extracted and sequenced. Identification of RNA editing events was done by following the workflow as previously reported. Essentially, STAR% aligner was used to match paired-end RNA seq reads to the human genome (hg19). The GATK workflow was followed to call variants in RNA-seq to identify all mutations in the RNA-seq libraries. We narrowed focus to those mutations within annotated mRNA transcripts, as well as restricting to solely A-to-G mutations in transcripts encoded by the forward strand and conversely T-to-C mutations in transcripts arising from the reverse strand. Additionally, all the mutations found using the above criteria were filtered using the dbSNP database. We took the sum of the filtered sets of RNA editing events from all RNA-seq libraries within the experiment and totaled the number of reads containing reference (A/T) and alternative (G/C) alleles from each library at each site. Beta-binomial distribution was performed to identify differences of edit frequencies between MIG and MSI2-ADAR, and those conditions with drugs added in the presence of each construct. Significant sites were determined by filtering for FDR-adjusted sites (Benjamini–Hochberg correction), using a p-value or FDR of < 0.05.MSI2 Hyper-TRIBE was performed as previously described . After PicoGreen quantification and quality control by Agilent BioAnalyzer, mRNA input was used for library preparation (TrueSeq Stranded mRNA LT Sample Prep Kit). Libraries were run on a HiSeq 4000 in a 100 bp/100 bp paired-end run, using the HiSeq 3000/4000 SBS Kit (Illumina). The average number of read pairs per sample was 100 million. Sequence data were aligned using STAR aligner to the human reference genome (version hg19). Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated and differential expression analysis was conducted using the DESeq software package. Differentially expressed genes were identified as those with FPKM greater than 1 showing differential expression greater than twofold (up or down) with an adjusted P values to account for multiple hypothesis testing to yield q values. Genes were determined to be significantly differentially expressed if they possessed a q value that was less than or equal to 0.05.Raw reads were first analyzed for quality control using FastQC. Reads were then aligned to the hg19 assembly of the human genome using the STAR aligner. The resulting BAM files had their reads counted using the summarizeOverlaps function from the GenomicAlignments package in R. A count table of reads mapping to genes was constructed, and a differential expression analysis was conducted using the DESeq software package. Before significance assessment, genes were filtered according to a reads per kilobase (RPK) value of 500 given the deep average depth of uniquely mapped reads  present in the samples. Statistical significance was determined with the negative binomial test using average read counts for DMSO, Ro, GSK-591, and combination for all genes passing the RPK filter. The Benjamini–Hochberg correction was applied to all nominal Enriched and depleted genes from the genome-wide CRISPR-Cas9 screen were analyzed for pathway enrichment using ClusterProfiler. DeSeq data and Ro-specific targets were analyzed using the Molecular Signatures Database  and using the ENRICHR program. For gene set enrichment analysis, we generated rnk files based on genes that were differentially expressed (log2fold change) in response to Ro, GSK591, or combination treatment against the MSigDB and against specific gene sets based on ENRICHR hits. We also generated rnk files for the CRISPR screen hits and ran these against the MSigDB and specific gene sets. Files for each GSEA result are found in the indicated Supplementary data files.6 cells were used per IP reaction) and washed twice with ice-cold PBS. Cells were lysed in ice-cold IP lysis buffer  for 30 min on ice and frozen at −80 °C immediately to aid the lysis. On the day of IP, the lysate was centrifuged to precipitate the debris. Supernatant was collected and incubated with 5 µg of anti-MSI2 antibody (Abcam) or Rabbit IgG (Millipore) overnight at 4 °C. RNA–MSI2 endogenous antibody complexes were pulled down using Dynabeads Protein A/G (Millipore) and washed five times in 100% IP lysis buffer, 70% IP lysis buffer and 30% PBS, 50% IP lysis buffer and 50% PBS, 30% IP lysis buffer and 70% PBS and 100% PBS. RNA was extracted using the phenol–chroloroform method and quantified by qRT–PCR.Z-138 cells were treated with 5uM of GSK-591 and Ro-0812 for 24 h were collected (20 × 102 First Strand Kit (Qiagen). CFX96 (Bio-Rad Laboratories). mRNA of human oncogenes and tumor suppressor genes were assessed using the PCR pathway array  according to the manufacturer’s instructions. Primers for TP53 (qHsaCED0045022), c-MYC (qHsaCID0012921), BCL-2 (qHsaCED0057245), SKA2 (qHsaCED0047540), HEXIM1 (qHsaCED0047867), and GAPDH (qHsaCED0038674) were purchased from Bio-Rad Laboratories. Expression of indicated genes were quantified by real-time PCR assays with SYBR Green dye and the CFX96 TouchTM Real-time PCR detection System.Total RNA was extracted with the RNeasy mini kit (Qiagen). A total of 1 μg of RNA was converted to cDNA using RTHEK-293T cells were transfected with pRL-CMV MYC 3′UTR, Firefly luciferase control and MSI2 and/or PRMT5 shRNA constructs or either the cells were treated with 5uM of GSK-591 and/or Ro-0812, in the indicated experiments. 48 h post-transfection, expression of Renilla and Firefly luciferase were determined by Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions.Lymphoma primary cells and cell lines were treated with GSK-591 and Venetoclax. After 72 h, apoptosis and cell death were determined using FITC Annexin V apoptosis detection kit according to the manufacturer’s instruction .6 cells were collected, fixed with 70% ethanol and then stained with propidium iodide (PI)/RNase staining solution  at room temperature for 15 minutes. Flow cytometric data were acquired using a FACSCalibur . Greater than 10,000 events were acquired. Doublets were excluded by gating out high FL3-W (width) cells.To determine the effect of GSK-591 and Ro combination on cell cycle, cell lines were incubated with these compounds for 72 h. 1 ×10Three hours after the last drug treatment, tumors were immediately fixed in fresh 4% paraformaldehyde rotating at 4 °C overnight. Fixed tissues were dehydrated and embedded in paraffin before 5 μm sections were put on slides. The tissue sections were deparaffinized with EZPrep buffer  and incubated with antibodies against Ki-67 , or c-MYC ; BCL-2  for 5 h, followed by 60 min of incubation with the corresponding biotinylated secondary antibodies. Slides were scanned with Pannoramic 250 Flash scanner  using 20X (0.8 NA) objective lens.t test was performed to determine significance for bar graphs, growth curves, and quantifications unless otherwise specified. Those tests resulting in p-values less than 0.05 were deemed significant. Error bars reflect the standard error of the mean (SEM) unless otherwise stated. All statistical analyses were carried out within the programs GraphPad Prism 8.0 and the R statistical environment.Statistical parameters and analysis are stated in corresponding figure Legends for each panel of data. The Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary Data FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11


Traditional crosslinked aero grade epoxy resins have excellent thermal-mechanical properties and solvent resistance, but they cannot be remolded, recycled, or repaired. Vitrimers can be topologically rearranged via an associative exchange mechanism, endowing them with thermoplasticity. Introducing dynamic bonds into crosslinked networks to obtain more sustainable thermosets is currently an interesting research topic. While recent research into vitrimers has indicated many advantages over traditional thermosets, an important shortcoming has been identified: susceptibility to creep at service temperature due to the dynamic bonds present in the network. In addition, designing aero grade epoxy vitrimers (similar to RTM6 resin) still remains a challenge. Herein, low creep aero grade epoxy vitrimer with thermal and mechanical properties similar to those of aero grade epoxy resins and with the ability to be recyclable, repairable, and reprocessable, has been prepared. In this manuscript, we demonstrate that aero grade epoxy vitrimer with reduced creep can be easily designed by the introduction of a certain fraction of permanent crosslinks, without having a negative effect on the stress relaxation of the material. Subsequently, the mechanical and relaxation properties were investigated and compared with those of classical aero grade epoxy resin. A high Tg (175 °C) epoxy vitrimer was obtained which fulfilled all mechanical and thermal specifications of the aero sector. This work provides a simple network design to obtain aero grade epoxy resins with excellent creep resistance at elevated temperatures while being sustainable. Epoxy resins are one of the most widely used thermoset resins for structural applications due to their high mechanical properties, chemical resistance to many solvents, high glass transition temperature (Tg) and resistance to creep at elevated temperatures ,2. HowevIn this sense, in the last 10 years, new approaches aim to convert thermoset resins into dynamic materials by introducing dynamic covalent bonds ,15,16,17Ea) [Ea offers high creep resistance a few tens of degrees below the reprocessing temperature, due to the high-temperature dependence of bond exchange. In this sense our group has designed a bunch of disulfide-containing epoxy vitrimers with tailor-made Ea, ranging up to 340 kJ/mol [Ea was only achieved in 2-amino phenyldisulfide containing epoxy vitrimers which offer lower Tg and thermal stability than vitrimers containing 4-amino phenyl disulfide.Despite the good properties associated with vitrimers, these materials have an Achilles’ heel: the possible creep at use conditions. Creep is the continuous, time-dependent deformation of a material under a certain load and temperature . While sEa) ,29,36,37® RTM6 is a widely utilized and aerospace-certified epoxy resin, which has been in service for 20 years, it would be helpful to obtain an epoxy vitrimer with the same mechanical and thermal properties in order to replace it. Everything inside commercial aircraft goes through a process called “certification”. Based on this background the aim of the research described in this paper was to obtain an epoxy resin comparable to HexFlow® RTM6 resin while being reprocessable, reparable, weldable, and recyclable to be commercially available for the aerospace industry. Here, for the first time, a simple and commercially available high Tg epoxy vitrimer ready to be used in the aeronautical sector has been prepared and thoroughly characterized. First, the curing kinetics, as well as thermal and mechanical properties, have been investigated. Secondly, the dynamic properties of the resin have been widely studied by stress relaxation and creep experiments to demonstrate its thermoformability and reprocessability.In the aerospace industry, epoxy resins are difficult to be replaced due to their good thermal and mechanical properties. The qualification process for new aerospace materials can take many years and is extremely expensive. Since Hexcel HexFlowN,N,N′,N′-Tetraglycidyl-4,4′-methylenebisbenzenamine (TGMDA) (MY21) and Bisphenol-F diglycidyl ether (DGBF) (PY306) based epoxy resins were purchased from Huntsman Advanced Materials. 4-Aminophenyl disulfide 98% (4-AFD), was purchased from Sigma-Aldrich and was used as received. RTM6 resin was purchased from Hexcel.−1. The glass transition temperature (Tg) was obtained as the inflection point of the heat flow step. The thermogravimetric measurements (TGA) were performed on a TA Instruments Q500 equipment controlled by TA Universal Analysis software under an air atmosphere at a heating rate of 10 °C min−1 from 25 °C to 600 °C. Thermogravimetric isothermal tests were carried out at 200 °C, 210 °C, 220 °C, and 230 °C under an air atmosphere. Thermomechanical experiments were performed using TA Instruments DMA Q800 equipment on rectangular samples (12.5 × 2 × 17.5 mm) on single cantilever beam from 25 to 250 °C. The temperature-dependent behavior was studied by monitoring changes in force and phase angle, keeping the amplitude of oscillation (15 µm) (within a linear viscoelastic region) and frequency (1 Hz) constant at a 3 °C min−1 heating rate. Tensile stress-relaxation experiments were also performed on a DMA Q800 instrument controlled by TA Universal Analysis software in tensile mode (6 × 1 × 38 mm). To maintain straightness, samples were initially preloaded at a force of 1 × 10−3 N and heated to testing temperature. Before starting with the test, samples were allowed 30 extra minutes to reach the thermal equilibrium. After that, the specimens were stretched by 1% and the deformation was maintained during the test. The decrease in stress over time at each temperature was recorded, and the stress relaxation modulus was calculated.Differential scanning calorimetry (DSC) measurements were performed using a DSC from TA Instruments (Discovery DSC25 Auto controlled by TRIOS software) over a temperature range from 25 °C to 200 °C under nitrogen at a scan rate of of 20 °C minEa was calculated, using the Arrhenius equation (Equation (1)):The relaxation times at each testing temperature were determined from the Maxwell equation, as the time required to relax 63% of the initial stress. With the relaxation times obtained at each temperature, the activation energy 12 Pa [From the Arrhenius relation, the topology freezing transition temperature (Tv) was obtained as the temperature at which the material reaches a viscosity of 1012 Pa .Creep experiments below the Tg were determined by a DMA Q800 instrument in dual cantilever mode. After soaking for 2 min at the testing temperature, 10 MPa (14% UTS (Ultimate tensile strength)) constant stress was applied for 25 min. Creep experiments above the Tg were determined by a DMA Q800 instrument in tensile mode. After soaking for 2 min at each testing temperature, 1 MPa constant stress was applied. The strain change was monitored over time. For the creep recovery experiment, the constant stress of 1 MPa was applied at 210 °C and 215 °C for 120 min, and a recovery step of 90 min was applied.Rheological characterization was performed using an AR2000ex rheometer controlled by TA Data Analysis software from TA instruments using a 25 mm plate-plate geometry and a gap of 1 mm. First, strain sweep experiments were performed to determine the linear viscoelastic regimen (LVR) of the materials. Time sweep experiments were carried out from 80 °C to 130 °C,) to determine gel point as the crossover of G′ and G″ (1% of strain and 1 Hz).−1 and flexural tests were performed according to ISO 178 standard.Mechanical characterization of both epoxy resins was performed using an INSTRON 3365 Long travel Elastomeric Extensometer controlled by Bluehill Lite software. Tensile strength measurements were carried out according to UNE-EN-ISO 527 standard, using dumbbell-type test specimens at an elongation rate of 1 mm·minThe reprocessing capacity of cured aero grade vitrimer was studied using 120 × 60 × 2 mm specimens. First, the sample was preheated at 200 °C for 5 min and then the preheated sample was placed between two Teflon-coated metal plates. The reprocessing of the sample was carried out on a VOGT hot press LABO PRESS 200T at 200 °C and 20 bar for 5 min. The demoulding of the sample was performed after cooling down below the Tg of the aero grade epoxy vitrimer.−1 (The synthesis of the aero grade epoxy vitrimer used in the present study was performed following the previously reported method by our group . Briefly−1 . Complet−1 .C) provides an idea of the network structure. According to the theory of rubber elasticity, νC can be experimentally obtained using Equation (2).r is the rubbery plateau at T g +  30 °C, A is the front factor often assumed to be unity, R is the gas constant (8.314 J·mol−1·K−1) and T is the absolute temperature in K. The average molecular weight between crosslinks (MC) can be also calculated from Equation (3).Our group previously demonstrated that repair, reprocessing, and recycling could be effectively achieved in medium Tg epoxy thermosets (130 °C) . HoweverAs can be seen in In order to study the thermal stability of prepared aero grade epoxy vitrimer, thermogravimetric measurements were carried out.−1 from 25 °C to isothermal conditions. Isotherms were measured under an air atmosphere for 20 min  of the cured samples was performed on a TA Instruments Q500 equipment under an air atmosphere at a heating rate of 30 °C·minr 20 min . Additior 20 min .Thermogravimetric analysis showed that developed epoxy vitrimer possesses thermal stability up to 210 °C (weight loss lower than 1% without Tg decrease) when exposed to 20 min under air atmosphere. From these results, it can be concluded that 210–220 °C is the maximum temperature at which the resin can be exposed under air atmosphere to assure no degradation of the epoxy vitrimer matrix. Thermal stability together with dynamic stress relaxation results will permit to the establishment of the time and temperature conditions for future thermoforming processes.One of the major drawbacks of the RTM6 resin is its inherent brittleness, ascribed to the highly crosslinked network and to the high internal stresses produced by volume shrinkage during curing. With the aim of reducing this brittleness, different approaches have been studied to increase toughness. In this sense, the introduction of a dynamic hardener could be a new approach to increasing the resin toughness. It has been demonstrated that polymer networks formed by transient crosslinks constantly reconfigure and dissipates energy, increasing the toughness of the resin. In developed aero grade epoxy vitrimer, the easier cleavage and rearrangement of aromatic disulfide bonds present in the 3D network, could be beneficial to stimulate the release of internal stresses that occurred in the curing of the network .Fracture toughness tests were performed by using SENB (Single-Edge Notched Bending) specimens in a 3-point bending configuration, according to the ISO 13586 test standard. Pre-notches were machined to a depth of 0.45 times the specimen width using a CEAST notching machine . Subsequently, the pre-notches were sharpened using a femtosecond laser (femtolaser) to a depth of approximately 0.5 times the width of the specimen. This process allowed the production of SENB specimens for 3-point bending. The SENB specimens were tested on a universal testing machine  with a support spacing of 48 mm and a crosshead displacement speed of 1 mm/min.QK can be calculated using Equation (4),Q is the maximum force, B is the thickness of the specimen, W is the width of the specimen and f is a geometrical calibration factor. The results of the QK calculation are shown in As can be seen from the force-displacement graphs in 1C, the critical stress intensity factor near the edge of a crack, and G1C, which is the energy needed to create two surfaces during the fracture propagation. 1C is improved from 0.62 Mpa·m1/2 to 1.02 MPa·m1/2, which corresponds to an increase of 64%. The increase in the fracture toughness of developed aero grade epoxy vitrimer can be attributable to the presence of dynamic disulfide bonds in the matrix together with lower Tg of the resin compared to RTM6 resin. There are two main parameters that describe the fracture toughness of a material: KIn order to study the processability of the 3R epoxy resin for the RTM process, the rheology of uncured resin was characterized. Taking into account the specifications for the RTM process for uncured resin, three different parameters were measured  and loss modulus (G″)) .In To study the effect of curing time at different temperatures a DSC analysis was carried out. The degree of cure at each temperature after a temperature ramp of 30 °C/min was calculated. Dynamic curing of both aero grade epoxy vitrimer and reference RTM6 was also performed in order to see the differences both in the curing exotherm and curing temperature . Table SThe heat of cure can be used to quantitatively determine the degree of curing of the resins. In fact, the degree of cure is defined as the ratio of the released heat up to a certain time by the total heat of the reaction. It ranges from 0, for uncured resin, to 100%, for completely cured resin. In Tensile and flexural tests were performed to characterize the mechanical properties of aero grade epoxy vitrimer and to compare it with reference RTM6 epoxy resin .Considering that developed epoxy vitrimer is focused on the aeronautical sector it is crucial to validate the mechanical properties in hot/wet conditions. Aircraft components manufactured from composite materials are typically exposed to hygrothermal environments which are well known to alter the physical and mechanical properties of fiber-reinforced composites. Epoxy resins are hydrophilic, which means that they absorb moisture from the environment. This makes epoxy resins susceptible to high water absorption. It is well known and studied that absorbed moisture lowers the mechanical properties of the materials. Water absorption into epoxy resins changes both chemical and physical properties through different mechanisms such as plasticization, crazing, hydrolysis, and swelling. Plasticization is the most important physical change that occurs due to the interaction of the water molecules with polar groups present in the epoxy resins and thus, decreasing the Tg. In order to take all these into account, the mechanical and thermal analysis of water absorbed samples has been also carried out. Both developed epoxy vitrimer and reference RTM6 were characterized at three different conditions:Dry/room temperature.HW2: Aged 14 days at 70 °C and 85% RH (relative humidity) and tested at 70 °C.HW4: Aged 14 days at 70 °C and 85% RH and tested at 120 °C.Water uptake measurements were carried out according to UNE-EN-ISO 4611. During the period of the aging study, gravimetric measurements were performed on balance. At selected times, different samples were removed from the chamber, dried superficially, and cooled to ambient temperature prior to their characterization. The samples were aged until a constant weight was obtained . It was RT and HW2 conditions, which means that the mechanical properties of both systems are similar at temperatures below Tg. However, if we compare both materials at HW4 conditions, epoxy vitrimer performed lower: −30% in tensile strength and −35% in flexural strength. The observed behavior at 120 °C is related to the Tg decrease of the resins due to water absorption. The water absorbed due to the aging of the samples until saturation decreases the Tg [As can be seen in s the Tg , reducinDue to the presence of dynamic hardener, developed aero grade epoxy resin was expected to show vitrimer behavior. The time and temperature-dependent relaxation modulus was tested by DMA to characterize its heat-induced malleability. In the following subsections, stress relaxation was studied first, followed by creep tests to end with thermoforming and reprocessing tests.−3 N.. After reaching the required temperature, 1% strain was applied and the relaxation modulus was monitored over time. In Ea) was calculated. The Arrhenius plot presents a linear correlation of ln (τ) with 1000/T in the measured temperature range (Ea) of 246 kJ·mol−1 was calculated. Activation energy (Ea) values are characteristic of each system and represent the sensitivity of the process to the temperature. For structural materials, high Ea is required in order to have high mechanical properties at working temperature while being reprocessable, reparable, and recyclable at temperatures above the Tg. In this sense, to reduce the creep at low temperatures and achieve low reprocessing time at high temperatures, high Ea is needed.During the stress relaxation test, in order to assure the straightness of the specimens, the samples were preloaded by applying 10re range  inset. F12 Pa·s [Tv) was calculated from the Maxwell equation: η = Gpτ* where η is the viscosity, Gp is the plateau modulus and τ* is the characteristic relaxation time. Using the equation above, τ* was calculated to be around 105 s. Tv could be calculated by the extrapolation of the Arrhenius fitted line  was also obtained. Tv is defined as the temperature at which the material reaches a viscosity of 1012 Pa·s ,42,43,44ted line  inset whAnother important parameter of vitrimers, besides stress-relaxation, is their resistance to creep. Compared to classical thermoset polymers, vitrimers are prone to creep at high temperatures above their Tg or Tv due to the exchange of dynamic bonds, especially if these exchange reactions are fast . HoweverTo assess good creep resistance of developed aero grade epoxy resin, creep recovery experiments were carried out at high temperatures and compared with reference RTM6 epoxy resin. The long-term performance of the material was predicted using the time-temperature superposition (TTS) principle .In order to compare the obtained results, due to the difference in the Tg of the material (175 °C for aero grade epoxy vitrimer and 210 °C for RTM 6 resin), the temperature behavior was normalized to the Tg of each of the materials. As can be seen in It is well known that the presence of permanent crosslinks could reduce the creep behavior of the developed aero grade epoxy vitrimer at T > Tg. With the aim to corroborate the heat-induced malleability at temperatures above the Tg, creep experiments were also carried out at 210 °C and 215 °C applying 1 MPa of constant stress. As can be seen in The dynamic behavior of aero grade epoxy network was exploited to demonstrate the capability to thermoform high Tg epoxy vitrimers.To validate the thermoforming capacity of developed aero grade epoxy resin, a fully cured 2 mm thick resin plate was hot-pressed in a mold with omega shape. First, the epoxy vitrimer was placed in a pre-heated oven at 200 °C for 5 min together with the mold used for thermoforming. Once the vitrimer and the mold were at the right temperature, the sample was gradually deformed at 36 N/s speed at 200 °C. After the desired deformation was reached, the vitrimer was kept in its new shape to cool down below the Tg to ensure full stress release due to the reorganization of dynamic disulfide bonds. This hot-press forming process is very fast compared to actual manufacturing processes for aero parts , thus having a tremendous impact on the aeronautics sector reducing the production time by at least 10 times.Finally, in order to confirm the reprocessing capabilities, the epoxy vitrimer was grinded to fine powder, which was then hot pressed into a mold by applying heat and pressure . Ea = 246 kJ/mol) of the network. This high Ea permits the design of resins and composites with high-temperature sensitivity, permitting to arrest of the dynamic behavior up to high temperatures and thus reducing the creep up to the Tg of the material. For the present aromatic disulfide-containing vitrimers, we have determined that up to 30% of permanent crosslinks can be introduced in the network while maintaining reprocessability. Our study provides a promising approach for limiting the non-desirable effects associated with creep in vitrimer applications, particularly for aerospace industry. In addition, the introduction of permanent crosslinks also enhanced the thermal and mechanical properties making the developed aero grade epoxy vitrimer the right candidate for introduction in the aero sector.Here we showed for the first time that an aero grade aromatic disulfide epoxy vitrimer with reduced creep can be obtained by incorporation of a certain fraction of permanent crosslinks. The epoxy vitrimer exhibited similar properties to widely used aero grade RTM6 aero resin while being reprocessable. The developed aero grade epoxy resin presents an acute temperature response due to the high activation energy (


The need for a new style of clinical trials, called decentralized clinical trials (DCTs), has been increasing as they do not depend on physical visits to clinical sites. DCTs are expected to provide a new opportunity to patients who cannot participate in a clinical trial due to geographical and time limitations. For the adoption of DCTs, it is essential that medical devices with Internet of Medical Things (IoMT) and Internet of Health Things (IoHT) based technologies are developed and commercially adopted. In this study, we aimed to identify the regulatory considerations when IoMT/IoHT-based technologies are used in DCTs or products developed using DCTs.To understand the study and development field of IoMT/IoHT comprehensively and panoramically, relevant papers published in Web of Science were searched online. Subsequently, a citation network was obtained and characterized as a cluster using a text mining method to identify IoMT/IoHT-based technologies expected to be utilized in DCTs or products developed using DCTs.Upon analysis of the top 15 clusters and subsequent 51 sub-clusters, we identified the therapeutic areas  and IoMT/IoHT-based technologies  that are expected to be used in DCTs. We also identified several considerations based on the current regulatory guidance.IoMT/IoHT-based technologies that are expected to be used or products developed using DCTs and key considerations made when they are used in DCTs were identified. The considerations could encourage conducting DCTs using IoMT/IoHT-based technologies. In the wake of the COVID-19 pandemic, there has been increasing interest in decentralized clinical trials (DCTs) that do not rely on visits to clinical sites to conduct clinical trials. There are two types of DCT-style studies. In one DCT-style, visits to clinical sites can be avoided altogether as several components can be implemented remotely, such as remote electronic informed consent and telemedicine. In the other style (hybrid style), not all study visits are decentralized; certain study visits need to be conducted at the clinical site as usual. For pharmaceutical companies, DCTs reduce subjects' inclusion period and the study population's bias by obtaining study participants from across the globe . TherefoThe advancement of information technology has played a key role in improving the implementation of DCTs. Thus, the adoption of Internet of Things (IoT) in the medical field, in the form of Internet of Medical Things (IoMT) and Internet of Health Things (IoHT), through concepts such as telemedicine, ePRO, and data collection by wearable devices, will enable further dissemination and promotion of DCTs. It is also expected that new IoMT/IoHT-based technologies will be developed while using the components of DCTs to be authorized as qualified medical devices by health authorities such as the FDA. However, considering that the field of IoMT/IoHT has been changing rapidly and the new IoMT/IoHT has been developing constantly, it is difficult to proactively identify the suitable IoMT/IoHT to utilize in DCT and the points to notice when the study sponsor wants to implement the IoMT/IoHT in DCTs.Therefore, first, we aimed to identify IoMT/IoHT-based technologies that are expected to be utilized in DCTs or products developed using DCTs by using horizon scanning based on bibliographic citation network analysis and text mining. Horizon scanning is a major topic in the strategic initiatives of the International Coalition of Medicines Regulatory Authorities (ICMRA), which is a voluntary coalition of medicines regulatory authorities. According to the concept notes of ICMRA Strategic Priority on Innovation, horizon scanning is broad-reaching information-gathering and monitoring activities to forecast impending products and technologies and potentially disruptive research methods .There are two main types of methods for collecting the data required to conduct well-programmed horizon scanning : expert-This study aimed to explore novel IoMT/IoHT-based technologies that may be applied in future DCTs using citation network analysis and text mining, both of which are foresight methods used for discovering early signs of emerging products and technologies. In addition, based on the results of citation network analysis and text mining, we aimed to examine the prospects for the utilization of IoMT/IoHT in DCTs and the issues and key considerations that can be assumed to be focused on operational and regulatory perspectives while developing novel IoMT/IoHT-based technologies to contribute to the popularization of IoMT/IoHT in DCTs.We utilized  and  as queries for analyzing papers published in the Web of Science literature database  to identify IoMT/IoHT-based technologies that are expected to be used in DCTs and products developed using DCTs.Aij represents the weight of the edge between i and j, i, ciis the community to which vertex i is assigned, δ-function δ is 1 if u=v and 0 otherwise, and where Labels corresponding to the size of the number of papers included were assigned in the clusters. The characteristics of each cluster were confirmed by extracting a summary of frequently cited academic papers and characteristic keywords in the cluster.i in a given cluster j, is given byFurthermore, we employed term frequency-inverse cluster frequency (TF-ICF) to extract the distinctive keywords of each cluster. TF is a measure of the significance of a term in a particular sentence, whereas ICF is a measure of the general importance of a term. The TC-ICF of a given term where N is the total number of sentences. Each cluster was labeled based on the resulting keywords and sentences.The visualization was converted to assume the relationships among these clusters after clustering the network and a large graph layout (LGL) based on a force-direct layout algorithm was used , 23. ThiRegulatory information for FDA , EMA 2525, MHLW We analyzed 144,977 papers obtained from the WoS database and discovered that 97,305 papers (67.1%) formed a citation network by extracting the largest connected component from all linkage components through direct citation of papers, and divided it into 141 clusters. Among the top 15 clusters listed in Because regulatory matters might be an issue for the utilization of IoMT/IoHT in DCTs, we retrieved relevant regulatory information in the US, EU, and Japan. via the Internet were developed. Both telehealth and remote monitoring are important elements of DCTs, and medical devices and infrastructure with these technologies and functions are expected to contribute to the standardization of DCTs. In response to the COVID-19 pandemic, telemedicine and digital health technologies (such as wearable devices) have been applied in the treatment of the COVID-19 to avoid the risk of further hospital-acquired infection. Based on the result of a rapid literature review by Bokolo Anthony Jnr, some considerations have been identified for applying telehealth and digital care solutions during the COVID-19 pandemic  of the obtained sub-clusters, we identified technologies and specific medical devices that are pivotal in the citation relationships that make up the sub-clusters. We subsequently categorized them according to therapeutic area. Remote monitoring of biological functions  using sensors and telemedicine, which are expected to be utilized not only in specific therapeutic areas, was found to be the most common . This means that IoMT/IoHT-based technologies, including wearable devices, will be available for use in daily life and clinical trials, including DCTs . We discpandemic –46.We focused on VR therapy as an IoMT/IoHT-based technology that is expected to be utilized and developed through DCTs. VR therapy, as the name suggests, involves the use of VR for medical purposes . VR techde novo premarket review pathway, a regulatory pathway for low-to-moderate-risk devices of a new type  . GuidancWe selected WoS as the database for the source of analysis; however, there are other databases, such as PubMed and Scopus, which were excluded. It is necessary to select a database for data source collection that is appropriate for each field of interest. Moreover, there is a time lag between the publication of papers in journals and those reflected in WoS, which causes a time gap in the understanding of the research field. In this study, we focused exclusively on IoMT/IoHT research trends as on the date when the article information was extracted from the database. Additionally, it is necessary to follow the changes in hub papers and other important papers in the research field in chronological order to understand the development of research fields and new technologies. We analyzed the potential IoMT/IoHT-based technologies, disease areas, and purposes of use based on the information in each sub-cluster; however, there is a possibility that subjectivity and bias were incorporated into the evaluation.In this study, we investigated the trends related to the adoption of IoT in the medical and healthcare fields (IoMT/IoHT) using citation network analysis and text mining methods. IoMT/IoHT and research fields that are expected to be utilized in DCTs, such as telemedicine, wearable devices, and VR, were identified. Based on the results, we established key considerations in using IoMT/IoHT in DCTs, such as how sponsors can ensure data quality from the IoMT/IoHT-based technology used in DCTs. To the contrary, considering that the field of IoMT/IoHT is evolving rapidly, some relatively recent studies are not included in the dataset used for citation network analysis; they are therefore not identified as hub papers because of having been published quite recently or having few citations. The findings of this study are expected to help in developing the new IoMT/IoHT using DCTs or in using the IoMT/IoHT in DCTs. For future studies, the works of literature and guidance related to DCTs and IoMT/IoHT will be retrieved and examined for continuous solutions to problems based on the latest information and knowledge.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MS contributed to the conception and design of the study. HS organized the database. HI collected data. HI, TT, and TS performed the data analysis. TS drafted the manuscript. HS and MS reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by AMED  Under Grant Number 21mk0101217h0001.TS was employed by Astellas Pharma Inc. The remaining 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.


Convolutional neural networks in the field of artificial intelligence show great potential in image recognition. It assisted endoscopy to improve the detection rate of early gastric cancer. The 5-year survival rate for advanced gastric cancer is less than 30%, while the 5-year survival rate for early gastric cancer is more than 90%. Therefore, earlier screening for gastric cancer can lead to a better prognosis. However, the detection rate of early gastric cancer in China has been extremely low due to many factors, such as the presence of gastric cancer without obvious symptoms, difficulty identifying lesions by the naked eye, and a lack of experience among endoscopists. The introduction of artificial intelligence can help mitigate these shortcomings and greatly improve the accuracy of screening. According to relevant reports, the sensitivity and accuracy of artificial intelligence trained on deep cirrocumulus neural networks are better than those of endoscopists, and evaluations also take less time, which can greatly reduce the burden on endoscopists. In addition, artificial intelligence can also perform real-time detection and feedback on the inspection process of the endoscopist to standardize the operation of the endoscopist. AI has also shown great potential in training novice endoscopists. With the maturity of AI technology, AI has the ability to improve the detection rate of early gastric cancer in China and reduce the death rate of gastric cancer related diseases in China. Gastric cancer (GC) is the fifth-most common malignant tumor and the third leading cause of cancer-related death in the world , 2. GastThe diagnosis of EGC is related to the ability of endoscopists to adequately analyze endoscopic images, a skill cultivated through extensive training over a long period . While tWith the rapid development of computer science and technology, artificial intelligence (AI) technology is maturing, allowing it to be used to improve accuracy in a variety of medical situations . The numAmong AI technologies, neural networks, represented by cirrocumulus neural networks, have demonstrated remarkable progress, achieving feats comparable to or even surpassing human beings in the field of image recognition. AI is not affected by subjectivity, fatigue, experience, or other factors. It performs medical image-assisted diagnoses well and has a high focus recognition rate. In addition, its learning ability is continuous and improves with increasing exposure to training data. AI has shown great potential in endoscopy, including in screening for EGC .We analyze current research trends in AI, EGC, and endoscopy by searching relevant topics in the Web of Science core database. The analysis results are presented with citeSpace drawings. In our search concerning endoscopy and EGC, we found 1,664 related articles, and 1,625 were used in the final analysis. In our search concerning endoscopy and AI, we found 392 related articles, and 354 were used in the final analysis. In our search concerning AI and EGC, we found 67 relevant articles, and 58 were used in the final analysis. We then combined these three search terms to perform retrieval again and found 55 relevant articles, and 40 were used in the final analysis. We analyzed the topics related to endoscopy and EGC, obtaining three figures –3. On anWe next analyzed the literature on endoscopy and AI and obtained two similar figures , 5. By cThis review will focus on these three aspects: convolutional neural networks, the dilemmas associated with EGC screening, and the feasibility of applying AI to GC screening.Machine learning and deep learning are considered two sub-technologies of AI . Deep leAn artificial neural network (ANN) is a monitoring model whose model structure is very similar to that of neurons in the human central nervous system , 18. NeuTo meet the need for increased performance, more and more complex neural networks are developed, resulting in the concept of deep learning. Deep learning works by progressively extracting higher-level features from raw input using multi-level structures . A deep At present, the most widely used and effective network is the convolutional neural network (CNN). It has shown great potential in many fields, such as pathological analyses, computed tomography, and magnetic resonance imaging analyses –28. A CNIf AI can be successfully combined with various clinical examinations, it will greatly improve clinical practice. However, at present this is a brand-new field, so further exploration and experimentation are necessary. In recent years, there have been numerous attempts to integrate AI into various medical fields, including endoscopy. The potential utility of this approach in GC screening is discussed below.Early gastric cancer is difficult to detect, as the early symptoms of GC are not obvious, and some patients do not actually show any early symptoms, while elderly people tend to avoid visiting the hospital for regular examinations . PatientAt present, only two nations have government-funded GC screening programs: Japan and South Korea . DespiteHowever, despite the country’s relatively high incidence of GC, China still lacks a countrywide screening program and the only way to identify EGC is through opportunistic screening . A domesEndoscopy is the most effective diagnostic method for gastric cancer and can improve the detection rate of EGC . DespiteSome studies have shown that the sensitivity of GC detection can be increased by training endoscopists to improve their operational skills and ability to identify lesions . In addiBecause of the uneven distribution of population and medical resources in China, the situation of endoscopy in China is more serious than that in some developed countries. The huge workload may result in our missed diagnosis rate being significantly higher than theirs. According to the census results of the number of practitioners of digestive endoscopy conducted in China in 2013, there is a huge gap in the number of endoscopists in China. Moreover, their technical level is not equal, and the doctors in economically developed cities have more medical resources and opportunities for intensive training than those in less developed cities. According to the census data, the number of digestive endoscopy physicians per million people in 20 provinces and cities is lower than the national average of 19. There are now only 30,000 endoscopists in China, despite a demand for endoscopy in the hundreds of millions. Such a big gap has left endoscopists in China with a huge workload, forcing them to reduce the examination time per patient in order to improve efficiency and relieve the pressure of work caused by a labor shortage. Studies have shown that the duration set aside for endoscopy and the rate of disease detection are positively correlated . ShorterIn addition, compared with other developed countries, the development rates of digestive endoscopy diagnoses and treatment technology in China are still quite low, and standardized training of endoscopists has not yet matured, resulting in a disparity of technical skills among endoscopists across the country. Operator factors significantly influence the outcome of endoscopy. Compared to other nations, China’s current condition has rendered the stability and sensitivity of endoscopy unreliable. In general, there are three dilemmas facing GC screening in China: (1) there is still a big gap between the development of digestive endoscopy technology in China and that of foreign countries and a systematic and standardized training system has not been implemented; (2) there is a serious shortage of endoscopists in China that is unable to meet the current demand for endoscopy in China; and (3) the accuracy of endoscopy cannot be guaranteed.Endoscopy screening for EGC is a difficult and time-consuming procedure, but it should not be taken lightly, as each missed diagnosis may cause patients to lose out on the most effective therapy option. Early identification and therapy are still the most effective treatments for GC. Endoscopists must therefore thoroughly examine each patient. However, humans are not machines, and long-term endoscopic operation can impair endoscopists’ discriminating capacity and impact the examination quality. The involvement of nurses can increase the rate of lesion identification and the quality of endoscopy by acting as a second observer during the procedure . As AI tFurthermore, an increasing number of studies have proven that trained CNNs can swiftly identify lesions with an accuracy equivalent to that of endoscopists. A research team from Japan created a model of a CNN-based system using a training model of 13,584 gastroscopic images of GC. The total sensitivity of the model reached 92.2%, and it only took 47 s to examine 2,296 detection images . CNN canThe Japan Cancer Research Foundation conducted a study comparing the speed and accuracy of endoscopic image recognition by artificial intelligence and endoscopists. Researchers trained a CNN-based model with 13,584 endoscopy images to work with 67 endoscopists to identify 2,940 images from 140 instances . The AI A similar experiment was carried out in the Department of Gastroenterology at the People’s Hospital of Wuhan University in China. They used an AI system designed by themselves to validate the results using 200 endoscopic pictures. Its accuracy, sensitivity, and specificity for identifying EGC were 92.5, 94, and 91%, respectively, compared to 89.7, 93.9, and 87.3% for experienced endoscopists . The DepDespite the limitations, that experiment and each of the others described above had their own innovations. At the same time, there are many similar retrospective experiments, all of which have verified the utility of AI in lesion identification and demonstrated the great potential of AI in endoscopy. Based on the above findings, we believe that AI can quickly identify lesions with accuracy, greatly reducing the current burden on endoscopists.Furthermore, there are many other aspects to AI that bear highlighting. For example, the AI system developed by the People’s Hospital of Wuhan University was able to divide gastroscopic images into 26 anatomical areas with an accuracy of 65.9%, which was comparable to the rate of 63.8% for experienced endoscopists, and reduced the rate of image sites missing by 15% in a comprehensive randomized controlled trial . In addiA successful case of applying AI to clinical practice was recently reported in the “EndoAngel.” This is an AI quality control auxiliary diagnosis system of digestive endoscopy based on a CNN model that can effectively monitor the blind area on GI imaging, assist in the detection of suspicious lesions in real time, improve the quality of endoscopy and improve the detection rate of GI tumor lesions; in addition, it is also equipped with a scoring training system for upper and lower gastrointestinal endoscopy. It is a fully functional AI product integrating quality control and auxiliary diagnostic functions. The People’s Hospital of Wuhan University has cooperated with 13 other hospitals to conduct a functional verification study of the EndoAngel for the early diagnosis of GC. The EndoAngel has a 92% diagnostic accuracy in EGC, and its main working mode is shown in The technological skill level of endoscopic physicians is disparate at present, and the operation is not sufficiently standardized, affecting the endoscope quality. The ADM system, based on a CNN and developed by the People’s Hospital of Wuhan University is intended to provide the following statistical quality indicators: colonoscopy time, cecal endoscopy intubation rate (CIR), adequate bowel preparation rate, polyp detection rate (PDR), adenoma detection rate (ADR), gastroscopy time, and gastric precancerous condition (GPC) detection rate. The system may also simultaneously analyze the quality of each endoscope and provide rapid feedback to the operator. Controlled experiments verified that the detection rate of precancerous lesions increased in the endoscopic group with AI feedback (3–7%) as well as in the control group (3.5–3.9%) . These fExtensive endoscopy cannot be performed in China at present. One reason for this is a lack of corresponding equipment in community hospitals, and another is the scarcity of endoscopists, with this latter reason being the main issue. Endoscopists are in short supply in China, being mostly centered in major hospitals; this means that even if rural hospitals have similar technology, no one is available to operate them. The advent of AI appears to be a game-changer. In terms of the sensitivity and accuracy of inspections, the present AI model based on a CNN appears to have the equivalent skill to professional endoscopists . If AI wThus far, retrospective trials of AI screening for early stomach cancer have yielded promising results. The precision and accuracy with which lesions are identified are equivalent to those of endoscopists. If AI were to be employed in the early stomach cancer screening process, it would significantly improve the poor detection rate of EGC in China . FurtherAll authors listed have made a substantial, direct, and intellectual contribution to the study, and approved it for publication.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.


Laryngeal squamous cell carcinoma (LSCC) is a prevalent malignant tumor of the head and neck with a dismal prognosis. Keratin17 (KRT17) has been proven to serve as an oncogene in various cancers, but it has never been explored in LSCC. We proposed to assess the impact and possible mechanisms of KRT17 in the development of LSCC.  Quantitative reverse transcription-PCR (qRT-PCR) was utilized to examine the mRNA levels. The Kaplan–Meier method was used to calculate the relationship between KRT17 expression and survival curves in LSCC patients. Cell counting kit-8 (CCK-8), colony formation, and flow cytometry assays were utilized to estimate LSCC cell proliferation. The migration and invasion abilities of LSCC cells were ascertained by wound-healing and transwell assays. Immunohistochemical and western blot assays were utilized to appraise protein levels. The xenograft tumor model was used to determine the effect of KRT17 on tumor growth. β-catenin signaling axes and found that these signaling pathways were largely blocked by KRT17 deletion.  In the present study, KRT17 was extremely high in LSCC tissues and cells and correlated with a poor prognosis. Inhibition of KRT17 weakens cell proliferative, migratory, and invasive abilities in LSCC and contributes to cell cycle arrest. Besides, we approved that knockdown of KRT17 extraordinarily restrained the xenograft tumor growth in vivo. We preliminarily investigated the role of KRT17 on the AKT/mTOR and Wnt/β-catenin signaling axes, illustrating KRT17 as a promising biomarker for making strides in LSCC treatment. Collectively, we uncovered that exhaustion of KRT17 suppresses LSCC progression through coordinating AKT/mTOR and Wnt/ Laryngeal squamous cell carcinoma (LSCC) is the second largest sort of head and neck malignancy, with extending recurrence and mortality –3. In spCurcuma amada, Humulus lupulus, and Hypericum perforatum can inhibit the expression of KRT17 in psoriatic skin [Keratin, one of the intermediate filament of the protein family, has a molecular weight of about 40–70 kDa and forms the cytoskeleton , 7. Keratic skin . KRT17, tic skin –16. In ttic skin . A latertic skin . In any β-catenin signaling axes. Our revelations propose that KRT17 may be an imperative marker of LSCC progression and unfavorable survival.In this study, we uncovered that KRT17 is elevated in LSCC tissues and is possibly associated with poor outcomes. KRT17 accelerates LSCC proliferation and invasion by means of enacting AKT/mTOR and Wnt/A total of 42 human LSCC and correspondent nontumor normal samples (5 cm distant from the tumor boundary) were obtained from the LSCC patients who experienced surgical resection in the First Affiliated Hospital, Huzhou University, The First People's Hospital of Huzhou hospital. All participants did not undergo preoperative radiotherapy or chemotherapy and signed an informed consent form. This study was endorsed by the Institutional Ethics Committee of the First Affiliated Hospital, Huzhou University, The First People's Hospital of Huzhou Hospital, and was undertaken in compliance with the Declaration of Helsinki.The cells and tissues were lysed utilizing Trizol reagent (Sangon Biotech (Shanghai) Co., Ltd., China). The PCR examination was accomplished utilizing PrimeScript RT reagent kit  and SYBR Premix ExTaq II (TaKaRa). The primer sequences are included in https://string-db.org) database based on key KRT17 coregulated genes in order to assess potential interactions between proteins of interest [A PPI network was constructed using the STRING , AKT , p-AKT , mTOR , p-mTOR , cyclin D1 , cyclin E , CDK2 , CDK4 , Snail , MMP7 , and GAPDH . Then, the membranes were encoded with a secondary antibody conjugated to HRP and finally subjected to measurement with an ECL kit .A RIPA lysate  was utilized to extricate total proteins, which were quantified by a BCA kit , followed by subjecting them to an SDS-PAGE gel and transferring them to PVDF membranes . Afterwards, blocking blots were performed with 5% fat-free milk, and the process was followed by incubation with the primary antibodies against KRT17 , E-cadherin , N-cadherin , Human LSCC cells  and bronchial epithelioid cells (16HBE) were obtained from iCell Bioscience Inc.  and cultured under the appropriate media and conditions as required by the instructions. Following the operational requirements of Lipofectamine 2000 , KRT17 shRNAs, and an appropriate negative control  were transfected into AMC-HN-8 and TU177 cells.μl CCK-8 (Beyotime) at the time of 24, 48, 72, and 96 h of incubation and then incubated for 4 h and detection of absorbance values at 450 nm using an enzyme reader .5000 AMC-HN-8 and TU177 cells were inoculated in each well of a 96-well plate, followed by the addition of 10 AMC-HN-8 and TU177 cells were inoculated into a 6-well plate and cultured for 2 weeks until the cells shaped into large clones. The complete medium was supplanted every 3 days. According to the requirements of crystal violet staining, clones were dyed, counted, and photographed.μL osmotic solution were included under dark conditions, and cell cycle distribution detection was conducted using a flow cytometer (BD Biosciences).AMC-HN-8 and TU177 cells were cultivated for 48 h, harvested by centrifugation, and fixed with 70% ethanol at 4°C for 12 h. DNA staining solution  and 5 When 6-well plate cell growth reached 90% confluence, the scratches were delivered with a 200 ul pipette tip and cultured without fetal bovine serum, and the gap size was measured 24 h later.5 cells/well) were included in the top chambers without serum medium, whereas the complete medium was included in the bottom chambers. After 24 h of incubation, cells moving to the bottom chambers were fixated with 4% paraformaldehyde, subsequently dyed with crystal violet, counted, and photographed.The abilities of cells to migrate or invade were determined in transwell chambers  without or pre-surfaced Matrigel. The AMC-HN-8 and TU177 cells  randomized in each group. Tumor volume was monitored at 3-day intervals utilizing a vernier caliper. Thirty-one days after injection, tumors were resected and weighed. Then, tumor tissues were fixed in formalin, embedded in paraffin, and cut into 4-μm-thick slices. The sections were incubated with the primary anti-Ki-67  and anti-KRT17  antibodies at 4°C for 12 h after dewaxing and antigen repair. In this way, HRP-conjugated secondary antibody was added, and the signals were generated with diaminobenzidine and photographed with microscopy.This study involved animal experiments that were endorsed by the institutional guidelines of the Committee of the First Affiliated Hospital, Huzhou University, The First People's Hospital of Huzhou Hospital, and were undertaken in compliance with the Declaration of Helsinki. 2 × 10t-test or one-way analysis of variance (ANOVA). LSCC patients were assigned to either low or high KRT17 expression groups based on the median value of KRT17 expressed in LSCC tissues, and their overall survival curves were ascertained following the Kaplan–Meier method. P < 0.05 was affirmed as representing statistical significance.Data are stated in terms of mean ± SD from at least three individual replicate trials. Statistics were obtained with SPSS 16.0 software, and graphical plots were generated with GraphPad Prism 8.0 software. Differences between two or among more groups were performed via Student's Previous studies have appeared that increased KRT17 expression in LSCC tissues . ConsistP < 0.001), T classification (P < 0.01), lymph node metastasis (P < 0.05), and clinical stage (P < 0.05). In any case, gender and primary position were not associated with KRT17 expression. To assist in assessing the clinical importance of KRT17 expression in LSCC, survival curves were utilized to compare the difference in overall survival between low and high KRT17 expression groups (P=0.414). The results uncovered that LSCC patients with high KRT17 expression had significantly lower overall survival than those with low KRT17 expression  to identify KRT17-related genes. As shown in β-catenin signaling-related proteins. Subsequently, we verified KRT17-mediated changes in the levels of proteins related to the above pathways in AMC-HN-8 and TU177 cells by western blot assay. The results recommended that exhaustion of KRT17 restrained the critical proteins of AKT/mTOR and Wnt/β-catenin pathways  and cell cycle-related proteins, counting CDK2, CDK4, and Cyclin E, was diminished in the sh-KRT17 group compared to the sh-NC group. Taken together, our discoveries uncovered that KRT17 actuated LSCC progression, at least in part by means of enactment of AKT/mTOR and Wnt/β-catenin axes.Next, we explored the mechanism by which KRT17 mediates LSCC progression. The previous pieces of evidence have unraveled the accelerating impacts of KRT17 on AKT/mTOR and Wnt/nin axes –24. To f Figures . Depletiβ-catenin axes. These discoveries uncover that KRT17 plays a part in the advancement of LSCC and is probably a prospective therapy candidate.Patients diagnosed with LSCC can be treated by surgery, chemotherapy, or radiotherapy, but the overall therapeutic impact is still not palatable, the long-term survival rate is low, and the prognosis is destitute. The occurrence and development of LSCC is considered to be the result of the imbalance between oncogenes and tumor suppressor genes. In this manner, effectively seeking gene-level therapeutic targets and potential mechanisms has become the focus of current research. In this study, we examined the relevance of KRT17 to LSCC. Our research recommended that KRT17 regulates growth, migration, and invasion in LSCC through the AKT/mTOR and Wnt/α pathway [Studies have appeared that KRT17 can directly participate in the regulation of an assortment of tumors. For example, KRT17 impacts the pathogenesis of cervical cancer by promoting the nuclear transport and degradation of protein P27 (Kip1) . Mihaela pathway . KRT17 w pathway . Moreove pathway , renal c pathway , pancrea pathway , and hep pathway . Li et a pathway , and KhaWe obtained high expression of KRT17 mRNA in LSCC by comparing 42 pairs of LSCC tissues with normal adjacent tissues, which is consistent with the present study . KRT17 hβ-catenin axes in LSCC has been extensively studied [β-catenin axis [β-catenin, which affirmed the inactivation status of the AKT/mTOR and Wnt/β-catenin axes. Consistently, the downstream proteins of the Wnt/β-catenin axis and the cell cycle-related proteins were also diminished after the exhaustion of KRT17.Activation of AKT/mTOR and Wnt/ studied –36. Pastnin axis , 22. In β-catenin axes, proposing KRT17 as a novel biological target in LSCC and giving unused considerations for the alter of treatment of LSCC.In conclusion, our study demonstrates that the knockdown of KRT17 restrains proliferation, migration, and invasion partly through AKT/mTOR and Wnt/