Medicago sativa) under drought stress to explore the photosynthetic responses of pod walls after 5, 10, 15, and 20 days of pollination  based on ultrastructural, physiological and proteomic analyses. Stomata were evidently observed on the outer epidermis of the pod wall. Chloroplasts had intact structures arranged alongside the cell wall, which on DAP5 were already capable of producing photosynthate. The pod wall at the late stage (DAP20) still had photosynthetic ability under well-watered (WW) treatments, while under water-stress (WS), the structure of the chloroplast membrane was damaged and the grana lamella of thylakoids were blurry. The chlorophyll a and chlorophyll b concentrations both decreased with the development of pod walls, and drought stress impeded the synthesis of photosynthetic pigments. Although the activity of ribulose-1,5-bisphosphate carboxylase (RuBisCo) decreased in the pod wall under drought stress, the activity of phosphoenolpyruvate carboxylase (PEPC) increased higher than that of RuBisCo. The proteomic analysis showed that the absorption of light is limited due to the suppression of the synthesis of chlorophyll a/b binding proteins by drought stress. Moreover, proteins involved in photosystem I and photosystem II were downregulated under WW compared with WS. Although the expression of some proteins participating in the regeneration period of RuBisCo was suppressed in the pod wall subjected to drought stress, the synthesis of PEPC was induced. In addition, some proteins, which were involved in the reduction period of RuBisCo, carbohydrate metabolism, and energy metabolism, and related to resistance, including chitinase, heat shock protein 81-2 (Hsp81-2), and lipoxygenases (LOXs), were highly expressed for the protective response to drought stress. It could be suggested that the pod wall in alfalfa is capable of operating photosynthesis and reducing the photosynthetic loss from drought stress through the promotion of the C4 pathway, ATP synthesis, and resistance ability. Increasing photosynthetic ability as a whole is essential for acquiring higher crop yields. Nonleaf green organs (NLGOs) make important contributions to photosynthate formation, especially under stress conditions. However, there is little information on the pod wall in legume forage related to seed development and yield. This experiment is designed for alfalfa ( Many scientists have previously reported that the silique shell of oil rape (Brassica napus) [Ricinus communis) [Cicer arietinum) [Glycine max) [Medicago sativa) [Oryza sativa) [Hordeum vulgare) [Triticum turgidum) [Photosynthesis is considered as the most important chemical reaction and provides over 90% of dry matter for crop yield formation ,2. Increa napus) ; the bolommunis) ; the podietinum) , soybeanine max) , and alf sativa) ; ears ofvulgare) , and wheurgidum) ; flowersurgidum) , stems [urgidum) , and roourgidum)  in some urgidum) .Zea mays) leaves [ppc, aat, mdh, me2, gpt, and ppdk, specific to NAD-ME type-C4 photosynthesis, have been identified in wheat caryopsis [2 was assimilated by C4 photosynthesis in the pericarp of wheat, but not in awn and glume [2 and heat or drought stress [Photosynthesis in leaves is sensitive to water deficit. Under water-deficit conditions, the flag leaves of cereal crops will wilt and senesce, while NLGOs are able to maintain relatively high water content due to some special features, including xeromorphic anatomy , lower s) leaves . One of ) leaves ,22,23. B) leaves . Some gearyopsis . Howevernd glume . A C4 pht stress ,26. AbovAlfalfa is widely cultured around the world to produce high-quality hay for feeding livestock, especially dairy cows. Seed producers have long focused on alfalfa seed yield increase. Moderate drought contributes to achieving higher seed yield during the flowering and seed maturation period. Nevertheless, little is known on the physiological response and the photosynthetic contribution of the pod wall in alfalfa under drought stress. Investigating and increasing the photosynthetic ability of NLGOs, especially under stress conditions, is a novel way to increase the photosynthetic ability of the whole plant and finally increase the grain yield. In this study, physiological, ultrastructural, and proteomic analyses were carried out to (1) investigate the photosynthetic characteristics of the pod wall in alfalfa, and (2) research the response mechanism of photosynthesis in the pod wall to drought stress. Stomata and epidermal hair were distinctly observed in the outer surface of the pod wall A,B. StomUnder WW, chloroplasts in the pod wall had the ability to photosynthesize from DAP5 to DAP20. Chloroplasts on DAP5 existed with the intact structure and were arranged close to the cell wall A. ChloroUnder WS, chloroplasts were able to produce photosynthate on DAP5 and DAP10, while the structure of chloroplasts was gradually damaged from DAP15 to DAP20. The chloroplasts had intact membrane structures and had already started to produce starch grains on DAP5 E, and thExcept for chloroplasts, the structure of other organelle or tissues changed under drought stress as well. The central vacuole was bigger in the cell under WW D, while p < 0.05) compared with WW  in the treatment of WS, while RuBisCo activities were decreased.PEPC and RuBisCo activities in pod walls both present a declining trend with the pod development . Howeverp < 0.05) for WW15 vs. WW10, and 101 proteins were significantly downregulated for WW20 vs. WW10  enrichment analysis, during pod development, most of the identified chlorophyll a–b binding proteins involved in the photosynthesis–antenna proteins were significantly downregulated in pod walls under WW or WS . The synUnder the treatment of WS, some proteins, including ribulose-phosphate 3-epimerase, ctosolic fructose-1 6-bisphosphatase, fuctose-1, 6-bisphosphatase, fructose-bisphosphate aldolase, glyoxysomal malate dehydrogenase, malate dehydrogenase, malic enzyme, and sedoheptulose-1,7-bisphosphatase, which are involved in carbon fixation of photosynthetic organisms, were significantly upregulated among different growth stages after pollination . In contThe synthesis of cytosolic fructose-1 6-bisphosphatase, fructose-1, 6-bisphosphatase, fructose-bisphosphate aldolase, and malic enzyme was inhibited by the drought stress on DAP10, and the synthesis of glyoxysomal malate dehydrogenase and ribose-5-phosphate isomerase A were both inhibited on DAP15 and DAP20, respectively . NeverthProteins involved in photosynthesis, including cytochrome b6-f complex iron-sulfur subunit, F0F1 ATP synthase subunit gamma, oxygen-evolving complex/thylakoid lumenal 25.6 kDa protein, and photosystem II oxygen-evolving enhancer protein, were significantly upregulated in the pod wall at different durations after pollination under WS, while light-harvesting complex I chlorophyll a/b binding protein, oxygen-evolving enhancer protein, photosystem I P700 chlorophyll a apoprotein a2, and photosystem II D2 protein were significantly downregulated . Under WFor the treatment of WS, some proteins, including oxygen-evolving enhancer protein, photosystem II oxygen-evolving enhancer protein, photosystem I reaction center subunit II, and photosystem I reaction center subunit N in the pod wall on DAP10, were significantly upregulated . HoweverMeanwhile, some proteins were expressed differently and significantly in the amino sugar and nucleotide sugar metabolism, the ascorbate and aldarate metabolism, the beta-alanine metabolism, the carbon metabolism, the starch and sucrose metabolism, the citrate cycle, the glycine, serine, and threonine metabolism, the linoleic acid metabolism, oxidative phosphorylation, phagosome, plant–pathogen interaction, proteasome, and the alpha-linolenic acid metabolism in pod walls under drought stress .2 to enter for operating photosynthesis as well. Previous research has shown that stoma was also found on other NLGOs, such as the exposed peduncles of wheat [Stomata were distinctly observed on the outer surface of the pod wall . Stoma aof wheat , the silof wheat , and theof wheat . In addiof wheat  and chicof wheat . Under Wof wheat ,22,28. HGossypium hirsutum) leaves and NLGOs, including bracts, stems and boll shells, decreased with bolls developing, and the decreasing rate of the concentration in stems and boll shells was lower than that in leaves at the late stage of boll [In the pod wall of alfalfa, chlorophyll a and chlorophyll b could be detected, and the concentration of chlorophyll a, chlorophyll b and total chlorophyll decreased with the development of pods . Similar of boll . The chl of boll ,20,23. E of boll . Althoug of boll ,23.Saccharum officinarum) [Cucumis sativus) [Six proteins identified in the pod wall were chlorophyll a–b binding proteins, which are the apoproteins of the light-harvesting complex of photosystem Ⅱ that existed on the membrane of chloroplasts . I3SZG9 cinarum)  and cucusativus)  under drsativus) .2 [Populus yunnanensis) [According to KEGG enrichment analysis for photosynthesis, the identified protein complex participates in the reactions taking place on the thylakoid membrane. Proteins, including PetC, PsbS, PsbQ, PsbO, and PsbC, were downregulated under WW , which m2 . PsaD, P2  and the 2 . The dowanensis)  and soybanensis) . Droughtanensis) .Under WW, PEPC, the key enzyme in the C4 cycle, was significantly upregulated in the pod wall on DAP15, compared with that on DAP10 . DroughtStarch grains were observed to be filling the chloroplasts in the pod wall on DAP10 under WS , which mACL gene could enhance drought resistance in tobacco (Nicotiana tabacum) [Identified proteins, including pyruvate dehydrogenase (PDH) E1 beta subunit and ATP-citrate lyase/succinyl (ACL)-CoA ligase involved in the citrate cycle, were upregulated in pod walls on DAP15 under WS . PDH protabacum) . Some ottabacum) , which ctabacum) .Identified proteins, including archaeal/vacuolar-type H+-ATPase subunit A, ATP synthase subunit beta, ATP synthase subunit alpha, and ATP synthase D chain, were upregulated in pod walls on DAP15 . Budak eVicia faba) [Arabidopsis thaliana) [CaLOX1 enhanced the resistance to drought stress in arabidopsis [Chitinase plays an important role in resistance to stress ,51. The ia faba) . Heat shhaliana) . In the haliana) . The ovebidopsis .In summary, the structure of chloroplast in the pod wall is damaged at the late stage of development under drought stress, but not at the early stage. The synthesis of some proteins involved in photosystem I, photosystem II, and the regeneration period of RuBisCo in the pod wall at the early stage and TCA cycle at the late stage are impeded under drought stress . NeverthThis study provides the ultrastructural, physiological, and proteomic changes in alfalfa pod walls under drought stress. The results suggest that the pod wall shows the capability of conducting photosynthesis and regulating the C4 photosynthetic pathway, ATP synthesis, and resistance metabolism to ensure the operation of physiological reactions under drought stress.M. sativa cv. Zhongmu No. 2 seeds were sown in the plastic pots  in a greenhouse in October 2015. Each pot was filled with a mixture soil of vermiculite, peat, and black soil by 2:1:1 . Four seedlings with a similar growth status were kept in each pot when the seedlings’ height was around 10 cm. They were equivalently and adequately watered every two days. All plants were cut till 10 cm, and then pots were moved out of and nearby the greenhouse, without any shelter, in April 2016. Every day, each pot was weighed with an electronic scale and watered to 7 kg. From 18 April (before visible bud stage), a drought stress treatment, denoted by WS, was started by watering each pot to 4.5 kg, while the control, denoted by WW, was still watered to 7 kg. Plants in WW and WS treatments were pollinated artificially every 5 days from 6 June till 21 June. After pollination, the flowers pollinated were marked with hang tags. On 26 June, the pod walls of marked pods with 3 replicates were collected on day 5 (DAP5), day 10 (DAP10), day 15 (DAP15), and day 20 (DAP20) after pollination under WW and WS treatments.The pod walls of DAP10 under WW and WS treatments were used as samples to observe the surface characteristics by using a scanning electron microscope . The pod walls of DAP5, DAP10, DAP15, and DAP20 under WW and WS treatments were used as samples for taking images with a transmission electron microscope  to observe ultrastructure characteristics. Pretreatments of the pod walls for observation by using the scanning and transmission electron microscopes were conducted according to .The pod walls on DAP5, DAP10, DAP15, and DAP20 under WW and WS treatments were sampled and cut into filaments, respectively. Chlorophyll a and chlorophyll b concentrations in each sample with three repetitions were determined by soaking in extracting solution, filling in the 10 mL centrifuge tube. The extracting solution was the mixture of acetone and absolute ethyl alcohol by the volume rate of 2:1. Absorbancy of extracting solution at 663 and 645 nm was determined by using a ultraviolet spectrophotometer  to calculate the concentration of chlorophyll a and chlorophyll b. The gross chlorophyll concentration was the sum of chlorophyll a and chlorophyll b.2SO4 (pH 7.8), 5% glycerol, 7 mM DTT, and 1 mM EDTA. The homogenate was filtered, and then the filtrate was centrifuged at 8000× g for 10 min at 4 °C. The supernatant was saved in an ice bath for subsequent enzyme assay. Photosynthetic enzyme activities were determined using an ultraviolet spectrophotometer  at 340 nm. The reaction solution for phosphoenolpyruvate carboxylase (PEPC) contained 0.1 mol L−1 Tris-H2SO4 (pH 9.2), 0.1 M MgCl2, 100 mM NaHCO3, 40 mM PEP, 1 mg mL−1 NADH and MDH [−1 GAPDH, 50 mM ATP, 50 mM phosphocreatine, and 160 U mL−1 phosphoglyceric kinase [The pod walls on DAP5, DAP10, DAP15, and DAP20 under WW and WS treatments were sampled with three repetitions. Each sample was homogenized in extracting solutions using a pestle and mortar in ice. The extracting solution contained 1 M Tris-H and MDH . The reac kinase .g for 15 min at 4 °C. The supernatant was collected, and the rest was centrifugated again, as above. The protein concentration was determined with a Bio-Rad Protein Assay kit based on the Bradford method, using BSA as a standard at a wavelength of 595 nm. All independent protein extractions were performed.The pod walls on DAP10, DAP15, and DAP20 under WW  and WS  treatments were collected and stored at −80 °C. Then, 0.05 g tissues of each sample with 3 repetitions were ground in liquid nitrogen before the addition of 200 μL plant total protein lysis buffer containing 20 mM Tris-HCl (pH7.5), 250 mM sucrose, 10 mM EGTA, 1% Triton X-100, protease inhibitor, and 1 M DTT. The mixture was incubated on ice for 20 min. Plant cell debris was removed via centrifugation at a speed of relative centrifugal force of 15,000× Briefly, 60 μg protein samples were reduced with 5 μL 1 M DTT for 1 h at 37 °C, alkylated with 20 μL 1 M iodoacetamide (IAA) for 1 h in the dark, and then digested with sequencing-grade modified trypsin (Promega) for 20 h at 37 °C. Digested peptides were separated with chromatography using an Easy-nLC1000 system (Thermo Scientific) autosampler. The peptide mixture was loaded on a self-made C18 trap column  in solution A (0.1% formic acid), then separated with a self-made Capillary C18 column , with a gradient solution B (100% acetonitrile and 0.1% formic acid) at a flow rate of 600 nL/min. The gradient consisted of the following steps: 0–10% solution B for 16 min, 10–22% for 35 min, 22–30% for 20 min, then increasing to 95% solution B in 1 min and holding for 6 min. Separated peptides were examined in the Orbitrap Fusion mass spectrometer , with a Michrom captive spray nanoelectrospray ionization (NSI) source. Spectra were scanned over the m/z range 300–4000 Da at 120,000 resolution. An 18-s exclusion time and 32% normalization collision energy were set at the dynamic exclusion window.The significance of differences between mean values of physiological parameters, including the chlorophyll concentration and the enzyme activities under WW and WS, were analyzed using an LSD test by software SAS version 8.0.t-tests were performed to determine the significance of changes between samples. A fold-change of >2 and p-value < 0.05 in at least two replicates were used as the thresholds to define differently accumulated protein species.RAW files of mass spectrometry were extracted using the MASCOT version 2.3.02 . Mass spectrometry data were searched, identified, and quantitatively analyzed using the software of Sequest HT and Proteome Discover 2.0 (Thermo Scientific). The database used in this study was uniprot-Medicago.fasta. Protein species with at least two unique peptides were selected for protein species quantitation, and the relative quantitative protein ratios between the two samples were calculated by comparing the average abundance values . Protein species detected in only one material (A-line or B-line), with at least two replicates considered to be presence/absence protein species. Additionally, Student’s


Cancer cachexia is a metabolic syndrome with alterations in gene regulatory networks that consequently lead to skeletal muscle wasting. Integrating microRNAs-mRNAs omics profiles offers an opportunity to understand transcriptional and post-transcriptional regulatory networks underlying muscle wasting. Here, we used RNA sequencing to simultaneously integrate and explore microRNAs and mRNAs expression profiles in the tibialis anterior (TA) muscles of the Lewis Lung Carcinoma (LLC) model of cancer cachexia. We found 1,008 mRNAs and 18 microRNAs differentially expressed in cachectic mice compared with controls. Although our transcriptomic analysis demonstrated a high heterogeneity in mRNA profiles of cachectic mice, we identified a reduced number of differentially expressed genes that were uniformly regulated within cachectic muscles. This set of uniformly regulated genes is associated with the extracellular matrix (ECM), proteolysis, and inflammatory response. We also used transcriptomic data to perform enrichment analysis of transcriptional factor binding sites in promoter sequences, which revealed activation of the atrophy-related transcription factors NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and microRNA expression profiles identified post-transcriptional regulation by microRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and the FoxO signaling pathway. Our integrative analysis of microRNA-mRNA co-profiles comprehensively characterized regulatory relationships of molecular pathways and revealed microRNAs targeting ECM-associated genes in cancer cachexia. Cancer cachexia is a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass that affects up to 80% of cancer patients, depending on the cancer type , and it Several studies have linked cachexia to increased plasma levels of proinflammatory cytokines such as interleukin (IL) 1β, IL6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) . These mRecent advances in mRNA transcriptome analysis have helped to unveil new molecular pathways, potential therapeutic targets, and biomarkers in cancer cachexia . Genome-Although informative, these previous transcriptomic studies addressing muscle wasting in cancer cachexia did not explore different levels of gene expression regulation by integrating mRNAs and miRNAs RNA-Seq data from the same set of muscle samples. Nevertheless, miRNA-mediated regulation of gene expression in cellular networks involves complex interactions among various miRNA targets through several mechanisms , which cIn the present study, we aimed to explore and integrate paired miRNA and mRNA co-profiles during skeletal muscle wasting in a mouse model of cancer-induced cachexia. This comprehensive analysis allowed the identification of new miRNA targets and regulatory strategies controlling gene expression in skeletal muscle atrophy. Although the atrophic muscles of cachectic mice showed high heterogeneity in the transcriptional profile, we successfully identified a set of differentially expressed genes uniformly regulated within these cachectic samples. We also characterized the regulatory relationships of molecular pathways, including miRNAs targeting extracellular matrix (ECM)-associated genes. Taken together, our results show that ECM-associated genes are dependent on inflammatory signaling and reveal miRNA-mRNA molecular networks that may play a role in the development of muscle wasting in cancer cachexia.ad libitum. Before inoculation into mice, LLC cells (ATCC® CRL-1642TM) were cultured in Dulbecco’s modified Eagle’s medium  supplied with 10% of fetal bovine serum  and 1% penicillin/streptomycin  and maintained in a 5% CO2, 37°C humidified incubator. After 3 days of acclimation to their environment, mice were randomly assigned into two groups. Twenty mice (LLC group) were inoculated subcutaneously with a total of 1.5 × 106 LLC cells (7.5 × 105 cells in 0.1 mL PBS in each flank). Ten mice (control group) were injected with equal volumes of 1X PBS.To generate the LLC model of cancer cachexia, we used 8-week old, healthy C57BL/10 male mice obtained from a breeding colony, maintained by our institutional animal care facility at the São Paulo State University . The animals were housed in cages under a 12 h light/dark cycle with food and water Lewis Lung Carcinoma and control mice were weighed daily and studied 22 days after LLC cells inoculation when the LLC group had developed overt cachexia. The animals were euthanized upon anesthesia with intraperitoneal ketamine and xylazine , and tumor diameter, tumor weight, and body weight were measured. The tibialis anterior (TA), soleus (SOL), and gastrocnemius (GAS) muscles were collected, weighed, and then snapped frozen in liquid nitrogen for further analyses. These muscles were chosen based on their fiber composition: TA muscle has a higher proportion of glycolytic fast-twitch fibers, SOL muscle has a higher proportion of oxidative slow-twitch fibers, and the GAS muscle, which comprises both fiber types.The study was performed following the guidelines of the Control of Animal Experimentation and Ethical Principles in Animal Research , under the approved protocol n° 702, emitted by the Institute of Bioscience of Botucatu Ethics Committee on Animal Use, from the São Paulo State University .RNA extraction was performed using the TRIZOL reagent , according to the manufacturer’s instructions. RNA was quantitated by spectrophotometry , and its integrity was ensured by obtaining RNA Integrity Number (RIN) > 8 . RNA samples were treated with DNA Free Kit  to remove genomic DNA contamination.We randomly selected samples (four control and six LLC mice) to construct the RNA sequencing libraries with the TruSeq Stranded Total RNA Sample Prep Kits , using 1000 ng of total RNA. These six LLC mice are those that survived the entire period of the experiment (22 days). Samples were indexed with adaptors and submitted for paired-end 2 × 100-bp sequencing using a HiSeq 2000 instrument . Sequencing was performed on ten RNA samples in one lane of the flow cell, following the manufacturer’s instructions. The lane produced ∼ 600 million raw paired reads. The data output in the fastq file format contained sequence information, including the sequencing quality . Average Phred scores ≥20 per position were used for alignment.The TruSeq Small RNA Sample Preparation kit  was used to prepare the miRNA-Seq libraries for the same set of samples used for mRNA-Seq, following the manufacturer’s instructions. miRNA libraries were 50 bp single-end sequenced using a HiSeq2000 instrument . Sequencing was performed on ten RNA samples in one lane of the flow cell.p-values ≤ 0.05. For assess mRNA transcript abundance, reads were converted to reads per thousand base pairs peak per million mapped reads (RPKM). For assessing miRNA abundance, the reads were converted to counts per million mapped reads (CPM). Finally, we used dot plots to demonstrate the expression of selected genes either previously associated with muscle atrophy in cachexia or that we hypothesized may be associated with muscle atrophy, based on the literature.Paired-end reads for mRNA were mapped to the mm10 genome using TopHat2  with theWe standardized the normalized counts of each gene and applied k-means clustering using Euclidian distance and with random initialization and 10000 executions, and finally selecting four clusters.1 was used to detect DNA motifs overrepresented in the promoter of the differentially expressed genes. Gene promoters were considering between nucleotides −300 and +50 relative to the Transcription Start Site (TSS). Significance was tested against CpG-content-matched promoters as background. Binding sites were considered significantly overrepresented when the p-value <0.01.Pscan web interface 1 was usep-value cut-off of 0.05 was used to identify enriched terms.Gene Ontology enrichment was performed using the ClueGO Cytoscape plugin , using aCandidate miRNA-mRNA targets relationships were predicted by at least one or more of the following target prediction algorithms (union set) extracted from mirDB , TargetS2, which also detects functional interactions among the corresponding genes. Network visualization was performed using the open-source software platform Cytoscape  were collected from control and LLC tumor-bearing mice. Collected samples were placed on the same slide to minimize staining differences; sections were incubated with a saturated picric acid solution followed by Picrosirius red (0.1% Sirius red in saturated picric acid) for 3 min, dehydrated, and mounted in Permount. Eight color pictures per sample were captured using a light microscope . The light intensity parameters used were the same for all samples. Picrosirius staining areas were assessed using the Image J software. As previously described, picrosirius staining areas were normalized for cell density to account for individual fiber atrophy . To quanI values were normalized to β-actin.Muscle proteins were extracted using Tris-Triton buffer  containing Protease Inhibitor Cocktail  and quantified by the Bradford method . Subsequt-test was applied to compare the groups. Statistical significance was considered achieved when the p-value was <0.05.Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the GraphPad Prisma software v 6.07 . For all statistical analyses not described elsewhere, Student’s As expected, all mice subcutaneously inoculated with Lewis Lung Carcinoma (LLC) cells developed cancer cachexia (LLC group) compared to mice injected with PBS (control group). The tumor was detected by palpation after 7 days of cell injection in LLC, after 15 days, the tumor site was visually identified as a skin projection; and after 22 days, when the animals were euthanized, tumor mass was observed under the skin. After euthanasia, the surgically exposed tumor was solid, vascularized, roughly spherical, measuring ∼2 cm in diameter, and weighing ∼4 g .Although the LLC cell line is highly tumorigenic, metastases were not visually identified. Five out of twenty mice died during the experiment (25%) . Consistn = 386) and II (n = 101) contain up-regulated genes, while clusters III (n = 157) and IV (n = 364) include down-regulated genes. Considering the gene expression variability within LLC samples, clusters II and IV contain genes that are uniformly regulated  was able to discriminate LLC and control samples . High heTrim63, Fbxo32, Ubc, Ubb, Psmd4, Psma7, Psmc2, Fbxo31, and Ube4a), autophagosome , and the translation inhibitor Eif4ebp1 (Il6ra), which presented a 5-log range in expression variability within LLC samples  and muscle metabolism and contraction  , ECM organization , and apoptotic process   . Clusterd Chac1) . Clusterd Chac1) . Remarkad Chac1) . Next, wd Chac1) , presentd Chac1) . This sed Chac1) . This seTrim63, Fbxo32, and Ubc - associated with the proteasomal degradation pathway - presented the highest abundance and degree of change in expression  transcript was highly abundant and is down-regulated cachexia  and abundance  of all transcripts . Abundanpression . AdditioEif4ebp1 , which hEif4ebp1 . We also and Mt2 , which h and Mt2 . We iden samples . Interescachexia . Also, aand Mmp8 .Gene ontology analysis revealed 33 terms that were clustered in eight main modules that are relevant to muscle wasting, which include genes associated with the scaffold, cellular metabolism, cellular signaling, cellular differentiation, cellular immune system process, cellular respiration, proteolysis, and ion regulation . NotablyFoxO1 and FoxO6 were up- and down-regulated, respectively , unfolding protein response (Xbp1), and the SMAD family .The transcriptional profile can provide a step toward the identification of key transcription factors that regulate gene expression. We performed an enrichment analysis of transcriptional motifs in the promoter sequences of 1008 differentially expressed genes . The proectively . The proectively . Additiond Stat3 . We founerodimer . We alsoerodimer ; among tMyf6, Myod1, Myog, Tcf12, Pbx1, lbx1, Nfix, and Nfic), lipid homeostasis (Rora and Rorc), energy metabolism (Med1), and muscle fiber-type specification   . Howeverpression .Out of 1915 mature miRNAs, 302 were expressed in skeletal muscle (mapped reads >32 in at least one of the sequenced samples). Eighteen miRNAs were differentially expressed (FDR ≤ 0.05 and |fold change| ≥ 1.5) in muscle wasting during cancer cachexia in comparison to controls . Interestingly, miR-29b-3p presented 22 potential targets, including many genes that encode proteins related to the ECM. Additionally, we found that repressed miRNAs do not share target genes. Furthermore, some genes such as Map2k6, Ptpn3, Mettl21c, Plxdc2, Ppargc1b, Rgs5, and Vegfa were found to be co-regulated by up to three upregulated miRNAs , which added higher precision and sensitivity for the identification of transcriptional and post-transcriptional events. Our transcriptomic data reliably differentiated muscle samples from cachectic and control mice. Notably, inter-individual variations in mRNA expression were evidenced in cachectic mice, which may be linked to individual genetic factors and stochastic tumor growth events that may determine the development and progression of muscle wasting. Studies in rodent models show heterogeneity in the occurrence of cachexia . This chE2f3, Yy1, and Creb3)  , unfoldid Creb3) , and musd Creb3) . Also, wing Mef2 . On the d Fbxo32 . Togethed Fbxo32 . The comd Fbxo32 . Also, id Fbxo32 . Moreoved Fbxo32 , furtherYY1  under the accession numbers GSE144567 (mRNAs) and GSE145393 (miRNAs).The datasets generated for this study can be found in the Gene Expression Omnibus (GEO) DataSets .GF, LM, PF, JG, MD-P-S, and RC: conceptualization. GF, JF, IV-J, LM, SC, PF, JG, and RF: data curation. GF, IV-J, SC, PF, and JG: formal analysis. GF, MD-P-S, and RC: funding acquisition. GF, JF, IV-J, LM, SC, PF, RF, SR, and RC: investigation. GF and RF: methodology. GF, MD-P-S, and RC: project administration. RC: resources and supervision. GF: software, visualization, and writing – original draft. All authors performend writing – review, editing, read the manuscript, and have agreed to be co-authors.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.


Yisui Qinghuang powder (YSQHP) is an effective traditional Chinese medicinal formulation used for the treatment of myelodysplastic syndromes (MDS). However, its pharmacological mechanism of action is unclear.  In this study, the active compounds of YSQHP were screened using the traditional Chinese medicine systems pharmacology (TCMSP) and HerDing databases, and the putative target genes of YSQHP were predicted using the STITCH and DrugBank databases. Then, we further screened the correlative biotargets of YSQHP and MDS. Finally, the compound-target-disease (C-T-D) network was conducted using Cytoscape, while GO and KEGG analyses were conducted using R software. Furthermore, DDI-CPI, a web molecular docking analysis tool, was used to verify potential targets and pathways. Finally, binding site analysis was performed to identify core targets using MOE software.  Our results identified 19 active compounds and 273 putative target genes of YSQHP. The findings of the C-T-D network revealed that Rb1, CASP3, BCL2, and MAPK3 showed the most number of interactions, whereas indirubin, tryptanthrin, G-Rg1, G-Rb1, and G-Rh2 showed the most number of potential targets. The GO analysis showed that 17 proteins were related with STPK activity, PUP ligase binding, and kinase regulator activity. The KEGG analysis showed that PI3K/AKT, apoptosis, and the p53 pathways were the main pathways involved. DDI-CPI identified the top 25 proteins related with PI3K/AKT, apoptosis, and the p53 pathways. CASP8, GSK3B, PRKCA, and VEGFR2 were identified as the correlative biotargets of DDI-CPI and PPI, and their binding sites were found to be indirubin, G-Rh2, and G-Rf.  Taken together, our results revealed that YSQHP likely exerts its antitumor effects by binding to CASP8, GSK3B, PRKCA, and VEGFR2 and by regulating the apoptosis, p53, and PI3K/AKT pathways. Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders characterized by bone marrow (BM) dysplasia, abnormal myeloid cell differentiation, peripheral blood cytopenia, and an increased risk of leukemic transformation . MDS res4S4) and arsenic trioxide (As2O3). As4S4 is an insoluble compound, while As2O3 is a soluble therapeutically active compound [Traditional Chinese medicine (TCM) has been used to treat various illnesses for thousands of years and is associated with fewer toxic effects compared with pharmacological drugs , 7. Yisucompound . AlthougNetwork pharmacology integrates systems biology with polypharmacology and is a suitable approach to characterize complex TCM formulations and identify their multiple targets and mechanisms of action , 11. We https://tcmspw.com/tcmsp.php) which is a unique systems pharmacology platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases. And, HerDing (http://210.107.182.73/TCMIDWebService/herdingDemo.jsp) database is a search engine that recommends herbs to treat diseases using genes and chemicals by integrating existing several databases and a text mining approach.The constituents of YSQHP were screened for using the traditional Chinese medicine systems pharmacology (TCMSP) , and the active compounds of YSQHP were screened for two drug-likeness indices OB  value of ≥30% and DL (drug-likeness) value of ≥0.18 for eliminating the ineffective compounds in herbs. Then, the active compounds omitted from the supplementary part were excavated by reading the literature.2O3 were identified using the STITCH database (http://stitch.embl.de/) [https://www.drugbank.ca/) [https://pubchem.ncbi.nlm.nih.gov/).The key targets of Asmbl.de/)  and the ank.ca/) . Then, ihttps://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM) (http://www.omim.org/) databases using the keyword, myelodysplasia syndrome. After duplicate data were removed, the proteins obtained from both databases were pooled using R software and converted into UniProt IDs.The potential therapeutic targets of MDS were screened for from the GeneCards (https://string-db.org/) based on a minimum required interaction score of 0.900, and it was visualized using Cytoscape.The relationship between the active ingredients of YSQHP and the predicted targets and active compounds were visualized by constructing the compound-target-disease (C-T-D) network using Cytoscape (v.3.6.1). The target-target (T-T) network between YSQHP and MDS was constructed using the STRING database (http://david.abcc.ncifcrf.gov). The genes were functionally annotated through the GO analysis, and significantly enriched terms with a P value <0.5 and FDR <0.5 were screened for. The KEGG (http://www.genome.jp/kegg/) database was used to identify enriched pathways, and data were analyzed and displayed using the GO plot package of R software (v.3.6.1). To further elucidate the mechanism underlying the therapeutic effect of YSQHP against MDS, selected targets were annotated for the apoptosis, p53, and PI3K/AKT signaling pathways  to identify the interacting genes, and the network was drawn using the KEGG parser.The signaling pathways associated with the therapeutic targets of MDS were then identified using DAVID  to simulate molecule docking and predict drugs with similar structure. Eleven effective compounds in YSQHP  were identified, and the structure of these 11 compounds was uploaded to the website for docking. Based on the optimistic model of the data mining results of this website, a docking score that was less than or equal to −8.4 was considered to indicate a potential interaction with the protein. To understand the frequency of the occurrence and the molecular functions of the potential targets, keywords, such as apoptosis, p53, and PI3K/AKT, were used to classify the functions of the proteins. Using the frequency of potential targets in the list, the top 25 proteins were selected and were used in the chemical-protein interactome (CPI) analysis. The molecular docking of indirubin, G-Rh2, G-Rf to GSK3B, CASP8, PRKCA, and VEGFR2 was verified using MOE software (v.2019.0102)..Drug-drug interactions were predicted via chemical-protein interactome or using DDI-CPI  were TP53, AKT1, JUN, MAPK1, STAT3, MAPK3, RB1, HDAC1, TNF, and CCND1.  The T-T network comprised 129 nodes and 545 edges, with an average node degree of 8.45 and an average local clustering coefficient of 0.459 . P53, AK  The GO analysis of the 129 overlapping targets showed that protein serine/threonine kinase activity, ubiquitin-like protein ligase binding, kinase regulator activity, ubiquitin protein ligase binding, and DNA-binding transcription activator activity (RNA polymerase II-specific) were the top 5 enriched functions. In addition, 17 proteins were associated with protein serine/threonine kinase activity with a gene ratio of 0.132, while 16 proteins were associated with ubiquitin-like protein ligase binding function . Thus, Y  The top 5 significantly enriched pathways among the 129 overlapping genes were PI3K/ AKT (33 proteins), apoptosis (26 proteins), cellular senescence (24 proteins), and cell cycle and the MAPK signaling pathways (15 proteins), as shown by the KEGG analysis . The p53  The top 25 proteins were identified based on the docking score and the target functions  . Further  CASP8, GSK3B, PRKCA, and VEGFR2 were identified through both the DDI-CPI and PPI analyses  and haveNetwork pharmacology is a novel field that integrates pharmacology, biochemistry, genomics, and bioinformatics . It is aTP53 mutations have a significantly higher risk of progressing to AML, compared to those with wild-type alleles [α2γ2) levels [MDS is a clonal hematopoietic disorder characterized by bone marrow failure that can develop into acute myeloid leukemia (AML). MDS progression involves multiple signaling pathways , 18. We  alleles . Therefo alleles . The p53 alleles –23. EHMT) levels . VCP is ) levels . KDM1A-m) levels . The YSQ) levels , the latCASP8 knockdown tumor cells via a CASP8-independent pathway. In addition, G-Rh2 inhibits the cisplatin-induced apoptosis of normal renal cells by downregulating the CASP8 expression [The molecular docking score of G-Rh2 and CSAP8 was −10.2, indicating that G-Rh2 likely induces CASP8-mediated apoptosis in MDS cells. CASP8 is expressed at low levels in malignant cells, and Xiao-Xi et al. showed that G-Rh2 can promote the apoptosis of pression . Therefopression , 28. CK2pression . PRKCA apression , 31. Higpression . Indirubpression , 34. YSQpression , 36. HCKpression , 38. PIMpression , 40. GSKpression .Serine/threonine kinase AKT mediates several pathways that regulate metabolism, proliferation, cell survival, growth, and angiogenesis. The AKT2/PI3K pathway is frequently activated in cancer cells and inhibits apoptosis . Given tYisui Qinghuang powder (YSQHP) is a red ginseng-supplemented derivative of Qinghuang powder (QHP) that has been used to treat MDS for over 30 years . YSQHP iCASP8, GSK3B, PRKCA, and VEGFR2 are the potential targets of YSQHP, which potentially inhibits the proliferation of malignant cells and stimulates hematopoietic stem cell differentiation. Furthermore, the antioxidant and antisenescent activities of red ginseng significantly contribute to the antitumor effects of YSQHP. Our findings should be validated experimentally.


Despite recent advances in colorectal cancer (CRC) treatment, a large proportion of patients show limited responses to therapies, especially in advanced stages. There is an urgent need to identify prognostic biomarkers and/or therapeutic targets in advanced stages, aiming to improve the efficacy of current treatments. We aimed to determine prognostic biomarkers in tumor tissue and circulation of CRC patients, with a special focus on T cell exhaustion markers. We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, KLRG1, TOX2, TOX3, Ki-67, and PRDM1 were elevated in CRC tumor tissues. We also investigated differences in gene expression between early and advanced disease stages. We found that TOX and potentially TIM-3, CTLA-4, VISTA, TIGIT, KLRG1, TOX2, SIRT1, Ki-67, and Helios mRNA levels in tumor tissue were elevated in advanced disease stages, suggesting their potential roles in CRC progression. In contrast, PD-1 and CD160 levels in tumor tissue were downregulated in advanced stages. In the circulation of CRC patients, mRNA levels of PD-1, VISTA and LAG-3 were higher than those of healthy individuals. Moreover, in circulation, PD-1, CTLA-4 and TIGIT mRNA levels were reduced in advanced stages. Interestingly, levels of PD-1 in both tumor tissue and circulation were reduced in advanced stages, suggesting that targeting PD-1 in patients with advanced stages could be less effective. Altogether, these findings suggest some potential T cell exhaustion markers that could be utilized as prognostic biomarkers and/or therapeutic targets for CRC. However, further investigations and validations in larger cohorts are required to confirm these findings.The online version of this article (10.1007/s00262-020-02593-w) contains supplementary material, which is available to authorized users. Cancer immunotherapies, including recombinant cytokines, immune checkpoint inhibitors (ICIs), adoptive T cell therapies, and vaccines, have revolutionized the treatment of several solid tumors and hematologic malignancies . ApproveAs cancer cells divide, they acquire genetic and epigenetic alterations giving rise to new cancer clones with different molecular profiles and inheritable traits favoring tumor growth/survival and immune response escape . These aIn this study, we investigated gene expression levels of various inhibitory ICs including PD-1, TIM-3, LAG-3, VISTA, CTLA-4, TIGIT, CD160, CD244, and KLRG1 in the tumor tissue of CRC patients. In addition, expression levels of markers related to T cell exhaustion, cell survival, senescence, proliferation and differentiation; for example, members of the TOX family, silent information regulator 1 (SIRT1), PRDM1 (gene name for Blimp1), Helios and Ki-67, were also determined in tumor tissues of CRC patients. These genes were selected based on their importance in cancer progression and T cell exhaustion as implicated by preclinical models and clinical data; however, their expression profiles in CRC and their expression patterns during the onset and progression of disease have not been addressed yet. Gene expression analyses to determine differences between early and advanced disease stages could be a useful tool to provide insights into the transcriptomic changes during disease onset and progression , 20. Ove6 cells in 1 ml of freezing medium , and stored in liquid nitrogen to be used in batches in subsequent experiments. Tumor tissues (TT) and paired-adjacent normal tissues (NT) were obtained from 70 CRC patients who underwent surgery at Hamad Medical Corporation, Doha, Qatar. TT and NT sections were identified by microscopic pathological examination by the pathologist. Typically, TT specimens were taken from tumor core, while normal tissue sections were taken away from the tumor margins and as deemed relevant by the pathologist. Tissue specimens were cut into pieces, snap-frozen in liquid nitrogen, and stored at − 80 °C to be used in subsequent experiments.PBMCs were isolated from peripheral blood samples of 30 healthy donors (HD) and 68 CRC patients by density gradient centrifugation using Histopaque-1077 . PBMCs were frozen in cryovials at a density of 5–10 × 10All patients included in the study were treatment-naïve prior to surgery and provided written informed consent prior to sample collection. Table Total RNA was extracted from PBMC of HDs and CRC patients, and NT/TT of CRC patients, using RNA/DNA/Protein Purification Plus Kit . Briefly, PBMCs were washed with 1 × phosphate-buffered saline (1 × PBS) and resuspended with lysis buffer. TTs and NTs were also resuspended with lysis buffer, but homogenized using a tissue homogenization set . RNA extraction was performed following the manufacturer’s protocol. RNA concentrations were measured using NanoDrop 2000 (Thermo scientific). 1 µg of RNA was reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit .−ΔΔCT, and normalized to β-actin. Sequences for the primers are shown in Supplementary Table 1.qRT-PCR was performed using QuantStudio 6/7 Flex real-time PCR system , for the genes listed in Supplementary Table 1, with PowerUp SYBR Green Master Mix (Applied Biosystems). Quantification of relative gene expression was determined using 2Ct values of target gene from the HDs were initially normalized to β-actin. The relative expression of target gene from each patient was calculated by normalizing the expression of each sample with average expression of the same gene from HDs, and subsequently 2−ΔΔCT method was applied. Additionally, for CRC tissues, the relative expression was calculated by normalizing the expression of each TT sample to its corresponding NT after the initial normalization to β-actin. Therefore, the relative fold change of each TT sample was calculated by keeping the value of corresponding NT sample as constant “1”. The statistical significance was calculated by performing unpaired and paired t tests for circulation and CRC tissues, respectively [For circulation, the ectively , 22.t tests were performed on samples that passed the Shapiro–Wilk normality test. Non-parametric Wilcoxon signed-rank tests or Mann–Whitney U tests were performed for samples that did not show normal distribution. Mann–Whitney U test was used to compare the differences between early (stages I and II) and advanced (stages III and IV) stages. Comparisons of gene expression among different tumor budding grades were performed using Kruskal–Wallis test one-way analysis of variance (ANOVA). A P value of > 0.05 was considered statistically nonsignificant. The P values are represented as follows;***P < 0.001,**P < 0.01,*P ≤ 0.05. Data are presented as mean ± standard error of the mean (SEM).Statistical analyses were performed using GraphPad Prism 8 software . Paired and unpaired Increased expression of multiple immune checkpoints and T cell exhaustion markers in the tumor tissue of cancer patients has been associated with the suppression of anti-tumor immunity and resistance to therapy , 23. TheWe found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, and KLRG1 were significantly higher in TT, compared to NT . PD-1, TIM-3, CTLA-4, LAG-3, and CD244 mRNA levels were relatively similar amongst the different grades of tumor budding . VISTA, TIGIT and KLRG1 showed a trend towards increased expression in patients with high grades of tumor budding . However, level of CD160 mRNA was significantly increased in patients with low grades of tumor budding . TOX, TOX2, TOX3, and TOX4 , in addition to SIRT1, Ki-67, Helios, and PRDM1 mRNA levels  showed no differences with grades of tumor budding.Collectively, these data suggest that mRNA levels of VISTA, TIGIT and KLRG1 in CRC tumor tissues could be elevated in CRC patients with high grades of tumor budding, which is considered as a risk factor for poor disease prognosis and metastasis in CRC patients. On the other hand, CD160 mRNA level was increased in patients with low grades of tumor budding. These data indicate that some IC expressions could be associated with tumor metastasis.Increased expression of T cell exhaustion markers, including ICs, in many cancer types has been previously reported . We have., showed that targeting TIGIT, using CRISPR/Cas9 system, in mouse colon cancer models inhibited tumor growth [., showed that KLRG1 positively regulates tumor growth/metastasis in animal models of breast and colon cancers, and melanoma, while its blockade enhances anti-tumor immunity [We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, and KLRG1 were elevated in CRC tumor tissue, implicating their potential contribution to CRC development and/or progression. Signaling pathways of these ICs have been associated with increased suppressive activity of T regulatory cells (Tregs), impaired activation of antigen-presenting cells and T effector cells, inhibition of T cell proliferation and reduced cytokine production –34, TIM-r growth . Greenbeimmunity .We also found that mRNA levels of TOX2 and TOX3 were significantly higher in CRC tumor tissues, suggesting their potential roles in CRC development and/or progression. The role of TOX proteins in T cell exhaustion has recently been highlighted in several studies , 14, 38.+ T cell exhausted phenotype in tumors [+ T cells [SIRT1, Ki-67, and PRDM1 mRNA levels were also elevated in CRC tumor tissues; these markers could contribute to CRC by promoting tumorigenesis, and inducing T cell exhaustion and functional impairment via indirect mechanisms –17, 39. n tumors . Moreoven tumors . Blimp-1n tumors . The expn tumors . Further T cells ; howeverAdditionally, we found that PD-1 mRNA levels in CRC tumor tissue and circulation were higher in early stages, suggesting that targeting PD-1 in CRC patients with advanced stages could be less effective. In contrast to the TME, the mRNA levels of CTLA-4 and TIGIT in the circulation of CRC patients at early stages were higher than those at advanced stages. This could suggest that T cells expressing CTLA-4 and TIGIT accumulate in the circulation of CRC patients during the onset of disease, until they are recruited to the TME as disease progresses, resulting in their accumulation within the tumor tissue.Levels of TIM-3, CTLA-4, TIGIT, TOX, TOX2, and SIRT1 genes in tumor tissue were significantly higher than that of the circulation; their expressions within the TME could be induced by tumor-mediated immunosuppression . These dTo our best knowledge, our study is the first one showing the differences in the expression of some T cell exhaustion markers between early and advanced disease stages in the TME and circulation of CRC patients. Collectively, our results could offer a therapeutic benefit and help in designing more effective treatment regimens for patients with advanced stages of CRC. However, further investigations are required to validate these findings in larger cohorts of patients. Additional studies are required to elucidate the mechanisms which regulate the expression of some of these markers in the tumor tissue and circulation of CRC patients.Supplementary Figure 1.A); TOX family members  (B) and other T cell exhaustion markers  (C) in CRC tissues with low (denoted as L), intermediate (denoted as I) and high (denoted as H) grades of tumor budding . Results are presented as fold change (FC) of gene expression in TT vs. NT. Means ± standard error of the means (SEM) are depicted on the scatter plots. (PDF 616 kb)Gene expression of immune checkpoints, T cell exhaustion and cancer progression-related markers in CRC tissues with different grades of tumor budding. Scatter plots show the expression levels of immune checkpoints  (Supplementary file2 (PDF 90 kb)Below is the link to the electronic supplementary material.


In this review, we first briefly introduce the individual components of m6A methyltransferases and explain their close connections to each other. Then, we concentrate on the extensive biological functions of m6A methyltransferases, which include cell growth, nerve development, osteogenic differentiation, metabolism, cardiovascular system homeostasis, infection and immunity, and tumour progression. We summarize the currently unresolved problems in this research field and propose expectations for m6A methyltransferases as novel targets for preventive and curative strategies for disease treatment in the future.M It was discovered in 1974, when Desrosiers and colleagues studied the methylation status of hepatoma cells mRNA using the polyadenosinic acid (PolyA) structure in eukaryotes [6A is an epigenetic mark, a heritable change driven by chemical modifications that alters gene expression without changing the nucleotide sequence [6A methylation modification is a reversible modification, which is methylated by m6A methyltransferases (writers), demethylated by m6A demethylases (erasers) and also recognized by m6A binding proteins (readers), participating in a series of biological processes [N6-methyladenosine , which play similar roles in life processes.Mn of RNA . This coresearch , 8. Althresearch , it doesresearch . METTL14research . These tresearch . WTAP loactivity . In mammactivity , while Zactivity . Moreove6A methyltransferases participate in germ cell maturation and preimplantation embryonic development in animals or plants and even in the reproduction of microorganisms [6A methyltransferases promoted oocyte development and meiosis. In murine and zebrafish oocytes, METTL3 mutations led to arrest in early developmental stage, suppressed maturation and caused defects in the maternal-to-zygotic transition [6A methyltransferases directly influence sex determination through selective splicing of specific genes in germ cells. Corresponding to METTL3, METTL4, WTAP, KIAA1429, Rbm15/15B and Zc3h13 in mammals, Ime4, dMETTL14, Virm, Fl(2)d, Nito and Flacc orthologues, respectively, together with a newly discovered unique conservative component, xio, form a functional methyltransferase complex that facilitates Sxl pre-mRNA splicing in Drosophila, suggesting that this complex has a role in sex determination [Drosophila also show flight defects and held out wings. However, fl(2)d, vir, and nito mutants die during larval stages, preventing the analysis of their adult phenotypes [Mrganisms . Althougrganisms , 18, witansition , 20  causes inadequate termination of their naive cell state and resistance to differentiation, which accounts for early embryonic lethality , 32 , which are DNA binding proteins  that can activate downstream neurotrophic factors to modulate synaptic plasticity and are thus closely related to impaired learning ability and memory formation [6A formation may improve learning ability and slow ageing- and/or disease-related memory loss  [The regulation of structure and function also includes cerebellar development and axonal regeneration. Some studies have shown precise spatiotemporal expression of the ms (CGCs)      . Lack of METTL14 conspicuously decreased the proliferation and induced premature differentiation of NSCs in vitro, suggesting that METTL14 enhances NSC self-renewal, ensuring the reserves of the neural stem cell bank, while analysis in vivo during cortical neurogenesis showed that a decrease in NSCs in the ventricular zone  was accompanied by fewer cortical neurons [From the perspective of neural stem cell research, some studies have revealed key roles for m neurons   . On the ntiation   involved in osteoclast differentiation and factors in bone-resorbing activity (Acp5 and Ctsk), upregulating the expression of the cellular fusion-specific gene Atp6v0d2, entrapping the Traf6 transcript in the nucleus, and subsequently suppressing the activation of MAPK, NF-κB and PI3K-AKT signalling pathways  , m6A abundance, extended mRNA lifetimes and increased expression [6A methyltransferases also act on lipid metabolism independently. Some studies have suggested that m6A methyltransferases negatively correlate with adipogenesis [Mtabolism  Among thtabolism   by mechanistically regulating p16 via m6A modification, thereby preventing arterial restenosis induced by intimal hyperplasia [Blood vessel physiology is complex and involves multiple molecules in multiple cells. METTL14 selectively hypermethylates the transcript of Klotho, a vascular system-protecting protein, promoting its degradation and attenuating the harmful expression of this protein induced by indoxyl sulfate in vascular calcification, thereby decreasing the vascular repair function. Interestingly, the forced expression of METTL14 in stressed human artery smooth muscle cells (HASMCs) has the opposite effect, which may predict the therapeutic potential of METTL14 in vascular calcification-involved diseases  Fig. 2bb2b. Howeerplasia   [6A methyltransferase complex promoted the translation of genes required for human erythropoiesis, including those encoding SETD histone methyltransferases, ribosomal components, and poly(A) RNA-binding proteins [Moreover, m (HSPCs)   , 81  , 83    . Moreoveproteins .6A methyltransferases play diverse roles in either restricting or modulating the lifecycles of viruses. We first focus on RNA viruses, which are classified into positive-sense, single-stranded RNA viruses, such as Flaviviridae. Zika virus (ZIKV) replication efficiency was enhanced after METTL3 or METTL14 knockdown, modifying its host mRNA landscape [6A methyltransferases in the DNA virus infection, in Kaposi’s sarcoma-associated herpesvirus (KSHV) and simian virus 40 (SV40), METTL3 reduced the post-transcriptional accumulation of the major viral lytic transactivator ORF50 [Bombyx mori nucleopolyhedrovirus (BmNPV) structural protein VP39 [6A methyltransferase in P. oryzae) [Recent studies suggest that mandscape  Fig. 3)6A methylandscape  andscape  andscape , 91 andscape , 94 andscape  or ORF50 –99 or ORF50  ein VP39   oryzae)  6A methyltransferases can regulate the immune response to infection [+ regulatory T cells (Tregs), SOCS targets the IL-2-STAT5 signalling pathway to sustain the suppressive functions of Tregs [In addition to acting directly on viruses, mnfection  Fig. 3)6A methylnfection  nfection  nfection  nfection  of Tregs  6A methyltransferases. METTL3 fostered pri-miR-65-3p processing in a microprocessor protein DiGeorge critical region 8-dependent manner to induce inflammatory pain and neuronal sensitization [Recent work on inflammation has yielded some intriguing mechanistic insights into how it might be affected by mtization . It alsotization . Corresptization , whereastization . In autotization , the efftization .6A methyltransferases in oncology has become a focus, and a variety of studies have led to breakthroughs in understanding and treatment.Recently, the function of m6A methyltransferases on the proliferation, invasion and metastasis of different tumours vary tremendously. In the majority of tumours of comparatively high incidence, such as digestive system tumours, including gastric cancer (GC) [6A methyltransferase components, namely, WTAP and KIAA1429, also play adverse roles in GC [6A methyltransferases remains controversial. Some studies have suggested that overexpression of METTL3 profoundly inhibited the proliferation, tumorigenicity and migration ability of glioma [The effects of mcer (GC) –118, colcer (GC) –121, hepcer (GC) , 123 andcer (GC) , as wellcer (GC) , 126, encer (GC) , thyroidcer (GC) , prostatcer (GC) , osteosacer (GC) , melanomcer (GC) , ovariancer (GC)  and morecer (GC) , HCC [12cer (GC)  and proscer (GC)  Fig. 4)6A methylcer (GC) , 117, LCcer (GC)  and ovarcer (GC)  the epitcer (GC)  es in GC , 136 andes in GC , 138 proes in GC , HCC [13es in GC , AML [83es in GC  and endoes in GC  es in GC  es in GC . Interesf glioma , 145, brf glioma  or bladdf glioma  by alterf glioma –153 or Wf glioma , similar6A methyltransferase plays a vital role in the proliferation, invasion and migration of cancer cells, its level can be used to judge the stage and prognosis of tumours. Survival analysis showed that METTL3 serves as a prognostic factor for poor outcomes for CRC [Because m for CRC , 143, GC for CRC , 133, pa for CRC , HCC [12 for CRC , 123 and for CRC  patients for CRC   for CRC , 155  for CRC , CRC [14 for CRC . In otheIn terms of treatment, while METTL3-silenced GSCs and pancreatic cancer cells showed enhanced sensitivity to irradiation , 161, ME6A is acknowledged as one of the most ubiquitous and abundant mRNA methylation modifications in eukaryotes. Therefore, m6A methyltransferases have attracted increasing attention due to their various functions in mediating growth and development, metabolism, behavioural activity and even disease development. In addition to these major aspects, their effects in other areas have been discovered, including drug toxicity [Mtoxicity  and cytotoxicity . In this6A methyltransferases are concerned, existing research on several major components (METTL3/METTL14/WTAP) has been relatively intensive, but studies on the later-discovered regulatory subunits, such as METTL16 [6A methyltransferases, demethylases and binding proteins. With the increasing number of discovered methyltransferases and binding proteins, it still unclear if any special m6A modification sites mediated by different methyltransferases; if any special m6A modification sites recognized by different binding proteins; if any special interactions among methyltransferases, demethylases and binding proteins. For instance, In addition to the basic structure and functional relationships mentioned in the introduction, Sorci M et al. illustrated multiple dimensions of mutual regulation in mRNA translation and stability, by which either knockdown or overexpression of METTL3 upregulated WTAP protein and influenced its homeostasis. In addition, WTAP upregulation can have a carcinogenic effect only in the presence of a functional m6A methylation complex [6A-dependent. Most m6A methyltransferases act as indispensable writers of N6-methyladenosine to play roles in biology. However, Qian JY et al. reported that KIAA1429 can change CDK1 transcript stability and extend its half-life to induce breast cancer [6A methyltransferase can regulate life processes in an m6A-independent manner. This possibility has attracted little attention but may eventually lead to improvements in this field. Thirdly, the bio-function of m6A methyltransferase remains contradictory. For instance, in osteogenesis, while most scientists have proved that METTL3 expression in BMScs is positively correlated with osteogenesis and negatively correlated with adipogenesis [6A methyltransferases. Currently, very few drugs are based on m6A methyltransferase function, and some related ideas are at the speculative stage. We anticipate an increased transition from mature in vitro cell experiments to in vivo studies and expansion of the development of some targeted clinical drugs. All these findings may reveal insights for developing novel preventive and curative strategies for related diseases.According to the current research foundation, the following points need to be addressed. First, as far as m METTL16 , METTL5, METTL16 , are few complex ; that ist cancer . Lin S et cancer , which sogenesis , 52, Yu ogenesis . In anotogenesis , which hogenesis . Similar


Many patients with the aggressive cancer diffuse large B-cell lymphoma (DLBCL) still respond poorly to treatment and suffer from relapsed or refractory disease. The identification of gene mutations that are responsible for the outgrowth of the relapsed tumor is crucial to understand the underlying mechanisms of therapy resistance. In this review, we provide a comprehensive overview of the affected genes and their biological functions in the context of therapy resistance. Furthermore, we discuss novel therapeutic strategies to treat patients with relapsed disease. We expect that the identification of these gene alterations in routine diagnostics holds great potential in guiding future therapy strategies in DLBCL.The majority of patients with diffuse large B-cell lymphoma (DLBCL) can be treated successfully with a combination of chemotherapy and the monoclonal anti-CD20 antibody rituximab. Nonetheless, approximately one-third of the patients with DLBCL still experience relapse or refractory (R/R) disease after first-line immunochemotherapy. Whole-exome sequencing on large cohorts of primary DLBCL has revealed the mutational landscape of DLBCL, which has provided a framework to define novel prognostic subtypes in DLBCL. Several studies have investigated the genetic alterations specifically associated with R/R DLBCL, thereby uncovering molecular pathways linked to therapy resistance. Here, we summarize the current state of knowledge regarding the genetic alterations that are enriched in R/R DLBCL, and the corresponding pathways affected by these gene mutations. Furthermore, we elaborate on their potential role in mediating therapy resistance, also in connection with findings in other B-cell malignancies, and discuss alternative treatment options. Hence, this review provides a comprehensive overview on the gene lesions and molecular mechanisms underlying R/R DLBCL, which are considered valuable parameters to guide treatment. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) and represents a heterogenous malignancy with respect to molecular alterations, morphology, clinical behavior and treatment response ,2. DLBCLIn order to understand the mechanisms that underly immunochemotherapy resistance in B cell malignancies, the genetic defects and biological processes associated with inferior treatment response are currently being investigated. In particular, deep sequencing of R/R DLBCL samples has provided new insight into the affected genes that contribute to the occurrence of the relapse-initiating clone. This review summarizes the current knowledge on the genetic lesions and underlying mechanisms associated with R/R DLBCL, and aims to provide an integrated framework of relevant targets for therapeutic intervention of R/R DLBCL.MYD88, CD79A/B, CARD11, TNFAIP3), and display chronic active BCR signaling, whereas those of the GCB subtype commonly display BCL2 and/or MYC gene rearrangements, as well as genetic lesions in EZH2 and PTEN [In an attempt to improve treatment outcome prediction and identify patients that may benefit from precision guided therapy, several molecular DLBCL classifications have been established. One of the earlier molecular classifications of DLBCL was based on gene expression profiling (GEP) related to the cell-of-origin, which resulted in the identification of two subtypes that comprise 80–85% of the cases: the activated B-cell like type (ABC) and the germinal center B-cell like type (GCB), with the remaining cases termed unclassified ,14. ABC/and PTEN ,18. DespMYC translocation in combination with a BCL2 and/or BCL6 translocation are classified as high-grade B-cell lymphoma (HGBL) [MYC/BCL2 or MYC/BCL6 translocations) and triple-hit (MYC/BCL2/BCL6 translocations) lymphomas (DHL/THL), and represent 5–10% of DLBCL patients [As recognized by the WHO in 2016, DLBCL cases with a a (HGBL) . These mpatients . Interespatients . Recentlpatients . CD79B mutations), BN2 (BCL6 fusions and NOTCH2 mutations), N1 (NOTCH1 mutations) and EZB (EZH2 mutations and BCL2 translocations), where BN2 and EZB subtypes showed better outcome [Whole-exome sequencing (WES), gene copy number analysis and high-throughput RNA sequencing (RNA-Seq) has resulted in novel DLBCL clusters related to treatment outcome. Schmitz et al. genetically dissected DLBCL into four subtypes: MCD  and the synthetic glucocorticoid prednisolone, together with rituximab. Chemotherapy resistance prototypically relates to multidrug resistance, changes in drug metabolism, inhibition of apoptosis, enhanced DNA repair and epigenetic modifications , while gMS4A1(CD20) gene mutations in de novo tumors and R/R disease [The addition of rituximab to standard chemotherapy has shown beneficial effects on DLBCL treatment outcome, but also generated specific resistance mechanisms. Rituximab exerts its therapeutic effects via binding to CD20, leading to (1) complement dependent cytotoxicity (CDC), and (2) antibody-dependent cellular toxicity (ADCC) . The thi disease ,35. Howe disease . IKZF1 deletions are linked to increased relapse risk [IKZF3 and TP53 have been identified as relapse drivers displaying increased mutation frequency in relapsed samples after immunochemotherapy [TP53 mutations are directly linked to chemotherapy resistance and these patients are now treated differently in order to improve treatment outcome [A successful approach, as shown in leukemias, to elucidate the molecular mechanisms that contribute to immunochemotherapy resistance and relapse development, involves high coverage WES analysis of tumor cell populations in both diagnosis and relapse samples. In the B-cell precursor acute lymphoblastic leukemia (BCP-ALL), pse risk ,38, whilotherapy . Notably outcome ,41,42,43The identification of relapse-associated mutations has resulted in the construction of clonal lineages from diagnosis to relapse for several cancers, including DLBCL ,46,47,48As described by Juskevicius et al. , these mMYC, BCL2, TP53 and members of the JAK-STAT signaling pathway. Other recurrent processes involve immune escape strategies and epigenetic regulation of the tumor genome. In the next paragraphs, we will elaborate on these pathways and describe their potential role in mediating therapy resistance.To identify genetic alterations that are responsible for inferior treatment outcome and relapse, several studies have investigated the genetic landscape in R/R DLBCL by targeted sequencing or WES . RelapseMYC, BCL2 and/or BCL6, and DEL are linked to inferior treatment response [As described earlier, HGBL with gene rearrangements affecting response ,20, althresponse ,56,57,58response ,60. Addiresponse . In contresponse .BCL2 are enriched in relapse, while certain variants are relapse-specific [BCL2 is more frequently altered in relapse, exhibiting almost doubled mutational frequencies [The anti-apoptotic protein BCL2 promotes cell survival in B cells, and many BCL2 family members play a crucial role in modulating cellular stress responses ,63. Indespecific ,46,52,53quencies ,52. Specquencies ,65.BCL2 translocation [BCL2 mutations are more prevalent in primary samples of relapsing patients than those of non-relapsing patients [BCL2 probably result in gain-of-function, thereby increasing its anti-apoptotic properties. The majority of these BCL2 alterations most likely play a direct role in R-CHOP therapy resistance and the outgrowth of the relapse-initiating clone  IGH-location ,67, and location . Diagnoslocation . Furtherpatients ,48. Thesng clone . As such effects ,72. Nota effects . MYC rearrangements relate to significantly inferior 5-year progression-free survival (PFS) and OS after R-CHOP treatment in patients with DLBCL [MYC mutations are more frequently observed at the time of relapse in paired diagnosis-relapse DLBCL samples, including several unique mutations that are not present at initial diagnosis [MYC mutations are more frequently detected in relapsed DLBCL [MYC mutations are often located in the N-terminus and are considered to reinforce MYC’s oncogenic potential [MYC have been observed in R/R DLBCL, and not in independent DLBCL primary cohorts [The MYC oncogene is a key player in B cell development and maturation, as it regulates the expression of multiple genes related to cell growth, differentiation, proliferation and survival . Overexpth DLBCL , and areth DLBCL , althougth DLBCL . On the iagnosis ,46,53. Aed DLBCL ,51,52,53otential . In addip14ARF/CDKN2A gene expression [L and MCL1 [TP53 mutations and BCL2 overexpression. Hence, the oncogenic and chemoresistance phenotype of p53 and BCL2 is enhanced by MYC gene alterations [MYC primarily induces apoptosis upon overexpression, which includes the activation of the p53 pathway through the suppression of pression . MYC alsand MCL1 ,83. Howeerations ,84. BCL6 chromosomal translocations are present in ~25% of DLBCL [BCL6 gene rearrangements have been associated with poor OS in DLBCL in a meta-analysis of 22 studies, which are prognostic mainly following rituximab-containing regimens [BCL2 and MYC, BCL6 alterations other than gene rearrangements are not reported as enriched in relapsed DLBCL. Interestingly, TP53 mutations are frequently observed in MYC/BCL2-rearranged lymphomas, while this is not the case for MYC/BCL6 lymphomas [TP53 alterations are preferred drivers, alleviating the requirement for certain BCL6 gene alterations.BCL6 facilitates the proliferation of germinal center B cells after T-cell dependent antigen stimulation and represses differentiation into plasma and memory cells . Distincof DLBCL ,25. BCL6regimens . In contymphomas . As BCL6ymphomas , and p53ymphomas , it coulTP53 represents the most commonly mutated gene in human cancer [TP53 gene, often clustering in the DNA-binding domain, which predominantly yield loss of wild-type p53 function, although gain-of-function activity has been described for several p53 mutants [TP53 gene aberrations are detected in 35% of the patients and are mostly associated with inferior prognosis [TP53 mutations (21%) and focal del (17p) TP53 gene deletions (29%), which frequently co-occur resulting in bi-allelic inactivation [TP53 mutations and deletions cluster within the A53 genetic subtype with a 5-year OS of 63%, representing an intermediate risk group [TP53 mutations on prognosis between the molecular subtypes, conferring no effect in the NOTCH2 group, but poor prognosis in the MYD88 subtype [Tumor suppressor p53 is involved in multiple key functions to guard the human genome against oncogenic transformation, and promotes cell cycle arrest, senescence, DNA repair and pro-apoptotic signaling under conditions of cellular stress ,93. TP53n cancer , where m mutants ,96. In Drognosis ,99,100. tivation ,101. In sk group . However subtype . TP53 gene aberrations at relapse [TP53 are clonally stable during the progression from diagnosis to relapse [TP53 mutations involves both the outgrowth of subclonal populations already present at diagnosis and the acquisition of novel relapse-specific mutations. Interestingly, ultra-deep sequencing has revealed the presence of TP53 mutations at very low allele frequency at time of diagnosis [TP53 mutations more often represents an intrinsic property of the initial tumor rather than de novo acquired [TP53 mutations in primary and relapsed samples of R/R DLBCL cohorts is higher than in independent primary DLBCL samples [n = 135), TP53 mutations were identified in 51% of the patients, whereas only 21% of primary DLBCL (n = 1200) showed TP53 mutations, highlighting the importance of TP53 alterations in relapse initiation [CDKN2A (INK4A/p14ARF) gene deletions (43% in relapse vs 15% in primary diagnosis), and mutations (30% in relapse vs 10% in primary diagnosis) [Most importantly, there is a significantly increased prevalence of  relapse ,44,51, w relapse . The inc samples ,51,52. Iitiation . Convergagnosis) ,103. TP53 mutations is known to cause resistance to a wide variety of anti-cancer drugs, including alkylating agents, anthracyclines, antimetabolites, and antiestrogens [miR-34a [TP53 gene alterations in mediating immunochemotherapy resistance of DLBCL.The presence of strogens . The undstrogens ,82. Howestrogens . In addi[miR-34a . Hence, L and MCL1, JUN and MYC [Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway occurs downstream of cytokine and growth factor receptor signaling and induces cell proliferation, differentiation and cell survival . JAKs ar and MYC . JAK1, STAT6, SOCS1 and downstream target PIM1 [SOCS1 mutations in DLBCL prognosis and relapse has been more complicated to unravel, which relates to the different functions of SOCS1 [SOCS1 pathogenic mutations confer reduced OS in R-CHOP-treated elderly DLBCL patients [SOCS1 mutations occur more frequently in relapsed DLBCL [NFKBIE [In DLBCL, mutations affecting the JAK/STAT pathway have been detected as relapse-enriched genetic alterations, including get PIM1 ,48,51,53of SOCS1 ,112, theof SOCS1 ,113. A rpatients , which fed DLBCL ,51,53. F [NFKBIE ,51, whic [NFKBIE ,116. ColHLA genes [CIITA encoding a transactivator of MHC-II gene expression [B2M gene alterations or relapse-specific variants, including non-synonymous mutations, frameshift indels, and focal deletions [B2M gene lesions impair MHC class I folding and transport to the cell surface, causing loss of (neo-)antigen presentation and thereby allowing immune escape [Evasion of immune surveillance is a critical step for DLBCL tumor development and relates to several mechanisms ,118, somLA genes , and in pression . Furthereletions ,51,52,53e escape . Alternae escape . B2M gene alterations have been linked to ICI resistance [B2M alterations may be important in ICI directed against the programmed death 1 (PD-1)/PD ligand 1 (PD-L1) interaction in R/R DLBCL [Attenuated B2M expression may also hold important consequences for therapies with immune checkpoint inhibitors (ICI), since proper tumor antigen presentation is required for the effective action of ICI. For instance, in melanoma, sistance ,123, altsistance . With ~2/R DLBCL ,126. The/R DLBCL . Moreove/R DLBCL . CD58 are associated with inferior prognosis in DLBCL [CD58 genetic alterations in matched diagnosis-relapse samples has been observed, including relapse-specific deletions and a frameshift indel in CD58 gene [CD58 mutations are more prevalent in diagnosis samples of patients with R/R disease [CD58 genetic lesions, indicating alternative pathways that deregulate CD58 function [Interestingly, a lack of B2M and reduced MHC-I expression often co-occurs with loss of CD58 cell surface expression in primary DLBCL . CD58 isin DLBCL . MoreoveD58 gene ,46, and  disease ,54. In afunction ,132. EZH2 and KTM2D, as well as the HAT genes CREBBP and EP300, are clearly linked to R/R DLBCL [Perturbation of epigenetic regulation is a common feature in DLBCL, with frequent gene alterations in histone methyltransferases (HMTs) and histone acetyltransferases (HATs) . These p/R DLBCL ,46,51,52EZH2 mutations have been identified as a driver for relapsed DLBCL [KMT2D mutations, which impair KMT2D/MLL2 protein function in mediating H3K4 mono- and di-methylation, have been reported as an early event in lymphoma development [KMT2D mutations are mostly stable between diagnosis and relapse, clonal expansions have been detected in relapsed DLBCL [KMT2D mutations have been reported as one of the most frequently observed relapse-associated gene alterations, present in 44% of relapsed DLBCL [KMT2D mutations occur more frequently in R/R DLBCL cohorts as compared to primary DLBCL [KMT2C gene encoding MLL3 have also been associated with relapse [Lymphoma-associated mutations affecting Polycomb protein EZH2 represent gain-of-function alterations, which enhance EZH2 methyltransferase activity leading to increased H3K27me3 and repression of transcription. ed DLBCL , with ined DLBCL ,51, and ed DLBCL ,34,44. Kelopment ,46. Althed DLBCL . As suched DLBCL . Similarry DLBCL . Mutatio relapse ,51,53, aCREBBP and EP300 [CREBBP and EP300 mutations have been detected [CREBBP mutations are present at higher frequency in R/R DLBCL cohorts, compared to primary DLBCL [TET2 and BRD4 [Other gene mutations that are clonally maintained in paired diagnosis-relapse samples involve the HAT genes nd EP300 ,44,46, wdetected ,52, whicdetected . Furtherry DLBCL ,52 Notabry DLBCL . Other rEZH2 mutations result in global H3K27me3 reprogramming that impacts B cell function and the surrounding immunological niche [An interesting observation that links these epigenetic proteins to relapse relates to their ability to functionally regulate key proteins associated with therapy resistance. KMT2D acts as a p53 coactivator as part of the ASCOM complex with ASC2, required for induction of endogenous p53 in response to DNA damage . Furtheral niche . In addial niche . Thus, tMultiple novel therapeutic agents are being developed to improve DLBCL treatment outcome, several of which target potential drivers of R-CHOP therapy resistance . In-deptL expression [PMAIP1/NOXA gene amplification [L and MCL1, such as PI3K delta inhibitor idelalisib, seems to be promising, where synergizing effects have been observed in DLBCL cell lines and in vivo mouse models [The BCL2 inhibitor (BCL2i) venetoclax has shown significant benefical activity in CLL and acute myeloid leukemia (AML) ,144,145,pression , or the fication . Therefoe models ,151. Thie models ,153, whiMYC translocation or amplification [Given the essential role of MYC in DLBCL/HGBL pathogenesis and its association with relapse, different therapeutic strategies are being developed to target MYC-dependent lymphomas . One appfication , and is fication ,154,160.TP53 mutations. One approach is to restore wild-type p53 function in tumor cells through structural reactivation of mutant p53 with small peptides, among which are PRIMA-1 and APR-246 (NCT04419389), as well as strategies targeting the MDM2/MDM4-p53 axis [BCL2 and TP53-mutant DLBCL cells, has been shown to enhance the cytotoxic effect of doxorubicin [There are several therapeutic strategies to target attenuated p53 activity in cancer, some of which may become beneficial for DLBCL patients harboring p53 axis ,162,163.p53 axis . Adminisorubicin . Howeverorubicin . A novelorubicin ,167. Othorubicin .Multiple JAK inhibitors (jakinibs) have been developed, which inhibit one or multiple JAK proteins, hereby interfering with upstream JAK-STAT signaling leading to reduced cell survival . BeneficCD58 and B2M result in loss-of-function, allowing lymphoma cells to escape immune responses. For reversing epigenetic silencing of CD58 expression, EZH2 inhibitors have been shown to be effective [B2M mutations in these patients.As immune escape is observed in both diagnosis and relapsed DLBCL, it is important to develop therapy strategies that overcome this critical issue. The DLBCL associated (epi-)genetic alterations in ffective . A propoffective . Howeverffective . These iffective ,176. Othffective . For ICIffective  and the ffective  have beeIn the past decade, different epigenetic therapies have been explored in cancer treatment, and several HDAC targeting drugs are now FDA approved for various hematological malignancies . HDAC inEZH2 mutations [KMT2D gene lesions, inhibition of KMD5/JARID1 has been proposed, which is known to counteract KTM2D by demethylating H3K4me3/2, and has been linked to cancer chemoresistance [KMT2D mutant GCB-DLBCL cell lines with concomitant diminished BCR signaling and altered expression of BCL2 family members [Specific therapeutic targeting of epigenetic modifiers has emerged for chromatin reader protein BRD4 for MYC-dependent DLBCL (BET-domain inhibitors) , and EZHutations ,192. Thesistance . Indeed, members . Thus, eDespite the development of many novel therapeutic modalities for DLBCL treatment, R-CHOP still represents the choice of first-line therapy for most DLBCL patients. However, resistance to R-CHOP remains a persistent problem, with knowledge still limited regarding the genetic alterations that drive immunotherapy resistance, resulting in the outgrowth of the relapse-initiating clone towards a full-blown relapse. In the past years, studies investigating the mutational landscape of R/R DLBCL have improved our insight on this topic. In line with the heterogeneity of the disease, the identified genes that represent relapse-enriched mutations show variability between the different studies outside the common targets described in this review. Part of this variation can be explained by the relatively small cohorts and the different sequencing platforms in these studies. Nevertheless, many of the identified genes can be assigned to specific pathways and biological functions, thereby revealing important mechanisms that define candidate R-CHOP immunochemotherapy resistance genes. Not surprisingly, many of the recurrent genes are linked to the regulation of apoptosis and cell proliferation, including BCL2 anti-apoptotic family members, MYC and p53, which highlights the importance for the therapeutic targeting of these proteins and associated pathways. However, there is limited evidence for beneficial effects of targeting these pathways by single targeting agents. For this reason, combinations of targeted therapies, such as inhibitors that may target multiple anti-apoptotic BCL2 family members, combined with drugs that affect MYC, p53 pathway, immune escape genes and/or epigenetic regulators may lead to synergizing effects and could be beneficial for R/R DLBCL patients. It is evident that (epi-)genetic alterations in these candidate R-CHOP resistance drivers are not shared by all relapsed tumors, implying that there is redundancy with other gene mutations that confer therapy resistance. The identification of these genetic relapse drivers will be essential to develop targeted therapies for this group of relapse-prone DLBCL patients. Hence, more extensive NGS analysis of paired diagnosis-relapse cohorts will be required to identify the remaining class of less prevalent relapse-enriched gene aberrations. Ideally in future routine diagnostics, the presence of these genetic alterations could be used as a diagnostic biomarker for immunochemotherapy resistance, and in this way guide alternative treatment choices.