Autophagy is involved in cancer initiation and progression but its role in uveal melanoma (UM) was rarely investigated. Herein, we built an autophagy-related gene (ARG) risk model of UM patients by univariate Cox regression and least absolute shrinkage and selection operator (Lasso) regression model and filtrated out nine prognostic ARGs in The Cancer Genome Atlas (TCGA) cohort. Survival and Receiver Operating Characteristic (ROC) Curve analysis in the TCGA and other four independent UM cohorts  proved that the ARG-signature possessed robust and steady prognosis predictive ability. We calculated risk scores for patients included in our study and patients with higher risk scores showed worse clinical outcomes. We found the expressions of the nine ARGs were significantly associated with clinical and molecular features (including risk score) and overall survival (OS) of UM patients. Furthermore, we utilized univariate and multivariate Cox regression analyses to determine the independent prognostic ability of the ARG-signature. Functional enrichment analysis showed the ARG-signature was correlated with several immune-related processes and pathways like T-cell activation and T-cell receptor signaling pathway. Gene set enrichment analysis (GSEA) found tumor hallmarks including angiogenesis, IL6-JAK-STAT3-signaling, reactive oxygen species pathway and oxidative phosphorylation were enriched in high-risk UM patients. Finally, infiltrations of several immune cells and immune-related scores were found significantly associated with the ARG-signature. In conclusion, the ARG-signature might be a strong predictor for evaluating the prognosis and immune infiltration of UM patients. Uveal melanoma (UM) is a type of malignant intraocular cancer derived from melanocytes with high metastasis rate and poor prognosis . Fifty pAutophagy, a cellular programmed-digestion process, has been reported related to several pathological process including malignancy progression. It is a special mechanism for cancer cells to obtain enough energy and nutriment in the hostile conditions of hypoxia, oxidative stress and nutrient starvation ,5. In reIn the present study, we collected five independent UM cohorts  and established an autophagy-related prognostic signature by using univariate Cox regression analysis and least absolute shrinkage and selection operator (Lasso) Cox regression in the TCGA training cohort. A risk score was calculated for each patient and by the median value of risk scores we dichotomized patients into low- and high-risk subgroups which showed different overall survival (OS) time and disease-free survival (DFS) time. Besides, functional enrichment analysis between low- and high-risk UM patients showed malignancy-related processes, pathways and signatures are associated with the high-risk patients. Immune microenvironment features, including multiple immune cells, machine-learning based scores, were also investigated for their associations with the ARG-signature in UMs.https://portal.gdc.cancer.gov/), and the related RNA expression and clinical information files of the other four cohorts  were downloaded from the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/) and previous published researches [http://autophagy.lu/clustering/index.html) [http://software.broadinstitute.org/gsea/msigdb) [RNA-seq profile and related clinical information of The Cancer Genome Atlas (TCGA) cohort were obtained from the TCGA website (searches . The basex.html)  and the /msigdb) . We inteFor the TCGA RNA-seq cohort, The Fragments Per Kilobase of transcript per Million (FPKM) values were transformed to Transcripts Per Kilobase Million (TPM) values according to an algorithm acquired in published researches ,17. For P<0.05) including 39 protective ARGs (HR < 1) and 94 risky ARGs (HR > 1). These OS-related ARGs were used to further construct the prognostic signature by Lasso Cox regression analysis. Finally, a 9-ARG signature was established and a risk score calculating formula, based on the related expression value and coefficients of the nine ARGs, was developed. The formula developed was as following:By taking the TCGA cohort as training cohort, we performed univariate Cox regression analysis to the 423 ARGs based on the survival information in the TCGA cohorts and we screened out 133 OS-related ARGs  in the present study. We used the R package ‘limma’ [P-value (Norm P) <0.05 and False Discovery Rate (FDR) < 0.25.Genes whose expressions were in significant differential levels between low- and high-risk UM patients with |log2 (fold change)| > 1 and  ‘limma’  to perfo ‘limma’  was usedhttps://gdc.cancer.gov/about-data/publications/panimmune).Based on the TPM expression values of UM patients in the TCGA cohort, the 22 immune cells infiltration proportions of each UM patients were calculated using ‘CIBERSORT’ R package. The ESTIMATE score, immune score, stromal score and tumor purity values of UM patients in the TCGA dataset were obtained from the website of PanCanAtlas Publications (https://www.r-project.org/).Two-sided log-rank test was applied to contrast the OS/DFS between different risk subgroups or expression levels of ARGs of UM patients in survival curves, visualized by R package ‘survival’. To evaluate the OS/DFS predictive power of the ARG-signature, receiver operating characteristic (ROC) curves were plotted by R package ‘timeROC’  and areaThe workflow of the development of the risk signature is shown as P<0.05), of which 39 ARGs were protective genes (HR < 1) and 94 ARGs were risky genes (HR > 1). Lasso Cox regression model was subsequently applied to the 133 OS-related ARGs to establish a prognostic model for the UM patients in the TCGA cohort. We successfully developed a prognostic signature based on nine ARGs  revealed that low-risk UM subgroup have higher survival rate and longer survival time compared with the high-risk subgroup D. And thFurthermore, associations between the 9-gene expression levels and clinical and molecular features (including risk score) were investigated. UM patients were in order with the risk score increasing and patients with higher risk were associated with lower DLC1, GABARAPL1, LMCD1, PRKCD, TUSC1 expressions, higher BNIP1, ITGA6 IKBKE and FKBP1A expressions, more Chr3/Chr8q loss, less Chr6p loss and higher tumor thickness, diagnostic age and clinical stage G.n=28) and GSE22138 (n=63) UM cohorts  was used to examined whether the ARG-signature could predict the DFS of UM patients. Like the results above, DFS analysis and patients’ distributions of DFS time indicated that the high-risk UM patients have a lower DFS rate and shorter DFS time , clinical stage , M status , T status  and risk stratification  were significantly associated with prognosis of UM patients, and gender  was not a prognostic predictor  in the univariate Cox regression analysis. The results of univariate Cox regression showed higher age  and the risk stratification  possessed independent prognostic roles  showed UM patients were in distinct directions based on all detected genes or 531 ARGs profiles in the TCGA cohort , which indicated that UM patients with different risk stratification harbored different gene expression status and autophagy status E,F.To investigate the inner molecular distinctions between low- and high-risk UM patients, DEGs between low- and high-risk patients were analyzed in the TCGA cohort. Differential expression analysis was first performed between low- and high-risk subgroups of UM patients in the TCGA cohort and 2369 DEGs were identified.In order to understand the main functional processes and pathways of the 2369 DEGs, we implemented GO and KEGG pathway to identify the dominating functions of them. Results of GO analysis revealed that functional enrichments of these DEGs included T-cell activation, lymphocyte activation, leukocyte activation and differentiation , plasma membrane protein complex, extracellular matrix, collagen-containing extracellular matrix , receptor regulator activity, channel activity, passive transmembrane transporter activity  and so on A. KEGG pTumor hallmarks were also investigated between low- and high-risk UM patients in the TCGA cohort by using gene set enrichment analysis (GSEA) method. We identified four tumor hallmarks including Angiogenesis, IL6-JAK-STAT3 signaling, Reactive oxygen species pathway and Oxidative phosphorylation were prominently enriched in high-risk UM patients C.In light of the results of functional enrichment analysis, we speculated that the ARG-signature was associated with the immune characterization of UM. The landscape of 22 immune cell infiltration proportions were shown in P=6.1⁁-5), Immune score  and Stromal score  and negatively associated with the Tumor Purity . These data demonstrated that UM patients with higher risk scores were infiltrated with more immune and stromal cells.Immune microenvironment-related scores, including ESTIMATE score, Tumor Purity, Immune score and Stromal score, were calculated for each patients using related algorithm. These immune microenvironment related scores were used to performed Pearson correlation analysis with the risk score to confirm the correlation between the ARG-signature and immune microenvironment status. Results showed that the risk score was positively associated with the ESTIMATE score  had been discovered as an oncogene and is overexpressed in over 30% of breast carcinomas samples and cell lines . It was in vitro ,36. Few In conclusion, totally 257 UM patients in five independent cohorts were included and used to develop and validate a prognostic predictive signature for UM patients. The ARG-signature showed a powerful predictive ability to the clinical outcomes in both training cohort and four external validation cohorts. Meanwhile, nine novel prognostic ARGs were identified associated with the clinical outcomes of UM patients and might provide some clues for further researches. Besides, UM patients with higher risk scores showed higher immune cell infiltration levels and tumor hallmarks enrichments.


Arf-expressing cells ameliorates cigarette smoke-induced lung pathologies in mice. Cigarette smoke induced the expression of Ink4a and Arf in lung tissue with concomitant increases in lung tissue compliance and alveolar airspace. The elimination of Arf-expressing cells prior to cigarette smoke exposure protected against these changes. Furthermore, the administration of cigarette smoke extract lead to pulmonary dysfunction, which was ameliorated by subsequent senescent cell elimination. Collectively, these results suggest that senescent cells are a potential therapeutic target for cigarette smoking-associated lung disease.Senescent cells accumulate in tissues during aging or pathological settings. The semi-genetic or pharmacological targeting of senescent cells revealed that cellular senescence underlies many aspects of the aging-associated phenotype and diseases. We previously reported that cellular senescence contributes to aging- and disease-associated pulmonary dysfunction. We herein report that the elimination of  Chronic obstructive pulmonary disease (COPD) is a severe and incurable disease with a relatively high prevalence and is currently one of the leading causes of death worldwide . A majorCdkn2a locus, which encodes two important tumor suppressors, namely, p16Ink4a and p19Arf (p14ARF in humans) . Fu. FuArf-e process . Hence, Arf-expressing cells exerted preventive effects against elastase-induced pathologies [We previously demonstrated that the elimination of hologies . The prehologies . While Ahologies ,49 and mArf-expressing cells ameliorated the CS- and CSE-induced pulmonary dysfunction in mice. These findings suggest emphysema-associated pathologies are exacerbated by cellular senescence, and that targeting senescent cells may be an effective approach for COPD therapy.Elimination of p19


Non-Hodgkin lymphomas (NHLs) are lymphoid malignancies of B- or T-cell origin. Despite great advances in treatment options and significant improvement of survival parameters, a large part of NHL patients either present with a chemotherapy-refractory disease or experience lymphoma relapse. Chemotherapy-based salvage therapy of relapsed/refractory NHL is, however, capable of re-inducing long-term remissions only in a minority of patients. Immunotherapy-based approaches, including bispecific antibodies, immune checkpoint inhibitors and genetically engineered T-cells carrying chimeric antigen receptors, single-agent or in combination with therapeutic monoclonal antibodies, immunomodulatory agents, chemotherapy or targeted agents demonstrated unprecedented clinical activity in heavily-pretreated patients with NHL, including chemotherapy-refractory cases with complex karyotype changes and other adverse prognostic factors. In this review, we recapitulate currently used immunotherapy modalities in NHL and discuss future perspectives of combinatorial immunotherapy strategies, including patient-tailored approaches. Non-Hodgkin’s lymphomas (NHL) comprise a group of more than 80 clinical entities according to the most recent WHO classification, which differ according to the cell of origin, clinical presentation, and prognosis . While cThe tumor microenvironment plays a critical role in lymphoma cell survival, growth, spread, and resistance to therapy. The landmark discovery that reactivation of tumor-suppressed cell-based immune responses can induce clinical remissions even in patients with chemotherapy refractory diseases led to the runaway development and testing of diverse new immunotherapy strategies and their combinations. Our better understanding of mechanisms of tumor immune escape and modes of action of anti-tumor immunity, together with unprecedented progress of genetic engineering enabled the concept and production of seemingly indefinite range of recombinant proteins and synthetic antibody constructs, including glycoengineered therapeutic monoclonal antibodies, bispecific antibody constructs, immune checkpoint inhibitors or activators, and genetically engineered T and NK cells carrying chimeric antigen receptors (CAR-T and CAR-NK cells) . In thisL and VH) responsible for epitope recognition, and a constant fragment crystallizable (Fc) region at the C-terminus that mediates various immune responses . I. I43]. I2/day Only autologous CAR T-cells are currently available for clinical use. However, custom-made manufacturing of autologous CAR T-cells is quite difficult from both clinical and logistic point of view. First, patients after multiple lines of treatments may not have enough T-cells for successful apheresis, or these cells may be of inferior quality. Second, the manufacturing procedure takes time, which may be critical especially for patients with biologically aggressive lymphomas. Though in theory, a CAR T-cell product may be available in two weeks after collection of starting material; current reality (at least in Europe) is around four weeks. Universal “off-the-shelf” CAR T cell (UCART) concept is therefore an attractive strategy. However, currently employed CAR T-cells have preserved native TCR receptors on their surface and consequently can elicit graft-versus-host disease (GvHD). Manufacturing of UCARTs thus requires additional engineering. T-cell receptor, or part of it, may be deleted by transcription-activator-like effector nucleases (TALEN) or CRISPR/Cas9 technologies, though currently employed protocols resulted only in partial TCR deletion and resulting products may still carry the risk of GvHD. Cooper et al. developed a CRISPR/Cas9 edited anti-CD7 T-cells, where both CD7 and TCRα are deleted . This prNK cells, in contrast to cytotoxic T lymphocytes, kill target cells in HLA-unrestricted manner and therefore do not elicit GvHD. NK cells recognize all class I HLA antigen-expressing tissues as “self” and kill only those tumor cells that lack them. CAR expression in NK cells results in targeted killing with theoretically minimal side effects. CAR design for NK cells possess different challenges compared to T-cell engineering. The viral transduction systems used for T-cells achieved less than 5% transduction in case of NK cells . TransduImmunomodulatory agents are small molecule cereblon-modulating chemical agents that have both direct anti-tumor effects and indirect (anti-angiogenic and immunomodulatory) activities . BesidesLenalidomide, a thalidomide derivative, is an orally available IMiD used for the treatment of multiple myeloma and several types of B-NHL. Its mechanisms of action are both direct-mediated through stimulation of E3 ubiquitin-ligase cereblon, and indirect-mediated through gain of function of immune effector cells, namely natural killer cells . In lymp2 regimen) versus placebo plus rituximab was 39.4 months and 14.1 months, respectively [2 regimen) demonstrated promising anti-lymphoma activity in patients with mantle cell lymphoma, diffuse large B-cell lymphoma and primary CNS lymphoma [2 regimen across B-NHL subtypes was a rationale for design and clinical testing of another promising combination of lenalidomide and obinutuzumab, a glycoengineered antibody with enhanced ADCC. A clinical trial of obinutuzumab and lenalidomide (GALEN) in R/R follicular lymphoma (NCT01582776) demonstrated that the GALEN regimen was active in previously-treated patients with manageable toxicity [The lenalidomide-mediated enhancement of NK cell functions results in anti-tumor synergy with therapeutic monoclonal antibodies, whose mode-of-action is based predominantly on the antibody-dependent cell-mediated cytotoxicity (ADCC). In the landmark phase 3 AUGMENT trial, median progression-free survival for lenalidomide plus rituximab  is a combination of fever, hypoxia, hypotension, and capillary leak syndrome, with or without other organ manifestations  . LaboratNeurotoxicity represents another specific adverse event associated with T-cell redirecting therapies. Neurotoxicity, or the immune effector cell-associated neurotoxicity syndrome (ICANS) may have distinct clinical manifestations including confusion, aphasia, apraxia, frontal release sings, meningism, palsy, seizures, cerebral edema, or coma. Expressive aphasia, especially the inability to name objects, is an early and quite specific symptom . VariousWithout any doubt, immunotherapy has radically changed our possibility to fight lymphomas including chemotherapy-refractory cases .It is evident that immunotherapy alone can induce durable remissions with a potential cure for many patients with otherwise resistant disease. Though immunotherapy may be effective even in late stages of disease, in these situations its efficacy may be compromised by patients’ poor performance status, destruction and exhaustion of cellular and humoral components of the immune system by previous lines of immunosuppressive therapies, and biologically highly aggressive disease. The use of immunotherapy at earlier clinical phases (or even in the front-line setting) might thus lead to increased response rates, better tolerability, and longer remissions. In the upcoming years, next-generation biologicals with better pharmacokinetic properties and improved safety profiles will further extend the indication of immunotherapy to even older and more comorbid patients. Since the clinical usage of allogeneic transplantation, it has become evident that the most effective setting for immunotherapy is eradication (or at least control) of minimal residual disease, while immunotherapy approaches appear less-effective in eliminating bulky tumor masses. Published data support this observation across diverse lymphoma subtypes and various other immunotherapy modalities. One of the most relevant pretreatment factors of CAR19 T-cells efficacy in DLBCL is tumor bulk. While PD-1 inhibition was largely ineffective as a salvage therapy in DLBCL patients, its administration as a maintenance after HDT-ASCT consolidation had clinical benefit. Anti-leukemic activity of the bispecific BiTE blinatumomab decreases with increasing pretreatment leukemic cell load. Use of immunotherapy in consolidative or maintenance setting, after prior debulking with chemotherapy or targeted agents and subsequent immunotherapy-mediated eradication of the residual lymphoma clone, may thus represent a feasible future approach to patients with newly diagnosed lymphoma. In follicular lymphoma or mantle cell lymphomas, where maintenance therapy had demonstrated large clinical benefit already with CD20 antibody rituximab, the innovative immunotherapy approaches might offer far better control of the residual lymphoma clone. Which immunotherapy modality will prevail in particular lymphoma subtypes will depend on the results of the currently ongoing clinical trials. It is also plausible that different immunotherapy agents will be used in combination(s) with each other, inducing synthetic lethality toward lymphoma cells.In conclusion, immunotherapy offers a curative perspective both to patients with NHL subtypes currently considered incurable and to those with curable diagnoses after the failure of currently used standard-of-care therapies. For the first time in history, cure for most patients with non-Hodgkin lymphomas seems to be an achievable goal.


As viruses have a capacity to rapidly evolve and continually alter the coding of their protein repertoires, host cells have evolved pathways to sense viruses through the one invariable feature common to all these pathogens—their nucleic acids. These genomic and transcriptional pathogen-associated molecular patterns (PAMPs) trigger the activation of germline-encoded anti-viral pattern recognition receptors (PRRs) that can distinguish viral nucleic acids from host forms by their localization and subtle differences in their chemistry. A wide range of transmembrane and cytosolic PRRs continually probe the intracellular environment for these viral PAMPs, activating pathways leading to the activation of anti-viral gene expression. The activation of Nuclear Factor Kappa B (NFκB) and Interferon (IFN) Regulatory Factor (IRF) family transcription factors are of central importance in driving pro-inflammatory and type-I interferon (TI-IFN) gene expression required to effectively restrict spread and trigger adaptive responses leading to clearance. Poxviruses evolve complex arrays of inhibitors which target these pathways at a variety of levels. This review will focus on how poxviruses target and inhibit PRR pathways leading to the activation of IRF family transcription factors. Germline-encoded pattern recognition receptors (PRRs) ‘sense’ infection by binding invariable chemical features of invading pathogens called pathogen-associated molecular patterns (PAMPs). Each PRR activates signal transduction pathways leading to gene expression, which orchestrates clearance of the specific type of pathogen from which the PAMP was derived. The principle PAMPs of viruses are their nucleic acids, which are distinguished from native host nucleic acids by cellular location and subtle differences in chemistry . In ordePoxviruses comprise a diverse family of large, enveloped, double-stranded DNA viruses. Their genomes range between 134 and 365 kb, which are spatially organized into central, relatively conserved regions which typically encode everything required for basic life cycle functions and diverse terminal regions with a higher degree of genetic plasticity where immunomodulatory proteins tend to be encoded. Approximately 130 to 328 open reading frames (ORFs) are bidirectionally encoded throughout their genomes . Group IPoxviruses have well-characterized immunoevasive and immunomodulatory strategies to suppress activation of the host innate immune system, seeking to sense them in order to drive effector responses leading to their clearance . These sPoxviruses appear to have a larger number of dedicated inhibitors focused on the inhibition of NFκB-activating pathways  but lessThe IRF family of transcription factors contains nine members in most vertebrates (IRF-1 to -9) ,11,12. AAlthough other IRF family members were initially thought to be responsible for TI-IFN induction, IRF3 and IRF7 are now considered to be the principle family members involved downstream of cytosolic nucleic acid receptors and nucleic acid sensing Toll-like receptors (TLRs)  . Whilst 385SSLENTVDLHISNSHPLSLTS405. This region is functionally separated into two sites: site I includes Ser385 and Ser386, whereas site II includes Ser396, Ser398, Ser402, Thr404 and Ser405. Although the precise residues modified in vivo remain controversial, initial phosphorylation in site II on Thr404 or Ser405 by TBK1 relieves a state of auto-inhibition and allows association with CBP/p300. In this quasi-active state, IRF3 is then primed to be fully activated by a second phosphorylation by TBK1 on Ser385 and Ser 386 within site I and then translocates into the nucleus [IRF3 is activated downstream of all anti-viral PRRs through phosphorylation by IκB kinase (IKK)-related protein kinases, TANK-binding Kinase (TBK1) and IKKε, resulting in its dimerization and translocation into the nucleus in association with CREB-binding protein (CBP)/p300 to bind and transactivate the expression of target genes, such as TI-IFNs ,18,19,20 nucleus ,22,23,24While the IKK complex that regulates canonical NFκB activation has been well-characterized, the precise nature of the equivalent IRF-activating complexes remains unclear. NFκB activation is controlled by an IKK complex which consists of a regulatory protein NFκB essential modulator (NEMO) and two IKK-family subunits, IKKα and IKKβ. Activation of this large multimeric complex begins with the binding of a ubiquitin-binding domain within NEMO to di-ubiquitin from TRAF6-generated ubiquitin chains, both free and conjugated, to multiple proteins in the signaling pathway. Biochemical and structural data suggest that this binding induces a conformational change within NEMO, which primes IKKβ for activation by allowing initial trans-auto phosphorylation, which in turn allows subsequent phosphorylation by transforming growth factor b-activated kinase−1 (TAK1), resulting in a fully active state (reviewed in ).tbk1-deficient mice phenotypically resembled p65-, IKKβ- and NEMO-deficient mice with profound liver apoptosis in utero [TBK1 was discovered based on its interaction with TANK  and was in utero ,29,30,31in utero . Subsequin utero ,18,33. Sin utero ,35. Howein utero .Multiple TNF Receptor Associated Factors (TRAFs) have been shown to be involved in IRF-activating pathways  , althougThe nature of the complex(es) formed for the activation of IRFs may be highly cell type-dependent. For example, recent evidence suggests that NAP1, TANK and TBKBP1 are not required for the RIG-I-like receptor (RLR) mitochondrial antiviral signaling (MAVS) pathway in HEK293T cells . AdditioNevertheless, analogous to how poxviruses have invested considerable evolution to targeting the IKK complex and NFκB directly , inhibitMCV is the only extant poxvirus that appears to have specifically adapted to infect humans ,57. MCV The VACV bcl−2-family protein N2 inhibits IRF activity directly at the level of nuclear IRF3  Figure . These aTLRs are the prototypical PRRs for PAMPs and damage-associated molecular patterns (DAMPs) and are widely conserved in animals . TLRs se−/- mice. This highlights the critical balance between preventative and over-zealous innate anti-viral responses [−/- mice, this suggests a TRIF-dependent TLR other than TLR3 may paradoxically be protective in VACV infections [TLR3 recognizes viral double-stranded RNA (dsRNA) in endosomes and triggers signaling through homodimeric interactions of the TLR3 Toll-IL1 Receptor (TIR) domain and the adapter TIR-domain-containing adapter-inducing IFNβ (TRIF), leading to recruitment of TRAF3 which undergoes K63-linked self-ubiquitination, resulting in complex formation with TBK1 and IKKε, which in turn phosphorylate and activate IRF3 . Involveesponses . As the fections . Consistfections . As TLR4fections  and vesifections , this sufections . There hfections ; howeverfections .TLR9, the first DNA-sensing PRR to be discovered , senses Consistent with the role of TLRs in sensing poxviruses, VACV encodes proteins that inhibit TLR-driven IRF activation . The VACOther poxviral proteins have unexpected roles in inhibiting TLR signaling to IRF activation. For example, E3 is a key virulence factor for VACV that has multiple activities in suppressing the host immune response and contains an N-terminal Z-DNA/RNA-binding motif as well as a C-terminal dsRNA-binding domain . The Z-DDespite the ability of TLRs to sense poxvirus infection, the attenuated modified vaccinia Ankara (MVA), which possesses several of the previously described inhibitors, still induces TI-IFNs in a TLR-independent fashion . ConsistThe importance of RLRs in managing poxviral infection is reflected in the fact that there is evidence that poxviral infection has played a role in positive selection of RLR families in different mammalian species over time . A ratioAlthough the physiological relevance of AT-rich dsDNA-sensing by RNA polymerase III in poxviral infections is unclear, additional cytosolic DNA sensors play a central role in the potency of cytosolic DNA, whether from viral infection or from aberrant host DNA localization, to drive IRF activation and induce TI-IFNs . Such DNWe have recently reviewed DNA virus inhibitors of the cGAS-STING pathway, including those of poxviruses . In addiCytosolic DNA-sensing pathways outside of cGAS-STING signaling are poorly defined, but multi-layered immune defense mechanisms for every PAMP are common. A cytosolic DNA-sensing mechanism in fibroblasts has been shown to be targeted by poxviruses for immune evasion; Ferguson et al.  showed tGiven the importance of these cytosolic sensing systems pathways for detecting poxviruses to drive TI-IFNs, as their pathways are further elaborated, along with new components and mechanisms that regulate them, we expect that additional as-yet undiscovered poxviral inhibitors that target them will also be identified.The induction of TI-IFNs by nucleic acid sensors is a critical feature of the response to poxviruses and indeed all viruses with differences in pathways employed, depending on the nature of the nucleic acids presented to the innate immune system during infection. These pathways have a rate-limiting reliance on IRF-family activation. Once secreted, TI-IFNs then drive IFN-stimulated gene expression in surrounding cells to induce the interference state, making uninfected cells non-permissive for the incoming virus. This IFN system aims to quarantine the virus and limit replication whilst assisting the emergence of a robust adaptive response needed for clearance. Consequently, poxviruses evolve highly efficient and, in some cases, multifunctional inhibitors which target IRF-activating pathways at multiple levels in order to prevent TI-IFN production, which would limit its spread. The extent to which they achieve this underlies the delicate balance between persistence, invasiveness and pathology that defines their presentation in disease.


Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs . In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures . Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids . In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category. Interactions between proteins and nucleic acids (DNA and RNA) are central to all aspects of maintaining and accessing genetic information. Nucleic acid-binding proteins are mostly composed of at least one DNA or RNA-binding domain where the interfacing with amino acids takes place in a specific or nonspecific manner . IdentifHomo sapiens [According to the Gene Ontology (GO) knowledgebase, there are 5037 nucleic acid-binding proteins (filtering GO:0003676 term by “protein”) with experimental evidence in  sapiens ,50,51. O sapiens ,52,53,54 sapiens , and Z-D sapiens .Amino acid composition of some nucleic acid-binding proteins was intensively studied at the beginning of the 70s, when Koichi Iwai et al. determined that “calf-thymus histones comprise five main types which differ in amino acid composition and electrophoretic mobility: A glycine-rich, arginine-rich histone ; a glutamic-acid-rich, arginine-rich histone (fe or III); a leucine-rich, intermediate type histone (f2a2 or IIb1); a serine-rich, slightly lysine-rich histone (f2b or IIb2); and an alanine-rich, very lysine-rich histone (f1 or I)” , by usinSeveral approaches are used to inspect the amino acid composition of nucleic acid-binding proteins. Basically, we can divide the methods into in vitro and in silico. In vitro approaches are necessary to obtain a sequence of the protein of interest. Although the development of large-scale genomic sequencing has greatly simplified the procedure of determining the primary structures of proteins, the genomic sequences of many organisms are still unknown, and also modifications such as post-translational events  may prevent proper determination of the protein sequence . Then, tNucleic acid-binding proteins are traditionally divided into two categories. The first category comprises proteins with the ability to bind DNA, and the second category comprises proteins that bind to RNA. This division is quite outdated, mainly because, from the historical perspective, proteins that bind RNA were typically considered as functionally distinct from proteins that bind DNA and studied independently. Interestingly, current gene ontology analyses reveal that DNA-binding is potentially a major function of the mRNA-binding proteins . Nonethe2+ binding in the largest and best-characterized subgroup of zinc-finger binding proteins named the Cys2His2 fold subgroup [Another, even more important division of nucleic acid-binding proteins is based on a so-called sequence or structure-specific type of binding. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs. One of such motifs, zinc-finger, binds DNA (or RNA) through specific interaction with nucleotides and sugar-phosphate backbone. Tandem repeating of slightly different zinc-finger motifs in protein then allows to recognizing its consensus nucleic acid-binding sequence specifically. Cysteine and histidine amino acid residues are crucially important to coordinate Znsubgroup ,78. OtheIn contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures  ,41,55,90As was shown above, proteins that preferentially recognize noncanonical nucleic acid structures often have a distinct amino acid composition with particular significant global enrichment and/or depletion of different amino acid residues. Noncanonical structures and proteins preferentially binding them often play a critical role in physiological molecular processes ,104,105,The scheme below depicts sequence and structure-specific nucleic acid-binding phenomena in a nutshell .Almost every year, multiple novel noncanonical nucleic acid-binding proteins are identified. This year was, for instance, found that Guanine Nucleotide-Binding Protein-Like 1 (GNL1) binds RNA G-quadruplex structures in genes associated with Parkinson’s disease , or thatThe global or local amino acid composition of nucleic acid-binding proteins is often overlooked and an unjustly underestimated parameter. Mainly statistically significant enrichment or depletion of particular amino acid residues may serve as a promising tool to predict novel proteins with a similar function, as it was confirmed e.g., for G-quadruplex binding proteins.


We report SPIN, an integrative computational method to reveal genome-wide intranuclear chromosome positioning and nuclear compartmentalization relative to multiple nuclear structures, which are pivotal for modulating genome function. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and putative associations with nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to other 3D genome features and genome function (transcription and replication timing). SPIN provides critical insights into nuclear spatial and functional compartmentalization.The online version contains supplementary material available at (10.1186/s13059-020-02253-3). In human and other higher eukaryotic cells, interphase chromosomes are organized spatially within the cell nucleus , 2, suchThe advent of whole-genome mapping methods for chromatin interactions such as Hi-C has shown that, at megabase resolution, chromosomes are spatially segregated into A/B compartments genome-wide . A/B comSeveral genome-wide mapping methods have enabled more direct examination between chromosome regions and specific nuclear compartments. In , DamID wHere, we develop a new computational method called SPIN  to identify genome-wide chromosome localization patterns relative to multiple nuclear compartments. SPIN integrates TSA-seq, DamID, and Hi-C in a unified framework based on hidden Markov random field (HMRF). As a proof-of-principle, we apply SPIN to TSA-seq (for nuclear speckles and nuclear lamina), DamID (for nuclear lamina and a marker of nucleoli), and Hi-C data to identify genome-wide spatial localization states in K562 cells. The “SPIN states” reveal new and detailed correlations with other features of genome structure and function, such as Hi-C subcompartments, topologically associating domains (TADs) , 17, hisThe overview of the SPIN method is illustrated in Fig. SPIN is different from previous methods for chromatin domain segmentation based on a hidden Markov model (HMM) , 21, 22 Note that in principle the input of SPIN on each genomic bin can also include functional genomic signals such as histone modifications, replication timing, and transcription levels. However, in this work, we explicitly limit the input signals to those that directly measure the spatial position of chromatin (TSA-seq and DamID) and use functional genomic data to evaluate the functional correlations of different SPIN states genome-wide.manuscript in prep.). Hi-C data for K562 are from AdditionaAdditional file 5 Table S3. GO analysis of genes near ChIP-seq peaks in different TF clusters based on the SPIN states. ChIP-seq peaks on each clusters are merged and used as input for GREAT to calculate GO term enrichment.Additional file 6 Review history.


Here we provide an overview of the main technologies  to enrich infiltrating or circulating T-cells in predefined neoantigen specificities (or tumor reactivity). The unique technical and regulatory challenges faced by such highly specialized and patient-specific manufacturing T-cell platforms are also discussed.Mutation-derived neoantigens are taking central stage as a determinant in eliciting effective antitumor immune responses following adoptive T-cell therapies. These mutations are patient-specific, and their targeting calls for highly personalized pipelines. The promising clinical outcomes of tumor-infiltrating lymphocyte (TIL) therapy have spurred interest in generating T-cell infusion products that have been selectively enriched in neoantigen (or autologous tumor) reactivity. The implementation of an isolation step, prior to T-cell  Building on the promising clinical outcomes achieved by TIL therapy in melanoma and cervical cancer  is therefore tightly linked to the identification of tumor-associated antigens, which are essential for tumor control.In the new age of personalized immune-oncology, tumor-infiltrating lymphocytes (TILs) generated from surgical resections, expanded l cancer , 2, effoAgainst this background, cancer neoantigens deriving from private mutations represent an ideal class of cancer antigens to target in that they are highly tumor-specific by nature, therefore reducing the potential induction of central and peripheral tolerance , 4. Mostin silico filtering, and immunogenicity evaluation, leading to private (and shared) neoantigen candidates - and cytomegalovirus (CMV)-specific], extending observations that many tumor infiltrates may be in fact not tumor-specific (+ T-cell receptor (TCR) repertoire is able to recognize autologous cancer cells. Yet, the frequency of CD8+ in TILs correlates with favorable prognosis, and increasing evidence has shown how a relatively limited set of neoantigen-specific T-cells from melanoma TILs can mediate tumor recognition, despite the tumor cells harboring hundreds of somatic mutations  tumor-associated antigen specificities is generally low , 21, 30.specific . A studyspecific  has asseutations –37. Collex vivo, limits a broader application of standard TIL therapies to other tumor types. In this regard, Cohen et al.  between circulating and infiltrating T-cells in ovarian cancer patients  tumor-antigen specificities may improve the response rates achieved to date by adoptive T-cell therapies. One can envision highly personalized and specialized platforms, which integrate tumor-antigen identification and the generation of T-cell infusion products with a predefined reactivity composition . Here, wfication . The two+ T-cell reactivities without restriction to functional parameters. MHC-based reagents have rapidly evolved from single fluorescent-labeled pMHC tetramers to increasingly advanced and optimized staining protocols with higher detection sensitivity . Most importantly, given that pMHC multimer production is quite time-consuming and has to be manufactured under Good Manufacturing Practice (GMP) conditions, generation of a library of pMHC multimers on a patient-specific basis may be cost prohibitive for clinical implementation. In this regard, UV-based peptide exchange technologies  T-cells has long been associated with effective tumor recognition and used as a functional readout to detect tumor-reactive T-cells (+ (and CD4+) T-cells upon specific IFN-γ secretion to be used for adoptive transfer. Another group has reported a GMP-grade isolation of protocol of polyclonal and polyfunctional antigen-specific T-cells from healthy donor PBLs, by IFN-γ labeling followed by FACS, using NY-ESO-1 as a model system  transfected with mutation-encoding RNA and showed that the expanded CD137+ fraction had indeed been enriched in neoantigen-specific T-cells. At this point in time, anti-CD137 and anti-IFN-γ are among the few clinical-grade commercially available antibodies for the selection of antigen-specific T-cells; several companies though now provide custom monoclonal antibody development and conjugation under GMP guidelines.The tumor necrosis factor receptor (TNFR) family member CD137 (or 4-1BB) has been initially characterized as a specific marker of TCR-induced activation of viral-specific CD8 T-cells –72. Follcreening , 73–75. On a different note, structural changes of activated integrins upon early TCR engagement can be exploited as the inside-out signal to detect functional T-cells . Dimitro+ and CD4+ T-cells recognize private mutated epitopes  and adds complexity and time  to an already labor-intensive TIL production pipeline. Less time-consuming and unbiased methods are therefore being evaluated to generate patient-specific T-cell products, which are clinically feasible for adoptive transfer.Using autologous tumor cells as targets circumvents the need for screening of immunogenic private or shared tumor antigens, while presenting to T-cells the complete range of naturally presented tumor antigens. In TIL production history, IFN-γ secretion has been exploited to prescreen which tumor fragments to expand: only TILs showing tumor reactivity above a predefined cutoff value were selected for downstream expansion and infused .+-sorted TILs demonstrated increased reactivity against shared antigens following overnight incubation in the presence of MHC-matched tumor cell lines. Importantly, the CD137-enriched fraction resulted in enhanced in vitro and in vivo antitumor reactivity  should also be taken into account when establishing melanoma –103. Howmelanoma –106.a priori identification of tumor-reactive TILs solely based on phenotypic profiling. The rationale behind this strategy lies in the fact that naturally occurring tumor-reactive TILs are chronically exposed to their cognate antigen in the tumor site, therefore expressing a defined set of surface activation- and/or exhaustion-associated markers, providing the opportunity for their direct isolation.Efforts from several groups are focusing on improving the + T-cells. CD103+ CD39+ TILs were sorted from tumor digests and expanded in vitro, resulting in increased cytotoxicity toward autologous tumor cells when compared to the respective single positive populations  enrichment and Starting from the isolation step itself, one has to consider not only the technical aspects of the sorting strategy but more importantly its compatibility with regulatory requirements. The choice of a FACS or MACS-based enrichment depends on several factors, including the number of cells in the source material (TILs or PBLs), the relative frequency of antigen-specific cells within, the level of purity needed for the final product. On one hand, magnetic beads are of lower technical complexity and clinical-grade isolation kits are already commercially available, on the other, FACS separation performs multiparameter analysis of single cells, achieving resolution and purity levels, which are not always possible by MACS. In addition, FACS analysis can characterize in real time the sorted bulk T-cell population (in terms of identity and purity), as a first in-process quality control. However, FACS is still not routinely applicable under GMP conditions, which require single-use and a closed fluidic system for clinical implementation.9 cells per patient). Several distributors supply culture bags or gas-permeable flasks, sterile tubing accessories, and welding to facilitate the conversion of research protocols to GMP closed manufacturing processes, where the risk of cross contamination has to be minimized.As the absolute cell counts of neoantigen-specific T-cells after isolation are extremely low for direct reinfusion, a rapid expansion procedure  of sorted cells is typically performed with allogeneic irradiated feeder cells in the presence of high-dose interleukin (IL)-2 and anti-CD3 . Dependiin vitro expansion. Indeed, during the REP phase, there can be interclonal competition resulting in an increased or decreased frequency of given specificities of interest compared to the starting culture. In this sense, enriching for tumor-specific T-cells at appreciable frequencies prior to up-scale would higher the chances of obtaining a final TIL product with adequate tumor reactivity upon infusion. The extensive expansion can also drive progressive T-cell differentiation and phenotype changes which may affect TIL in vivo persistence, homing, and proliferative capacity shortly after transfer, as these cells reencounter cognate antigens within the tumor microenvironment  and exhaustion; both are peripheral tolerance mechanisms restricting an escalating, therefore potentially damaging, immune response. Melanoma TILs undergoing intense polyclonal TCR stimulation during REP have been shown to be more sensitive to AICD when cocultured  control –121. In siveness –124.in vivo and respond to tumor antigen restimulation upon transfer and how their gene signature correlates to clinical benefit should be studied systematically on a larger number of patients receiving the same infusion regimen.Overall, how infused antigen-specific TIL clonotypes persist + T-cells as compared with standard TILs generated from the same patients.Further pre-sensitization approaches could help increase neoantigen-specific T-cell frequency in starting TIL (or PBL) cultures and facilitate downstream sorting of the population of interest. Our group has reported that the addition of synthetic peptide pools of all predicted class-I neoantigens can improve conventional TIL generation in ovarian cancer . Primed Finally, while it is tempting to focus on private neoantigens deriving from single point mutations, driver genes (such as RAS and BRAF) recurrently affected by mutation or fusion events across individuals and cancer types would be expected to yield semiprivate (or even “shared”) neoantigens. This seems to be especially the case for hematological malignancies, where immunogenic neoantigens have been reported to be mutated in up to 30% of patients , 126, alThe identification of neoantigens as drivers of successful antitumor immunity is offering exciting new opportunities for cancer immunotherapies, including making T-cell infusion products highly individualized for more effective treatment. Moving forward, patient-specific T-cell enrichment technologies will need to be integrated into clinically compliant pipelines. In this respect, the first Food and Drug Administration (FDA) approval of the first adoptive cell therapy  represents a huge achievement in the immune-oncology field and will hopefully pave the way for further approvals of personalized immunotherapies.VB wrote the manuscript and made the figures. VB, AH, and GC contributed to the concept, discussion of content, and editing of the manuscript. All authors contributed to the article and approved the submitted version.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.


Arabidopsis thaliana, 8-nitro-cGMP was reported to mediate NO-dependent signaling pathways controlling abscisic acid (ABA)-induced stomatal closure, however, its derivative 8-mercapto-cGMP (8-SH-cGMP) was later shown as the active component of hydrogen sulfide (H2S)-mediated guard cell signaling. Here we present a survey of current knowledge on biosynthesis, metabolism and biological activities of nitrated nucleotides with special attention to described and proposed functions of 8-nitro-cGMP and its metabolites in plant physiology and stress responses.Nitration of diverse biomolecules, including proteins, lipids and nucleic acid, by reactive nitrogen species represents one of the key mechanisms mediating nitric oxide (NO) biological activity across all types of organisms. 8-nitroguanosine 3′5′-cyclic monophosphate (8-nitro-cGMP) has been described as a unique electrophilic intermediate involved in intracellular redox signaling. In animal cells, 8-nitro-cGMP is formed from guanosine-5′-triphosphate by a combined action of reactive nitrogen (RNS) and oxygen species (ROS) and guanylate cyclase. As demonstrated originally in animal models, 8-nitro-cGMP shows certain biological activities closely resembling its analog cGMP; however, its regulatory functions are mediated mainly by its electrophilic properties and chemical interactions with protein thiols resulting in a novel protein post-translational modification termed S-guanylation. In  Nitric oxide (NO) is a crucial gaseous signaling molecule which plays vital roles in a broad spectrum of physiological and developmental processes throughout the plant life, including germination, development of leaves, roots and reproductive organs, stomata movement and plant senescence . NO alsoin vitro  belongs to reactive sulfur species with recognized signaling role across a wide range of organisms. In animals, H2S biosynthesis is controlled by two crucial enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Knockdown of CBS and CSE resulted in elevated 8-nitro-cGMP concentrations, indicating that its activity might be regulated by sulfur species , the primary sensor proteins of intracellular cGMP known to control an array of cellular reactions . S-guany2S, polyamines and methyl jasmonate  channels. Increased levels of RNS, produced by ROS and NO reactions, resulted in 8-nitro-cGMP production triggering increased levels of cytosolic Ca2+ which activated the SLAC1 anion channels to promote stomata closure. In the following study, the 8-nitro-cGMP metabolite 8-SH-cGMP was also observed to induce closure of stomata pores  of the target proteins. Polyamine spermine was reported to induce autophagy in plants, mediated by increased generation of ROS and NO required for effective triggering of autophagy (Alternaria alternata toxin in tobacco BY-2 cells (in vitro was inhibited by combined treatment with cGMP and NO donor, but it is not known if this effect was mediated by 8-nitro-cGMP (in vitro and in vivo.In plants, autophagy mediates selective destruction of viruses as well limits infection by bacterial and filamentous pathogens. Emerging evidence indicates that autophagy is a key regulator of plant innate immunity and contributes with both pro-death and pro-survival functions to antimicrobial defenses, depending on the pathogenic lifestyle . The rolnstrated . Sulfideutophagy . Combine-2 cells . In Arabtro-cGMP . Given tTo date, only two scientific reports investigated the functions of 8-nitro-cGMP and 8-SH-GMP in plant cell signaling and regulation. Further progress in this field may be hampered by several experimental obstacles: the pure chemical standards of described compounds are not commercially available, their synthesis requires dubious purification and analytical techniques requires mass-spectrometry instrumentation. A reporter system has been developed for specific and sensitive detection of cyclic nucleotides including cGMP in bacteria and plants; however, if this system shows response also to cGMP derivatives has not been tested . It shouMP and LL designed the manuscript. MP wrote the initial version of the manuscript. LL read and corrected the final version.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.


Insects are arguably the most successful group of animals in the world in terms of both species numbers and diverse habitats. The sesquiterpenoids juvenile hormone, methyl farnesoate, and farnesoic acid are well known to regulate metamorphosis, reproduction, sexual dimorphism, eusociality, and defense in insects. Nevertheless, different insects have evolved with different sesquiterpenoid biosynthetic pathway as well as products. On the other hand, non-coding RNAs such as microRNAs have been implicated in regulation of many important biological processes, and have recently been explored in the regulation of sesquiterpenoid production. In this review, we summarize the latest findings on the diversity of sesquiterpenoids reported in different groups of insects, as well as the recent advancements in the understanding of regulation of sesquiterpenoid production by microRNAs. In insects and crustaceans, sesquiterpenoid hormones including farnesoic acid (FA), methyl farnesoate (MF) and juvenile hormone (JH) regulate the development, metamorphosis and reproduction . The begSAM-dependent juvenile hormone acid O-methyltransferase (JHAMT) , which will form MF catalyzed by a  (JHAMT) . In inse (JHAMT) , honeybe (JHAMT) , locusts (JHAMT) , and pea (JHAMT) , MF is o (JHAMT) . Direct  (JHAMT) . Moreove (JHAMT) . For ins (JHAMT) ; while i (JHAMT) . Further (JHAMT) . In the  (JHAMT) . In the  (JHAMT) ; and in  (JHAMT) . It is w (JHAMT) .A special feature of insects is that they have evolved with distinct modes of metamorphosis, including hemimetaboly (incomplete) and holometaboly (complete) . These bDrosophila melanogaster, sesquiterpenoids exert status quo function to prevent metamorphosis in the early larval stage  or its paralog named Germ-cell expressed (Gce) in Drosophila, which encodes a transcription factor of the bHLH-PAS family  on the promoter region of Krüppel homolog 1 (Kr-h1) to activate transcription , Broad-complex (Br-C), ecdysone-inducible proteins E75 and E93, which subsequently inhibit 20E biosynthesis in the prothoracic gland (Spok by binding to the Kr-h1 binding site (KBS) and turn on the methylation which in turns also leads to the suppression of ecdysone biosynthesis  to form a heterodimer  synthesis in the fat body  ILP2 and ILP3 by AKT pathway to phosphorylate the fork head transcription factor (FOXO) and induce Vg expression  and adenovirus E2 factor-1 (E2f1)  . JH actig oocyte . In the of Kr-h1 .Acyrthosiphon pisum, enhanced sesquiterpenoid degradation by juvenile hormone esterase (JHE) accounts for the lower JH titer during short-day conditions that produces sexual morphs, in contrast to the higher JH titer in parthenogenetic morphs during long-day conditions  via JH-Met-Kr-h1 signaling and promotes Vg synthesis and ovary development during short-day period, while low JH titer initiates reproductive diapause and promotes lipid storage in the fat body instead of Vg synthesis during the long-day period  can impose female development via promoting expression of fruitless (fru), doublesex (dsx), and transformer (tra). Male development occurs when sxl is turned off  stimulate the production of queens and upregulate Vg gene expression. The effect of JH in ants is interpreted as mimicking the effect of hibernation   . In methh1 level . On the nificant . However ovaries . These sZootermopsis nevadensis show that the JH-Met and transforming growth factor beta (TGFβ) pathways are involved in the ecdysteroid synthesis for molting in soldier formation  result in inhibition of cantharidin biosynthesis, suggesting degradation of JH-III is essential in producing potential precursors of cantharidin  by HSP70/HSP90 chaperone machinery to form mature RNA-induced silencing complex (RISC) after strand selection  have been implicated in regulation of many important biological processes . In canoelection . Recentlorphosis , and trend let-7 .A. aegypti, four JH biosynthetic enzyme genes including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), diphosphomevalonate decarboxylase (PP-MevD), aldehyde dehydrogenase (ALDH), and farnesyl-pyrophosphate synthase (FPPS) were in silico predicted to be potentially regulated by miRNAs  infected larvae. Methoprene treatment decreases expression of Br-C Z2 and increases expression of these miRNAs miR-8 and miR-429 which could target Br-C Z2 (In  Br-C Z2 , implyinDrosophila, miRNA bantam has been found to interact with JHAMT both in silico, in vitro, and in vivo (bantam in the brain decreases expression levels of JHAMT.; The knockdown of bantam increases the expression level of JHAMT (bantam mutants demonstrates decreased sesquiterpenoid levels and male genital defects. bantam mutant phenotypes can be rescued by exogenous sesquiterpenoid application (bantam and other miRNAs on JHAMT and Met have also been tested both in silico and in vitro, revealing a conserved system of miRNAs in regulation of sesquiterpenoids established in the arthropod ancestor (In  in vivo . The oveof JHAMT . Hormonalication . In otheancestor ; Table 2SSKT, SL, CL, and JH wrote the first draft of the manuscript. All authors proofread the final version of the manuscript.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.


Two groups also differed in rate of females  and leukemia . After a median follow-up of 29.1 months, 5-year OS rate was 63.1%. Cox proportional-hazard regression evaluated an adjusted risk of post-transplant mortality related to depression: OS decreased sequentially from no depression (adjusted hazard ratio [aHR] = 1) to pre-transplant depression only , and to having both depression and anxiety disorder  groups. Pre-transplant anxiety (anxiety only) did not have significant influence in OS. Additional medical and psychiatric care might be necessary in patients who experienced depression, especially with anxiety, before allo-HSCT.Studies investigating association of depression with overall survival (OS) after allogeneic hematopoietic stem cell transplantation  yielded conflicting results. A nationwide cohort study, which included all adult patients  who received allo-HSCT from 2002 to 2018 in South Korea, analyzed risk of pre-transplant depression in OS of allo-HSCT. Subjects were followed from the day they received allo-HSCT, to occurrence of death, or last follow-up day . Median age at allo-HSCT for depression and non-depression groups were 50 and 45 ( However, allo-HSCT is a highly aggressive and demanding medical supports with a significant risk of mortality3. Besides primary hematologic disorder and disease relapse after transplantation, transplant-related mortality is an important obstacle influencing treatment outcome of allo-HSCT4. Thus, careful assessment of risk and accurate prediction of mortality are necessary before undertaking allo-HSCT5.Allogeneic hematopoietic stem cell transplantation  provides curative therapy for patients with malignant and nonmalignant hematologic disorders6. Studies showed that more than 20% of patients receiving allo-HSCT experience clinically significant depression before undergoing transplantation7. Besides its negative impact on the quality of life, depression is also associated with poor treatment survival outcome in patients receiving allo-HSCT8. Thus, most stem cell transplantation centers require patients to receive a comprehensive psychiatric evaluation as a part of the pre-transplant process9. Numerous prognostic scoring systems recognizes depression as an important risk factor of post-transplant mortality10.The Hematopoietic cell transplantation-specific comorbidity index (HCT-CI), one of the most important prognostic scoring systems in allo-HSCT, acknowledged that depression increases post-transplant mortality with hazard ratio of 1.811.Depression is a common comorbidity of oncological and chronic hematologic diseases13. A study comprising of 123 patients suggested that existing diagnosis of depression before allo-HSCT were not related to post-transplant OS14. In contrast, a more recent study showed that, among 190 patients receiving allo-HSCT, depression before transplantation predicted higher 1-year  and 3-year mortality 15. Thus, studies containing larger cohorts are needed to confirm temporal association between pre-transplant depression and OS of allo-HSCT.Studies investigating the impact of pre-transplant depression on overall survival (OS) of patients receiving allo-HSCT have yielded conflicting resultsp = 0.004)16. However, the study neither used a validated measurement nor diagnostic assessment to define pre-transplant depression. Rather, the study merely used retrospective chart review of a single question reported in the CIBMTR database; was there “clinically significant depression requiring treatment?” Inevitably, the diagnosis of depression was prone to reporter bias. To overcome this bias, the study excluded transplant centers having more than 10 transplants without reports of both depressed and nondepressed patients to the registry. By doing so, selection bias became another important limitation. Co-occurrence of anxiety disorder and depression are very common in patients receiving allo-HSCT17. By neglecting possible overlapping impact of pre-transplant anxiety, it is still uncertain whether depression truly increased post-transplant mortality independent from the anxiety. Whether anxiety disorder causes additive risk in post-transplantation mortality risk among patients with depression history is another important issue needing investigation. Patients with leukemia are known to have a higher post-transplant mortality than patients with non-malignant hematologic disease19. Thus, subgroup analysis comprising more homogenous group, leukemia patients only, are needed to address detrimental effect of pre-transplant depression in OS of allo-HSCT. Lastly, CIBMTR is an US based registry, so studies containing a large sample size must also be repeated in diverse ethnicities to confirm impact of pre-transplant depression in outcomes of allo-HSCT.A recent large sized cohort  study, using data from the Center for International Blood and Marrow Transplant Research (CIBMTR), showed that pre-transplant depression was associated with lower OS  database containing data of majority of allo-HSCTs performed in South Korea (hereafter “Korea”). We first replicated previous findings by investigating association of pre-transplant depression in the long-term survival of allo-HSCT in Asian population (Koreans). Beyond replication, we extended previous work by analyzing independent risk of pre-transplant depression, separating possible overlapping effect of anxiety, in OS of allo-HSCT. Whether negative impact of pre-transplant depression in allo-HSCT is augmented by co-morbid anxiety was also evaluated. In addition, sub-group analysis of patients with leukemia was conducted to confirm risk of pre-transplant depression in allo-HSCT among higher risk group. Lastly, landmark analyses were performed at 100 days post-transplant to exclude patients at high risk of early mortality post-transplant and to evaluate the impact of prognostic variables on OS of patients still alive at these time points.Between January 2002 and December 2018, a total of 7,170 adult patients who received allo-HSCT were included in this study (Table After a median follow-up of 29.1 months (range 0 to 207), the estimated overall survival (OS) rates at 5 years were 63.1% , and to both depression and anxiety group . However, anxiety only group did not have significant influence in OS.The factors found to be significant in the univariable analysis were all included in the final multivariable analysis Table . The posp < 0.001), and neither depression only nor anxiety only group showed statistical significance.Among 7,170 adult patients who received allo-HSCT, 6,391 patients were alive at the designated 100 days landmark and were included in the landmark analysis , and to both depression and anxiety disorder group .In the multivariable analysis of total and landmark cohort, hematologic disease had the highest aHR   to having depression only (aHR = 1.202), and then to having both depression and anxiety (aHR = 1.231). However, in the landmark analysis at 100 days post-transplantation, OS after transplantation was significantly lower only in patients having both depression and anxiety (aHR = 1.253).22. However, only one large sized longitudinal cohort study conducted in the US using CIBMTR data showed that depression history decreases the OS with aHR of 1.1316. To the best of our knowledge, this is the largest nationwide cohort study conducted in Asia, or outside US, showing the risk of pre-transplantation depression in OS of allo-HSCT. We replicated previous research and showed that pre-transplant depression decreases OS with aHR of 1.167, which was very similar to the HR reported by El-Jawahri et al. By carefully sub-classifying patients according to their anxiety disorder history, we were able to eliminate overlapping impact of pre-transplant anxiety from that of depression. Thus, our findings more specifically confirmed that pre-transplant depression independently decreases OS of allo-HSCT regardless of anxiety disorder. We also extended previous work by showing that OS decrement was more pronounced in patients who had both depression and anxiety before allo-HSCT (aHR = 1.202).Numerous studies suggested association of pre-transplantation depression with lower OS in allo-HSCT23. Consequently, it is difficult to accurately distinguish normal anxiety or adjustment reaction from that of true morbid anxiety24. Thus, patients having anxiety reaction could have been over-diagnosed as having anxiety disorder in our cohort, which might have led to decreased significance in OS. In line with this hypothesis, patients in our cohort had twice more history of anxiety than depression. Previous study also showed that patients having pre-transplantation depression more concordantly experienced clinically significant depression even after transplantation, whereas patients having pre-transplant anxiety tended to experience anxiety symptoms less continuously26. Thus, depression might had longer impact than anxiety in OS of patients receiving allo-HSCT. In addition, landmark analyses performed at 100 days post-transplant, which excluded patients at high risk of early mortality post-transplant, showed that OS was significantly lower only in patients who had both depression and anxiety disorder. Thus, prospective studies which include more complete predictors of mortality in allo-HSCT are needed to confirm complicated relationship among depression, anxiety, and survival outcome after allo-HSCT.Interestingly, we did not detect significant association between anxiety disorder and OS of allo-HSCT. Anxiety is a frequent response to threat, so it is commonly found in patients having severe medical conditionsOur results have additional strengths. First, by conducing a nationwide cohort study, we included all adult patients who received allo-HSCT from 2002 to 2018. We were able to prevent selection bias and minimize recruitment setting effect, so our results have higher generalizability. Second, a more stringent definition was used to determine pre-transplant depression. Rather than using a single questionnaire or retrospective chart review, all patients with pre-transplant depression visited hospital due to exact diagnosis of depression [ICD-10 code for major depressive disorder (F32 × or F33 ×)]. Thus, we further prevented selection and reporting bias. Lastly, by conducting a subgroup analysis comprising of patients with leukemia only, we eliminated underlying hematologic disease as a possible co-factor and confirmed the negative impact of depression, and additive effect of anxiety, in post-transplant mortality.27. Studies showed that depression is often accompanied by inflammatory diseases including irritable bowel syndrome, type 2 diabetes, arthritis and autoimmune disorders, which can activate the peripheral and central inflammatory response28. Furthermore, other studies illustrated that depression and acute graft-versus-host-disease (GVHD) share common pro-inflammatory reactions: increment of inflammatory cytokines  and shifting toward T helper 1 response system30. Likewise, past study showed that pre-HCT depression increased the incidence of acute GVHD16. Poor treatment adherence due to depression is another important cause of higher mortality31. However, additional prospective studies focusing on biological mechanism are needed to define common pathway linking depression, and anxiety, with that of post-transplantation mortality in patients receiving allo-HSCT.The pathophysiological basis of pre-transplantation depression having a negative impact in OS of patients receiving allo-HSCT is still obscure. Nevertheless, our findings could be predicted by association of depression with immune system dysregulationsSeveral limitations should also be noted. First, occurrence of depression was based on clinical diagnosis with hospital visit records rather than using objective depressive scale measurements. As a result, we were unable to assess mortality difference depending on the depression severity. Since the depression severity is not known, some patients in the non-depression group may have had depression but have been undiagnosed. Second, we were also unable to investigate antidepressant effect, or treatment effect, in the risk of mortality. Likewise, depression episodes after transplantation were not considered. Thus, the effect of depression recurrence or chronicity in the post-transplant mortality were not studied. Third, whether time to diagnosis from depression and anxiety or duration of their illnesses influenced OS were not analyzed. Fourth, unbalanced baseline characteristics between depression group and non-depression group could harbor possible bias. Fifth, the cause of death, and relapse related and non-relapse related mortality according to depression were not investigated. In addition, other important clinical factors such as GVHD and infection episodes after transplantation, pre-transplantation conditioning, performance score, remission status at time of transplant, type of donor, smoking history, and alcohol status were not considered. Lastly, although Korea has a mandatory public health insurance system providing a comprehensive medical coverage to all residents of Korea, socioeconomic status could still have influenced the OS.In conclusion, pre-transplant depression, independent from anxiety, decreased OS of adult patients receiving allo-HSCT. The mortality risk was higher in patients who had both depression and anxiety than patients having neither. Same trend was noted in patients who received allo-HSCT due to leukemia. Thus, our results suggest that patients having depression, especially both depression and anxiety, before allo-HSCT have a higher risk of post-transplantation mortality needing more intensive care.34.The Korean National Health Insurance Service (KNHIS) is a mandatory public health insurance system of Korea and offers comprehensive medical coverage to all residents of Korea. Data were acquired from the KNHIS database, which contains all medical claims for the population covered under the KNHIS. The database has been widely used in various epidemiological studies and is described in detail elsewhereAdult patients (≥ 18 years) who underwent allo-HSCT between 2002 and 2018 were identified using KNHIS database procedure codes X5061 for bone marrow and X5063 for peripheral blood allo-HSCT. For those who received second allo-HSCT, we used first allo-HSCT data as their baseline values. As a result, nearly all allo-HSCTs performed in Korea during the study period were included in the analysis. Comorbidities including hypertension, diabetes mellitus, chronic obstructive pulmonary disease, cerebro- or cardiovascular disease, and anxiety disorder were also extracted using International Classification of Diseases, Tenth Revision (ICD-10) codes. This study was approved by the institutional review board of Seoul St. Mary’s Hospital, Seoul, Korea (KC19ZNSI0396). Consent from individual subjects were not needed because the study used publicly open, anonymous data.The primary endpoint of this study was OS difference after allo-HSCT according to pre-transplant depression history. In order to utilize more conservative definition of depression, patients having ICD-10 code for major depressive disorder (F32 × or F33 ×) and visited hospital due to the same code within five years prior to the allo-HSCT was defined as having pre-transplant depression. Difference between the two groups (depression group and non-depression group) in baseline demographic and clinical characteristics were compared using student’s-T test for continuous variables and Chi-squared test for categorical variables.To investigate independent risk of pre-transplant depression, and prevent overlapping impact of anxiety, we also identified patients who were diagnosed with and visited hospital due to anxiety disorder (F30 ×) before allo-HSCT. Thus, patients were subdivided in to four groups; no history of depression and anxiety disorder (none), depression history without anxiety (depression only), anxiety disorder without depression (anxiety only), and having both depression and anxiety disorder (both depression and anxiety).p < 0.10 in univariable analysis were included in the single multivariable model. Thereafter, the factors found to be significant in this single multivariable model with depression variable were all included in the final multivariable model. For all statistical analysis, we used R statistical software .Retrospective cohorts were followed from the day they received allo-HSCT to the occurrence of death or the last follow-up day , whichever came first. The OS rate represents the proportion of patients who were alive at the specified time after the date of transplantation and was associated with death due to any cause. Cox proportional-hazard regression evaluated risk of post-transplant mortality related to depression. All risk factors with All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.Supplementary Information.


Our results strongly indicate that MRTF-A is an important regulator of the balance between osteogenesis and adipogenesis of hASCs through its role in mediating the cytoskeletal dynamics. These results provide MRTF-A as a new interesting target for guiding the stem cell differentiation in tissue engineering applications for regenerative medicine.Previous studies have demonstrated that myocardin-related transcription factor A (MRTF-A) generates a link between the dynamics of the actin cytoskeleton and gene expression with its coregulator, serum response factor (SRF). MRTF-A has also been suggested as a regulator of stem cell differentiation. However, the role of MRTF-A in human mesenchymal stem cell differentiation remains understudied. We aimed to elucidate whether MRTF-A is a potential regulator of human adipose stem cell (hASC) differentiation towards adipogenic and osteogenic lineages. To study the role of MRTF-A activity in the differentiation process, hASCs were cultured in adipogenic and osteogenic media supplemented with inhibitor molecules CCG-1423 or CCG-100602 that have been shown to block the expression of MRTF-A/SRF-activated genes. Our results of image-based quantification of Oil Red O stained lipid droplets and perilipin 1 staining denote that MRTF-A inhibition enhanced the adipogenic differentiation. On the contrary, MRTF-A inhibition led to diminished activity of an early osteogenic marker alkaline phosphatase, and export of extracellular matrix (ECM) proteins collagen type I and osteopontin. Also, quantitative Alizarin Red staining representing ECM mineralization was significantly decreased under MRTF-A inhibition. Image-based analysis of Phalloidin staining revealed that MRTF-A inhibition reduced the F-actin formation and parallel orientation of the actin filaments. Additionally, MRTF-A inhibition affected the protein amounts of  In briesotropy) , 39.2 in CellBIND 6-well plate (Corning) and cultured 7 days in BM, OM, or AM supplemented with 20 μM CCG-1423 or 12 μM CCG-100602. Samples were washed with PBS (Lonza), lysed with 2x LAEMMLI sample buffer , and separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis. After separation, the proteins were transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membrane using Trans-Blot Turbo Ready-to-assemble transfer Kit (Bio-Rad). The PVDF membranes were blocked with 5% nonfat milk powder in Tris-buffered saline (TBS) supplemented with 0.05% Tween 20 (Sigma-Aldrich). Membranes were incubated with primary antibodies followed by washing steps  and secondary antibody incubation  by comparing the inhibited conditions to the control condition without the inhibitors. MRTF-A inhibitor effects on cell proliferation , ALP activity (4 donors), quantitative Alizarin Red staining (3 donors), and image-based analysis of actin (2 donors) and ORO (4 donors) were evaluated. All quantitative results are presented as mean and standard deviation (SD). Nonparametric Kruskal-Wallis and Mann-Whitney tests were performed using GraphPad Prism version 5.02 , followed by Bonferroni post hoc test. The cells were identified as mesenchymal based on their plastic adherence, differentiation potential towards adipogenic and osteogenic lineages, and the surface marker expression pattern conveying the criteria given by the International Society for Cellular Therapy . The expWe first examined the role of MRTF-A in hASC adhesion and viability by using two inhibitor molecules (CCG-1423 and CCG-100602) in BM, OM, and AM cultures. A selection of concentrations of both inhibitors was used to find optimal concentrations for cell viability. Based on the representative LIVE/DEAD-stained images of adherent hASCs in Cell proliferation, analyzed with CyQUANT assay, aligned with the viability results. Figures μM CCG-1423 or 10 or 12 μM CCG-100602. The representative ORO- and DAPI-stained fluorescence images are shown in μm in diameter to demonstrate the adipogenic maturation  expression, but both inhibitors had a downregulating effect on leptin (LEP) in AM condition.Adipogenic differentiation was further assessed with ICC staining of adipogenic marker Plin1 . The inhμM CCG-1423, whereas the next-generation inhibitor CCG-100602 reduced ALP significantly with both concentrations. Additionally, the gene expression of an early osteogenic marker gene runt-related transcription factor 2a (RUNX2A) was decreased with CCG-1423 when analyzed with qRT-PCR  and Golgi network . In OM, The ability of the hASCs to differentiate further towards osteoblasts was evaluated by AR staining of calcium deposits at day 21 for the assessment of ECM mineralization Figures . AR staiβ-actin, α-SMA, pMLC, and MLC at day 7  adipocytes , 19. Thein vivo and in vitro that the bone development and expression of osteogenic markers, respectively, were negatively affected by the loss of MRTF-A function in mice [RUNX2A gene expression was hindered with CCG-1423.Next, we asked if the enhanced adipogenic differentiation with MRTF-A inhibition was linked to a reciprocal reduction in the osteogenic potential of hASCs. Our goal was to carefully study whether early osteogenesis, ECM production, and matrix mineralization were regulated by MRTF-A. Unlike adipogenesis, the relation of MRTF-A and osteogenesis has been demonstrated previously only in one study to our knowledge. Bian and coworkers showed  in mice . SRF kno in mice . LikewisProduction and secretion of extracellular proteins and mineralization of ECM are an important part of skeletal development, bone remodeling, and homeostasis . Therefoα-SMA and MLC indicating to SRF-dependent regulation. Importantly, these cytoskeletal changes were related to the observed moderate enhancement of adipogenesis in BM condition. The adipogenic culture supplements and MRTF-A inhibition caused the hASCs to adopt more spread morphology with reduced parallel alignment of the actin filaments. Furthermore, the observed significant reduction in actin polymerization but relatively unaffected cellular protein level of β-actin may suggest that MRTF-A inhibition altered the ratio between G-actin and F-actin. Similar inhibitory effect on F-actin was also demonstrated recently in human intestinal myofibroblasts with CCG-100602 [Finally, the actin cytoskeleton and synthesis of actin-related proteins were studied to elucidate the role of MRTF-A in regulating the differentiation fate decision of hASCs by mediating the actin dynamics. Dramatic cytoskeletal changes have been reported to occur early in the differentiation process of the fibroblastic and spindle-like mesenchymal precursor cells into mesenchymal lineages to drive the formation of specialized tissues , 14, 50.G-100602 . During G-100602 . Thus, tin vitro. MRTF-A translates the cytoskeletal changes to gene transcription via SRF and provides an essential temporarily coupled regulatory signaling node in stem cell differentiation. This study adds to the knowledge on the regulation of differentiation lineage commitment in human stem cells and provides further insight into molecular targets for pharmacological intervention to guide the differentiation fate into the desired direction.In summary, we have provided evidence that MRTF-A transcription cofactor is an important regulator of the inverse balance between adipogenesis and osteogenesis of hASCs. Our results showing that MRTF-A inhibitors enhance the lipid droplet formation and maturation indicate that MRTF-A is a negative regulator of adipogenesis. Reciprocally, our novel findings of reduced osteogenesis as a response to MRTF-A inhibition highlight the necessity of MRTF-A activity on the osteogenic outcome of hASCs


While increasing longitudinal evidence suggests that negative age views accelerate cognitive decline and increase dementia risk, we know little about such co-variance dynamics on a daily basis. We make use of subjective age and working memory performance data obtained six times a day over seven consecutive days as people went about their daily routines from 123 young-old  and 42 old-old  adults. Notably, multilevel models revealed considerably-sized short-term intra-individual variation of subjective age and working memory within days and these short-term within-day fluctuations in subjective age and working memory were coupled as expected. Hence, increased subjective age went along with lowered working memory confirming previous research. However, the respective between-day associations appeared reversed. Given this evidence of correlated short-term variability, we also discuss implications of different change dynamics that might explain moment-to-moment versus day-to-day associations between subjective age and working memory.


EGFR) mutations are disappointing. The biological mechanisms underlying immune escape and both unresponsiveness and resistance to immunotherapy in EGFR-mutant NSCLC patients have been partially investigated. To this regard, lung cancer immune escape largely involves high amounts of adenosine within the tumor milieu with broad immunosuppressive effects. Indeed, besides immune checkpoint receptors and their ligands, other mechanisms inducing immunosuppression and including adenosine produced by ecto-nucleotidases CD39 and CD73 contribute to lung tumorigenesis and progression. Here, we review the clinical results of immune checkpoint inhibitors in EGFR-mutant NSCLC, focusing on the dynamic immune composition of EGFR-mutant tumor microenvironment. The adenosine pathway-mediated dysregulation of energy metabolism in tumor microenvironment is suggested as a potential mechanism involved in the immune escape process. Finally, we report the strong rationale for planning strategies of combination therapy with immune checkpoints blockade and adenosine signaling inhibition to overcome immune escape and immunotherapy resistance in EGFR-mutated NSCLC.Despite the relevant antitumor efficacy of immunotherapy in advanced non-small cell lung cancer (NSCLC), the results in patients whose cancer harbors activating epidermal growth factor receptor ( EGFR) mutations and Anaplastic Lymphoma Kinase (ALK) gene translocation, have improved the outlook in terms of both progression-free survival (PFS) and overall survival (OS), showing a promising future for advanced NSCLC therapy  characterized instead by durable response (Tyrosine kinase inhibitors (TKIs) in oncogene-driven tumors and immunotherapy are the two major evolving strategies in the treatment of NSCLC , 7. To ded NSCLC . Despiteresponse .The use of immune checkpoint inhibitors (ICIs) has revolutionized the management of patients with non-oncogene addicted NSCLC in both first- and second-line settings, showing an unexpected long-term effectiveness and a good toxicity profile ; on the EGFR mutations and TKIs naive (EGFR mutations in NSCLC may lead to a different responsiveness and outcomes to ICIs (EGFR-TKIs could modulate immune responsiveness to cancer by shaping the tumor microenvironment (TME) and enhancing ICIs benefit , programmed-cell death-1 (PD-1) or its ligand PD-L1, in NSCLC harboring Is naive . In addi to ICIs . It is n benefit . Therefo ongoing , 16.EGFR-mutated cancer cells represent a crucial hallmark of immunosuppression , known as a powerful immunosuppressive nucleoside  (The relevant success of the immunotherapy associated with favorable safety profile in advanced lung cancer treatment –23 suggerations) . In the rations) , while Nrations) –33.EGFR-TKIs, fail : 0.67; p < 0.001], but not in those EGFR mutated , although no clear conclusions can be drawn due to the limited number of patients as part of subgroup analyses.Unfortunately, after failure of first-line TKIs, patients with g cancer , 36. BasEGFR-mutant NSCLC patients with PD-L1 expression of at least 1%, confirming once again the failure of immunotherapy alone for EGFR mutant patients  the efficacy of PD-1 inhibitors tended to be lower as compared to EGFR wild-type patients (EGFR-mutant NSCLC (+/CD8+ tumor infiltrating lymphocytes (TILs), an immune metabolic reprogramming process and a lower mutation burden  provided significant clinical benefit  ; median PFS was 10.3 months with ABCP vs. 6.1 months with BCP, associated with a similar and good safety profile , natural killer (NK) cells, T-regulatory cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), involved in the increased release of immunoregulatory soluble factors such as cytokines and exosomes, as summarized in To this regard, recently it has been reported that the TME of + TILs in an EGFR-mutated NSCLC group compared with a wild-type EGFR group  whose expression is regulated by the MEK/ERK pathway  also emerged as a promising predictive biomarker of ICIs efficacy . TMB is  low TMB .EGFR oncogene pathway induces the release of several immunosuppressive factors to accomplish evasion of the host anti-cancer immune response. On the other hand, the inefficient killing of tumor cells is mostly due to a direct effect of EGFR-mutated lung cells through the over-production of negative modulators of immune cells including tumor necrosis factor-β (TGF-β), interleukin-10 (IL-10), vascular endothelial growth factor (VEGF), indolamine 2,3-dioxygenase (IDO), C-C chemokine ligand 2 (CCL-2), arginase (ARG)-1, and adenosine. The abundant immunosuppressive environment promotes both conversion and proliferation of CD3+CD4+CD25− cells into CD4+CD25+FoxP3+ Tregs population, leading to immune suppression mediated by tumor release of EGFR-containing exosomes , CD203α (ecto-nucleotide pyro-phosphatase phosphodiesterase 1) and CD73  and from TGCA. According to these evidence, EGFR-mutant adenocarcinomas displayed >2-fold increased expression of CD73 compared to wild type, and this mechanism might, at least in part, explain their poor responsiveness to immunotherapy   .EGFR-mutated NSCLC who have developed resistance to EGFR-TKIs, although this study had some limitations such as the limited number of patients analyzed  in the treatment of advanced NSCLC are summarized in EGFR-mutated NSCLC.Finally, the current development of anti-CD73-based strategies and relative ongoing clinical trials in monotherapy or combination with immune checkpoint inhibitors as anti-PD-(L)1 mAbs or targeted therapies 1 monotherapy has been shown to have minimal activity in EGFR-mutant NSCLC and therefore should only be considered after all other therapies that have been shown to be more effective in this patient population, such as EGFR-TKIs, platinum-doublet chemotherapy, and probably docetaxel plus anti-angiogenic drug (Ramucirumab®), have been exhausted.Lung tumor remains the most aggressive and life-threatening cancer, although significant therapeutic improvements have been obtained in the last years. To this regard, immunotherapy combinations are promising strategies aimed at restoring the anti-cancer immunity as well as overcoming innate and adaptive resistance to ICIs. In the era of successful immunotherapy, while non-oncogene addicted advanced NSCLC obtains a great survival benefit, the effectiveness of immunotherapy in The identification of additional biomarkers that are predictive of benefit to anti-PD-1 mAbs would be an important advance in our understanding, especially considering the potential lethal immune-toxicities and high cost of ICIs.EGFR-mutated NSCLC TME is required. The “uninflamed” TME and the low TMB are typical features of EGFR-mutated NSCLC, potentially explaining the impaired response to immunotherapies.Therefore, a better understanding of biological mechanisms involved in tumor progression and immune evasion as well as of the dynamic hallmarks of EGFR oncogene activation, over-expression of immunosuppressive molecule such as CD73 and reduced expression of IFNγ signature, and this may explain, at least in part, the limited responsiveness to immunotherapy in EGFR-mutated NSCLC. Despite these preliminary evidence, the exact relevance of CD73-adenosine signaling in both EGFR-mutated tumor cells and infiltrating immune cells to the efficacy of immune checkpoint inhibitors remains unclear. Anyway, in the preclinical studies the pharmacological antagonism of CD39/CD73—adenosine signaling potentiated anti-tumor responses in preclinical models that otherwise failed to respond to anti-PD-1/PD-L1 inhibition. These data agree with observations that blockade of other immune checkpoint inhibitors over-expressed in the TME, such as CTLA-4, TIM-3, TIGIT can enhancing IFNγ production by CD8+ TILs following anti-PD-1 inhibition.To this regard, an interesting hallmark of NSCLC-harboring oncogenic driver mutations, leading to immune escape, and conferring primary immune resistance, is the reprogramming of energy metabolism mediated by CD39/CD73—adenosine signaling. In order to reinforce the critical role of adenosine signaling in tumorigenesis, several trials have demonstrated that CD73 over-expression by cancer cells correlated with poor prognosis in NSCLC patients. Furthermore, in advanced NSCLC recent findings identified a novel relationship between EGFR-mutated NSCLC are needed to target the dysregulated immunometabolism in the TME, in order to overcome primary resistance to immunotherapy.In conclusion, adenosinergic signaling is emerging as a powerful immune-metabolic checkpoint in advanced NSCLC. Hence, further pre-clinical data and clinical trials aiming at translating adenosine signaling inhibition strategies in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.CG received honoraria as speaker bureau and advisory board member from Astra Zeneca, BMS, MSD, and Roche. 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.