Ardisia kteniophylla A. DC, widely known as folk medicinal herb and ornamental plant, has been extensively investigated due to its unique leaf color, anti-cancer and other pharmacological activities. The quantitative real-time PCR (qRT-PCR) was an excellent tool for the analysis of gene expression with its high sensitivity and quantitative properties. Normalizing gene expression with stable reference genes was essential for qRT-PCR accuracy. In addition, no studies have yet been performed on the selection, verification and stability of internal reference genes suitable for A. kteniophylla, which has greatly hindered the biomolecular researches of this species. In this study, 29 candidate genes were successfully screened according to stable expression patterns of large-scale RNA seq data that from a variety of tissues and the roots of different growth stages in A. kteniophylla. The candidates were then further determined via qRT-PCR in various experimental samples, including MeJA, ABA, SA, NaCl, CuSO4, AgNO3, MnSO4, CoCl2, drought, low temperature, heat, waterlogging, wounding and oxidative stress. To assess the stability of the candidates, five commonly used strategies were employed: delta-CT, geNorm, BestKeeper, NormFinder, and the comprehensive tool RefFinder. In summary, UBC2 and UBA1 were found to be effective in accurately normalizing target gene expression in A. kteniophella regardless of experimental conditions, while PP2A-2 had the lowest stability. Additionally, to verify the reliability of the recommended reference genes under different colored leaf samples, we examined the expression patterns of six genes associated with anthocyanin synthesis and regulation. Our findings suggested that PAP1 and ANS3 may be involved in leaf color change in A. kteniphella. This study successfully identified the ideal reference gene for qRT-PCR analysis in A. kteniphella, providing a foundation for future research on gene function, particularly in the biosynthesis of anthocyanins. Ardisia kteniophylla A. DC, an evergreen shrub from the genus Ardisia (Primulaceae family), was mainly found in South China, North Vietnam, Laos and Thailand, and commonly known as a traditional folk medicine to treat rheumatism and injuries from muscles and fractures in China  has been widely used in fields such as genome sequencing, RNA seq, ChIP sequencing, and has become a commonly used biotechnology for its high resolution, low cost, and high sensitivity . Most reis aurea , Alternaxeroides , Zingibeficinale  and applficinale . To screficinale , NormFinficinale , BestKeeficinale , and ΔCtficinale , were coficinale . Consequpilosula , Scutellcalensis , Allium  sativum , and so A. kteniophylla. Subsequently, 29 candidate genes were successfully screened and their expression levels were further determined via qRT-PCR in 14 experimental samples. Finally, five commonly used statistical algorithms—delta-CT, geNorm, BestKeeper, NormFinder, and the comprehensive tool RefFinder were used to determine the stability of those candidates and rank them. Furthermore, to confirm the reliability and applicability of the reference genes, we analyzed the expression patterns of target genes related to anthocyanin regulation and synthesis. This work represents the initial systematic investigation of stable reference genes in A. kteniophylla, serving as a fundamental basis for future research on the molecular mechanisms underlying biological functions in A. kteniophylla, with a particular focus on the expression of genes related to anthocyanin synthesis.Herein, we used a comprehensive expression profile of large-scale RNAseq data that from a variety of different tissues and the roots of different growth stages to select for the most stable genes expressed in A. kteniophylla utilizing RNA-seq data from various tissues, such as mainroot, fibrous root, root nodule, stem xylem, stem phloem, leaf, flower, and fruit, as well as roots of varying growth stages, including 1 month old, 3 months old, 6 months old, 1 year old, 2 years old, and 3 years old, which has yet to be published by our laboratory. A total of 42 data sets were used to calculate fragments per kilobase per million reads (FPKM), mean values (MV) and standard deviations (SD) for each gene. To identify genes with high expression levels and stability, we selected genes with MV values greater than 30 and ranked them based on their SD values from the smallest to the highest. The top 500 genes were further filtered on the basis of functional annotations to identify 29 candidates with commonly known functions. The heatmap were generated using TBtools software  filled with soil mixture (peat: perlite = 1:1) and grown in the greenhouse of South China Botanical Garden, Chinese Academy of Sciences . Healthy six-month-old seedlings were divided into different groups and subsequently receive abiotic stress and hormone treatments.4 , 1 mmol AgNO3 , 200 μmol MnSO4 , 100 μmol CoCl2 , Drought (20% PEG6000), low temperature , heat stress ,Waterlogging (sinking 1-2cm underwater), Wounding  and Oxidative stress . All control and experimental plants had three biological replicates. Prior to RNA extraction, the samples were gathered and frozen immediately in liquid nitrogen, then stored at -80°C.A total of fourteen experimental groups were performed: including 100 μmol MeJA , 100 mM ABA , 100 μmol SA , 100 mmol NaCl , 200 µM CuSO−1 agarose gel electrophoresis, while the concentration and purity of RNAs were determined using a NanoDrop 2000c spectrophotometer . The RNAs were deemed acceptable if they exhibited an optical density of A260/280 ≈ 1.8–2.1 and A260/230 ≈ 1.7–2.2. The HiScript®II Reverse Reverse Transcriptase  was employed to synthesize single-strand cDNA from 1.0 µg of total RNA in a 20 µL volume system, following the manufacturer’s protocols. Prior to use, all cDNA samples were diluted with nuclease-free water (1:20) and stored at -20°C.The Fastpure® Universal Plant Total RNA Isolation Kit  was utilized to extract total RNAs from each collected sample in accordance with the recommended protocols of the supplier. Subsequently, the integrity and quality of the total RNA samples were assessed through 1 g LE) and regression coefficient (2R) were assessed using the diluted cDNA, as described earlier  and standard deviation (SD). The candidate genes were arranged in ascending order based on their SD, and 29 genes with MV exceeding 30 and possessing functional annotations were chosen  was assessed and the results, presented in E values for each primer pair ranged from 94.08% (ADF) to 104.87% (ATP). Additionally, the regression coefficient (2R) values varied between 0.9836 for α-TUB-3 and 0.9999 for EIF-1A.In order to identify optimal reference genes for gene expression profiling of A. kteniophylla, their expression levels were assessed across various treatment conditions via qRT-PCR (UBC2 (2.01), SAM5 (2.04), and UBA1 (2.04) were the most stable reference genes, while α-TUB-3 (3.64), HSP70 (3.83) and PP2A-2 (9.26) were the most unstable. BestKeeper evaluate the stability of candidates by calculating the CV and SD of the Ct (cycle threshold) values of the candidate genes, and the genes with the lowest CV and SD (CV ± SD) considered to be the most stable. As depicted in UBA1 (3.49 ± 0.75), CYP1 (4.11 ± 0.80) and PP2A-1 (3.77 ± 0.81) were the most stable reference genes, while PP2A-2 (17.43 ± 4.78), RPL13 (12.94 ± 2.75) and α-TUB-4 (7.91 ± 2.36) were found to be the least stable. GeNorm ranked the candidates according to expression stability M values, which are the average paired variation of a certain gene with all other candidate genes, with lower M values indicating more stable expression , hormone treatment  and abiotic stress . The results depicted in UBA1 and UBC2 exhibit the highest stability as reference genes for heavy metal stress, while PP2A-2 displays the least stability  at seedling stage, which was mainly caused by the difference in anthocyanin content. At present, numerous studies have suggested that anthocyanins were widely distributed pigments in plants, and their biosynthetic pathways and regulatory mechanisms have been widely studied in model plants and ornamental plants (UBC2 and UBA1) in A. kteniophylla were the most suitable for gene expression studies, while PP2A-2 was not recommended as the reference gene. To determine the reliability of the reference genes selected for normalization, we selected six key genes related to anthocyanin synthesis and regulation as target genes  for normalization with the most stable reference gene or combination as well as the least stable reference gene.As shown in l plants . Based oet genes 5 (incluUBC2 and UBA1) were used alone or in combination under different color leaf samples, while the expression pattern of the most unstable expression reference gene (PP2A-2) obtained was significantly different from those of the stable references used alone or in combination. For example, when normalized with the most suitable reference genes UBC2 or UBA1 and their combination, the expression levels of PAP1 was highest in the red leaves, followed by semi red leaves and lowest in green leaves. However, the expression patterns of PAP1 were quite different from those of the optimal reference gene when normalized with the unstable gene PP2A-2, which PAP1 gene showed the highest expression level in the semi red leaves, followed by green leaves and lowest in red leaves . Meanwhile, the expression patterns of PAP1 and ANS3 were similar: red leaf > semi-red leaf > green leaf, indicating that these two genes may play a significant role in the formation and change of leaf color in A. kteniophylla. In contrast, the transcription factor TT8 and structural genes ANS1 may not have performed.Furthermore, this study found that among the six key genes that may be potentially related to anthocyanin synthesis in different leaf color samples, the transcription factor d leaves Figure 4A. kteniophylla, as a significant medicinal and ornamental plant, has drawn more attention of researchers due to its showy leaf color, anticancer and other pharmacological activities. In recent years, high-throughput sequencing has become increasingly popular, RNA sequencing based on genome sequence has been widely used to detect gene expression, which provides a basis for the study of molecular biology studies of A. kteniophylla. Additionally, qRT-PCR was a method of high sensitivity, accuracy, and throughput for analyzing gene expression, required the ideal reference genes as a prerequisite to assure the accuracy and dependability of the results. So far, the development of stable reference genes in A. kteniophylla has received little attention, despite evaluating and validating reference genes in a variety of plants. Furthermore, the establishment of reference genes for stable expression in A. kteniophylla using RNA-seq datasets has not been previously reported.A. kteniophylla various tissues and growth years. In the present study, appropriate reference genes were selected using expression profile data to quantify genes in A. kteniophylla, with special attention to the application of reference genes in the study of anthocyanin synthesis and regulation related genes during the color change process of A. kteniophylla leaves. Mughal et al. , MV (FPKM mean), MFC (maximum multiple change of FPKM), and CV (coefficient of variation), for assessing candidate expression stability  to improve the reliability of reference gene evaluation  possess completely distinct expression levels and stability characteristics , as a key enzyme gene in anthocyanin synthesis pathway, catalyzed the conversion of leucoanthocyanidins to anthocyanins (PAP1 (R2R3-MYB family) and TT8 (bHLH family) genes can positively regulate structural genes in the anthocyanin pathway and promote anthocyanin synthesis and accumulation  in three distinct colored leaves of A. kteniophella seedlings. This normalization involved the use of the two most stable reference genes, their combinations, and the least stable reference gene. The findings revealed that the employment of the most stably expressed reference genes (UBC2 and UBA1) either individually or in conjunction facilitated the clear expression patterns of all target genes across the various colored leaves samples.In g leaves . Additiog leaves . Moleculocyanins . Transcrmulation . To ascePP2A-2) showed complete inconsistency with the results of the stable gene and significant changes occurred. This clearly demonstrated the impact of using unstable reference genes on qRT-PCR results, and also indicated that the relative expression level of target genes varies with the selection and quantity of references. Previously, similar results have been studied in multiple species, including Codonopsis pilosula  can be found below: BioProject, PRJNA1026808.WW: Conceptualization, Formal Analysis, Methodology, Validation, Writing – original draft, Writing – review & editing. XZ: Data curation, Formal Analysis, Methodology, Software, Visualization, Writing – review & editing. XXX: Data curation, Formal Analysis, Methodology, Validation, Writing – review & editing. XCX: Data curation, Methodology, Software, Visualization, Writing – review & editing. LF: Data curation, Formal Analysis, Investigation, Visualization, Writing – review & editing. HC: Conceptualization, Formal Analysis, Funding acquisition, Resources, Writing – review & editing.


In the last twenty years, we have witnessed a paradigm shift in the treatment and prognosis of acute myeloid leukemia (AML), thanks to the introduction of new efficient drugs or approaches to refine old therapies, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents, and Venetoclax, the optimization of conditioning regimens in allogeneic hematopoietic stem cell transplantation and the improvement of supportive care. However, the long-term survival of non-M3 and non-core binding factor-AML is still dismal. For this reason, the expectations for the recently developed immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor strategies, successfully tested in other hematologic malignancies, were very high. The inherent characteristics of AML blasts hampered the development of these treatments, and the path of immunotherapy in AML has been bumpy. Herein, we provide a detailed review of potential antigenic targets, available data from pre-clinical and clinical trials, and future directions of immunotherapies in AML. In the last twenty years, the knowledge of mechanisms of leukemogenesis and molecular/genetic features of acute myeloid leukemia (AML) has rapidly progressed, allowing a precise characterization of the disease and several updates on AML classification ,2. In thThe introduction in clinical practice of new drugs, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents (HMA) and Venetoclax (VEN) ,7, the eThus, the recent and successful introduction of immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor (CAR) strategies, in lymphoid malignancies has raised hopes for their possible use in AML as well.Furthermore, other immunologic therapies showed efficacy in AML, and some of them are established as gold standards in different settings. The most illustrative example is HSCT: after HSC engraftment, the donor immune system is capable of targeting the residual leukemic cells, giving life to the so-called “Graft-versus-Leukemia” (GvL) effect . Key chaFLT3+ AML cells, which causes an increase in CD8+CD107a+IFN-γ+ T cells and is capable of eradicating leukemia cells , morphologic leukemia-free state [MLFS] and partial response [PR]) of 22% and a composite complete response  rate of 19%. Infusion-related reactions (IRR)/CRS occurred in all thirty patients treated (grade ≥ 3 in 13.3%)  infusion), an overall response rate  .In 2021, the report of the dose expansion cohort was published. cCR rate was 20%, with an ORR of 24%. IRR/CRS were observed in 96% of patients (grade ≥ 3 in 4%). Grade ≥ 3 neutropenia and thrombocytopenia occurred in 2% and 6%, respectively . A trialThe phase 1 dose escalation study evaluating AMG330 as a continuous IV infusion in patients with R/R AML showed a cCR rate of 11.4%, with an 89% treatment discontinuation rate for disease progression, adverse events, and/or patient requests. Grade ≥ 3 CRS was observed in 31.4% . The stuIn 2019, the results of AMG673, a CD33 × CD3 BiTE genetically fused to the N-terminus of a single-chain IgG Fc region , phase I study were presented: an ORR of 22.2% with a cCR rate of 3.7% were observed. CRS occurred in 50% of patients (≥grade 3 in 13.3%) .CD123 was also targeted by a BiTE, Vibecotamab; the phase I dose-escalation study, enrolling 104 AML, 1 B-cell ALL, and 1 chronic myeloid leukemia (CML), showed, at the higher dose tested (0.75 µg/kg), a cCR rate of 13.7%; CRS occurred in 58.5% of patients (8% grade ≥ 3) . A phaseA combination with conventional therapy was tested with a second CD123 × CD3 BiTE: APVO436. This drug was at first evaluated as monotherapy in relapsed/refractory (R/R) AML or MDS. In the AML cohort, 2 of the 34 evaluable patients (5.8%) obtained CR. Overall, IRR/CRS were observed in 50% of patients (≥grade 3 in 13%) . An updaRecently, new antigen targets are being explored in ongoing clinical trials: Tepoditamab (MCLA-117), a BiTE targeting CD3, and CLEC12A, an antigen present on leukemic stem cells and leukemic blasts whose expression seems stable throughout diagnosis, treatment, and relapse , showed,Since the early 2010s, efforts have been made to redirect NK-cell activity against AML blasts. In 2013, Wiernick et al. observed that CD16 × 33 BiKE and ADAM17 inhibitors can overcome inhibitory signals mediated by class I major histocompatibility complex (MHC) recognition and CD16 clipping, resulting in potent NK cell killing of CD33+ AML targets . In the More recently, the role of CD16 × CD33 BiKE was investigated in pediatric CD33+ AML and ALL patients. NK cells from the patients profited from restoration of effector functions by BiKE treatment, which, thus, could constitute a promising new option for supporting maintenance therapy or “bridging” consolidation chemotherapy before HSCT . The onlCAR-T cells are constructs composed of an antigen recognition extracellular domain, a transmembrane domain, and an intracellular domain consisting of a co-stimulation domain and cluster of differentiation 3   Figure . The varThe first clinical trial published in 2015, testing a CD33-directed CAR-T, enrolled one R/R AML patient. A decrease in blasts was observed without a clinical response; furthermore, IRR occurred .In 2019, a CAR-T directed against NKG2D , a receptor that transmits activation signals in NK-cells and activates the immune system against NKG2D ligands, expressed in AML blasts and poorly expressed in healthy tissues , was tesIn 2021, a new trial evaluated a CD33-directed CAR-T in 3 patients; again, none of them met criteria for response to treatment. Two patients developed CRS .In the same year, one case of a CD38+ AML refractory to chemotherapy combined with an anti-CD38 monoclonal antibody (Daratumumab) successfully treated with CD38-directed CAR-T was reported . AlthougIn the 2022 ASH meeting, several encouraging new data were presented.2 and cyclophosphamide 500 mg/m2 days −5 to −3; cohort 2). No response was observed in cohort 1; among the 10 AML patients enrolled in cohort 2, the cCR rate was 20% with an ORR of 30%. Overall, CRS occurred in 70.8% of patients (≥grade 3 in 4.2%) [PRGN-3006, a CD33-directed CAR-T with IL-15 membrane bound, was tested in 20 R/R AML, 1 chronic myelomonocytic leukemia (CMML) with ≥5% blasts, and 3 high-risk MDS without (cohort 1) or with lymphodepletion (fludarabine 30 mg/min 4.2%) .UCART123v1.2, an universal, anti-CD123 allogeneic CAR-T product obtained by the ex vivo knockout of the genes of the T-cell receptor (TCR) alpha constant and CD52 to minimize the risk of GvHD and allow the use of anti-CD52-directed therapy as part of lymphodepletion, was tested in combination with Alemtuzumab (anti-CD52) in 16 patients: 1 patient (6.2%) achieved MLFS, and 1 patient had MRD-negative CR. CRS occurred in 93.7% of patients (≥grade 3 in 18.7%) .An anti-CD123 CAR-T was also tested in 12 R/R AML pediatric patients in a phase I dose escalation trial; the ORR was 16.7%, with one patient (8.3%) achieving CR. No CRS occurred .In 2020, the first case of successful CAR-T targeting CLL1 , also targeted by bispecific antibody-based strategies (see above), was reported in a patient with secondary AML . In 2022CLL1 was also successfully targeted in the pediatric population; in 2021, a phase I interim analysis showed an ORR of 81.8% with a cCR rate of 72.7% (50% achieving MRD negativity) . In 2022Recently, anti-CLL1 CAR-T with stimulatory domains was tested: 4 patients were treated with CD28/CD27-equipped anti-CLL1 CAR-T, while 3 were treated with 4-1-BB-equipped anti-CLL1 CAR-T. In the first group, 3/4 patients achieved MRD-negative CR, while in the second one, 1 pt achieved MRD-positive CR and one pt achieved MRD-negative CR; CRS was experienced by all patients without ≥grade 3 events . Other CLL1-directed CAR-T trials are currently ongoing .To date, early results from only one trial testing CAR-NK in R/R AML patients have been presented.8, 1.2 × 109, or 1.8 × 109 cells per round after the precondition with Fludarabine (30 mg/m2) and Cytoxan 300–500 mg/m2 for 3 days to 5 days, determined by tumor burden at baseline).An anti-CD33 CAR-NK was tested in 10 heavily pre-treated patients  in a phase I dose escalation trial , whose targeted CAR-T was able toefficiently kill CD7+ AML cells and CD7+ primary blasts of R/R-AML patients in vitro and significantly inhibit leukemia cell growth in a xenograft mouse model ;CD19 (NCT04257175), occasionally expressed in AML blasts  ;FLT3 and DNMT3A-mutated AML and already successfully targeted by CAR-T constructs in pre-clinical studies [CD44v6 (NCT04097301), overexpressed in  studies ;CD70 (NCT04662294), expressed on most leukemic blasts but with little or no expression in normal bone marrow samples, is already targeted by the human monoclonal antibody Casatuzumab ;FLT3 , whose targeted CAR-T showed potent inhibition of leukemia proliferation in xenograft models ;LILRB4  (NCT04803929), highly expressed in AML with monocytic differentiation and suspected to be involved in tumor escape, maintains an immunosuppressive milieu for tumor cells ;CD276 (NCT04692948), expressed in AML with monocytic differentiation as well, whose targeted CAR-T exhibited efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML . Ongoing clinical trials are exploring new targets, such as:The comprehensive knowledge of tumor microenvironment and mechanisms of therapy escape led to the development, since the early 2010s, of drugs targeting the broad landscape of cells and molecules around the tumor cells, first in solid tumors than in hematologic malignancies . In a siCD47 is a transmembrane protein broadly expressed in human cells and on many types of tumors, where it plays a pivotal role in immune escape; in fact, the binding to its ligand, SIRPα , triggers a “don’t eat me” signal to the macrophages, inhibiting phagocytosis . In the In AML, CD47 is overexpressed and correlates with a poor prognosis ,94. MajeA second T cell-dependent mechanism in vivo was documented by Tseng et al., who showed that treatment with an anti-CD47 antibody had a greater effect in wild-type mice with established tumors than in T cell-deficient nude mice with equivalent tumor burden .The first drug targeting SIRP/CD47 interaction was CC-90002, an anti-CD47 monoclonal antibody, whose results were presented in 2019: among the 24 R/R AML patients and 4 high-risk (HR) R/R MDS patients treated, no objective response was documented, with an 82% rate of treatment-emergent adverse events .TP53-mutated AML patients achieved cCR. The combination therapy was well tolerated, with a safety profile similar to AZA monotherapy [In the same year, the first results of another anti-CD47 antibody, Magrolimab, in combination with AZA were presented. 25 newly diagnosed (ND) AML patients ineligible for induction chemotherapy were enrolled; among the 16 patients evaluable for response, 11 obtained an objective response , and 5 presented stable disease. Interestingly, 5/6 otherapy .TP53-mutated AML, the ORR was 71%  [The 2021 update of the study confirmed the promising results, with a 65% ORR ; in the subset of 5% MLFS) . A phaseSurprisingly, the ENHANCE study, testing Magrolimab plus AZA in HR-MDS, was recently discontinued due to futility based on a planned analysis ; these rA trial testing Magrolimab plus Atezolizumab  combination therapy (NCT03922477) is ongoing.Lemzoparlimab is an anti-CD47 antibody endowed with a red blood cell (RBC) sparing property and unique binding epitope, potentially differentiating itself from other CD47 axis-targeting therapies. Preliminary results from the phase I study, presented in 2020, showed a 20% cCR rate (MLFS) with an acceptable safety profile (no patient developed a treatment-related serious adverse event) . The phaIn 2022, Evorpacept, a fusion protein containing two engineered high-affinity CD47 binding domains of SIRPα linked to an inactive Fc region of human immunoglobulin was designed to promote the phagocytosis of tumor cells and was tested in combination with VEN and AZA in R/R AML or ND HR AML. A blast decrease was observed in all patients, with an objective response in all ND AML patients and 44.4% of R/R AML patients. Among the 14 patients enrolled, 1 grade 3 CRS occurred . Programmed cell death protein 1 (PD1) is a cell surface receptor expressed on several types of T cells  and other immune cells  and has a fundamental role in countering positive signals through the TCR and CD28 by engaging its ligands PDL1 and/or PDL2 . Thus, sSeveral drugs targeting this pathway, blocking PD1  or PDL1 , have been successfully tested and approved by the FDA in solid cancers and hematologic malignancies ,111, altThe first data on Nivolumab in combination with AZA dates back to 2017; the early results, on 53 R/R AML patients, showed an ORR of 35% with a cCR rate of 14%. Furthermore, the median overall survival (5.7 months) was superior to historical survival with AZA-based salvage protocols from the same institution. ≥grade 3 immune-related adverse events (IrAE) were observed in 14% of patients and responded rapidly to steroids .TP53 mutation was more frequent in non-responders (p = 0.06). Six patients had seven grade 3–4 IrAEs with an overall acceptable safety profile [In the same year, the results of the therapy combination with Cytarabine and Idarubicin as first-line therapy in a cohort of ND AML and HR MDS (30 patients with AML 2 with HR MDS) were presented: the cCR rate was 72%. More than grade 3 IrAE occurred in 5 patients . The fin profile .Nivolumab was also tested as maintenance therapy. In 2018, it was tested in 14 patients with HR AML  in CR, with a 6 and 12 month CR duration of 79% and 71%, respectively. Five patents experienced ≥grade 3 IrAE . In 2022In a similar fashion, Pembrolizumab was tested in different clinical settings: the combination with AZA was investigated in R/R AML and older ND AML by Gojo et al., who presented the preliminary results at the 2019 ASH meeting. The R/R AML cohort achieved an ORR of 17.2% (cCR 13.8%), while the ND AML cohort achieved 58.8% (cCR 47%). Grade 3/4 IrAE was observed in 9 (24%) patients in the first cohort and 3 (14%) patients in the second one . The combination with Decitabine (DEC) was tested in a cohort of 10 R/R AML patients, of whom 3 obtained cCR. The toxicity profile of the combination therapy was consistent with that expected from DEC alone, with the exception of three patients who suffered IrAE, likely as a consequence of Pembrolizumab . The combination with chemotherapy was explored in R/R patients by Zeidner et al.: 37 patients were enrolled to receive Pembrolizumab after high-dose cytarabine in a phase II study. The ORR was 46%, with a cCR rate of 38%; the administration of Pembrolizumab was associated with 5 IrAE, and no severe toxicity or delay in hematologic recovery were observed . In a foFinally, Pembrolizumab was tested as maintenance therapy after autologous HSCT in non-favorable-risk AML patients in the first CR or beyond who were ineligible for alloSCT. Among the twenty patients enrolled, after a median follow-up of 80 months, 14 patients remain alive, with 10 in continuous remission. Treatment was well tolerated, with only 1 non-relapse death .In 2022 the results of a large randomized phase II trial evaluating older patients with AML receiving AZA with or without Darvalumab were published. Although the therapy combination did not raise safety concerns, it failed to achieve a better response  . Trials testing anti-PDL1 are currently ongoing ; no results are available at the moment.T cell immunoglobulin and mucin domain 3 (TIM3) is a transmembrane protein expressed on immune cells  that binds multiple ligands. One of the most known is Galectin-9 , which plays an important role in immune cell regulation. Zhu et al. showed that Gal-9, through the TIM3 link, was able to induce intracellular calcium flux, aggregation, and death of T helper cells, and administration of Gal-9 in vivo resulted in selective loss of interferon-gamma-producing cells . In the The detection of TIM3 in myeloid malignancies cells, in particular MDS  and AML The first anti-TIM3 antibody, Sabatolimab, was tested in combination with HMA in a cohort of ND or R/R AML patients. The combination Sabatolimab plus DEC achieved an ORR of 41% in ND AML patients and 25% in R/R AML patients, whereas Sabatolimab plus AZA, tested only in ND AML patients, obtained a 27% ORR. Three IrAE were observed in the DEC cohort vs. none in the AZA cohort . In the study update in 2021, in a cohort of 40 evaluable patients with ND AML, the ORR was 40.0%. The response rate was consistent in AML patients bearing an adverse-risk mutation . The combination was safe and well tolerated, with grade ≥ 3 adverse events similar to HMA alone . Trials evaluating new clinical settings, such as AML in MRD+ CR after HSCT (NCT04623216), and therapy combinations, such as Sabatolimab plus AZA/VEN , are currently ongoing.CD70 is a molecule expressed in activated T cells and dendritic cells that, by binding its ligand, CD27, a costimulatory receptor of the tumor necrosis factor superfamily, constitutively expressed on lymphocytes and hematologic stem cells, promotes lymphocyte expansion and cell survival ,138. FurThus, as also briefly discussed earlier, the CD27/70 pathway represents a good target to address AML; in fact, Riether et al., in in vitro studies, documented strong proliferation inhibition blocking CD27 and CD70 .Casatuzumab, an anti-CD70, has been tested in combination with AZA in ND AML patients ineligible for chemotherapy in a phase I/II trial. The ORR in patients treated at the phase II dose was 37.9% . Seven patients (18.4%) reported infusion-related reactions, including two with grade 3 events. There were 10 fatal treatment-emergent adverse events; none were considered drug-related .In 2021, the preliminary results of the therapy combination Cusatuzumab plus AZA/VEN in ND AML patients ineligible for chemotherapy were presented: among the evaluable 42 patients, the ORR was 92.9% . Apart from the IrAE (11.4% of patients with 2.3% at grade ≥ 3), the safety profile was consistent with that previously reported for the AZA/VEN regimen .CTLA4 (cytotoxic T-lymphocyte-associated protein 4) is a transmembrane protein member of the immunoglobulin gene superfamily expressed by both CD4+ and CD8+ T cells, which, interacting with its ligands, CD80 and CD86, inhibits immune response . InteresIpilimumab, an anti-CTLA4 antibody, was first tested as post-HSCT salvage therapy in various malignancies, including 2 AML patients; no effect was observed . In 2023A phase I trial investigating Ipilimumab in combination with donor lymphocyte infusion (DLI) for myeloid malignancies relapsed after HSCT is ongoing (NCT03912064).CD200 is a cell membrane glycoprotein of the immunoglobulin supergene family, present on both cells of myeloid/lymphoid origin. Its receptor, CD200R, is expressed on cells of the monocyte/myeloid lineage and some T-cell subsets [ subsets . CD200/C subsets ,148. On  subsets ,150,151. subsets ,153. A tLAG-3 (lymphocyte activation gene-3) is a type I transmembrane protein with four Ig-like domains structurally similar to CD4 expressed on activated T cells. The bind with its ligand, MHC II, leads to the inhibition of T-cell functions; in fact, its blockade has been demonstrated to reinvigorate exhausted T cells and strengthen anti-infection immunity [immunity . Multiplimmunity ,155,156.Immunotherapy is based on mechanisms of immune escape and responsiveness. Such mechanisms can be different in the pre-leukemic and transforming stages of diverse subsets of leukemia. Failure to receive immunotherapy can be triggered by diverse disease etiologies and by chemotherapy resistance and immune evasion. Furthermore, the complex and disease-specific microenvironment may contribute significantly to the differency in efficacy observed between AML and lymphoid disorders . In thisGiven the genetic and epigenetic heterogeneity of pediatric and adult AML, immunotherapy has still not provided a paradigm-shifting breakthrough. The accumulation of genetic mutations and alterations of pathways defining anti-tumor activity are also very different across subsets of leukemia, which might explain different levels of response. However, the safety profile has been good for most of the new drugs, and the therapy was well tolerated by patients, who were commonly elderly and subjected to a heavy previous burden of therapy. In addition, IrAEs were usually low-grade and easily treated with steroid therapy. Concerning the anti-leukemic activity, some compounds showed encouraging results, but, unfortunately, there are still only a few randomized comparative studies, which are warranted to clarify the actual benefits and to frame these strategies in the most appropriate clinical subsets.The ongoing trials evaluating drugs targeting new markers and addressing different patient subsets allow us to envision the possibility, for immunotherapies, of expanding both safe chemo-free strategies and first-line combination therapies in AML.


Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies. Primary9/L . Childre9/L . Older a9/L ,6. ITP is considered an immune-mediated disease and platelet autoantibodies (PAA) are thought to be important actors . Testinggagitalics is not necessary, but it must be consistent in the whole papper), polymerase (pol), and envelope (env), flanked between two regulatory long terminal repeats (LTRs) [Human endogenous retroviruses (HERVs) originate from ancestral infections of somatic cells with subsequent integration into germ line of primates millions of years ago. During evolution, the continuous mutations have blocked the production of infectious virions. However, HERVs maintain their retroviral structure with three principal genes: group associated antigens (s (LTRs) . Most HEs (LTRs) ,16, reprs (LTRs)  and syncs (LTRs) , are esss (LTRs) ,20. Posts (LTRs) ,22,23,24s (LTRs) ,26. Theis (LTRs) ,26,27,28s (LTRs) ,30,31,32s (LTRs) ,34; theis (LTRs) , reminisHERV expression is modulated by environmental factors via epigenetic mechanisms. Tripartite motif containing 28 (TRIM28), also called KAP1 or TIF1-β, is a nuclear co-repressor of Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs) ,36. SET Despite the wealth of data supporting the role of HERVs, TRIM28, and SETDB1 in eliciting and/or maintaining autoimmune diseases, no studies explored their expressions in patients affected by ITP. Therefore, the aims of this study were to assess and compare the transcriptional levels of pol genes of HERV-H, -K, and -W, the three retroviral families most widely studied ,21; env All children and adolescents affected by CITP . This kit provides treatment with DNase during the first step of RNA extraction. To exclude contamination with residual genomic DNA, total RNA extracts were directly amplified without reverse transcription. RNA concentration and purity were spectroscopy determined  at absorbance of 260 and 280 nm. RNAs were stored at −80 °C until use.2 25 mM, 1 μL of RNase inhibitor 20 U/l, 2 μL ImpromII (Promega), 2 μL mix dNTPs 100 mM (Promega), 0.4 μL random hexamers 250 μM (Promega), and dd-water in a final volume of 20 μL. The reaction mix was carried out in a GeneAmp PCR system, 9700 Thermal Cycle  under the following conditions: 5 min at 25 °C, 60 min at 42 °C, and 15 min at 70 °C for the inactivation of enzymes; the cDNAs were stored at −20 °C until use.A total of 400 ng of RNA was reverse-transcribed with 2 μL of buffer 10× 4.8 μL of MgCl2, 200 nmol of specific probes, and 500 nmol of specific primers.Transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of SYN1, SYN2, and HWERV-W; and TRIM28 and SETDB1 were achieved as previously described in detail ,24,33,34−ΔΔCt method [All amplifications were performed in a 96-well plate at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and at 60 °C for 1 min. Each sample was run in triplicate. Relative quantification of target gene transcripts was performed using the 2t method . GAPDH wt method ,24,33,34p < 0.05 was taken to be statistically significant.One-way ANOVA test was used to compare the age between patients with CITP and the two subgroups (B1 and B2) of HC. Mann–Whitney test was used to compare the transcripts of pol genes of every HERV family; env genes of SYN1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 between children with CITP and control children. Spearman correlation test was performed to estimate the correlations concerning transcription levels of each HERV sequence and TRIM28 or SETDB1 in every group of children. Statistical analyses were performed using Prism software, version 7 . In all analyses, p = 0.1998). As reported in p = 0.4111); Group B2: median 21.8, IQR 21.5–22.4 (p = 0.1432).The transcription levels of housekeeping gene GAPDH were similar between patients with CITP and HC: Medians and IQR 25%–75%: Group A: median 22.3, IQR 21.8–22.7; Group B1: median 22.1, IQR 21.6–22.5 . When expressions of HERV-env genes from patients on eltrombopag alone were compared to those from HC, only SYN1 showed significantly higher values . Medians and IQR 25%–75% of SETDB1: on eltrombopag: median 1.64, IQR 1.27–1.80; no treatment: median 1.80, IQR 1.46–2.17 (p = 0.3397). The median transcriptional levels of TRIM28 and SETDB1 were lower in the 11 patients on eltrombopag treatment alone as compared to the 18 patients with no treatment, but they did not reach statistically significant differences, with borderline r TRIM28 . MediansCurrent results highlight for the first time that children and adolescents with CITP exhibit higher transcriptional levels of pol genes of HERV-H, -K, and -W and env genes of SYN1 and SYN2 in whole blood as compared to age-matched healthy subjects. Some clues that endogenous retroviruses could be involved in inducing alterations in platelet count can be found in the literature. Retrovirus-like particles were detected in lysates of purified platelets ,44. ReveThe cause and the clinical relevance of the enhanced expression of HERV sequences in CITP children remain elusive. Several experimental studies demonstrate that defective transcription of TRIM28 and SETDB1 may lead to increased HERV activation, via DNA hypomethylation and limited chromatin condensation ,39. The Viral infections often precede the onset of primary ITP by a few weeks. The virus-driven stimulation of the immune system is thought to be highly implicated in inducing targeted injuries ,55. SeveThe number of HERVs with high mRNA levels in CITP patients suggests that their upregulation is a wide and complex phenomenon. As regards the single HERV genes, pol genes of HERV-H, HERV-K, and HERV-W contribute to the production of reverse polymerase, that catalyzes the mRNA retro-transcription into the DNA. This may result in new mutations or dysregulated transcription of cellular genes. It must be underlined that the primers and probes we used revealed preserved sequences of many pol genes from different HERV families, which, however, did not allow outlining of the effects of single HERV loci. SYN1 and SYN2 share syncytial and immunomodulatory properties. They target T-cell activation and significantly suppress T-cell functions ,20. SYN1Eltrombopag is a nonpeptide thrombopoietin receptor agonist used for treatment of CITP as a highly effective and well-tolerated therapy for achieving hemostatic platelet counts ,70. A thEpigenetic processes encompass chromatin-based mechanisms in the regulation of gene expression with no structural DNA changes. Environmental factors can cause epigenetic alterations, such as those induced by TRIM28 and SETDB1. TRIM28, through binding to lysine residues of target proteins, causes their phosphorylation and proteasome-driven degradation. TRIM28 recruits SETDB1 for SUMOylation, a post-translational event involved in several cell functions, such as protein degradation, RNA splicing, and transcriptional inhibition ,72. TRIMBetter understanding of the physiopathology of ITP and the failure of tolerance checkpoints responsible for autoantibody formation may open the way to new diagnostic and prognostic approaches and innovative therapeutic strategies. For instance, early detection and longitudinal analysis of the variables here taken into consideration might predict acute or chronic evolution of ITP and allow a patient-tailored therapy. A number of anti-HERV therapeutic strategies might be adopted, such as specific anti-RNAs, monoclonal antibodies, cytotoxic T lymphocytes against HERV antigens, and antiretroviral therapies ,78,79. H


Mounting evidence indicate that cuproptosis, a novel form of programmed cell death, contributes to cancer development and progression. However, a comprehensive analysis regarding the expressions, functions, and regulatory network of cuproptosis-related genes is still lacking. In the present work, cuproptosis-related genes, upstream miRNAs and lncRNAs, and clinical data of breast cancer from TCGA database were analyzed by R language including Cox regression analysis, correlation calculation, ROC curve construction, and survival evaluation, and were further verified by public-available databases. Chemosensitivity and immune infiltration were also evaluated by online tools. SLC31A1 was significantly increased in breast cancer samples than those in normal tissues. SLC31A1 was negatively related to a favorable outcome in breast cancer, and the AUC value increased with the prolongation of follow-up time. LINC01614 and miR-204-5p were potential upstream regulators of SLC31A1. Moreover, SLC31A1 was significantly positively correlated with different immune cells infiltration, immune cell biomarkers, and immune checkpoints in breast cancer. SLC31A1 was a potential cuproptosis-related gene in breast cancer, which was significantly upregulated and was able to predict diagnosis, prognosis, chemosensitivity, and immune infiltration. LINC01640/miR-204-5p/SLC31A1 might be a significant and promising axis during cuproptosis in breast cancer. Though great progress has been made in screening, diagnosis, and treatment of breast cancer during the past few decades, the overall survival remains poor in breast cancer patients2. Therefore, it is significant to investigate molecular mechanisms of breast cancer and discover new targets and therapeutic strategies to improve clinical outcomes.Breast cancer is one of the leading causes of death among females3. A recent publication revealed that intracellular copper accumulation contributed to the aggregation of mitochondrial lipoylated proteins, leading to a novel form of programmed cell death termed cuproptosis3. Accumulating evidence suggest that dysregulated copper level is responsible for oncogenesis and cancer progression4. Unlike oxidative stress-related cell death such as apoptosis, ferroptosis, and necroptosis, studies of cuproptosis in cancer prognosis and immunity are still lacking.Copper is one of the fundamental materials in various biological processes including mitochondrial respiration8. Researchers have confirmed that aberrant regulation of these cuproptosis genes were closely related to cancer development, immune response evaluation, and prognosis prediction11. To date, the expressions, functions, prognostic values, and regulatory network of cuproptosis-related genes and their correlation with immune landscape in breast cancer have not been fully elucidated. In the present work, several cuproptosis-related genes were collected. Using R language and multiple network databases, expression levels, prognostic and diagnostic values of cuproptosis-related gene SLC31A1 were analyzed in breast cancer. Potential upstream miRNAs and lncRNAs of SLC31A1, significance of SLC31A1 in chemosensitivity and immune response were also evaluated. This study might provide important information to uncover the role of cuproptosis-related gene SLC31A1 in breast cancer and might offer a new strategy to treat breast cancer through intervening the LINC01614/miR-204-5p/SLC31A1 axis.Recently, several copper metabolism genes, including FDX1, SLC31A1, ATP7B, and CDKN2, have been reportedhttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) generates over 2.5 petabytes of genomic, epigenomic, transcriptomic, and proteomic data. These data are publicly available for anyone to download and analyze during cancer research. R is a freely downloadable language and environment for statistical computing and graphics12. R provides a wide variety of statistical and graphical techniques, and is highly extensible. In the present study, a total of 1097 clinical data, 1027 miRNAs, 1226 mRNAs and 1226 lncRNAs were downloaded from TCGA database, patients with no prognostic information or incomplete gene expression data were excluded to ensure accurate measurement. Differential analysis, univariate Cox analysis, and correlation analysis were also performed by using the limma, pheatmap, survival, and timeROC packages in R language.TCGA (http://starBase.sysu.edu.cn/), an online tool updated to provide the most comprehensive analysis of miRNA-mRNA and miRNA-lncRNA interaction networks from large-scale CLIP-Seq data, was used to evaluate the expression levels of cuproptosis-related genes, miRNAs and lncRNAs13. In the present work, connection of SLC31A1-miRNA, SLC31A1-lncRNA, miRNA-lncRNA, and SLC31A1-immune landscape in breast cancer was accessed by starBase.starBase (http://gepia.cancer-pku.cn/) is a web server to profile gene expressions between tumor tissues and normal samples from the TCGA and GTEx projects and to explore correlations between gene expressions and patients survival14. In the present study, expression levels and prognostic values of cuproptosis-related genes in breast cancer were analyzed by GEPIA. Correlations among SLC31A1, immune genes, and immune cells biomarkers in breast cancer were also confirmed by using GEPIA database.GEPIA  is a web-based platform for evaluating the relevance between gene expressions and survival15. In the present work, Kaplan–Meier plotter was employed to analyze the clinical prognostic significance of cuproptosis-related genes, miRNAs, and lncRNAs in breast cancer patients, including overall survival.Kaplan–Meier plotter (http://dna00.bio.kyutech.ac.jp/PrognoScan/) is a large database for assessing the biological relationship between gene expressions and prognostic information including relapse free survival (RFS) and distant metastasis free survival (DMFS) in breast cancer patients across a large collection of publicly available cancer microarray datasets16.PrognoScan (http://bioinfo.life.hust.edu.cn/GSCA/#/) is an integrated platform for cancer analysis at genomic, pharmacogenomic and immunogenomic levels17. In the present study, GSCA was used to analyze cuproptosis-related genes expressions, tumor immune infiltration, drug sensitivity, and their associations with clinical outcomes.GSCA (http://cistrome.shinyapps.io/timer/) is a web server for systematic analysis of tumor-infiltrating immune cells across multiple cancer types18. In the present work, TIMER was employed to comprehensively evaluate the correlation of SLC31A1 with immune cells markers, tumor-infiltrating immune cells and immune check points in breast cancer.TIMER  is an online resource for cancer miRNome interactive analysis and visualization based on the human miRNome data from 33 cancer types in TCGA19. In the present study, CancerMIRNome was applied to plot ROC curves and survival analysis, by using the upstream miRNAs of cuproptosis-related genes SLC31A1 filtered by R.CancerMIRNome (http://ctrdb.ncpsb.org.cn/) is a database designed for researchers to collect and evaluate patient-derived clinical transcriptomes with cancer drug response20. In the present work, CTR-DB was employed to discover the role of SLC31A1 in predicting chemotherapeutic sensitivity in breast cancer.CTR-DB (http://www.string-db.org/). Then, pathway enrichment analysis was performed. Cytoscape (http://cytoscape.org/) was used to construct the possible functional network.Protein–protein interaction (PPI) information and networks for SLC31A1 gene were analyzed by using the Search Tool for the Retrieval of Interacting Genes (STRING) database , and then transcribed into cDNA by using the Hiscript II qRT SuperMix . Quantitative real‐time PCR was carried out by using the AceQ qPCR SYBR Green Master Mix  on a ViiA 7 Real-Time PCR System . Primer sequences used for PCR are shown in Supplementary Table All the results in this study were automatically analyzed by the above-mentioned online database or R language. P-values < 0.05 or logrank P-value < 0.05 was defined as significant difference.The ethics committee of the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University approved the present study. Collection and analysis of clinical samples were conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Changzhou No.2 People’s Hospital. Informed written consent was received from all patients.23. A total of 17 cuproptosis-related genes were differentially expressed between breast cancer samples and normal tissues by using the limma package and pheatmap package in R language (Fig. In the present work, 1097 clinical data, 1027 miRNAs, 1226 mRNAs, and 1226 lncRNAs were downloaded from TCGA database. Patients with no prognostic information or incomplete gene expression data were excluded to ensure accurate measurement. In all 1076 female breast cancer patients with the mean age of 58.1 ± 12.9 ranging from 26 to 90, there were 16.9% stage I, 55.8% stage II, 24.3% stage III, and 3% stage IV. By reviewing the publications, a number of cuproptosis-related genes were enrolled for evaluation, consisting of ATP7A, ATP7B, CDKN2A, DBT, DLAT, DLD, DLST, FDX1, GCSH, GLS, LIAS, LIPT1, LIPT2, MTF1, NFE2L2, NLRP3, PDHA1, PDHB, and SLC31A1Prognostic values of two cuproptosis-related genes in breast cancer, namely CDKN2A and SLC31A1, were then evaluated. First, overall survival analysis was performed by using the survival package in R language. In particular, no statistical prognostic value of CDKN2A in breast cancer was observed Fig. A–F. TakeGene expression of SLC31A1 in breast cancer samples and adjacent normal tissues were analyzed. Expression level of SLC31A1 was increased in breast cancer samples with respect to normal tissues (Supplementary Fig. 24. To determine whether cuproptosis-related gene SLC31A1 could be modulated by corresponding miRNAs, upstream miRNAs of SLC31A1 were predicted. First, differentially expressed miRNAs of breast cancer were comprehensively profiled through the limma package in R language, and univariate Cox regression analysis was performed on the obtained 191 down-regulated miRNAs. A total of 8 miRNAs were enrolled, followed by Pearson correlation calculation between SLC31A1 and 8 miRNAs using the limma package in R language. Only 6 miRNAs, namely miR-125b-1-3p, miR-204-5p, miR-3613-3p, miR-383-5p, miR-3926, and miR-4491, were negatively correlated (Table As known to all, miRNAs are a class of single-stranded and noncoding RNA that serve as posttranscriptional regulators of gene expressionPotential upstream lncRNAs of the miR-204-5p/SLC31A1 axis were subsequently predicted. As described above, differentially expressed lncRNAs of breast cancer were comprehensively listed using the limma package in R language, and univariate Cox regression analysis was performed on the obtained 137 up-regulated lncRNAs. A total of 32 lncRNAs were enrolled, followed by Pearson correlation analysis between miR-204-5p and 32 lncRNAs (Table Since cuproptosis-related gene SLC31A1 was highly expressed and served as a promising diagnostic and prognostic marker in breast cancer, it is necessary to evaluate the role of SLC31A1 in predicting chemotherapeutic response during breast cancer treatment. By using the online tool GSCA, it was found that SLC31A1 modulated the chemosensitivity of multiple drugs Fig. A. In ter+ T cells, macrophages, neutrophils, and dendritic cells. Besides, correlation analysis of SLC31A1 and immune cells biomarkers indicated that SLC31A1 was positively correlated with various immune cells biomarkers (Table Role of SLC31A1 in immune response was also determined. Interestingly, levels of infiltrated immune cells gradually elevated along with the increase of SLC31A1 copy number Fig. A. In parrs Table . Given trs Table B–D, GEPIrs Table E–G, and rs Table H–J showe1. Precise surgery, adjuvant chemotherapy, and even immune therapy have greatly improved the overall survival, but prognosis of breast cancer patients remains unsatisfied2. Current anti-cancer therapy is based on clinical, pathological, and molecular features, and a novel approach to predict diagnosis, prognosis, and chemosensitivity is urgently required. Therefore, discovering some more promising and effective biomarkers and a significant signaling axis is of crucial importance.Breast cancer is the most common cancer among women worldwide3. Mounting evidence indicate that cuproptosis contributes to cancer development and progression4. However, a comprehensive analysis regarding the expression patterns, diagnostic and prognostic values, and regulatory network of cuproptosis-related genes is still lacking and need to be deeply investigated. By using R language and multiple online databases on 17 candidate cuproptosis-related genes, CDKN2A and SLC31A1 were found to be increased in breast cancer samples with respect to normal tissues. After analyzing and confirming their prognostic values in breast cancer, SLC31A1 was selected as the most potential gene responsible for cuproptosis. SLC31A1 is a trans membrane protein maintaining copper homeostasis that has been recently reported to negatively contribute to cisplatin resistance in various cancers including osteosarcoma and epithelial ovarian cancer26. In the present study, SLC31A1 expression was significantly decreased in non-response groups with respect to response groups, when breast cancer patients were treated with conventional chemotherapy regimens including FEC plus docetaxel, TA, and FEC plus paclitaxel. Opposite to the above results, Takeda et al.27 reported that SLC31A1 was predominantly expressed in triple-negative breast cancer resistant to TA-based neoadjuvant chemotherapy. Thus, there is a major need to evaluate SLC31A1 expression in different molecular subtypes of breast cancer treated with different chemotherapy regimens.Cuproptosis is a novel form of programmed cell death triggered by accumulation of intracellular copper level and aggregation of mitochondrial lipoylated proteins24. In the present work, upstream miRNAs of SLC31A1 were comprehensively predicted, after which Cox regression analysis, correlation calculation, ROC curve construction, and survival evaluation were performed. Among the candidate miRNAs, miR-204-5p was considered as the most potential upstream miRNA of cuproptosis-related gene SLC31A1. Several publications have shown that miR-204-5p acts as a tumor suppressor in many types of cancer including colorectal cancer, glioma, and thyroid carcinoma30. However, in breast cancer, studies on miR-204-5p expression show conflicting results, suggesting a possible dual regulatory role. While some investigations supported a tumor suppressor role of miR-204-5p in regulating proliferation, metastasis, and immune microenvironment remodeling in basal-like breast cancer cells31, other studies showed up-regulation of miR-204-5p in breast cancer tissues and the pro-proliferative effect of miR-204-5p in luminal breast cancer cells32. Given that no relevant reports on miR-204-5p and SLC31A1 are currently available, future studies investigating the regulation of miR-204-5p in SLC31A1 are needed in different breast cancer subtypes with distinctly different biology.Research over the past few years has indicated that miRNAs are a class of single-stranded and noncoding RNA with a capacity for posttranscriptional regulation of gene expression33. In the present work, LINC01614 was identified as the potential upstream lncRNA of miR-204-5p, after a series of lncRNAs prediction, Cox regression analysis, correlation calculation, and survival evaluation. Previous studies have showed that LINC01614 functioned as an oncogenic lncRNA in several malignancies, such as pancreatic cancer, papillary thyroid carcinoma, and gastric cancer36. In breast cancer, lncRNA transcriptional landscape identified LINC01614 as a non-favorable prognostic biomarker. Highest expression of LINC01614 was observed in luminal and HER2 + breast cancer subtypes, while lowest expression was in basal-like breast cancer subtype37. Moreover, LINC01614 was found to regulate epithelial-mesenchymal transition and tamoxifen sensitivity in luminal breast cancer cells38. Up to now, there is no direct evidence reporting the regulation of LINC01614 on miR-204-5p. It is therefore desired that further attention be drawn to this field.In recent years, lncRNAs have been reported to take part in various biological processes via regulating multiple molecules including miRNAs39. In the present study, SLC31A1 was significantly associated with infiltration degree of immune cells and levels of immune cells biomarkers. In addition, SLC31A1 was positively related to the expressions of immune checkpoints CD274 and CTLA4, suggesting that targeting SLC31A1 might be a potential way to improve immunotherapy efficacy in breast cancer.Tumor microenvironment, composed of cancer cells, surrounding fibroblasts, and tumor-infiltrating immune cells, play an essential role in oncogenesis and immunotherapy efficacyThe present study has some significant advantages. First, while most of the publications reporting SLC31A1 were based on limited data derived from online tools, the current work utilized R language to obtain TCGA breast cancer information in a more synchronous and comprehensive manner. This approach offered more rigorous analysis and more convincing results. Second, the present study is the first to propose that LINC01640/miR-204-5p/SLC31A1 might be a significant and promising axis during cuproptosis in breast cancer, whereas other bioinformatics analysis only focused on the expression levels, prognostic and diagnostic values, and immune regulation of cuproptosis-related gene SLC31A1. There are also certain limitations to be thoroughly considered. On one hand, the detailed molecular subtypes and treatment information were not available for data analysis, which might have potential influence on the results; on the other hand, statistical analysis was based on data derived from public-available databases, and further biological experiments and clinical verification are required in the future.In summary, this study found that SLC31A1 was a potential cuproptosis-related gene, which was significantly upregulated and was able to predict diagnosis, prognosis, chemosensitivity, and immune infiltration in breast cancer. LINC01640/miR-204-5p/SLC31A1 might be a significant and promising axis during cuproptosis in breast cancer.Supplementary Figure 1.Supplementary Table 1.


Thermogenic fat, consisting of brown and beige adipocytes, dissipates energy in the form of heat, in contrast to the characteristics of white adipocytes that store energy. Increasing energy expenditure by activating brown adipocytes or inducing beige adipocytes is a potential therapeutic strategy for treating obesity and type 2 diabetes. Thus, a better understanding of the underlying mechanisms of thermogenesis provides novel therapeutic interventions for metabolic diseases. In this review, we summarize the recent advances in the molecular regulation of thermogenesis, focusing on transcription factors, epigenetic regulators, metabolites, and non-coding RNAs. We further discuss the intercellular and inter-organ crosstalk that regulate thermogenesis, considering the heterogeneity and complex tissue microenvironment of thermogenic fat. Obesity is a chronic and complex condition resulting from an imbalance of excessive energy intake and insufficient energy expenditure, and it is tightly associated with type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease (NAFLD), and other metabolic diseases . Adiposeuncoupling protein 1 (Ucp1), which uncouples oxidative respiration from ATP synthesis, resulting in energy dissipation as heat  forms a transcriptional complex with PRDM16 to induce brown fat cell determination and differentiation . Early Bdroplets . Interfedroplets . Claussndroplets . Loft etdroplets . Zinc fidroplets . Zfp423 droplets . Dempersat cells . Taken tAdipogenesis is involved with complicated epigenetic remodeling that mainly include histone modification and DNA methylation, the two fundamental processes that play crucial roles in the regulation of gene expression and genome stability. In general, Histone modifications modulate chromatin structure, influencing gene accessibility and transcriptional activity, while DNA methylation directly modifies the DNA sequence, leading to gene silencing. A lot of studies have demonstrated the roles of epigenetic modulators in regulating the formation and function of thermogenic adipocytes Table 2.PPARγ and then promote adipocyte differentiation and white adipocyte browning  and histone acetyltransferase p300 (P300), which catalyze histone acetylation of H3K27, improve the expression of browning . Generald Prdm16 .Ucp1 and Pgc1α to inhibit their expression  exert their influences on thermogenesis through deacetylation of H3K27ac . HDAC1 apression . Ferrariand Ucp1 . Howeverby HDAC3 . Bagchi by HDAC3 . Other hby HDAC3 , 81, 82. adipose . Shuai ed Prdm16 . Moreoved Prdm16 , were fod Prdm16 .transformation related protein 53 (Trp53), a repressor of thermogenic program   in order to activate thermogenic program and induce white adipocyte browning  . Similaric genes . Tateishipidemia . Pan et browning . Moreovepression . AltogetNon-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in the development and physiology of white, brown and beige adipocytes, and non-coding RNAs themselves can serve as markers of different adipocyte tissue depots Table 3.fibroblast growth factor 21 (Fgf21) expression through repressing the expression of transducer of ErbB-2.1 (Tob1), which further promotes white fat cell browning and BAT thermogenesis (fibronectin type III domain-containing protein 5 (Fndc5) expression   . MiR-30bogenesis . Ge et apression , while Fpression . MiR-106ipocytes . Giroud ipocytes . miR-133browning , 98. Micbrowning . MiR-193Runx1t1) , while FRunx1t1) . The detPgc1α’s mRNA elements  and increasing thermogenic genes expression  for 1 month exhibited increased brown adipocyte mass and activity in mice, since BBR promotes the DNA demethylation of pression . Dietarypression . Chlorogpression . Lone etpression , 126. Elpression . Epicatepression .Apart from the aforementioned nutrients and small molecules that regulate thermogenesis of brown and beige adipocytes, there are other metabolites performing the similar functions, including flavan-3-Alcohol, fucoxanthin, irisin, leptin, luteolin, Menthol Neuregulin 4 (Nrg4), Prostaglandin (PG), Purple Sweet Potato (PSP), Quercetin, Resveratrol, Rice Bran, Sesamol, Taurine, Telmisartan, and 3-Hydroxydaidzein –153, 155As extensively discussed in a recent review , thermog-/-Vegfr2 mice showed impaired angiogenesis as well as reduced browning of iWAT, which is modulated through the endothelial cells-derived platelet-derived growth factor-CC (PDGF-CC)-induced signaling pathway, since administration of PDGF-CC upregulated the expression level of Ucp1 and promoted browning of iWAT both in mice and humans  cells, lymphocytes, dendritic cells, neutrophils, eosinophils, T cells, and mast cells, which play an important role in regulating metabolic homeostasis , 178. ThUcp1 expression in iWAT of mice, mainly though the direct adhesion between α4-integrin in activated M1 macrophage and vascular cell adhesion molecule 1 (Vcam-1) in adipocytes -deficient or macrophage-specific interleukin-4 receptor α (Ilr4α) knockout mice exhibited impaired BAT thermogenic response, suggesting the positive role of M2 macrophages in BAT thermogenesis, which is further supported by the specific depletion of Ilr4α in myeloid cells of mice , another group of adipose resident immune cells, also activate M2 macrophage and regulate thermogenesis in brown and beige adipocytes . Activatrol mice . In a rerol mice , which srol mice , 194.Il-4 expression, and blocking IL4/IL13 signaling abrogates METRNL-induced browning of iWAT in mice  gene in adipocytes, and regulates the balance of energy via decreasing food intake and inducing energy expenditure  neurons in the hypothalamus suppress iWAT browning in mice . Mechani in mice . In contondition . UnderstFst is also induced in brown adipocytes in response to cold  is a circulating peptide hormone, which is mainly expressed in the liver in response to starvation or exercise and induced in BAT and WAT when fasted or exposed to cold environment both in mice and humans . FGF21 n to cold –228.On the other hand, brown adipocytes secrete batokines to regulate the functions of the liver. As discussed above, FGF21 mediate the bi-directional crosstalk between BAT and the liver in mice , 222. BeUcp1-/- mice exhibits decreased capacity to clear succinate from both the liver and the circulation, thus driving liver inflammation through the interaction with stellate cells and macrophages , a lipokine secreted from BAT when exposed to cold or exercise in mice and human, increases skeletal muscle fatty acid oxidation and uptake , 250. 12The gastrointestinal tract (GI tract) plays a very important role in thermogenesis through gut microbiota or directly secreting factors from intestinal cells . In a stBesides gut microbiota, the GI tract also secrete various factors to regulate thermogenesis. Secretin, secreted by the gut and upregulated during fasting, increases lipolysis and inhibits glucose uptake in mice . Li et ain vivo. For example, recent study has shown that thermogenesis could be induced through local hyperthermia therapy, mainly through the HSF1-A2B1 transcriptional axis (Understanding of the development route of thermogenic fat will provides novel therapeutic interventions for metabolic diseases. In this review, we discussed the regulatory network of thermogenic fat at the molecular and cellular levels, respectively. The molecular regulation of thermogenic fat mainly involves transcriptional regulation, epigenetic regulation, non-coding RNA regulation and metabolic reprogramming. Among these regulators, PPARγ, PRDM16 and PGC1α represent the core regulators, as most of the other regulators regulate the thermogenesis depending on them. Besides, thermogenic fat is also educated by other cell types within adipose depots or other organs. These complex and comprehensive regulatory networks help to maintain the functionality of thermogenic fat in response to kinds of changes of the environment. This holds a promising strategy for inducing artificial thermogenesis to counteract obesity nal axis . HoweverAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Here, we report the identification of ARE sequences in the promoter regions of genes encoding several epigenetic regulatory factors, such as histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and proteins involved in microRNA biogenesis. In this research, we study this possibility by integrating bioinformatic, genetic, pharmacological, and molecular approaches. We found ARE sequences in the promoter regions of genes encoding several HDACs, DNMTs, and proteins involved in miRNA biogenesis. We confirmed that NRF2 regulates the production of these genes by studying NRF2-deficient cells and cells treated with dimethyl fumarate (DMF), an inducer of the NRF2 signaling pathway. In addition, we found that NRF2 could be involved in the target RNA-dependent microRNA degradation (TDMD) of miR-155-5p through its interaction with Nfe2l2 mRNA. Our data indicate that NRF2 has an epigenetic regulatory function, complementing its traditional function and expanding the regulatory dimensions that should be considered when developing NRF2-centered therapeutic strategies.The epigenetic regulation of gene expression is a complex and tightly regulated process that defines cellular identity and is associated with health and disease processes. Oxidative stress is capable of inducing epigenetic modifications. The transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) is a master regulator of cellular homeostasis, regulating genes bearing  The cells of multicellular organisms have the same genetic content, yet their functions are diverse because they express different genes. These differences are due in large part to epigenetic mechanisms that lead to heritable and stable changes in gene expression programs that occur through alterations in chromatin structure. Epigenetic gene regulation provides an adaptive layer of control of gene expression to enable the organism to adjust to a varying environment by eliciting histone modifications, DNA methylation, and gene silencing via microRNAs (miRNAs) ,2. EpigeOxidative stress arises from an imbalance between reactive oxygen species and the cell’s antioxidant capacity, leading to an accumulation of ROS and a disruption of the epigenetic state of the cell. In turn, oxidative damage triggers epigenetic changes in the chromatin structure, histone modifications, DNA methylation, and DNA-binding proteins . One of However, until now, whether NRF2 influences epigenetic processes by modulating the expression of DNA methyltransferases (DNMTs), histones deacetylases (HDACs), and miRNAs has not been investigated. Here, we identify for the first time several HDACs, DNMTs, and genes encoding proteins involved in the biogenesis of miRNAs. Additionally, we present evidence that NRF2 could modulate levels of specific miRNAs through target-directed miRNA degradation (TDMD), further expanding the functional toolkit of NRF2.https://genome.ucsc.edu/cgi-bin/hgTables) (accessed on 3 February 2023) containing the locations of every transcript and its RefSeq accession number in the genome. To identify regulatory elements, a combined segmentation BED file was generated by concatenating Combined Segmentations [NFE2L2, MAFF, MAFK, MAFG, and BACH1 or their mouse orthologs were downloaded and intersected with the extended transcripts of HDAC1, HDAC2, HDAC3, SIRT1, DNMT1, DNMT3A, DNMT3B, DROSHA, DGCR8, DICER1, and TARBP2 genes or their mouse orthologs. Then, the sequences of the ChIP-seq peaks were extracted using pybedtools from the FASTA file of the hg19 human genome  and are previously described [−/−Keap1 and +/+Keap1 MEFs were provided by Dr. Ken Itoh . Mouse embryonic fibroblasts (MEFs) and mouse hippocampus-derived HT22 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 80 μg/mL gentamicin . All cell lines were mycoplasma-free, as ascertained through regular tests. Cells were changed to serum-free DMEM without antibiotics 16 h before the addition of dimethyl fumarate (DMF-20 μM) treatment .escribed ,25,26. K−ΔΔCT method, with normalization of the raw data based on the geometric mean of Actb and Gapdh (Applied Biosystems), encoding housekeeping proteins. All PCR amplifications were performed in triplicate.Total RNA extraction, reverse transcription, and quantitative polymerase chain reaction (qPCR) were performed as detailed in previous articles . Primer The expression vector pcDNA3.1/V5HisB-mNRF2ΔETGE was described in McMahon et al. . pEF-ΔNRTransient transfections of MEFs or HT22 cells and luciferase assays were performed as described in . pTK-Ren3VO4, 1 mM NaF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin). Whole-cell lysates were loaded for SDS-PAGE. Immunoblots were performed as described in [Whole-cell lysates were prepared in RIPA-Buffer  and Nfe2l2 (eleven ASOs). Incubation of whole-cell lysates with biotinylated ASOs was followed by incubation with Streptavidin-coupled Dynabeads™ . RNAs were isolated from the pull-down materials, and qPCR was performed. Briefly, whole-cell lysates were prepared using RIPA buffer with a cocktail of protease and phosphatase inhibitors . One milligram of whole-cell extract was incubated with 1 μg of either Nfe2l2 ASO or control LacZ ASO for 16 h at 4 °C, whereupon RNA complexes were isolated with M-280 Streptavidin Dynabeads (Invitrogen) for 2 h at 25 °C. Total RNA was isolated with Trizol-Choloroform and cDNAs were synthesized with the Maxima Reverse Transcription kit following the manufacturer’s protocols ; real-time quantitative (q)PCR analysis was then performed using a PCR kit according to manufacturer’s instructions , and the mRNA abundance was calculated via the 2−ΔΔCT analysis method, using Gapdh mRNA levels as the control transcript for normalization. miRs were reverse-transcribed using the MiR-X kit  and quantified via qPCR analysis using U6 as the normalization control RNA.To identify ® Green PCR Kit . We employed hsa-miR-128-3p miRCURY LNA (YP00205995), mmu-miR-155-5p miRCURY LNA (YP02119303), and miR-103a-3p miRCURY LNA (YP00204063) PCR Assays . Data analyses were based on the ΔΔCT method, with normalization of the raw data based on UniSp6 . All PCR amplifications were performed from at least triplicate samples.Total RNA extraction was performed using the miRNeasy Mini Kit . For reverse transcription and qPCR, we used miRCURY LNA SYBRt-tests. A one-way ANOVA with post-hoc Newman–Keuls test was used.Data are presented as the mean ± SEM. To determine the statistical test to be used, we employed GraphPad Instat 3, which includes the analysis of the data to a normal distribution via the Kolmogorov–Smirnov test. In addition, statistical assessments of differences between groups were analyzed  by performing an unpaired Student’s The epigenome is comprised of modifications to chromatin, including histone modifications and DNA methylation. One of the main histone modifications is acetylation, which is often a necessary precursor to other modifications, such as phosphorylation, methylation, and ubiquitylation. Acetylation is controlled by the opposing functions of two families of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are involved in key biological functions, such as transcription, cell cycle, autophagy, DNA damage repair, stress responses, and senescence ,29. HDACHDAC1, HDAC2, HDAC3, SIRT1, DNMT1, DNMT3a, and DNMT3b.First, to define comprehensively the role of NRF2 in the transcriptional regulation of HDACs and DNMTs, we searched for putative ARE sequences in the ChIP-Atlas database, an integrative database covering almost all public data archived in the Sequence Read Archive of NCBI, EBI, and DDBJ, using ChIP-seq data  of the hMAFG, MAFF, MAFK, BACH1, and NFE2L2. We used Python-based bioinformatic analysis to scan this binding region for the consensus ARE, as established in the JASPAR database [TGACNNNGCN) described by [The ChIP-Atlas database includes experimental data from chromatin immunoprecipitation (ChIP) analysis of the ARE-binding transcription factors database ,35. Depedatabase ,37. Thesribed by . As show+/+Nfe2l2 and −/−Nfe2l2 mice  derived from −/− mice A. These −/− mice B, furthe−/− mice . We trea−/− mice C. These −/− mice D. Taken Dnmt1, Dnmt3a, and Dnmt3b mRNAs in NRF2-deficient MEFs. We observed that the levels of Dnmt1 and Dnmt3b mRNAs were significantly reduced, while the changes in Dnmt3a mRNA were more modest (p = 0.07)  A. These  = 0.07) B. We the = 0.07) C,D; furtBesides histone acetylation and DNA methylation, miRNAs  reduce the stability and translation of target mRNAs and are also epigenetic regulators. ROS can modulate miRNA biogenesis at many levels, and several enzymes and components of the miRNA processing machinery can be affected by ROS . TherefoDGCR8, DROSHA, DICER1, and TARBP2 genes. We observed that, especially in the human genome  of messenger RNAs (mRNAs) govern their stability, localization, and expression . miRNAs Nfe2l2 mRNA, by using the Targetscan (119 miRNAs) and miR DB (55 miRNAs) databases (Nfe2l2 mRNA (NM_010902.4) were designed in order to pull down the endogenous Nfe2l2 mRNA. Whole-cell lysates from HT22 cells were then prepared and incubated with either Nfe2l2 ASOs or control LacZ ASO, whereupon RNA complexes were isolated with streptavidin Dynabeads (Invitrogen). As shown in Nfe2l2 mRNA was highly enriched in Nfe2l2 ASO samples compared to that in LacZ ASO samples. We then measured the levels of all the predicted miRNAs  and SIRT1, DNMTs, and proteins involved in miRNA biogenesis, DROSHA, DGCR8, DICER1, and TARBP2. Our data further suggest that NRF2 may be involved in the regulation of the levels of certain miRNAs through TDMD. These new data are of great potential relevance to the pharmacological regulation of NRF2 as a therapeutic strategy for various pathologies since epigenetic changes triggered by NRF2 will also have to be weighed.NRF2 is a pleiotropic transcription factor capable of regulating the expression of genes involved in different processes, including xenobiotic, redox, and carbohydrate metabolism, inflammation, and proteostasis  Figure . TherefoOur studies suggest that when NRF2 levels are decreased in pathologies, this reduction may lead to decreased expression of Type-I HDACs and increased expression of genes that were repressed under physiologic conditions. Similarly, NRF2 deficiency would also lower DNMT levels, in turn inducing the expression of genes that were previously repressed. On the other hand, NRF2 inducers, such as sulforaphane, were found to induce NRF2 regulation at the epigenetic level, mainly associated with DNA methylation . Epigene, and neurodegenerative diseases [NFE2L2 promoter have been documented [NFE2L2, reduced the translation of NRF2, and increased the levels of intracellular ROS [The results presented in this study can be potentially relevant to a wide range of pathologies where epigenetic mechanisms are of particular importance, such as cancer ,53, allediseases ,56. In ccumented ,57,58, wcumented . Along tcumented . In the ular ROS . Howeverular ROS . Therefoular ROS .Outside of cancer, type I and II HDAC inhibition mediated by Trichostatin A (TSA) activated transcription factor NRF2 and protected against cerebral ischemic damage. On the other hand, SIRT1 activation was also found to induce the NRF2 signaling pathway, with beneficial effects in focal cerebral ischemia , indicatNfe2l2  or have a more general functions, as well as whether there are differences between the various brain cell types.Oxidative stress is one of the main inducers of the NRF2 pathway, and thus, its activation is linked to the induction of oxidative stress-associated miRNAs, the so-called “redoximiRs”, such as miR-27a-3p and miR-155-5p ,78. In H2l2 mRNA B. There 2l2 mRNA B. Here, icroglia ,84. Furticroglia , supportThis study demonstrates for the first time that the molecules involved in epigenetic gene regulation, namely HDACs, DNMTs, and miRNA biogenesis factors, are under the control by NRF2, expanding the field of action of this transcription factor.