Pediatric acute myeloid leukemia is a clonal disorder characterized by malignant transformation of the hematopoietic stem cell. The incidence and the outcome remain inferior when compared to pediatric ALL, although prognosis has improved in the last decades, with 80% overall survival rate reported in some studies. The standard therapeutic approach is a combined cytarabine and anthracycline-based regimen followed by consolidation with allogeneic stem cell transplantation  for high-risk AML and allo-SCT for non-high-risk patients only in second complete remission after relapse. In the last decade, several drugs have been used in clinical trials to improve outcomes in pediatric AML treatment. Pediatric acute myeloid leukemia (AML) is a heterogeneous clonal disorder characterized by malignant transformation of the hematopoietic stem cell (HSC). Pediatric AML is less common than acute lymphoblastic leukemia (ALL); while ALL accounts for 80% of all pediatric acute leukemias, AML barely reaches 15–20%. Pediatric AML has a bimodal age distribution, with higher incidence in patients younger than 2 years and a second peak in adolescents up to 10 to 20 years old [Pediatric AML is generally characterized by poorer prognosis compared to ALL although outcomes have improved in the recent years. The 3-year overall survival (OS) is 70% or even higher ,3,4. NotWhile our current understanding of pediatric AML may be influenced by adult studies, substantial differences in the mutational landscape and tolerance to treatment have emerged between children and adults .The realUnderstanding the biology of pediatric-AML-specific gene mutations may enable new and more specific up-front treatment approaches in order to increase the rates and the duration of the remission and to guarantee more therapeutic options in case of relapse. The aim of our review is to analyze the efficacy and safety of new therapeutic agents already approved for adult AML and translate them for application to pediatric high-risk AML, focusing on molecular pathways and potential therapeutic indications.While the majority of pediatric AML occurs de novo, there is an increased incidence of AML reported in certain hereditary disorders such as Fanconi anemia, Kostmann syndrome, Shwachman–Diamond syndrome, and Diamond–Blackfan anemia, which may be related to alterations of DNA repair or detoxification of reactive oxygen species genes to pathogenetic variants in telomere biology genes or ribosome function .ChildrenAML is derived from a clonal proliferation and the abnormal differentiation of myeloid stem cells as a consequence of two sequential genetic events. This process is known as the two-hit model of leukemogenesis . In AML,The WHO classification for hematological malignancies, revised in 2016, integrated genetic characteristics, such as karyotypes and molecular aberrations, with morphology, immunophenotype, and clinical presentation, but has limited application in children, since cytogenetic and genetic abnormalities are uncommon as compared to adult AML . As suchNew discoveries in AML genetic alterations were applied to pediatric patients, improving risk stratification and therapeutic approaches. The TARGET project, an analysis of the molecular aberrations in pediatric AML, showed that the mutation rate is lower in pediatric than in adult AML, with different somatic aberrations including structural changes, aberrant DNA methylation, and specific pediatric mutations . In partFusion genes specifically identified in pediatric AML and associated with a grim prognosis include CBFA2T3-GLS2 and NUP98-NSD1.CBFA2T3-GLS2 is a chimeric transcript derived from a cryptic inversion of the telomeric region of chromosome 16 and expressed especially in non-DS FAB M7 AML. This gene mutation is present in patients younger than 5 years old and is characterized by high bone marrow blast count and extra-medullary involvement . In contDiagnostic approaches in pediatric AML may be influenced by experience in adults . TreatmeFLT3 mutation is also associated with dismal prognosis in adult AML  and remaFew changes were observed over the last forty years in the treatment algorithm of both pediatric and adult AML. The standard therapeutic approach is a combined cytarabine and anthracycline-based regimen followed by consolidation with allogeneic stem cell transplantation  for high-risk AML and allo-SCT for non-high-risk patients only in second CR after relapse ,33.In thPhysiological gene expression is regulated by epigenetic chromatin modifications, such as DNA methylation and histone acetylation ,35. AlteThe second retrospective study was are port of eight children and young adults with a median age of 4 years with r/r AML that evaluated the efficacy of low-dose decitabine. Three of eight patients reached a complete response, with the best responses observed after a median of 2.5 cycles (range 1–4 cycles). In this study, there were no severe side effects, except for neutropenia. An episode of febrile neutropenia occurred, but the overall infection rate was low . Moreoven = 12; ALL n = 2) and showed promising results in terms of a high complete remission rate  [The first pediatric phase I study evaluating azacytidine combined with intensive fludarabine plus cytarabine-based chemotherapy(NCT01861002) included14 patients with r/r acute leukemia  .A phase II trial in patients younger than 21 years with de novo AML to evaluate the eventual improvement in OS and event-free survival (EFS) (NCT03164057) through epigenetic priming with DNMT inhibitors prior to chemotherapy is currently ongoing. Decitabine was also investigated in a randomized two-arm phase II clinical trial (NCT01177540) comparing standard chemotherapy with or without decitabine in patients with AML aged1–16 years. Although there was a good tolerability profile, there was no difference in CR rate between the two arms .Treatment in vitro with the histone deacetylation (HDAC) inhibitors, vorinostat and panobinostat, was associated with the activation of tumor-suppressor genes and cancer cell death . In adulInteresting results were reported from a phase I trial (NCT02676323) evaluating the safety, pharmacokinetics, and pharmacodynamics of panobinostat in 17 pediatric patients with relapsed AML; 8 out of 17 patients (47%) achieved CR and 6 (75%) achieved undetectable MRD status .The efficacy of vorinostat in pediatric relapsed AML is currently being evaluated in an ongoing clinical trial (NCT03263936); the results represent the first step in assessing of the utility of an HDAC inhibitor in pediatric patients.Epigenetic treatment should be considered for a special group of pediatric AML patients carrying MLL translocations. The MLL fusion protein recruits a histone methyl transferase (HMT), DOT1L, resulting in the aberrant methylation of MLL gene targets and enhanced expression of leukemia-associated genes . Since tPreclinical studies demonstrated that the addition of pinometostat to conventional chemotherapy might sensitize MLL-associated leukemia cells to chemotherapy, by way of DOT1L inhibition. Nevertheless, a phase I trial of pinometostat, a small-molecule DOT1L inhibitor, conducted in children with r/r MLL-associated AML (NCT02141828) demonstrated only a transient reduction in leukemic blast counts . A phaseImmune-based therapy is a promising approach to overcome resistance to conventional chemotherapy in AML. Different types of immunotherapies are being evaluated in AML, including monoclonal antibodies, T-cell therapy, and cancer vaccines .In pediatric AML, the most relevant immune-based therapy approach consists of targeting surface antigens, particularly CD33  and CD123 (IL3Rα), both highly expressed, although not exclusively, by AML cells .Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody conjugated to a cytotoxic agent, calicheamicin, with a potent antitumor effect against CD33-expressing cells . InitialIn adult high-risk AML patients, no benefit was observed in clinical trials combining GO with conventional therapy ; howeverCD123, the IL-3 receptor α-chain, is also expressed on the surface of AML blasts in the majority of cases . CD123-tFurthermore, PD-1 inhibition was also evaluated in adult patients with relapse after HSCT to restore donor chimerism .Very few studies evaluating checkpoint inhibitors in pediatric AML are currently available; a phase I/II (NCT03825367) trial is currently evaluating the efficacy of nivolumab combined with azacytidine in pediatric r/r AML.In contrast to lymphoid neoplasms, where different surface antigens such as CD19, CD22, and recently, CD20 and CD23 were targeted for cellular therapy chimeric antigen receptor T (CAR-T) cells leading to very good results, this treatment strategy has not yet been successfully applied to treatment for patients with AML . The maiSeveral phase I trials focused on the clinical evaluation of CD33 CAR-T cells in adults with r/r AML, and there have been a number of case reports and pilot studies showing the use of CAR-T cells in AML . A phaseTrials evaluating the efficacy of anti-CD123 CAR-T are currently open or have recently been completed for patients with r/r AML. In pediatrics, two phase I trials (NCT02159495 and NCT04318678) are currently evaluating the efficacy of CD123-directed CAR-T cells in r/r AML patients older than 12 years up to 21 years of age .CPX-351, a liposomal combination of daunorubicin and cytarabine in a 5:1 molar ratio, is approved by the EMA for the treatment of adults with newly diagnosed, therapy-related AML (t-AML) or AML with MDS-related changes. The efficacy and safety of CPX-351 in children was established in the last year, and in March 2021, the FDA approved a revised label for CPX-351 to treat newly diagnosed t-AML or AML with MDS-related changes in pediatric patients aged 1 year and older.Two different clinical trials, theCPX-MA-1201 trial (NCT01943682) and AAML 1421 (NCT02642965), explored the effectiveness of CPX-351 in pediatric AML. CPX-MA-1201, a phase I study of CPX-351 monotherapy, reported CR in two patients and one undetectable MRD among six treated pediatric patients with r/r AML. The AAML 1421 (NCT02642965), a phase I/II evaluating the effectiveness of CPX-351 combined with fludarabine, cytarabine, and granulocyte colony-stimulating factor(FLAG) in 39 pediatric patients in first relapse, reported CR in 20 (54%) patients, 5 CR with partial recovery of platelet count , and 5 CR with incomplete blood count recovery (14%) with no differences in terms of age-related toxicity profile for the drug .FurthermC-KIT (CD117), a tyrosine kinase receptor expressed by hematopoietic progenitors , plays aA Chinese trial (NCT03173612) is currently assessing efficacy of dasatinibin CBF pediatric AML harboring c-kit mutation, but the results are still pending.The Ras proteins H, K, and N are small GTPases involved in cellular signaling pathways, such as MAPK and PI3K/mTOR, that drive cell proliferation, differentiation, and growth. Ras may be a potential therapeutic target . CompounFMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase receptor expressed by immature hematopoietic cells but is lost during cellular differentiation and plays a critical role in stem cell proliferation, survival, and differentiation .In pediatric AML, the incidence of internal tandem duplication(ITD) and tyrosine kinase domain (TKD) FLT3 mutations are similar, including the pediatric-specific FLT3-TKD mutations subgroup. FLT3-ITD mutations are associated with a dismal outcome . While iFirst-generation FLT3 inhibitors midostaurin, sorafenib, lestaurtinib, and sunitinib are promiscuous multi-kinase inhibitors that show activity not only against FLT3 but also against other kinases, such as KIT, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor (VEGF), leading to increased toxicity associated with off-target inhibition .p = 0.078) and 91% vs. 70% (p = 0.007) in two subgroups, respectively. EFS rates at 3 years were 57.5% vs. 34.3% (p = 0.007), but no significant differences were observed in 3-year OS between the two treatment arms , probably due to the initiation of TKI therapy after withdrawal from protocol therapy or at time of relapse [Midostaurin has been extensively tested in adult AML. Based on results of the RATIFY trial, it was approved in adults with de novo FLT3-mutated AML combined with induction chemotherapy . To date relapse .Interestingly, lestaurtinib was evaluated in the AAML06P1 trial, but this phase I/II study was closed before efficacy could be assessed . More poIn adults, gilteritinib, the newest third-generation FLT3 inhibitor, has become the new standard of care for management of r/r FLT3-mutated AML, based upon greater efficacy and less toxicity compared to standard chemotherapy alone .Two ongoCrenolanib is a potent and selective inhibitor of FLT3, PDGFRα/β, and KIT, showing encouraging activity in adult AML. In pediatric FLT3-mutated hematologic malignancies, NCT02270788, a phase I trial, is currently open to evaluate feasibility combined with sorafenib.Another group of molecular aberrations of increasing interest in myeloid malignancies are activating mutations of Janus kinases  . RuxolitBased on these results, future clinical studies will clarify the role of target agents combined with standard chemotherapy. One of these is currently ongoing and will evaluate the efficacy and feasibility of combination chemotherapy with target agents according to the result of targeted deep sequencing in pediatric patients with r/r solid tumor or AML (NCT02638428).KIPI and p53 [The proteasome is a large protein complex responsible for the degradation of most cellular proteins under physiological conditions, regulating cellular processes, such as cell survival and signaling . One of  and p53 ,117,118. and p53 . Indeed,p =0.236), and the 3-year OS was 63.6% ± 4.5% vs.67.2% ± 4.3% (p =0.356), respectively. In addition, subgroup analyses by risk group stratification showed similar outcomes for both low- and high-risk patients. In particular, the 3-yearDFS and OS for low-risk patients were 52.9% ± 3.7% and 74.1% ± 3.4%, respectively, while for high-risk AML they were 27.8% ± 6.6% and 36.9% ± 7.6%, respectively [Based on this, the Children’s Oncology Group developed the phase III AAML1031 trial to evaluate whether the addition of bortezomib to standard chemotherapy improved survival in pediatric patients with newly diagnosed AML . All patectively .Neddylation involves post-translational enzymatic conjugation of an ubiquitin-like protein, NEDD8 , to lysine residues on the target protein. Enzymatic cascades in this process involve NEDD8-activating enzyme E1 (NAE), NEDD8-conjuagating enzyme E2, and substrate-specific NEDD8-E3 ligases. This process regulates tumor-suppressor and oncoprotein activity and is dysregulated in several malignancies, including AML, making it an attractive therapeutic target .In partiPevonedistat (MLN4924) is a novel NAE inhibitor that blocks the first step of the neddylation cascade and has single-agent anti-proliferative activity in adult r/r AML .There is an ongoing phase I trial (NCT03813147)to evaluate the side effects and efficacy of pevonedistat combined with azacytidine, fludarabine phosphate, and cytarabine in patients up to 21 years with r/r AML and MDS.TP53 aberrations are rare in pediatric AML . AlternaCurrently, a phase I study (NCT03654716) is evaluating the efficacy of the dual MDM2/MDMX inhibitor, ALRN-6924, as a possible treatment for refractory solid tumor, brain tumor, lymphoma, or leukemia  in pediatric patients and young adults (aged from 1 to 21 years old).Venetoclax is a highly selective oral inhibitor of the B-cell lymphoma-2 (BCL-2) protein, an anti-apoptotic member of the BCL-2 family of proteins also including BCL-XL, MCL1 (myeloid cell leukemia sequence 1). Overexpression of BCL-2 and other anti-apoptotic proteins has been documented in different type of malignancies, including AML ,130.Venetoclax was approved by both the FDA and the EMA combined with azacytidine, decitabine, or low-dose cytarabine for de novo AML in adults 75 years old or who have comorbidities precluding intensive induction chemotherapy, based on a CR rate of 70% .To date, there are limited data on the efficacy of venetoclax in pediatric AML and MDS, but many trials are ongoing to evaluate the efficacy and possible toxicity in this population .In the single-center experience of the Children’s Hospital of Colorado, venetoclax was administered in combination with azacytidine to pediatric r/r AML or high-risk MDS, showing good responses with three CR. Although a larger patient cohort is needed to confirm the safety and efficacy of venetoclax-based regimens for pediatric AML, this report demonstrated promising rates of remission induction, also in patients who are refractory to cytotoxic chemotherapy .CBFβ/MYH11, RUNX1-RUNX1T1, and CEBPA, while no benefit was reported for FLT3-AML [Moreover, Karol et al. reported exciting results of a phase I trial combining venetoclax with low- and high-intensity chemotherapy in 38 patients aged 2–22 years with r/r AML or acute leukemia of ambiguous lineage: a CR rate of 70% and overall response rate (ORR) of 80% was reported among 20 treated patients. As noted by the authors, this response rate is consistent with observations made by the two major studies in relapsed AML from the European and American pediatric oncology cooperative groups. Additionally, favorable outcomes with venetoclax were reported for genetic subtypes of pediatric AML including mutated FLT3-AML .IDH1/2 mutations occur in approximately 2–4% of pediatric AML, mostly in adolescents. Mutations in IDH1/2 impair cellular differentiation, generating high levels of 2-hydroxyglutarate that inhibit various components of the epigenetic machinery. Currently, NCT02813135 is ongoing to evaluate the activity of enasidenib as a single agent in10pediatric r/r IDH2-mutated AML cases. Moreover, a phase II trial, NCT04203316, is investigating the safety of enasidenib in pediatric r/r AML. No clinical trials with ivosidenib are open in pediatric AML, but NCT04195555 studies the efficacy of ivosidenib in treating pediatric patients with solid tumors or histiocytic disorders with IDH1 genetic mutations ,137.Menin inhibitors are novel, targeted agents currently in clinical development. Menin has a tumor-suppressor function in endocrine glands and is critical for leukemogenesis in a subgroup of mixed-lineage leukemia (MLL), driven by the rearrangement of the KMT2A gene, which encodes an epigenetic modifier. Menin inhibitors prevent the binding between MLL and menin, halting the oncogenic function of the KMT2A complex. The first trial in humans is NCT04065399, a phase I-II trial, which analyzes the efficacy and dose-limiting toxicities of a small molecule, SNDX-5613, in adults and children with r/r acute leukemia. Fifty-four patients recruited had a median of three prior therapies, and 45 patients were NMP1 or KMT2A mutated. The trial has demonstrated that the composite CR rate in the last subgroup of AML was 44% (20/45 patients) ,139.To date, the gold standard in the treatment of pediatric patients with AML is multiagent chemotherapy. For pediatric patients with high-risk features and those who relapse, allo-SCT is standard in first- and second-CR, respectively. Clinical trials are critical for advancing the treatment of pediatric patients with AML. Advances in cancer genomics and research in stem cell biology are providing new information about leukemogenesis and pharmacogenetics. Potential therapeutic approaches are advancing personalized treatment strategies, which may modify the clinical course of this pathology.


The novel sodium-glucose co-transporter 2 inhibitor (SGLT2i) potentially ameliorates heart failure and reduces cardiac arrhythmia. Cardiac fibrosis plays a pivotal role in the pathophysiology of HF and atrial myopathy, but the effect of SGLT2i on fibrogenesis remains to be elucidated. This study investigated whether SGLT2i directly modulates fibroblast activities and its underlying mechanisms.2+ fluorescence imaging, and Western blotting were applied to human atrial fibroblasts. Empagliflozin  reduced migration capability and collagen type I, and III production. Compared with control cells, empagliflozin (1 μmol/L)- treated atrial fibroblasts exhibited lower endoplasmic reticulum (ER) Ca2+ leakage, Ca2+ entry, inositol trisphosphate (IP3), lower expression of phosphorylated phospholipase C (PLC), and lower intracellular pH. In the presence of cariporide (an Na+-H+ exchanger (NHE) inhibitor, 10 μmol/L), control and empagliflozin (1 μmol/L)-treated atrial fibroblasts revealed similar intracellular pH, ER Ca2+ leakage, Ca2+ entry, phosphorylated PLC, pro-collagen type I, type III protein expression, and migration capability. Moreover, empagliflozin  significantly increased left ventricle systolic function, ß-hydroxybutyrate and decreased atrial fibrosis, in isoproterenol -induced HF rats.Migration, proliferation analyses, intracellular pH assay, intracellular inositol triphosphate (IP3) assay, Ca2+ release, extracellular Ca2+ entry and the profibrotic activities of atrial fibroblasts.By inhibiting NHE, empagliflozin decreases the expression of phosphorylated PLC and IP3 production, thereby reducing ER CaThe online version contains supplementary material available at 10.1186/s12933-023-01756-0. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are novel class of anti-diabetic agents that reduce the risk of cardiovascular death and hospitalization in patients with heart failure (HF) and type 2 diabetes , 2. SGLT2+) signaling pathway plays a critical role in fibrogenesis and induces proliferation, collagen production, migration, and myofibroblast differentiation capabilities of fibroblasts [+ binding site of the Na+/H+ exchanger (NHE) thereby decreasing NHE activity and lowering the intracellular pH [The calcium (Caroblasts –16. SGLTlular pH .2+ through endoplasmic reticulum (ER) Ca2+ leakage or Ca2+ influx [Increasing intracellular pH has been proven to induce cytosolic Ca+ influx , 19. NHE+ influx , 21 and + influx –24. Incr+ influx . Dapagli+ influx . Accordi2. Cells from passages 4 to 6 were used to avoid possible variations in cellular function. The SGLT2 was shown to be present in the cells by western blot . The fibroblasts were seeded on uncoated culture dishes as monolayers in FGM™-3 Cardiac Fibroblast Growth Medium-3 BulletKit  at 37 °C with 5% COMigration of atrial fibroblasts was studied using a wound-healing assay. Briefly, cells were plated in 6-well plates and treated with empagliflozin  or NHE inhibitor  for 48 h in serum free culture medium for 72 h. Six hours before the end of the treatment, cells were scraped using the tip of a P200 pipette tip. Each area of the gap was assessed using Image J 1.45 s software . The net migration area after 6 h was subtracted from that at the time of the initial scratch.Atrial fibroblast proliferation was measured using a commercial MTS kit  as previously described . In brieWestern blotting was performed as described previously . Atrial +.Intracellular pH was calculated using a Cell Meter Fluorometric Intracellular pH Assay Kit  following the manufacturer’s instructions. In brief, atrial fibroblasts were seeded onto a 96-well culture black plate at a density of 3000 cells/well. After growing to confluence, the cells were incubated with empagliflozin (1 μmol/L) or cariporide (10 μmol/L) for 6 h. The cells were loaded with pH-sensitive cell-permeable fluorescent dye 20,70-biscarboxyethyl-5,6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) in Hanks’ buffer with 20 mM HEPES and 4.17 mM sodium bicarbonate for 1 h at 37 °C and 5% CO2, in dark. After subsequent washing with phosphate-buffered saline (PBS), fluorescence was measured at excitation/emission wavelengths (Ex/Em) of 505/535 nm and 430/535 nm on a SpectraMax M2 fluorimeter . The ratio of fluorescence at 505/535 nm and 430/535 nm was converted to a pH unit with Spexyte Intracellular pH Calibration Buffer Kit (AAT Bioquest). Atrial fibroblasts loaded with BCECF-AM were incubated with a range of calibration buffer (pH 4.5 − 8.0) at 37 °C for 10 min with nigericin (10 mmol/L), a proton ionophore that can modulate the intracellular pH with external pH in the presence of 100 − 150 mmol/L K2+ was measured with a ratiometric Ca2+ indicator Fura-2 using a fluorescence plate reader as described previously [3 cells/well, after incubation overnight, the cells were treated with or without empagliflozin (1 μmol/L) or cariporide (10 μmol/L) for 48 h. Cells were then stained with 5 μmol/L Fura-2 acetoxymethyl ester  in a Ca2+-free solution with (in mmol/L) KH2PO4 1.2, NaCl 120, MgSO4 1.2, KCl 5.4, HEPES 6, glucose 10 (pH 7.40) for 30 min at 37 °C in a 5% CO2 incubator. Measurements of intracellular Ca2+ were performed every 2 s with a fast switching of the excitation wavelengths 340 and 380 nm and a constant emission wavelength of 510 nm using a CLARIOstar PLUS Microplate Reader  equipped with two injectors and analyzed with a CLARIOstar MARS software (BMG Lab Technologies). The intracellular Ca2+ concentration in each well was expressed as the fluorescence ratio of F340/F380 and the changes of baseline to peak calcium amplitude as well as the area under curve (AUC) of Ca2+ tracing were calculated. The decay time of Ca2+ entry (T50) was calculated from the peak to the 50% of the decay.The intracellular Caeviously . Atrial 2+ was recorded for 2 min in Ca2+-free buffer, followed by co-treatment with the ER Ca-ATPase inhibitor  for ER Ca2+ store depletion. After the intracellular Ca2+ surge from the thapsigargin-induced ER Ca2+ leak returned to a steady state, the extracellular Ca2+ concentration was increased to 2 mmol/L to measure Ca2+ entry. The change in intracellular Ca2+ (∆ F340/F380) from the steady state of extracellular Ca2+-free to the plateau state of thapsigargin cotreatment was defined as the amount of ER Ca2+ depletion and the change from the steady state of post-ER Ca2+-induced intracellular Ca2+ surge to the plateau state under 2 mmol/L Ca2+ solution was used to represent Ca2+ entry.Baseline intracellular CaCell lysates of atrial fibroblasts treated with or without empagliflozin (1 μmol/L), or cariporide (10 μmol/L) were assayed for IP3 production using a human IP3 ELISA kit  according to the manufacturer’s instructions. Protein concentrations from the cell lysate of each treatment were used for normalization.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the local animal ethics review board (LAC-2021-0223). Animal studies are reported in compliance with the ARRIVE guidelines [HF induction was conducted as described previously . Male Wiidelines  and withidelines .Echocardiography was performed before euthanasia. Rats were sedated with 2% isoflurane (in oxygen), placed in the left lateral decubitus position, and scanned using a commercially available echo scanner  using a 10S phased array pediatric transducer and a cardiac application with high temporal and spatial resolutions. The transmission frequency was 10 MHz; the depth 2.5 cm; and the frame rate 225 frames/s. LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV posterior wall thickness (LVPW), and heart rate (HR) were measured. The fractional shortening (%) was measured as (LVEDD-LVESD)/LVEDD × 100.Blood serum was collected before euthanasia, and assayed for ß-OH using a ß-OH fluorometric assay kit  according to the manufacturer’s instructions.Atrial fibrosis analysis was performed per a previously described method with modification . In brieP < 0.05 was considered statistically significant.All quantitative data are expressed as mean ± standard error of the mean. A paired or unpaired t–test for normal distribution, Mann–Whitney rank-sum test for non-normal distribution, and one–way ANOVA or repeated-measures ANOVA or ANOVA on ranks with a post hoc Fisher’s least significant difference (LSD) test were used to compare atrial fibroblasts under different conditions. A Compared with control cells, empagliflozin (1 or 5 μmol/L)-treated atrial fibroblasts exhibited lower migration capability, and lower pro-collagen type I and III protein expression in a dose-dependent manner -treated atrial fibroblasts exhibited lower thapsigargin-induced ER Ca2+ leakage, extracellular Ca2+ entry, and T50 during Ca2+ entry phase  atrial fibroblasts exhibited lower intracellular pH but had a similar expression of NHE1 protein Fig. . In the As shown in Fig. Serum levels of ß-OH revealed that isoproterenol-treated rats receiving empagliflozin have higher serum β-hydroxybutyrate than HF treated with vehicles and healthy control rats before euthanasia Fig. B. Masson2+ homeostasis by inhibiting NHE activity in human atrial fibroblasts, thereby reducing their pro-fibrotic cellular activities. Cardiomyocytes with NHE overexpression had a higher intracellular pH. Mice with NHE overexpression exhibited HF and cardiac fibrosis, which can be improved by the NHE1 inhibitor cariporide [Empagliflozin has been demonstrated to improve cardiac fibrosis in various HF experimental models , but theriporide . In thisriporide . In addi2+ release or Ca2+ influx [+ and Ca2+ in ventricular myocytes [Elevated intracellular pH activates the PLC/IP3 receptor signaling pathway, thereby inducing ER Ca+ influx . Pro-fib+ influx . In the + influx . Empaglimyocytes . In the 2+ content reduction in diabetic myocytes [2+ leakage [2+ leakage. Inhibition of ER Ca2+ leakage interrupts the downstream signaling of pro-fibrotic cytokine [2+ release, suggesting that it downregulated the PLC/IP3/ER Ca2+ release signaling pathway by decreasing intracellular pH through the inhibition of the NHE, leading to decreased collagen production. Ca2+-dependent activation of PLC has been proven in various cells [2+ solution increases PLC activity whereas perfusion with a Ca2+ channel blocker decreases PLC activity [+ by directly docking at the late sodium channel (Nav1.5), inhibiting NHE influx, and reverse mode of NCX thereby decreasing intracellular Ca2+ content in cardiomyocytes [2+. Hence, the effect of empagliflozin on lowering PLC activity may also be caused by the decreased cytosolic Ca2+ in empagliflozin-treated atrial fibroblasts. The gateways of Ca2+ entry include Orai channels, Transient receptor potential (TRP) channels, voltage-operated Ca2+ channels, or NCX. Attenuated Orai channel signaling reduces the collagen production capabilities of atrial fibroblasts [2+ can activate Orai channels [2+ overload through the L-type Ca2+ channel (a kind of voltage-operated Ca2+ channel) in cardiomyocytes [2+ emptying, PLC activity, and extracellular Ca2+ entry. Besides, cariporide with and without empagliflozin reduced ER Ca2+ leakage, and extracellular Ca2+ entry of atrial fibroblasts to a similar extent, suggesting that empagliflozin may decrease suggesting that empagliflozin decreased the cytosolic Ca2+ through inhibition of NHE signaling pathway.Our previous study found that empagliflozin attenuated sarcoplasmic reticulum (SR) Camyocytes . Besides leakage , suggestcytokine  The presus cells , 42. Peractivity . SGLT2i myocytes , 44, 45.roblasts . Transforoblasts . The empchannels , 49. TRPchannels , 51. A pmyocytes . In the 2+ extrusion can be conducted by Ca2+ efflux through the plasma membrane ATPases (PMCAs) and forward mode Na+/Ca2+ exchangers on the cell membrane, or by pumping Ca2+ back to ER through SR Ca2+-ATPase (SERCA) on the ER membrane. Increasing intracellular pH has been proven to inhibit the ER Ca2+ reuptake capability of SERCA [2+ entry phase. Besides, cariporide with and without empagliflozin shortened the decay time of the Ca2+ entry phase of atrial fibroblasts to a similar extent. Hence, empagliflozin might enhance SERCA function and increase the ER Ca2+ reuptake thereby shortening the decay time of the Ca2+ entry phase of atrial fibroblasts via its effect on decreasing intracellular pH.Cytoplasmic Caof SERCA . In the 2+ and decrease the cardiac contraction. However, previous studies have shown that empagliflozin attenuated HF and increased Ca2+ content in diabetic cardiomyocytes, which were supposed to arise from its enhancing effects on SERCA expression and L-type calcium currents [2+ content in cardiomyocytes). Isoproterenol activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling and induces aberrant Ca2+ spark frequency, thereby inducing arrhythmogenesis in failing heart [2+ channels [+ channels [+ channels [2+ spark frequency and attenuated CaMKII activities in failing cardiomyocytes [The inhibition of NHE by empagliflozin may reduce intracellular Cacurrents . Reducticurrents . Moreovecurrents , 55. Theng heart , 57. Empchannels , Na+ chachannels , and K+ channels , suggestchannels . Empaglimyocytes . Hence, myocytes , 40, 60.myocytes . Isoprotmyocytes , 63. In DM patients treated with SGLT2i exhibited higher levels of ketone (β-OHB) , 65. SGLMore severe atrial fibrosis is associated with a higher incidence of atrial fibrillation . EmpagliThere were a few limitations in this study. First, SGLT2i improved end-systolic and end-diastolic pressure–volume relationships in diabetic animal models according to pressure–volume loop experiments . However2+ release, extracellular Ca2+ entry and the profibrotic activities of atrial fibroblasts.In conclusion, as summarized in Fig. Additional file 1 : Fig. S1. SGLT2 protein is expressed in human atrial fibroblasts.


DNA double-strand breaks (DSBs), the most deleterious DNA lesions, are primarily repaired by two pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ), the choice of which is largely dependent on cell cycle phase and the local chromatin landscape. Recent studies have revealed that post-translational modifications on histones play pivotal roles in regulating DSB repair pathways including repair pathway choice. In this review, we present our current understanding of how these DSB repair pathways are employed in various chromatin landscapes to safeguard genomic integrity. We place an emphasis on the impact of different histone post-translational modifications, characteristic of euchromatin or heterochromatin regions, on DSB repair pathway choice. We discuss the potential roles of damage-induced chromatin modifications in the maintenance of genome and epigenome integrity. Finally, we discuss how RNA transcripts from the vicinity of DSBs at actively transcribed regions also regulate DSB repair pathway choice. Genome integrity is repeatedly challenged by a variety of endogenous and environmental stresses including, but not limited to, replication stress, reactive oxygen stress (ROS), ionizing radiation (IR), ultraviolet (UV) light, and various chemicals, all of which can induce DNA lesions . Among t2 phases of the cell cycle  wrapped by 147 base pairs of DNA . The unsThe dynamics of histone PTMs on chromatin is precisely controlled by proteins that write and erase these modifications . EnzymesIn a broad sense, the chromatin landscape comprises not only DNA and histone proteins but also nascent RNA transcripts that interact with the chromatin, as well as the modifications on these nucleic acids and proteins . The spatiotemporal dynamics of these factors accurately control DNA replication, gene transcription and genome stability. DSBs occur in the context of different chromatin landscapes, which in turn activate DDR signaling pathways to remodel chromatin structure and modulate nucleosome organization such as histone variant exchange and histone post-translational modification, to facilitate DSB repair . SpecifiUpon DSB induction, a cascade of histone and protein PTMs are induced which are critical for recruitment of DNA repair proteins. Initially, the PIKK family kinase ATM is recruited to the chromatin and activated through its interaction with NBS1 in the MRE11-RAD50-NBS1 (MRN) complex , followeWhether or not DNA end resection occurs is the critical decision step in DSB repair pathway choice, as extensive DNA end resection necessitates repair by HR and blocks NHEJ . Meanwhi2 and G1 phases (Drosophila melanogaster tumor-suppressor protein l (3)mbt, and JMJD2A/KDM4A, the human histone demethylase, bound to methylated H4K20 and masked the histone interface in undamaged chromatin to prevent 53BP1 from binding to the chromatin , newly synthesized H4 histone was found to be exclusively unmethylated at lysine 20 (H4K20me0), which is a signature of post-replicative chromatin  methyltransferase catalyzes mono-methylation of H4K20 (H4K20me1) and subsequently the suppressor of variegation 4–20 homologue ½ (SUV4-20H1/2) converts the mono-methylation to di- and tri-methylation (H4K20me2/3). In this manner, most of the H4 lysine 20 is methylated by G1 phase  in BARD1 was identified to recruit BRCA1 to DSB sites through its binding to H2AK15ub (In vitro pull-down assays showed the interaction between GST–BARD1 (ARD–BRCT) and recombinant nucleosome variants were strongly stimulated by H2AK15ub but inhibited by methylation on H4K20 , an ufmylation E3 ligase, was identified to be recruited to DSB sites by the MRN complex . The recs repair . It is lH3K9me3 promotes HR repair and reduces NHEJ, seemingly by a combination of mechanisms. H3K9me3 is enriched at heterochromatin regions, but also increases at DNA damage sites to form transient repressive chromatin in euchromatin . SeveralIn facultative heterochromatin, the repressive histone mark H3K27me3 is increased after DSB induction . This ocDrosophila lysine demethylase 4a (dKDM4a) is recruited to DSBs in pericentromeric heterochromatin, but not in euchromatin, and promotes the demethylation of heterochromatic histone marks, H3K9me3 and H3K56me3, to limit HR in heterochromatin domains -mediated CENP-A assembly onto an epigenetically engineered human kinetochore  was characterized as an essential factor in HR repair . RNAPIIIM phases , which iM phases , whereasSB sites . InteresSB sites . At DSB SB sites . ParadoxSB sites , which msceGFP in the direct-repeat GFP (DR-GFP) reporter was replaced with a tetracycline-inducible (Tet-On) promoter (sceGFP was actively transcribed, HR efficiency was markedly enhanced although HR products were still detectable when transcription was inactive (ASCL1, HR products were dramatically increased when transcription was active. Further, RNA transcripts were shown to stimulate HR by annealing with DNA mediated by the RAD51-associated protein RAD51AP1 (via different mechanisms because the generation of R-loops is independent of DNA end resection but is induced at RNA-DNA hybrids that forms on ssDNA generated by resection (1 phase (During HR repair, a ssDNA/Rad51 nucleoprotein filament invades the donor dsDNA homolog and forms a heteroduplex DNA joint called the D-loop . When thpromoter . When scinactive . In agreRAD51AP1 . RAD51APRAD51AP1 . At sub-RAD51AP1 . R-loopsesection . In cent(1 phase . These s1 phase, the pre-existing H4K20me1/2 on chromatin is recognized by 53BP1 to promote DSB repair by NHEJ. However, H4K20 is unmethylated on the post-replicative chromatin in S/G2 phase, which enhances the recruitment of BRCA1-BARD1 to facilitate HR repair. We have learned that in euchromatin and heterochromatin regions histone PTMs that promote transcriptional activation such as H3K36me and H3K4me2 and those that promote transcriptional repression such as H3K9me3, both promote DSB repair by HR through interactions with distinct factors. Intriguingly, we now appreciate that DSB-induced RNAs are involved in DSB repair regulation. The nascent RNA transcripts can also form R-loops with DNA ends at the breaks to promote HR repair.In the last two decades, our knowledge of how epigenetic modifications regulate DSB repair, and DSB repair pathway choice has grown exponentially. Our knowledge of the interplay between the pro-NHEJ factor 53BP1 and pro-HR heterodimer BRCA1-BARD1 has increased extensively. The recruitment of 53BP1-RIF1-Shieldin to DSBs plays a critical role in promoting NHEJ repair. We now have an improved appreciation of how pre-existing and DSB-induced histone PTMs play pivotal roles in regulating DSB repair pathway choice in different phases of the cell cycle: Upon DSB induction, RNF168 is recruited to DSB sites to monoubiquitylate H2AK15, which is a prerequisite for both 53BP1 and BRCA1-BARD1 recruitment. In GIt remains largely unknown how the intricate chromatin modification network, namely the chromatin landscape, is orchestrated to regulate DSB repair pathway choices. More studies are required to address whether other histone modifications, such as crotonylation and glycosylation, also contribute to DSB repair pathway choice. In addition, it is tempting to speculate whether DSB-induced small RNAs, diRNAs/DDRNAs, and dilincRNAs can stimulate heritable epigenetic changes on DNA molecules, such as DNA methylation, in the chromatin landscape surrounding the DSB sites, which may act as an “epigenetic memory” to prime the repaired region for more efficient repair if DSBs occur at the same sites. Indeed, DSBs do initiate the transient recruitment of the DNA methyltransferases DNMT1 and DNMT3B, in a SIRT1 dependent manner, and subsequent methylation at promoter regions .1 but also in G2 phase where both HR and NHEJ repair pathways are functional (1 phase without the presence of sister chromatids. Utilization of HR repair at repetitive sequences is thought to be detrimental to genome stability as it usually leads to repeat contraction and expansion (1 counteracts the engagement of alternative mutagenic repair pathways, resulting in the prevention of chromosomal abnormalities (Why do the histone PTMs involved in DSB repair pathway choice, regardless of whether they occur in euchromatin or heterochromatin regions, tend to promote HR repair? Does this indicate that NHEJ repair is the default mechanism of DSB repair throughout the cell cycle and specific histone modifications are required to overcome the NHEJ default to promote HR? Indeed, NHEJ was found, by live imaging and fluorescent labeling of components in HR and NHEJ repair pathways, to be the dominant repair pathway not only in Gnctional . Why thexpansion . Howevermalities .Interestingly, the highest occurrence of HR is at the peak of active replication with the longest half-lives of DSBs in mid S phase , where s


The acute myeloid leukemia (AML) patients obtain limited benefits from current immune checkpoint blockades (ICBs), although immunotherapy have achieved encouraging success in numerous cancers. Here, we found that V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint, is highly expressed in primary AML cells and associated with poor prognosis of AML patients. Targeting VISTA by anti-VISTA mAb boosts T cell-mediated cytotoxicity to AML cells. Interestingly, high expression of VISTA is positively associated with hyperactive STAT3 in AML. Further evidence showed that STAT3 functions as a transcriptional regulator to modulate VISTA expression by directly binding to DNA response element of VISTA gene. We further develop a potent and selective STAT3 inhibitor W1046, which significantly suppresses AML proliferation and survival. W1046 remarkably enhances the efficacy of VISTA mAb by activating T cells via inhibition of STAT3 signaling and down-regulation of VISTA. Moreover, combination of W1046 and VISTA mAb achieves a significant anti-AML effect in vitro and in vivo. Overall, our findings confirm that VISTA is a potential target for AML therapy which transcriptionally regulated by STAT3 and provide a promising therapeutic strategy for immunotherapy of AML.The online version contains supplementary material available at 10.1186/s13045-023-01410-y. Despite encouraging responses elicited by immune checkpoint blockades (ICBs) have been shown in many types of cancers, the acute myeloid leukemia (AML) patients obtain little benefit from current ICBs , 2. HoweWe found that VISTA was significantly higher in AML cells and was the most correlative immune checkpoint for overall prognosis of AML patients basing on TCGA database Fig. D, E. TheBasing on above results, we assumed STAT3 inhibitor can act as an immunomodulator in AML immunotherapy. On this account, we designed a novel STAT3 inhibitor W1046 (Fig. + T cells and CD8+ T cells (Fig. Emerging evidences reported that combination of immunomodulator and immune checkpoint blockade generally has synergistic effect and maximizes benefits –12. GiveOverall, our study firstly suggested that VISTA is a promising target for AML immunotherapy, regulated by STAT3 transcriptionally. A novel and selective STAT3 inhibitor W1046 was discovered and showed significant anti-tumor efficacy in AML. Combination of VISTA mAb and STAT3 inhibitor demonstrated remarkable anti-leukemia efficacy in AML by restoring T cells activation in vitro and in vivo, which shed a light on a new therapeutic strategy for AML therapy.Additional file 1: Supplementary figures S1–S5 and supplementary materials and methods.


Upon herbivory, Ca2+ waves travel long distances to transmit and convert the local signal to a systemic defense program. Reactive oxygen species (ROS), Ca2+ and electrical signaling are interlinked to form a network supporting rapid signal transmission, whereas the Ca2+ message is decoded and relayed by Ca2+-binding proteins . Monitoring the generation of Ca2+ signals at the whole plant or cell level and their long-distance propagation during biotic interactions requires innovative imaging techniques based on sensitive sensors and using genetically encoded indicators. This review summarizes the recent advances in Ca2+ signaling upon herbivory and reviews the most recent Ca2+ imaging techniques and methods.In plant–insect interactions, calcium (Ca The plant–insect interaction dates back to prehistorical times . The evoThe recognition and appropriate response to the attacking enemy occur within a few minutes and require the activation of specific pathways most of which finally lead to gene activation  . Upon in2+), a universal second messenger the intracellular variation in concentration of which has been associated to the plant perception of biotic stress [2+ concentration ([Ca2+]cyt) elevation involves multiple channels and pathways regulating local and long-distance [Ca2+]cyt signals cyt were observed only after herbivory cyt increase propagates the systemic wound signaling cyt, ROS production and Vm depolarization [N-linolenoyl-l-glutamine (GLN18:3) induce a RBOHD and RBOHF-dependent ROS burst in several plant species [Wounding may also induces a drop in hydraulic pressure  in response to herbivory. The expression of the Ca2+-sensing aequorin system in transgenic soybean (Glycine max) suspension cells and the effects of herbivore-wounding in Lima bean (P. lunatus) leaves show increasing amounts of H2O2 that correlate with a higher [Ca2+]cyt [G. biloba leaves damaged by S. littoralis [P. vittata [2+] fluxes and H2O2 were detected in a tobacco variety (G140) infested by the tobacco aphid M. persicae. Even in this case, H2O2 accumulation depended on Ca2+ influx, which were both increased in a long period of aphid feeding [Several plant species produce hydrogen peroxide  [2+ permeable channels in membranes [2+ signaling are the consequence of the combined effect of wounding and the secretion of salivary elicitors from the feeding insect [Acyrthosiphon pisum produce two types of saliva that mediate their interactions with plants which are based on Ca2+ signaling-related responses [S. littoralis ventral eversible gland contains elicitors able to trigger both Ca2+ signaling and H2O2 production in Arabidopsis [Laodelphax striatellus) secretes salivary proteins into its plant host during feeding that enhance plant resistance to insects by inducing Ca2+ and ROS signaling pathways [Manduca sexta) OS induce Ca2+-dependent ROS production in tomato (Solanum lycopersicum) protoplasts [T. urticae) were found to trigger the [Ca2+]cyt influx and the generation of ROS in kidney bean plants (P. vulgaris) cyt; this event is associated to the opening of K+ channels, with particular action on ligand-gated rectified K+ channels, which in turn are correlated to ROS production [2+]cyt, and Arabidopsis loss of function mutants were found to have an impaired increase in [Ca2+]cyt in response to eATP, thus demonstrating a link between eATP and Ca2+ signaling cyt increase in receiving plants cyt increased in Arabidopsis mesophyll cells after being treated with the volatile monoterpene alcohol linalool in the attempt to increase plant resistance to diamondback moth (Plutella xylostella) cyt and production of H2O2, demonstrating the ability of plants to detect and respond to volatiles emitted by non-plant organisms [Other interesting effects of plant–insect interactions on Carganisms .2+ message needs to be decoded and relayed. This task is brought by Ca2+-binding proteins (CBPs) [2+-binding motif called the EF-hand [2+-binding protein (LsECP1) in the small brown planthopper Laodelphax striatellus exhibits Ca2+-binding activity. Knocking down LsECP1 in L. striatellus prompted a higher level of [Ca2+]cyt in rice fed plants, whereas overexpression suppressed wound-induced H2O2 accumulation cyt causes the breaking down of the CAM1-WRKY53 complex, then the detachment of WRKY53 reduces the JA content by downregulating the LOXs gene expression cyt variations cyt in the systemic defense-related signaling in plants . Al. Al2+ re results ,123. In  results . The dev signals . In planiosensor . Howevern plants , includin plants , pathogen plants , salt stn plants  and wounn plants  was succn plants . Anyway,levation . They harganisms .2+ indicators, including GCaMP3 [2+-responsive signal and their simpler microscope requirements [2+ sensor Yellow Cameleon NES-YC3.6 [2+ signals in flg22- and chitin-induced experiments on a cellular scale. Here the use of GECO1 allowed to visualize well defined [Ca2+]cyt oscillations in both epidermal and guard cells cyt sensor showed a rise in [Ca2+]cyt at the wounding position within 2 sec [2+]cyt signal was observed moving from younger to older leaves and vice versa [2+]cyt upon S. littoralis feeding on a rosette leaf. The signal propagates quickly through the vasculature (2+ signaling with the use of reporter dyes and especially with the more recent genetically encoded Ca2+ indicators, which can be very helpful to decode both abiotic and biotic responses in plants.In the last decade, the approach to Ca in vivo ,144,145.stresses . In factcumented , but itscumented . S. littin 2 sec . Furtherce versa . By follce versa , we obseculature . Thus, i2+ variations are among the earliest events during plant–insect interactions. Ca2+ waves arise by the wounded plant to transmit at long distance and to convert local signals into a systemic defense program. In this review, we showed that the Ca2+ signaling is accompanied by other important rapid signals, with particular reference to ROS and electrical signaling. The interdependence of these three networked events is clear in plant-biotic interactions and have been demonstrated several times in plant–insect interactions. In order to be decoded and relayed, the Ca2+ message requires proteins able to bind the ion and trigger the signal transduction pathways that lead to plant responses to biotic attack. We described the role of calmodulin, calmodulin-like proteins, Ca2+-dependent protein kinases, annexins and calcineurin B-like proteins in plant–insect interactions. An important role in the study of Ca2+ signaling is the ability to visualize in real-time the generation of Ca2+ signals both at the single cell and whole plant level. Innovative imaging techniques have been developed based on sensitive sensors and using genetically encoded indicators that open new ways in the deciphering of the Ca2+ signaling in plant–insect interactions.Ca


Background: Atrial fibroblasts activation causes atrial fibrosis, which is one major pathophysiological contributor to atrial fibrillation (AF) genesis. Klotho is a pleiotropic protein with remarkable cardiovascular effects, including anti-inflammatory, anti-oxidative, and anti-apoptotic effects. This study investigated whether Klotho can modulate the activity of human atrial fibroblasts and provides an anti-fibrotic effect. Methods: Cell migration assay and proliferation assay were used to investigate fibrogenesis activities in single human atrial fibroblasts with or without treatment of Klotho . Calcium fluorescence imaging, the whole-cell patch-clamp, and Western blotting were performed in human atrial fibroblasts treated with and without Klotho  to evaluate the store-operated calcium entry (SOCE), transient receptor potential (TRP) currents, and downstream signaling. Results: High dose of Klotho  significantly reduced the migration of human atrial fibroblasts without alternating their proliferation; in addition, treatment of Klotho  also decreased SOCE and TRP currents. In the presence of BI-749327 , Klotho  could not inhibit fibroblast migration nor suppress the TRP currents. Klotho-treated fibroblasts  had lower phosphorylated phospholipase C (PLC) (p-PLCβ3 Ser537) expression than the control. The PLC inhibitor, U73122 , reduced the migration, decreased SOCE and TRP currents, and lowered p-PLCβ3 in atrial fibroblasts, similar to Klotho. In the presence of the U73122 , Klotho  could not further modulate the migration and collagen synthesis nor suppress the TRP currents in human atrial fibroblasts. Conclusions: Klotho inhibited pro-fibrotic activities and SOCE by inhibiting the PLC signaling and suppressing the TRP currents, which may provide a novel insight into atrial fibrosis and arrhythmogenesis. Atrial fibrosis plays a significant role in both electrical and structural remodeling. Cardiac fibroblasts, which account for about 75% of total cardiac cells, can synthesize extracellular matrix (ECM) and modulate electrical characteristics of cardiomyocytes while they are coupling to cardiomyocytes [Atrial fibrillation (AF) is the most common clinical arrhythmia that raises the risk of major adverse cardiovascular events and has become a growing public health problem . The patmyocytes . Klotho, a type 1 single-pass transmembrane protein containing a large extracellular domain, is present in many organs, including the heart ,5. Solub2+-signaling in both cardiac fibroblasts [TRP channels, consisting of 6-transmembrane polypeptide subunits that assemble as tetramers forming cation-permeable pores, are extensively expressed in the heart and are responsible for Caroblasts ,15,16. Troblasts . Among troblasts ,19. Xie roblasts . However2 at 37 °C as previously described [The NHCF-A human atrial fibroblasts cell line was purchased from Lonza Research Laboratory , and all fibroblasts were seeded as monolayers on uncoated culture dishes containing Dulbecco’s modified Eagle’s medium , including 100 U/mL penicillin-streptomycin  and 10% fetal bovine serum , in a humidified atmosphere containing 5% COescribed . Human aA wound-healing assay was used to analyze the migration of human atrial fibroblasts treated with or without Klotho in different concentrations  6 h after a cell monolayer in a six-well plate was craped with a P200 pipette tip. To explore the role of TRPC6 and intracellular signaling underlying the effects of Klotho in fibroblasts activity, we evaluated the migration in control, Klotho 100 pM-treated, BI-749327 1 μM -treated, combined Klotho and BI-749327-treated, U73122 1 μM -treated and combined Klotho and U73122-treated human atrial fibroblasts for 48 h. Each gap area was assessed using the ImageJ software . The net migration areas after 6 h were subtracted from that at the time of the initial scratches.As previously described, we used a commercial MTS kit  to measure the proliferation of human atrial fibroblasts . Human aWe used the whole-cell patch-clamp technique to measure the ionic currents in human atrial fibroblasts with and without the administration of Klotho , BI-749327 , or U73122  by using an Axopatch 1D amplifier  at 35 ± 1 °C. The tip resistance of borosilicate glass electrodes containing pipette solution used in this study was 3–5 MΩ.2, 0.2 mM Na3GTP, buffered at 100 nM free Ca2+ with 10 mM BAPTA, and 10 mM HEPES (titrated with CsOH to pH 7.2), and the external solution contained 140 mM NaCl, 5 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (titrated with NaOH to pH 7.4). We depolarized the human atrial fibroblasts by using triangular voltage ramps from –120 mV to +120 mV with a 200 ms pulse duration . Considering TRP to be a non-selective cation channel, peak inward (-110 mV) and peak outward (+110 mV) current density were used to analyze the current amplitude.As described previously, the TRP current in human atrial fibroblasts was measured in the voltage-clamp configuration . For rec2+-free solution containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM glucose, 6 mM HEPES, and 8 mM taurine (PH 7.40) for 40 min at 36 °C. We used a Polychrome V monochromator  mounted on an upright Leica DMI 3000B microscope  with dual-excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm to acquire Fura-2 fluorescence images. The MetaFuor software version 7.7.6.0  was used to analyze Fura-2 images and the relative level of intracellular Ca2+ was represented by the ratio of fluorescence due to excitation at 340 nm (F340) to 380 nm (F380). To measure Ca2+ entry, all fibroblasts were initially superfused in the Ca2+-free solution for 2 min, following thapsigargin (2.5 μM) application. After thapsigargin induced Ca2+ release from the endoplasmic reticulum (ER), the extracellular solution was changed from a Ca2+-free solution to a solution containing 2mmol/L Ca2+ to measure Ca2+ entry via store-operated channels activated by ER Ca2+-store depletion. The Ca2+ entry was represented by the change in intracellular Ca2+ from a Ca2+-free solution to a 2 mmol/L Ca2+ solution (ΔF340/F380). The human atrial fibroblasts were loaded on 1 cm glass-bottom chamber slides with the Fura-2-acetoxymethyl ester  and Pluronic F-127  in a CaTM software . All targeted bands were normalized to the glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH)  to confirm equal protein loading.Human atrial fibroblasts with and without the administration of Klotho  or U73122  were homogenized and centrifuged in the buffer. We separated proteins electrophoretically on a polyacrylamide gel electrophoresis with 4% to 12% sodium dodecyl sulfate and electrophoretically transferred them to a polyvinylidene difluoride membrane that was equilibrated with sodium dodecyl sulfate as described previously . We probt-test, Mann–Whitney rank-sum test, or one-way analysis of variance (repeated measures or non-repeated measures) with Tukey’s post hoc test to compare human cardiac fibroblasts under different treatment conditions. Statistical significance was defined as a p-value < 0.05. Statistics analyses were carried out using SPSS Statistic 18.0 software  and SigmaPlot 12 software .Means and standard errors of the means were used to describe all continuous variables. We used an unpaired Compared with control human atrial fibroblasts, a high dose of Klotho (100 pM)-treated atrial fibroblasts exhibited lesser migratory capability and lower expression of procollagen type IA1 and procollagen type III A,B. Howe2+ entry (SOCE) plays an essential role in the cellular calcium signaling and modulates cell migration [2+ entry (SOCE) than control fibroblasts -treated fibroblasts, PLC inhibitor -treated fibroblasts, and combined Klotho (100 pM) and PLC inhibitor (1 μM)-treated fibroblasts had a lower ER release and a lower SOCE than control fibroblasts with a similar extent . MoreoveAF is the most common arrhythmia in clinical practice and contributes to cardiovascular adverse events such as heart failure and ischemic stroke . The ess2+ is essential for the regulation of the activation of fibroblasts [2+ in pulmonary vein cardiomyocytes [2+ release and the subsequent Ca2+ entry. These findings exhibited an association between inhibition of intracellular Ca2+ through SOCE and suppression of fibroblast functions, such as collagen synthesis and migration, in fibroblasts treated with Klotho (100 pM).Previous studies demonstrated that cytosolic Caroblasts ,41,42, amyocytes . The pre2+ signaling plays a vital role in cardiac fibrosis, including the synthesis of extracellular matrix proteins and migration [2+ signaling. Among numerous non-selective cation channels regulating the Ca2+ entry and subsequent extracellular signal-related kinase pathways [2+ signaling and mediate fibrogenesis in AF [2+ entry and SOCE [2+ regulation in response to mechanical stretch, which contributes to the structure and electrical remodeling [3) and diacylglycerol (DAG). Consequently, DAG activates protein kinase C, which promotes atrial fibrosis through modulating fibroblast function and stimulating collagen synthesis, and IP3 activates the IP3 receptor and induces rapid release of the luminal Ca2+ from the ER [2+ activates the stromal interaction molecule that subsequently triggers SOCE through promoting Ca2+ release-activated Ca2+ channel protein 1, forming a complex with TRPCs, contributing to extracellular Ca2+ influx [2+ influx [Intracellular signaling of cardiac fibroblasts that govern fibroblast behavior is complex, among which Caigration . Recentlpathways ,24,44, Tis in AF ,44. TRPCand SOCE . Nikolovmodeling . PLC sigm the ER . Depleti+ influx ,49. In a+ influx ,51. In o+/K+-ATPase, TRPV5, TRPC6, and renal outer medullary potassium channel 1 [2+ release and the subsequent extracellular Ca2+ entry to a similar extent. The above findings suggest no synergetic inhibitory effects of Klotho and U73122 on atrial fibroblasts. Thus, Klotho might exhibit its inhibitory effects on fibroblast migration, collagen synthesis, and SOCE by inhibiting the PLC signaling. Klotho is a coreceptor of fibroblast growth factor (FGF) receptors, and it triggers signal propagation of FGFs, such as FGF19, FGF21, and FGF23 . Loss ofhannel 1 . A previhannel 1 . In the 3R and following stromal interaction molecule activation. Second, human atrial fibroblasts from passages 4–6 were used in this study, and their fibroblast characteristics might be different from those in patients. Third, several conditions such as aging and heart failure could enhance cardiac fibrosis and AF genesis. Future experiments from pathological animal models are useful to confirm Klotho’s potential in reducing atrial fibrosis and AF prevention.First, although we found that Klotho modulates PLC signaling and subsequent SOCE, the detailed mechanisms underlying Klotho’s regulation in human atrial fibroblasts are not fully elucidated. More experiments are mandatory to clarify the modulatory effects of Klotho on IPKlotho modulates the pro-fibrotic activities and intracellular calcium homeostasis in human atrial fibroblasts through its inhibitory effect on the PLC pathway and decreases SOCE by suppressing TRPC6 currents, which may provide a novel therapeutic insight into atrial fibrosis and AF genesis.Klotho decreased migration and expressions of procollagen type IA1 and procollagen type III in human atrial fibroblasts.Klotho reduced TRP currents, mainly due to the decreased TRPC6 currents, and subsequently modulated fibroblasts activity.2+ release and SOCE in human atrial fibroblasts.Klotho inhibited the PLC pathway and decreased thapsigargin-induced ER Ca


NPM1-mutated acute myeloid leukemia (AML) is one of the most common subtypes of AML in patients with a normal karyotype. In the recently introduced International Consensus Classification, detection of gene mutations typically associated with myelodysplastic syndrome (MDS) is considered an adverse biomarker in AML patients. However, the impact of these gene mutations occurring in the setting of AML with NPM1 mutation and without FLT3-ITD mutation, a favorable subtype, is unclear. Furthermore, correlation between minimal measurable disease (MMD) with survival in the context of these co-mutations also remains unclear. This study aims to address these issues. We found that patients with or without MDS-related gene mutations treated intensively had a similar MMD rate; however, the former group had a higher relapse rate and shorter progression-free survival (PFS).NPM1 mutation is unclear. Methods: Using a cohort of 107 patients with NPM1-mutated AML treated with risk-adapted therapy, we compared survival outcomes of patients without MDS-related gene mutations (group A) with those carrying concurrent FLT3-ITD (group B) or with MDS-related gene mutations (group C). Minimal measurable disease (MMD) status assessed by multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and/or next-generation sequencing (NGS) were reviewed. Results: Among the 69 patients treated intensively, group C showed significantly inferior progression-free survival  but not overall survival  compared to group A. Though groups A and C had a similar MMD rate, group C patients had a higher relapse rate (p = 0.016). Relapse correlated with MMD status at the end of cycle 2 induction (p = 0.023). Survival of group C patients was similar to that of group B. Conclusion: MDS-related gene mutations are associated with an inferior survival in NPM1-mutated AML.Background: The impact of gene mutations typically associated with myelodysplastic syndrome (MDS) in acute myeloid leukemia (AML) with  NPM1 mutation and a normal karyotype comprises 30% of AML and is one of the most common subtypes [FLT3-ITDhigh  > 0.5) were previously designated as “intermediate-risk” based on the European LeukemiaNet (ELN) risk stratification [NPM1-mutated AML with FLT3-ITD co-mutation as intermediate-risk group regardless of AR [Acute myeloid leukemia (AML) with subtypes ,3. This fication ,5, the nss of AR .NPM1 mutation even if the mutation was detected [ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 genes denotes MDS-related gene mutations. Studies have shown their prevalence in secondary AML [NPM1-mutated AML is less clear. Additionally, AML with TP53 mutation and WT1 mutation has been shown to be aggressive [NPM1-mutated AML is also unclear. To address this issue, we designed and performed the current study comparing survival of three subgroups of NPM1 mutated AML: group A, without MDS-related gene mutations; group B, with FLT3-ITD mutation regardless of AR; and group C, with MDS-related gene mutations. For this study, TP53-mutated cases were included in group C, as it is also commonly seen in secondary AML [WT1 but without FLT3-ITD were excluded from group C and designated as group D.Historically, patients who developed AML preceded by a history of myelodysplastic syndrome (MDS) or with cytogenetic aberrations associated with MDS  were designated as AML with myelodysplasia-related changes (AML-MRC) rather than AML with detected ,8. A patdary AML . Howeverdary AML ,10,11. CNPM1- and MDS-related genes.Pretreatment variables such as age, performance status, white blood cell (WBC) count, and molecular genetic data currently represent the best-established, albeit imperfect, predictors of response to therapy . IncreasNPM1 mutation. MMD status assessed by various combinations of MFC, PCR, and NGS was correlated with mutation profiles as well as survival.In the current study, we evaluated 107 consecutive patients of de novo AML with NPM1. AML with other recurrent genetic aberrations or secondary/transformed AML was excluded [NPM1 mutation. These abnormalities have not been shown to affect prognosis [We searched databases in MD Anderson Cancer Center and Northwestern University Feinberg School of Medicine from 1 January 2012 through 31 December 2018 for patients diagnosed with de novo AML associated with mutated excluded . All patrognosis ,7,16,17,rognosis . ClinicaFLT3-ITD mutation were treated with FLT3 inhibitor. A subset of intensively treated patients received allogeneic hematopoietic stem cell transplantation (HSCT).Patients were treated based on clinical assessment and institutional protocols. Patients who were treated intensively received either Fludarabine + Ara-C + G-CSF + Idarubicin (FLAG-IDA), Cladribine combined with Idarubicin and Ara-C (CLIA) with or without venetoclax, or standard “3 + 7”. Alternatively, non-intensive regimens included hypomethylating agents (HMA) with or without venetoclax or Cladribine or low-dose Ara-C alternating with decitabine. FLT3 or IDH inhibitors were added in the appropriate context. All patients with Initial and post-treatment bone marrow aspirate samples were processed using standard methods and analyzed using FACS Canto II 8-color instruments . Details of the assay have been previously described ,21. The FLT3-ITD and tyrosine kinase domain (TKD) mutations and NPM1 mutations were identified using PCR-based methods followed by capillary electrophoresis on Genetic Analyzer , as described previously [NPM1 mutations. High-throughput sequencing was performed using a MiSeq sequencer , and data were analyzed using MiSeq Reporter Software. A 28-gene NGS-panel assessed full exons for mutations in the following genes: ABL, EGFR, GATA2, IKZF2, MDM2, NOTCH1, RUNX1, ASXL1, EZH2, HRAS, JAK2, MLL, NPM1, TET2, BRAF, FLT3, IDH1, KIT, MPL, NRAS, TP53, DNMT3A, GATA1, IDH2, KRAS, MYD88, PTPN11, and WT1. In a subset of cases, an 81-gene panel was also performed .Survival distributions were estimated by the Kaplan–Meier method (Log-rank test) using Graph-Pad software ; unpaired DNMT3A, IDH1/2, ASXL1, TET2, or SRSF2 were considered as underlying clonal hematopoiesis of undetermined significance (CHIP). Survival was determined from the date of diagnosis to date of death from any cause or last follow-up.Complete remission (CR) and CR with incomplete blood count recovery (CRi) are defined according to the standard criteria ,30. For Statistical analyses were performed by two biostatisticians independently.The mutation profiles of all 107 cases are summarized in p < 0.0001) and MMD was more frequently detected in 26/34 (76.47%) patients (p < 0.0001)  ( 0.0001) . At lastp = 0.0009) (p = 0.023)  and 13  patients at EOC1 and EOC2, respectively, seen across all the three subgroups  at a similar rate . However 0.0009) . Among t= 0.023) .FLT3-ITD showed significantly higher relapse rate and worse survival than patients of group A (p < 0.0001) but not OS (p = 0.055) than group A (p = 0.46), PFS (p = 0.28), or OS (p = 0.89) . Survival curves were censored for patients who received HSCT from the date of transplantation  . In addiantation .NPM1 mutation, and eight patients acquired additional mutations not seen at initial diagnosis, including FLT3-ITD in five, FLT3-TKD in two, and WT1 in two. Additionally, six patients who initially had a normal karyotype developed an abnormal karyotype, and 5/6 died within 12 months -related mutation profiles into the risk stratification.NPM1 have a sensitivity level of 0.01–1%, it was significant whenever abnormal variants were detected. However, samples obtained at EOC1 often and sometimes EOC2 are hypocellular, potentially limiting MFC assay sensitivity. Thus, not unexpectedly, we observed discordant results between MFC and molecular methods.Detectable MMD at EOC2 but not EOC1 correlated with relapse. This finding supports the notion that delayed blast clearance is an adverse risk factor  and the DNMT3A, ASXL1, TET2, IDH1/2, and SRSF2 are now recognized as CHIP-like mutations that can persist during CR [DNMT3A mutation was shown to have no adverse impact on survival but more likely correlated with MMD positivity and potentially increased the risk for relapse or secondary AML [uring CR ,35. In curing CR ,37. The NPM1-mutated AML have implications in patient survival in the current era of risk-adapted therapy. In addition to FLT3-ITD, concurrent MDS-related gene mutations are associated with an inferior outcome, supporting their recognition in NPM1-mutated AML. The mechanisms of this phenomenon remain to be explored, but they are most likely related to persistence of underlying preleukemic clone and unremitted risk for genetic alterations in affected patients despite achieving CR.The mutation profiles of


Methods: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. Results: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. Conclusions: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca2+ handling machinery.Background: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca Breast cancer represents the most widespread cancer in women and accounted for about 685.000 deaths in 2020. This death toll has been forecasted to rise to 7 million by 2040 . Triple Platelet-derived extracellular vesicles (PEVs) are gaining growing interest as mediators of platelet function in different physiological and pathological contexts, from haemostasis to cardiovascular diseases ,5. In thMedium-large PEVs (henceforth PEVs for brevity) have been widely studied in the frame of the platelet–cancer crosstalk. Their levels in the circulation are increased in oncological patients bearing diverse types of cancer, including cutaneous malignant melanoma , colorec2+ handling machinery supports several cancer hallmarks, including tissue invasion and metastasis [2+ concentration ([Ca2+]i) in MDA-MB-231 cells can be elicited by inositol-1,4,5-trisphosphate (InsP3) i i ,18. HereThe triple negative breast cancer line, MDA-MB-231, was provided by Professor Livia Visai . Cancer cells were periodically checked to verify the absence of bacterial contamination and were maintained in DMEM supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, split every two days, and used for the experiments within 10 passages. The count of vital cells was determined by Trypan Blue staining and phase contrast microscopy analysis.8/mL in HEPES buffer  supplemented with 1 mM CaCl2, 0.5 mM MgCl2 and 5.5 mM glucose. To induce the release of PEVs, platelets were stimulated with the physiological agonist thrombin (0.2 U/mL) for 30 min at 37 °C under constant stirring. Platelets were pelleted by low-speed centrifugation  and the supernatant was then centrifuged at 18,500× g for 90 min at 10 °C to collect medium-large PEVs, which were finally resuspended in HEPES buffer. The protein content of the different preparations of PEVs was determined by BCA assay.Human blood platelets were purified from healthy donors as recently described . Upon th2+ imaging was used to measure intracellular Ca2+ signals in PEV-treated cancer cells i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm [ratio (F340/F380)]. An increase in [Ca2+]i causes an increase in the ratio i was evaluated by using the Grynkiewicz equation, as shown elsewhere i  to cause passive ER Ca2+ efflux through Ca2+-permeable leakage channels. Subsequently, 1.8 mM Ca2+ is restituted to external Ca2+ to monitor SOCE through Orai1 channels that have previously been activated by STIM1 upon the depletion of the ER Ca2+ store. 2+]i under 0Ca2+ conditions, which reflects ER Ca2+ releasing ability in the absence (Ctrl) and presence of PEVs. PEVs caused a significant (p < 0.05) elevation in CPA-evoked ER Ca2+ mobilization  to cleave phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and InsP3 i evoked by the re-addition of extracellular Ca2+  (2+ store with CPA (30 µM)  cells A–C. The lar Ca2+ . Pharmacnoeuvres ,37,40,63 (50 µM) D,F; and  (30 µM) E,F.3-induced ER Ca2+ mobilization and SOCE that could potentiate the pro-migratory Ca2+ response to serum stimulation.In agreement with previous reports on other cancer cell types ,52, seru2+ handling machinery potentiates serum-induced migration upon pretreatment with PEVs, we evaluated MDA-MB-231 motility in the absence (Ctrl) and presence of specific InsP3R and SOCE inhibitors. The pharmacological blockade of InsP3Rs with 2-APB (50 µM) significantly inhibited serum-migration in MDA-MB-231 cells pre-treated with PEVs , whereas this process was not affected by SOCE inhibition with BTP-2 (20 µM) . We confirmed that PEVs potentiated p38 MAPK and MLC2 phosphorylation (2+-sensitive Pyk2 (3R inhibition with 2-APB (50 µM) and to SOCE blockade with BTP-2 (20 µM)  A,B. Nextrylation C–F, whilive Pyk2 . We then (20 µM) C–F. Thes2+]i may occur upstream of p38 MAPK and MLC2 recruitment i is required to trigger the release of PEVs from the plasma membrane of activated platelets i in MDA-MB-231 cells, with this being initiated by InsP3-induced ER Ca2+ release and maintained by SOCE activation [2+ response to PEVs is unlikely to engage the Ca2+-dependent molecular machinery that potentiates migration during the late stages of PEV stimulation. These pieces of evidence prompted us to assess whether long-term exposure to PEVs stimulates the p38 MAPK and MLC2 signalling pathways and potentiates MDA-MB-231 cell migration through remodelling the Ca2+ handling machinery.A sustained increase in [Calatelets . Prelimir injury . Furthertivation . Neverthtivation . Therefo2+]i or the basal plasma membrane permeability to Ca2+, which is mediated by a yet-to-be-identified Ca2+-permeable route in MDA-MB-231 cells, while it is mediated by Orai1 channels in low-migrating MCF-7 cells i has long been known to mediate the pro-migratory effect of serum on cancer cells [2+]i in this highly migrating breast cancer cell line. Pharmacological manipulation confirmed that serum-evoked intracellular Ca2+ release in MDA-MB-231 cells was inhibited by interfering with PLC activity with U73122 by inhibiting InsP3Rs with 2-APB and by depleting the ER Ca2+ pool with CPA. Furthermore, serum-evoked extracellular Ca2+ entry was dampened by BTP-2 and Pyr6, two highly specific inhibitors of Orai1. These findings concur with the involvement of InsP3Rs and SOCE in the Ca2+ signal evoked by serum in MDA-MB-231 cells, as also documented in other types of cancer cells i. Under the same conditions, we failed to detect any effect with regard to PEVs on the activation of the Ca2+-sensitive focal adhesion kinase Pyk2, whose role in driving the breast cancer metastatic outgrowth was previously documented [2+ sources which can engage the same Ca2+-dependent effectors [3Rs. Alternatively, InsP3-induced ER Ca2+ release could recruit additional Ca2+-dependent effectors of cell motility that are not coupled to SOCE and remain to be identified. Future work is mandatory to assess this issue, but InsP3-induced ER Ca2+ release is clearly required to potentiate p38 MAPK and MLC2 phosphorylation and to stimulate migration in PEV-treated MDA-MB-231 cells.An increase in [Caer cells ,31,52,53er cells  and thater cells ,53,86, ier cells . As expeer cells ,26,32. T to PEVs . In agreigration ,18,19,20igration  and furtcumented . The seeffectors ,89,90. H2+ transport system in the highly aggressive TNBC cell line, MDA-MB-231, thereby leading to an increase in InsP3-induced Ca2+ release and SOCE amplitude. In particular, the larger Ca2+ mobilization from the ER is required to potentiate serum-induced migration through the recruitment of p38 MAPK and MLC2. These findings lay the foundation for targeting the Ca2+ handling machinery not only to prevent breast cancer cell stimulation by pro-oncogenic chemical mediators and physical signals [Herein, we provide the first evidence that long-term exposure to PEVs induces a remarkable alteration in the Ca signals ,37,55,56


Still, the mechanisms governing such interplay are poorly understood. Here, we review the recent data on mechanisms underlying the brain cGMP signaling and its interconnection with Ca2+ signaling. We also discuss the recent evidence stressing the importance of such interplay for normal brain function as well as in Alzheimer’s disease.Cyclic guanosine monophosphate (cGMP) is a ubiquitous second messenger and a key molecule in many important signaling cascades in the body and brain, including phototransduction, olfaction, vasodilation, and functional hyperemia. Additionally, cGMP is involved in long-term potentiation (LTP), a cellular correlate of learning and memory, and recent studies have identified the cGMP-increasing drug Sildenafil as a potential risk modifier in Alzheimer’s disease (AD). AD development is accompanied by a net increase in the expression of nitric oxide (NO) synthases but a decreased activity of soluble guanylate cyclases, so the exact sign and extent of AD-mediated imbalance remain unclear. Moreover, human patients and mouse models of the disease present with entangled deregulation of both cGMP and Ca Cyclic guanosine monophosphate (cGMP) is one of the ubiquitous second messengers of the body and brain. It targets a manifold of downstream pathways, thus eliciting a broad variety of cellular/tissue effects. The role of cGMP has been extensively studied in the periphery, where it has important physiological functions in the vasculature, heart, pulmonary arteries, and gastrointestinal tract . The dys2+ concentration ([Ca2+]i) enhancing the production of cGMP i is low due to the constant removal of Ca2+ from the cytosol. The extrusion mechanisms include the plasma membrane Ca2+-ATPase (PMCA pump), the transmembrane Na+/Ca2+ exchanger, as well as the sarco-/endoplasmatic reticulum Ca2+-ATPase (SERCA pump), mediating the reuptake of Ca2+ into the endoplasmatic reticulum (ER). The Ca2+ and the cGMP signaling pathways are interconnected. The most obvious link is the Ca2+-dependent NO production by nNOS and eNOS described above. Both constitutively active forms of NOS have a binding site for calmodulin. For nNOS, it was shown that at low [Ca2+]i, calmodulin (CaM) is either free or pre-bound to nNOS with its C-terminus. Upon an increase in [Ca2+]i, also the N-terminus of CaM is binding to nNOS, enabling the activation of this enzyme. Upon sequestration of intracellular Ca2+, Ca2+ dissociation from the N-terminus of CaM likely causes nNOS deactivation i, but downregulates the efflux of Ca2+ via PMCA i trigger the generation of NO by constitutively active NOSs, and NO, as a freely diffusible molecule, supports RyR-mediated Ca2+ release in further cells/subcellular compartments. Finally, rises in [Ca2+]i were shown to release ANP from astrocytes by means of Ca2+-dependent exocytosis i by modulating the release of Ca2+ from the intracellular stores and the Ca2+ influx/efflux over the cell membrane. Although it is known that both signaling pathways are deregulated in AD, the interplay between the two has been largely neglected.cGMP signaling controls the function of major cell types of the brain parenchyma and the vasculature both alone and in an interplay with intracellular Ca2+ and cGMP sensors [2+]i and cGMP at the subcellular resolution and over different stages of AD. This is extremely important because early neuronal hyperactivity [2+]i but also NO and cGMP production, and decreased cGMP could enhance neuronal hypoactivity, found in late-stage AD [Given the complexity of the molecular mechanisms described above as well as the cellular players involved, this interplay must be studied in vivo. Current developments in the field of optical Ca sensors ,213,214 activity ,215 likestage AD ,217.2+ signaling.Depending on the exact outcome of the longitudinal preclinical studies, individualized treatment options taking into account the sex , disease stage, and the level of neuroinflammation can be developed. Fortunately, future therapies can choose from a rich cGMP-modulating toolkit containing BBB-permeant PDE inhibitors , NO-GC stimulators , etc. The hypothesis-driven development of such therapeutics is, however, impossible without a clear understanding of the direction and the extent of the modulation of NO/cGMP/cGK signaling pathway in aging and AD and its cell type-specific interplay with the intracellular Ca


Plasmacytoid dendritic cells are the main type I interferon producing cells in humans and are able to modulate innate and adaptive immune responses. Tumor infiltration by plasmacytoid dendritic cells is already described and is associated with poor outcomes in cancers. In hematological diseases, Blastic Plasmacytoid Dendritic Cells Neoplasm (blastic counterpart) is well described, but little is known about tumor infiltration by mature plasmacytoid dendritic cells described in Myeloid Neoplasms . This review provides a comprehensive overview of plasmacytoid dendritic cells in Myeloid Neoplasms.Plasmacytoid dendritic cells (pDC) are the main type I interferon producing cells in humans and are able to modulate innate and adaptive immune responses. Tumor infiltration by plasmacytoid dendritic cells is already well described and is associated with poor outcomes in cancers due to the tolerogenic activity of pDC. In hematological diseases, Blastic Plasmacytoid Dendritic Cells Neoplasm (BPDCN), aggressive leukemia derived from pDCs, is well described, but little is known about tumor infiltration by mature pDC described in Myeloid Neoplasms (MN). Recently, mature pDC proliferation (MPDCP) has been described as a differential diagnosis of BPDCN associated with acute myeloid leukemia (pDC-AML), myelodysplastic syndrome (pDC-MDS) and chronic myelomonocytic leukemia (pDC-CMML). Tumor cells are myeloid blasts and/or mature myeloid cells from related myeloid disorders and pDC derived from a clonal proliferation. The poor prognosis associated with MPDCP requires a better understanding of pDC biology, MN oncogenesis and immune response. This review provides a comprehensive overview about the biological aspects of pDCs, the description of pDC proliferation in MN, and an insight into putative therapies in pDC-AML regarding personalized medicine. WHO Classification of Tumours of Heamatopoietic and Lymphoid Tissues . Mor. MorRUNX+ blasts from pDC-AML were able to differentiate in pDC, with a greater rate of pDC obtained with pDC-AML blasts than CD34+ cord blood progenitors and blasts from mutRUNX1 AML without pDC excess. No pDC differentiation was observed with wtRUNX1 AML without pDC excess [Furthermore, ex vivo culture highlighted that CD34C excess ,102.mutRUNX1 AML and pDC-AML [Epidemiologic data highlighted predominant skin involvement in pDC-AML compared to AML without pDC excess, a greater proportion of adverse ELN risk, and poorer outcomes, including lower induction therapy rates, a greater relapse rate, and a lower overall survival median at 18.8 months vs. 36.7 months [ pDC-AML . This alOn the other hand, L-asparaginase anti-leukemic affect is extremely well known in acute lymphoblastic leukemia . It is aRecently, an anti-pDC strategy was investigated in pDC-AML, using the anti-CD123-directed diphtheria toxin tagraxofusp-erzs, which was able to eliminate the pDC population in pDC-AML. However, anti-leukemic activity was limited with only a reduction in the blast rate with tagraxofusp-erzs  and AML TET2, IDH1, IDH2, and FLT3 mutations [Immunotherapy is another strategy to modulate the tumor microenvironment and interactions between pDCs and T-cells. In AML and myelodysplastic syndromes, IDO1 expression has been identified as an independent adverse prognostic factor , able toutations .+ T-cell activity in AML, which expressed PD-1 [RAS mutations were associated with T-cell expression of PD-1 and TIGIT [+CD73−TOX+TCF-1lowCD8+ T-cells phenotype was increased in AML, and TIGIT blockade could improve T-cell cytotoxicity [Treg proportion in AML BM is also higher than in healthy donors. The expression of ICOSL by AML blasts promotes Treg accumulation in BM and blockade of ICOS signaling impaired Treg generation and retarded AML progression . Epigenesed PD-1 . The GITsed PD-1 . ASXL1 lnd TIGIT . The partoxicity . CD47 bltoxicity . Currenttoxicity ,148. Of toxicity . In additoxicity . All tho+ T-cells reinjection strategies used in cancers have been optimized by transgenic T-cell receptor T-cells engineering (tgTCR T-cells), which enables us to recognize TAA specifically presented by selected MHC molecules [Optimization of cancer immunotherapy could implicate DC modulations or targeting . Moreoveolecules ,153,154.olecules , but furRUNX1 mutation rate was higher in pDC-AML than in all AML, but RUNX1 mutations were more frequently reported in de novo AML and M0-AML in comparison with all AML and pDC-AML [RUNX1 mutation [mutRUNX1 AML and mutRUNX1 pDC-AML, with ASXL1, DNMT3A and RAS pathway mutations [RUNX1 mutation rate in pDC-CMML was not increased and only concerned 3–12% of pDC-CMML versus 2–20% of pDC-poor CMML [mutRUNX1 M0-pDC-AML seemed to have a particular clonal evolution with a pDC proliferation driven by RUNX1 mutation, contrary to pDC-CMML.The  pDC-AML ,157,158.mutation . Furtherutations . Howeveroor CMML . Thus, dRUNX1 mutations are mainly a loss of function [IRF7 expression and increased antigen presentation and ZEB 1 and 2 expression loss [+ T-cell and Treg can cause inflammatory disorders [On the other hand, function ,158,160.function , leadingion loss ,161,162.ion loss . MYD88-Nion loss ,165. Dysion loss . In addiisorders ,168. RUNisorders ,170.mutRUNX1 AML [+ T-cells cytotoxicity [Targeting RUNX family abnormalities could be an interesting strategy in pDC-AML. Indeed, BET induces a TCF4 amplification loop . The use1mut AML ,176, and1mut AML  and the toxicity . FurtherFinally, the regulation of pDC by fatty acid and lipid metabolism was also reported . Indeed,A better understanding of pDCs biology, oncogenesis and immune response have permitted us to identify pDCs as a potent target in myeloid neoplasms. Through their tolerogenic activity, pDC are able to modulate innate and adaptive immune responses in myeloid neoplasm, responsible for adverse outcomes. Systematic research of pDC infiltration and pDC markers in BM at diagnosis or at relapse of myeloid neoplasm could predict adverse outcomes and may allow more appropriate treatments to be found. Regarding precision medicine, treatment targeting pDCs could be an interesting way to optimize outcomes observed in MPDCP associated with myeloid neoplasms, but there is still a need for further investigations. In the future, adoptive therapies such as CAR T-cells, tgTCR T-cells, as well as BiTE and other new targeting strategies and immunotherapies might be used as adjuvant treatments in the early stages of the disease.