IKZF1 have been frequently delineated in B‐lineage acute leukaemia (B‐ALL) but rarely elucidated in acute myeloid leukaemia (AML). IKZF1 mutations confer a poor prognosis in AML, and hotspot mutations of IKZF1, N159Y and N159S tend to occur in B‐ALL and AML respectively. However, the pathogenesis of IKZF1 N159S in AML and IKZF1 lineage susceptibility are largely unknown.Genetic mutations of IKZF1‐mutated AML patients were evaluated. Multi‐omics analysis and functional assays were performed in vitro using IKZF1 mutations knock‐in AML cell lines.The genetic and clinical characteristics of IKZF1 were identified in 475 newly diagnosed AML (non‐M3) patients. Based on RNA sequencing, three classes of IKZF1‐related AML were defined, including 9 patients (39.13%) with IKZF1 N159S mutations, 10 (43.47%) with CEBPA mutations and 4 others (17.39%). IKZF1 N159S may define a unique subgroup with higher HOXA/B expression and native B‐cell immune fractions. Gene expression data of multiple knock‐in cell lines indicate that the lymphocyte differentiation‐related MME and CD44 kept high expression in IKZF1 N159Y but were downregulated in N159S. CUT&TAG sequencing showed that IKZF1 N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis of IKZF1 N159S may depend on the deregulation of several cofactors, such as oncogenic MYC and CPNE7 targets.23 (4.84%) small sequence variants of IKZF1‐related AML, which may promote an in‐depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML.Collectively, we dissected the molecular spectrum and clinical features of  IKZF1 bared unfavourable prognostic significance in the leukaemogenesis of AML, especially for the hotspot IKZF1 (p.N159S) point mutation.Genetic mutations of IKZF1 (p.N159S) point mutation occurs preferentially in AML with poor prognosis and defines a rare molecular subtype with unique gene expression.IKZF1 (p.N159S) mutation exhibits a more aggressive disease progression potentially by deregulation of NOTCH signalling, VEGF, CPNE7 and MYC‐related factors.Recurrent  Recently, based on RNA‐sequencing (RNA‐Seq) data of 655 AML patients, the largest multi‐centre cohort in China, we have illuminated eight meta‐subgroups of AML, which provides a comprehensive framework to pinpoint rare molecular subtypes in AML, among which, recurrent IKZF1 mutations was discovered in this cohort.Acute myeloid leukaemia (AML) is a group of the most aggressive haematopoietic malignancies in adults associated with development blockage and over‐proliferation of myeloid lineage cells.IKZF1 encodes IKAROS, which is a critical transcription factor for lymphopoiesis and immune haematopoiesis,IKZF1 are commonly involved in the pathogenesis of ALL, especially B‐ALL. Deletion of exons 4−7 of IKZF1 leads to the generation of Ik6 heterodimer, which interferes with normal B‐lymphocyte signalling and promotes B‐ALL development.IKZF1 have even been more widely reported in the unfavourable molecular subtypes of B‐ALL, such as in nearly 40% of BCR::ABL1/‐like cases.IKZF1 N159Y is a newly identified rare subtype of B‐ALL with unique gene expression profile, characterised by significant upregulation of the transcriptional coactivator YAP1, SALL1 and ARHGEF28B, and downregulation of the B‐cell receptor signalling and JAK‐STAT signalling pathways.IKZF1 mutations are predisposed to T, B, and myeloid cell combined immunodeficiency and AL.IKZF1 in ALL, IKZF1 mutations have rarely been reported in AML. A recent study reported that the frequency of IKZF1 mutations in AML was 2.6% (5/193), which were significantly associated with SF3B1 and biCEBPA mutations.IKZF1 mutations were retrospectively identified in OHSU , TCGA  and TARGET  cohorts. Among these 13 IKZF1 mutations, 5 were N195S mutation .IKZF1 with unique gene expression profiles in AML. Among them, IKZF1 N159S was a recurrent hotspot mutation, which closely clustered in a subset of patients with myelodysplasia‐related mutations and upregulated gene expression of the HOXA/B family genes.HOXA‐related AML, which suggests the emerging synergistic role of IKAROS in high‐risk AML.Although a large number of studies have elucidated the important pathogenic role of IKZF1 mutations. Firstly, genetic alterations of IKZF1 are common and widely reported in B‐ALL, however, the genomic landscape, molecular classification and clinical significance are scarcely investigated in a large AML cohort. Meanwhile, certain IKZF1 mutations tend to occur in B‐ALL (p.N159Y) or AML (p.N159S) have been reported but the potential mechanism of the IKZF1‐related lineage susceptibility is rarely studied.IKZF1 in 383 AMLs that we previously published and 92 newly diagnosed primary AMLs excluding patients with PML::RARA (M3). In addition, via comparison of gene expression profiles as well as in vitro investigation, we proposed to understand the underlying leukaemogenesis of IKZF1 in AML. We hope our work will serve as a novel reference for future research on leukaemia‐lineage susceptibility, and as a starting point for a new, genetically based disease classification that will contribute to the improved risk stratification and ultimately better treatment outcomes.To date, a few critical issues remain to be addressed to further explore the pathogenesis and improve the prognosis of AML patients with 22.1IKZF1 mutations is provided  and significance was defined as p < .05.Wilcoxon rank‐sum test was used for calculating the statistical significance when comparing of two groups while Kruskal‐Wallis test was used for more than two groups, paired‐samples two‐tailed Student's 2.4The funders have no role in the study design, data collection, data analysis, data interpretation or report writing.Additional methods are outlined in 33.1IKZF1 N159S occurred in 1.89% (9/475) of AML, accounting for 39.13% (9/23) of all IKZF1 mutations in this cohort.The similarity of the patient samples based on gene expression data was visualised in a t‐Distributed Stochastic Neighbour Embedding (t‐SNE) analysis, which showed an overall separation of AML samples according to different genetic abnormalities defined by the 5th edition of the World Health Organization (WHO) Classification of Hematolymphoid TumorsIKZF1 N159S were clustered into myelodysplasia‐related/‐like (G5) subgroup, while the majority of other IKZF1 mutant loci displayed a similar gene expression profile to biallelic CEBPA or ‐like  subgroup, which incorporated not only biCEBPA, but also moCEBPA mutations with loss of heterozygosity and several CEBPA WT cases.IKZF1 aberrations were recognised including IKZF1 N159S, biCEBPA/‐like and others  to those in the ELN adverse risk group  and the variant allele frequency (VAF) of IKZF1 mutations appeared to be higher in IKZF1 N159S‐positive cases  and core markers including HOXA/B, MYCT1, PAWR and BEND4.IKZF1 N159S hotspot mutations in this work , p53‐mediated apoptosis (CCDC8) and activator TGF‐beta pathway (RBPMS). We also provided a comparison of IKZF1‐positive gene expression profile with other non‐IKZF1 AML using the same 1267 genes expression, and further demonstrated that IKZF1 N159S may define a separate molecular subtype with unique GEP in all our AML cases, even within the subcluster of myelodysplasia‐related/‐like AML  IKAROS.IKZF1 resulted in significant inhibition of cell growth and proliferation coinciding with its tumour‐suppression function, as measured by CCK‐8 assays in leukaemia cells K562 and U937. In contrast, cells with N159S and N159Y expression showed more aggressive cell proliferation; however, G158S expression cells showed a similar growth level to that of the WT IKZF1 cells, suggesting that G158S potentially retains the normal tumour‐suppression function of IKAROS, while N159S/Y could partially impair this function , the deregulated gene sets can be divided into six subclasses, namely, C1‐C6. These subclusters of genes were enriched in WT, WT/G158S, N159Y/S, N159S and N159Y, respectively. Among them, we identified at least 23 genes, that is, RELN, CXCR1, IGSF9B, ONECUT2, TRIB1 and MAP3K20, that were specifically upregulated in N159S mutant cell line  targeting MYC and CPNE7 were used to knockdown the expression level of MYC and CPNE7 in N159S‐expressing cells  is a group of highly genetic heterogeneous diseases with various prognosis. Functionally, gene mutations could be classified into different types, as exemplified by the dysregulation of transcription factors (TFs), that is, IKZF1 were firstly reported in aggressive B‐ALL subtypes, that is, BCR::ABL1/‐like, which were characterised by the high relapse rate and short survival duration.IKZF1 mutations were poorly characterised in the research field of AML. Only a few studies have reported the rare IKZF1 mutations in acute myeloid leukaemia with a low frequency.KMT2A translocations.IKZF1 (IKAROS) in disease fate determination has been rarely explored.With the advent of the era of high‐throughput sequencing and precision medicine of AL, genetic mutations of IKZF1 sequence variants (n = 23) in AML was elucidated. Three major classes of IKZF1 mutations were identified including IKZF1 N159S , biCEBPA/‐like IKZF1  and others (n = 4). Most of them may result in abnormal genome binding patterns and gene expression regulations via impacting the DNA‐binding domain of IKZF1. Besides, the hotspot IKZF1 N159S mutations frequently coexisted with RUNX1 and spliceosome mutations. These comutations are common in the myelodysplasia‐related/‐like AML, particularly in elderly populations. Prognosis‐wise, AML patients with IKZF1 N159S had an extremely poor prognosis, even worse than TP53 mutation/complex karyotype AML in our cohort. According to the available drug response data, combination therapy of demethylation agents and Homoharringtonine (HHT)/all‐trans‐retinoic acid (ATRA) showed potential sensitivity in IKZF1 N159S‐positive AML  of BCR::ABL1‐like B‐ALL was also upregulated in IKZF1 N159S‐related AML.IKZF1 N159S. These specifically enriched pathways may partially interpret the unique immune microenvironment and clinical prognosis of IKZF1 N159S, providing a wealth of therapeutic targets/pathways for this high‐risk AML subtype.Gene expression profiles indicated that compared with biIKZF1 genetic mutations. IKZF1 N159S exerted aberrant regulation of cell apoptosis, cell cycle and cell viability, which indicated the perturbation of the tumour‐suppression function of IKZF1. We then compared the gene expression profiles of hotspot mutations IKZF1 N159S, G158S and N159Y and found that B‐lymphoid development markers, such as MME and CD44, were highly expressed in N159Y‐positive but downregulated in N159S‐positive cell line. RUNX2 was also highly expressed in IKZF1 N159Y knock‐in AML cells, which was reported to be abundant in the haematopoietic stem/progenitor cells in mice, and the upregulation of RUNX2 led to the repression of myeloid differentiation.RUNX1T1, a well‐known factor for haematopoietic differentiation and myeloid development, was highly expressed in IKZF1 N159S knock‐in AML cells instead of IKZF1 N159Y.Next, we performed in vitro functional experiments to investigate the cellular effects of CPNE7, VWDE, LTK and IGSF9B, of IKZF1 N159S‐positive AML. Among these, we speculate that the inherent characteristics of recurrent IKZF1 N159S mutation might lead to a more deteriorative evolution of AML via upregulating MYC targets and CPNE7, which were directly regulated by IKZF1 N159S. Inhibitors of MYC have been widely exploited, that is, 10058‐F4, APTO‐253. Therefore, the employment of MYC inhibitors could bring new therapeutic ideas for patients with IKZF1 N159S mutations.Based on gene expression profiling and CUT&TAG sequencing of knock‐in human cell lines, we identified several potential common and specific pathways, that is, JAK‐STAT, NOTCH signalling pathway and B‐cell development, and genes, that is, IKZF1 and defined the significant function of IKZF1 N159S in AML. The clinical and biological significance of IKZF1 variants were explored both in patients and cell line level, highlighting the key role of IKZF1 in the pathogenesis, treatment, and stratification of AML. Furthermore, it may appear as an important molecular target for tailored treatment, as exemplified by MYC inhibitor. These findings may further promote the accurate classification and improvement of prognosis for patients with IKZF1‐positive AML, which may also exert potential impact on other tumour researches involving immune microenvironment and lineage switching mechanism.Taken together, our data identified the mutation spectrum of SY and LJF conceived and designed the study. WY and ZYY performed the experiments. WY, LJF and ZYL analysed and interpreted data. CWY, WY, STF, YW, ZJN and ZYM collected the clinical data; WY and LJF wrote the manuscript. CWY, ZYY and SY revised the paper. All authors reviewed the data and paper and approved the final version of the manuscript.This work was supported by the National Natural Science Foundation of China (No. 82270116) and the Shanghai Municipal Education Commission‐Gaofeng Clinical Medicine Grant Support (No. 20161406).The authors declare no competing interests.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


FLT3‐ITD)–positive AML cells in vitro, and significantly inhibiting AML progression in vivo. Mechanistically, ACC010 and HHT cooperatively downregulated MYC and inhibited FLT3 activation. Further, when HHT was added, ACC010‐resistant cells demonstrated a good synergy. We also extended our study to the mouse BaF3 cell line with FLT3‐inhibitor‐resistant FLT3‐ITD/tyrosine kinase domain mutations and AML cells without FLT3‐ITD. Collectively, our results suggested that the combination treatment of ACC010 and HHT might be a promising strategy for AML patients, especially those carrying FLT3‐ITD.Bromodomain‐containing protein 4 (BRD4) inhibitors have been clinically developed to treat acute myeloid leukemia (AML), but their application is limited by the possibility of drug resistance, which is reportedly associated with the activation of the WNT/β‐catenin pathway. Meanwhile, homoharringtonine (HHT), a classic antileukemia drug, possibly inhibits the WNT/β‐catenin pathway. In this study, we attempted to combine a novel BRD4 inhibitor (ACC010) and HHT to explore their synergistic lethal effects in treating AML. Here, we found that co‐treatment with ACC010 and HHT synergistically inhibited cell proliferation, induced apoptosis, and arrested the cell cycle in FMS‐like tyrosine kinase 3‐internal tandem duplication  and homoharringtonine (HHT) synergistically inhibited cell proliferation, induced apoptosis, and arrested the cell cycle in AML with  AMLacute myeloid leukemiaBRD4bromodomain‐containing protein 4FLT3‐ITDFMS‐like tyrosine kinase 3‐internal tandem duplicationHHThomoharringtonineshRNAshort hairpin RNATKDtyrosine kinase domainTKItyrosine kinase inhibitorTKI‐Rtyrosine kinase inhibitor resistance1Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is characterized by genetic heterogeneity . Under tFLT3 internal tandem duplication (FLT3‐ITD) is the most common mutational form in AML and occurs in approximately 25% of AML and 30% of cytogenetic normal AML [FLT3‐ITD have a high incidence of relapse and relatively short survival duration. Current evidence suggests that FLT3‐ITD constitutively activates FLT3 kinase activity to promote the proliferation of AML [FLT3‐ITD alone is not sufficient to generate AML, the role of a therapeutic target has broadened the treatment options for AML patients [FLT3‐ITD/TKD mutations. However, TKI resistance (TKI‐R) may emerge in acquired FLT3‐TKD mutations or in the case of high levels of FLT3 ligand [FMS‐like tyrosine kinase‐3 (FLT3) is a receptor tyrosine kinase normally expressed in hematopoietic stem and progenitor cells which plays a critical role in early hematopoiesis . FLT3 inrmal AML . AML patpatients . Consist3 ligand , 8. ThusMYC [MYC activation and aberrant transcriptional elongation [FLT3‐ITD, BRD4 inhibitors, alone or in combination with FLT3 inhibitors, also exhibited excellent proliferation inhibition [in vitro experiments, their clinical outcome is unsatisfactory when administrated as single agents, along with the issue of the development of resistance. It has been reported that the mechanism of BRD4 inhibitor resistance in AML was mainly associated with WNT/β‐catenin–mediated c‐MYC reactivation after suppression, so targeting the WNT/β‐catenin–c‐MYC axis possibly could help overcome BRD4 inhibitor resistance [BRD4 belongs to the family of BET  proteins, called chromatin “readers” that bind to the acetylated lysines on histone proteins, which has an important impact on transcriptional regulation of multiple important oncogenes, including MYC . BRD4 haongation . In AML hibition . To datehibition , 13. Howsistance .Cephalotaxus hainanensis. Over the past few years, our research team has devoted great efforts to understand its effect and mechanism in AML [de novo AML [FLT3‐ITD via downregulating FLT3 expression and inhibiting FLT3‐mediated downstream signaling activation [c‐MYC activation by directly binding the NF‐κB‐repressing factor, whereas HHT also reduced the protein expression of CTNNB1, which is known as an intracellular signal transducer in the WNT signaling pathway [FLT3‐ITD–positive AML.Homoharringtonine (HHT), a classic antileukemia drug in China, was originally isolated from the m in AML , 17, 18.novo AML , 16. Bec pathway . TherefoFLT3‐ITD in vitro and in vivo. The combination inhibited cell proliferation by inducing apoptosis and arrested the cell cycle at the G0/G1 phase. Mechanistically, downregulation of MYC and inhibition of the FLT3 pathway may account for the synergistic effects. Furthermore, synergistic effects were also shown in cells resistant to ACC010 or FLT3 inhibitors. Moreover, the synergistic effects of this combination could also be expanded to some subtypes of FLT3‐ITD–negative AML. Therefore, our studies suggested that ACC010 plus HHT might be a promising combination regimen to treat AML, especially FLT3‐ITD–positive AML.Because BRD4 inhibitors and HHT could downregulate MYC through different pathways, and the downregulation of WNT/β‐catenin by HHT presumably could help overcome resistance to BRD4 inhibitors, we attempted to combine a novel BRD4 inhibitor ACC010 and HHT in AML cells. In this study, we demonstrated that the combination of ACC010 and HHT had significant synergistic effects in AML with 22.1in vitro assays and in 0.5% sodium carboxymethyl cellulose (CMC‐Na) for in vivo assays. HHT was purchased from Med Chem Express  and was dissolved in phosphate‐buffered saline for in vitro and in vivo assays. JQ1 was purchased from Sigma‐Aldrich  and was dissolved in dimethyl sulfoxide. OTX015 was purchased from Selleckchem  and was dissolved in dimethyl sulfoxide. The antibodies GAPDH, FLT3, p‐FLT3, STAT5, p‐STAT5, PARP, caspase‐3, caspase‐7, CDK2, CDK4, CDK6, P21, P27, RB, p‐RB, AKT, p‐AKT, ERK1/2, p‐ERK1/2, MYC, and APC were purchased from Cell Signaling Technology . β‐Actin antibody was purchased from Proteintech .ACC010 was kindly provided by Jiangsu Aidea Pharmaceutical Co., Ltd. . The synthesis of ACC010 was described in the patent CN106132968B Fig. . ACC010 2.2FLT3‐ITD–positive AML cell lines MV4‐11 (RRID: CVCL_0064) and MOLM13 (RRID: CVCL_2119) were a kind gift from R. Bhatia  and were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS . THP1 (RRID: CVCL_0006), OCI‐AML2 (RRID: CVCL_1619), OCI‐AML3 (RRID: CVCL_1844), KG1‐α (RRID: CVCL_1824), HL‐60 (RRID: CVCL_0002), U937 (RRID: CVCL_0007), JURKAT (RRID: CVCL_0065), and K562 (RRID: CVCL_0004) cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences . KASUMI‐1 (RRID: CVCL_0589) cell line was gifted by C. Saijuan . These cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% FBS. All human cell lines have been authenticated using STR profiling within the last 3 years and that all experiments were performed with mycoplasma‐free cells. As for human samples, the experiments were undertaken with the understanding and written consent of each subject. Human data have been performed in accordance with the Declaration of Helsinki and have been approved by Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (IIT20220135B). Mononuclear cells from AML patient samples (n = 16) were obtained from the First Affiliated Hospital of Zhejiang University  between 2021 and 2022, isolated by Ficoll–Hypaque (Sigma‐Aldrich) density gradient centrifugation and cultured in RPMI 1640 medium with 10% FBS. Testing for FLT3‐ITD and other genetic mutations was performed at the First Affiliated Hospital of Zhejiang University . Patient characteristics can be found in Table 2.34 cells/well, and primary AML cells were seeded in 96‐well plates with 1 × 105 cells/well. Cells were treated with variable concentrations of ACC010 and/or HHT for 48 h. Cell viability was assayed by CellTiter‐Lumi™ Luminescent Cell Viability Assay Kit  according to the manufacturer's instructions.Acute myeloid leukemia cell lines were seeded in 96‐well plates with 1–2 × 102.4−1) was added to each well at every 24 h, varying from 0 to 96 h, and the cells were incubated for an additional 4 h at 37 °C before absorbance was measured at 490 nm.Cells were seeded in 96‐well plates (5000 cells/well), which were treated with single agents or a combination regimen. MTS solution measuring 10 μL  , following the manufacturer's instructions. Cells were collected after treatment with drugs for 48 or 72 h. Apoptotic cells were analyzed by flow cytometry after incubation with Annexin‐V FITC and propidium iodide (PI) for 30 min at room temperature using FACScan™ flow cytometer . Cell cycle assay was also detected by flow cytometry using PI DNA staining from MULTI SCIENCES. After treatment with drugs for 48 h, the cells were harvested, fixed overnight with 75% ethanol at 4 °C, and then incubated with DNA staining for 30 min at room temperature. Analysis was conducted by FACScan™ flow cytometer (Becton Dickinson).2.6g for 15 min at 4 °C, and a BCA reagent (Thermo Fisher Scientific) was used to determine the protein concentration of the cellular supernatant. Western blotting was performed using 4–12% SDS/PAGE gel and cellular proteins were transferred onto a preactivated PVDF membrane . Then membranes were blocked with 5% nonfat milk for 1 h and incubated overnight with primary antibodies at 4 °C. Subsequently, the membranes were washed thrice with TBST and incubated with secondary antibodies  for 1 h at room temperature. The target proteins were visualized using FDbio‐Femto ECL  and analyzed using image lab™ software . The densitometry quantification was performed using the imagej software .Cells were lysed in RIPA buffer  with protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific) on ice for 30 min. Next, the cell lysate was centrifuged at 12 000 2.7GAPDH .MYC .APC .DVL1 .FZD5 .GSK3B .Total RNA was extracted from the cells using the TRIzol reagent . Reverse transcription was performed using RNA PCR core kit (TaKaRa). Quantitative real‐time PCR was carried out using SYBR Green qPCR mastermix (TaKaRa). Analysis was performed with Bio‐Rad CFX96 (Bio‐Rad). GAPDH was used as internal control. The sequences of the primers were as follows:2.86) were injected into each mouse via the tail vein. After 6 days, cell engraftment was assessed after intraperitoneal injection of luciferin (Promega). Then mice were randomly assigned to four groups according to the intensity of the luciferin signal by using the IVIS Imaging System . Each group was treated as follows: 0.5% CMC‐Na , 20 mg·kg−1 of ACC010 , 0.5 mg·kg−1 of HHT , or 20 mg·kg−1 of ACC010 and 0.5 mg·kg−1 of HHT (administered per the single‐agent group). Mouse body weight was determined once a week. The growth of the leukemia cells was monitored every week by using an IVIS. The survival curve of the mice was analyzed by graphpad prism 9 .For AML xenografts, female NCG (NOD/ShiLtJGpt‐Prkdcem26Cd52Il2rgem26Cd22/Gpt) mice, aged 5–6 weeks, were purchased from GemPharmatech Co., Ltd  and were raised in the Experimental Animal Center of Zhejiang Chinese Medicine University Laboratory Animal Research Center. Mice were exposed to a 10/14 h light–dark cycle, kept under normal room temperature and fed standard pellet food and tap water. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee . And all animal experiments were performed following animal use guidelines and ethical approval. In this study, MOLM13‐luciferase cells  were designed and cloned into a modified pLKO.1‐puro‐shRNA plasmid. The sequence of sh1 was GGAAGTGGAAGAGAATAAA. The sequence of sh2 was GATTACTATAAGATCATTA. To knock down APC, MV4‐11 was transfected with mCherry protein containing shRNA or control lentiviruses cloned into pLVX‐shRNA2 vector. The sequence of NC was AGCGTGTAGCTAGCAGAGG. The sequence of sh1 was CCCAGTTTGTTTCTCAAGAAA. The sequence of sh2 was TAATGAACACTACAGATAGAA. For MYC overexpression, the coding DNA sequence was cloned into the pCDH1‐MSCV‐MCS‐EF1‐GreenPuro vector and then transfected into MV4‐11. BaF3 cells were stably expressing FLT3‐ITD or FLT3‐ITD/F691L or FLT3‐ITD/D835V utilizing PCDH lentivirus vector containing those mutations.To knock down 2.10graphpad prism 9 and expressed as mean ± SEM. Statistical significance was assessed using two‐tailed Student's t‐tests to compare means between two groups . calcusyn software  was used to calculate combination index. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination. Assessment of compound synergy was also conducted by Bliss scoring utilizing DrugComb online web application tool (https://drugcomb.fimm.fi). The combination effect is additive if the Bliss score equals 0, whereas the combination effect is synergistic if the Bliss score is positive. Survival was analyzed using the Kaplan–Meier method and analyzed using a log rank test.Data were analyzed using 33.1FLT3‐ITD) were both of the most sensitive to ACC010 and HHT, whereas U937 was relatively resistant to both drugs in AML cell lines , ACC010 group (20 mg·kg−1), HHT group (0.5 mg·kg−1), and combination group (20 mg·kg−1 of ACC010 and 0.5 mg·kg−1 of HHT). We injected each mouse with 1 × 106 MOLM13‐luciferase cells. Six days after injection, when leukemia cells were engrafted in the bone marrow, we started the aforementioned drug administrations. All mice had equal tumor burdens before being administered any treatment. Mice treated with a combination of ACC010 (20 mg·kg−1) and HHT (0.5 mg·kg−1) had significantly lower leukemia tumor burdens on day 13 and day 20 than mice treated with vehicle or ACC010 or HHT alone  variability in patients. BRD4 inhibitor OTX015 is the first compound that has undergone a phase I clinical trial in AML . The results demonstrated that although the clinical response to OTX015 was observed in AML patients, OTX015's therapeutic index (the ratio between its toxicity and therapeutic dose) was narrow. Patients who achieved remissions ultimately relapsed after 2–5 months [in vitro and in vivo. Furthermore, evidence suggested that MYC provided a possible inherent mechanism for this combination, and HHT might help overcome resistance to BRD4 inhibitors via downregulation of the WNT/β‐catenin pathway. Our results demonstrated that BRD4 inhibitor‐resistant AML cells with MYC overexpression or APC knockdown were also sensitive to BRD4 inhibitors when combined with HHT.It is noteworthy that targeting BRD4 is a promising strategy in AML therapy. Several structure‐based inhibitors are being developed afterward. Although BRD4 inhibitors showed remarkable antileukemia activity in 5 months . Other r5 months . Another5 months . Our stuFLT3 mutated subtype in AML, which is associated with poor prognosis. Current guidelines recommend earlier incorporation of targeted agents to achieve deeper remissions after detecting FLT3mut at diagnosis. The clinical application of FLT3 inhibitors has improved outcomes in AML patients with FLT3 mutations. However, as mentioned before, FLT3 inhibitors are faced with a complication that drug resistance compromises their efficacy in clinical therapy. Previous studies reported that resistance mechanisms include the acquisition of secondary FLT3‐TKD mutations. Among those mutations, F691 and D835 in FLT3‐ITD were found to be substantial barriers to disease control in AML patients treated with FLT3‐TKIs [FLT3‐ITD in addition to FLT3‐TKD mutations.In this article, we focused on the FLT3‐ITD–positive AML patients, with an overall response rate of 78% [FLT3‐ITD–positive cells and re‐sensitized FLT3‐TKI–resistant cells; BRD4 inhibitor JQ1 had synergistic lethal effects in AML and could help overcome FLT3‐TKI resistance; HHT could help to inhibit the growth of drug‐resistant clones and increase the antileukemia effects in FLT3‐ITD AML [FLT3‐ITD AML. This regimen did not include FLT3‐TKIs but also still presented significant killing effects. In addition, observations suggest that the combination can be utilized in AML cells with FLT3‐TKD mutations. Further studies are needed to confirm whether the combination of BRD4 inhibitors and HHT can enhance the efficacy of FLT3‐TKIs to help overcome resistance. Another FLT3‐TKI resistance mechanism was considered to be related to high levels of FLT3 ligand, which could lead to the activation of the FLT3‐MAPK pathway and thus confer resistance to FLT3 inhibitors [Currently, some clinical trials involving combination regimens with FLT3‐TKIs in AML have completed, including the combination of sorafenib and 5‐azacytidine, which is proven to be effective in untreated e of 78% . Anothere of 78% . Meanwhi‐ITD AML , 29, 30.5FLT3‐ITD mutation. With MYC overexpression or APC knockdown to generate cells resistant to BRD4 inhibitor, we found that HHT could enhance the efficacy of the BRD4 inhibitor to help overcome resistance. Furthermore, we extended this regimen to cells with FLT3‐TKD mutation and cells without FLT3‐ITD. The experimental results revealed the potential of ACC010 combination with HHT in the therapy of AML patients harboring FLT3‐TKD who did not respond well to classic FLT3 inhibitors. Meanwhile, the combination regimen could treat AML patients without FLT3‐ITD mutation. Moreover, our results support that other BRD4 inhibitors to combine with HHT for AML therapy.Our study showed a significant synergistic effect of the combination of BRD4 inhibitors and HHT in AML with The authors declare no conflict of interest.YQ, XZ, and SHM performed the research. JJ designed the research study and supervised the experiments. XH, JSH, HYT, and JS provided specific experimental advice, technical support, and reagents. WWW, XJL, QL, WLY, FLL, JJP, YTZ, and YCZ collected clinical samples and analyzed the data. YQ and XZ wrote the paper. JJ reviewed the paper. All authors read and approved the final manuscript.https://publons.com/publon/10.1002/1878‐0261.13368.The peer review history for this article is available at Fig. S1. Chemical structure of ACC010 and the target protein in AML cell line. A. Chemical structure of ACC010 described in the patent CN106132968B. B. Expression of BRD4, MYC, CDK6 and BCL2 were analyzed by Western blot after treated with ACC010 or JQ1 in MV4‐11. C‐D. The IC50 of ACC010 was analyzed in MV4‐11 BRD4 knockdown cell. ** for p < 0.01, *** for p < 0.001.Click here for additional data file.Fig. S2. Algebraic estimate and Bliss score analysis in AML cell lines and patients. A. The combination index (CI) of ACC010 and HHT in AML cell lines was calculated using CalcuSyn software after 48 hours of drugs’ treatment. B. The combination index (CI) of ACC010 and HHT in AML patients was calculated using CalcuSyn software after 48 hours of drugs’ treatment. C. The Bliss score analysis of combinations in AML cell lines and patients was conducted utilizing DrugComb online web application tool (https://drugcomb.fimm.fi).Click here for additional data file.Fig. S3. Lethal effects of ACC010 and HHT against normal samples and effects on apoptosis and cell cycle in primary AML cells. A. normal samples which from healthy donors or CD34+ hematopoietic stem cells were treated with variable concentrations of ACC010 or HHT for 48 hours and cell viability was analyzed. B. Apoptosis in primary AML cells induced by two drugs or their combination utilizing FCM after incubation with Annexin‐V and PI. C. Cell cycle analysis by flow cytometry in primary AML cells cultured with drugs for 48 h.Click here for additional data file.Fig. S4. Synergistic effects of ACC010 and HHT on ACC010‐resistant leukemia cells and FLT3‐ITD/TKD BaF3 cell. A. AML cell lines were treated with ACC010 for 48 hours and IC50 were showed. B. MYC mRNA levels in indicated leukemia cell lines after 2 h, 6 h, 12 h, 24 h and 48 h of ACC010 treatment (2 μM), relative to DMSO‐treated cells. C. Bliss score analysis of combinations in MY4‐11 MYC‐OE cells. D. mRNA levels of WNT/β‐catenin pathway genes was detected in AML cell lines. The bars represented average of ACC010 resistant AML cell lines  or sensitive cells . * for p < 0.05. E. Bliss score analysis of combinations in MY4‐11 APC‐knockdown cells. F‐G. JURKAT and K562 cells were treated with variable concentrations of ACC010 or HHT for 48 hours. Cell viability, CI value and Bliss score were analyzed then. H. Bliss score analysis of combinations in BaF3 cells transfected with NC, WT, FLT3‐ITD, FLT3‐ITD/F691L and FLT3‐ITD/D835V.Click here for additional data file.Fig. S5. JQ1 and OTX015 combined with HHT in AML cell lines. A. AML cell lines were treated with JQ1 for 48 hours and IC50 were showed. B. AML cell lines were treated with OTX015 for 48 hours and IC50 were showed. C. Bliss score analysis of co‐treatment with JQ1 and HHT in AML cell lines utilizing DrugComb online web application tool (https://drugcomb.fimm.fi). D. Bliss score analysis of co‐treatment with OTX015 and HHT in AML cell lines utilizing DrugComb online web application tool (https://drugcomb.fimm.fi).Click here for additional data file.Table S1. Characteristics of primary AML patients.Click here for additional data file.


Cardiovascular diseases (CVD), with high morbidity and mortality, seriously affect people’s life and social development. Clinically, reperfusion therapy is typically used to treat ischemic cardiomyopathy, such as severe coronary heart disease and acute myocardial infarction. However, reperfusion therapy can lead to myocardial ischemia reperfusion injury (MIRI), which can affect the prognosis of patients. Studying the mechanisms of MIRI can help us improve the treatment of MIRI. The pathological process of MIRI involves many mechanisms such as ferroptosis and mitophagy. Ferroptosis can exacerbate MIRI, and regulation of mitophagy can alleviate MIRI. Both ferroptosis and mitophagy are closely related to ROS, but there is no clear understanding of the relationship between ferroptosis and mitophagy. In this review, we analyzed the relationship between ferroptosis and mitophagy according to the role of mTOR, NLPR3 and HIF. In addition, simultaneous regulation of mitophagy and ferroptosis may be superior to single therapy for MIRI. We summarized potential drugs that can regulate mitophagy and/or ferroptosis, hoping to provide reference for the development of drugs and methods for MIRI treatment. Cardiovascular diseases (CVD), including hypertension, coronary heart disease and myocardial infarction, not only pose a serious threat to human health but also bring heavy economic burden to patients . CoronarAs we know, mitochondria are extremely important since the heart needs efficient oxidative metabolism . MitochoFerroptosis, a new form of cell death is identified as a primary mechanism of MIRI . Ferroptvia regulating mitophagy and/or inhibiting ferroptosis, providing reference for the treatment of MIRI. This study was a mechanism-oriented review that explored the innovative relationship between mitophagy and ferroptosis participated in the development of CVD, which could provide new insight for CVD treatment and bring hope to patients by improving clinical efficacy, improving patient prognosis and increasing the quality of life of CVD patients.Although with advanced development of drug management, CVD remain the most common cause of death worldwide , which rLiterature searches were conducted in the PubMed, Web of Science and Chinese National Knowledge Infrastructure databases. In addition to articles published since 2017, earlier articles were also considered. The keywords used were as follows: myocardial ischemia reperfusion injury, ferroptosis, mitophagy, myocardial ischemia reperfusion injury and ferroptosis, mitophagy and myocardial ischemia reperfusion injury, iron metabolism, the mechanism of ferroptosis, the mechanism of mitophagy. As our work gradually unfolded, we then searched literature by keywords HIF and mitophagy, HIF and ferroptosis, NLRP3 and mitophagy, NLRP3 and ferroptosis, mTOR and mitophagy, mTOR and ferroptosis, natural compounds with mitophagy and/or ferroptosis. After removing duplicate articles and the articles with little relevance, 151 articles were selected for this review.etc.  can be absorbed directly via heme/folate transporter 1 at the apical membrane of intestinal epithelial cells (3+) in food is partly reduced and dissolved by gastric acid and ascorbic acid, and the rest is reduced to Fe2+ by cytochrome B, which is then transported to intestinal epithelial cells by divalent metal-ion transporter 1  for absorption. Subsequently, after traversing the basolateral membrane via ferroportin 1, Fe2+ is oxidized to Fe3+ by Hephaestin (HEPH), and then binds with transferrin (TF) to form TF-Fe3+ complex for utilization by organs (3+ complex binds with the transferrin receptor (TFR) and enters the endosome through endocytosis, Fe3+ is released from the TF-Fe3+ complex, then is reduced to Fe2+ by STEAP3 and crosses the endosomal membrane into the cytoplasm by DMT  forms except a small amounts of iron are lost through skin exfoliation, gastrointestinal exfoliation, and urine and bile excretion . After eal cells . Non-hemy organs . After tm by DMT . The imp with TF .etc. When the body is in a pathological condition of iron overload, the capacity of plasma transferrin to bind iron is saturated, leading to the accumulation of non-transferrin bound iron  first acquire hydrogen atoms in PUFA to produce Lipid ROS (L-). Then, the Lipid radicals react with the oxygen molecule to form Lipid peroxyl radicals (LOO-). Lipid peroxyl radicals extract hydrogen atoms from other PUFA to form a new LOO- and Lipid hydroperoxide (LOOH). LOO- can continuously react with PUFAs, which makes the lipid peroxidation of PUFAs have the characteristics of cascade reaction  and phosphatidylethanolamine (PE) are oxidatively decomposed. PUFA is the main component of phospholipids in cell and organelle membranes, it is also an important substrate for the synthesis of PE, the main component in the inner layer of the phospholipid bilayer. PUFA has a high affinity with ROS. ROS of hydroxyl radicals (OH•) and hydrogen peroxide   . When GSm (GSSG) .γ-glutamylcysteine under the catalysis of glutamate-cysteine ligase. Next, GSH is formed from γ-glutamylcysteine and glycine under the catalysis of GSH synthase. Also, GSH can regulate glutamate-cysteine ligase through negative feedback  and a heavy-chain subunit SLC3A2 , which are linked by an extracellular covalent disulfide bond and play different roles. System Xc- imports extracellular cystine in exchange for intracellular glutamate at a ratio of 1:1 . Intracefeedback .When system Xc- dysfunction occurs, the cell redox becomes unbalanced. A study of glioma cells showed that knockdown of SLC7A11 increased ROS production and decreased glutathione production, resulting in increased cell death under oxidative and genotoxic stress, and overexpression of SLC7A11 leads to increased resistance to oxidative stress . The sysWe have analyzed the relationship between mitophagy and MIRI as well ferroptosis and MIRI respectively, and the mechanism of ferroptosis. Mitophagy also has complex regulatory mechanisms. Classic mitophagy pathways include PINK1/Parkin, BNIP3/Nix and FUNDC1 pathway . Mitophaα, HIF-2 α, and HIF-3 α, is a heterodimer transcription factor that plays a key role in mediating adaptive responses to hypoxia. HIF is closely associated with ferroptosis. In the hypoxic environment, HIF-1 is involved in the increase of TFR gene transcription in Hep3B human hepatoma cells (α potentiates oxidative cell death by increasing cellular iron (α induced by a widely used plasticizer (di (2-ethylhexyl) phthalate, DEHP) lead to ferroptosis in Leydig and Sertoli cells  was used, mitochondrial morphology and function were preserved and cell death was prevented, despite the GPX4 inhibition and lipid peroxidation remained . Therefoβ/IL-18 in response to numerous danger signals and pathogens (β and IL-18 contents, iron deposition in myocardial tissue were significantly increased, while the GPX4 expression was significantly decreased (NLRP3 is strongly associated with ferroptosis due to the level change of ROS. The NLRP3 inflammasome is a critical component of the innate immune system and inflammation, mediating caspase-1 activation and secretion of the pro-inflammatory cytokine IL-1 athogens . In a swecreased . In an Hecreased . Therefoetc. (etc., PINK1 accumulates on the outer membrane of injured mitochondria, recruiting Parkin from the cytoplasm to the mitochondrial and activating it. Activated Parkin ubiquitinated mitochondrial membrane proteins allow mitochondria to be recognized and swallowed by autophagic vesicles, and eventually fuse with lysosomes and be degraded (NLRP3 is also associated with CVD, including atherosclerosis, MIRI, heart failure, etc. . The ROSetc. . Mitophaetc. . Therefoetc. . In normdegraded . NLRP3 idegraded . Althougvia FUNDC1 both have been reported (As a serine-threonine kinase, mTOR, which is made up of mTORC1 and mTORC2, is an essential controller in cell growth and metabolism. In addition, mTOR is also involved in ferroptosis regulation. In tumor cells, mTOR inhibition can lead to GPX4 degradation and promotes ferroptosis . When mTreported . This inreported . Sometimreported . Therefoα/BNIP3 pathway (Considering the important role of inhibiting ferroptosis and regulating mitophagy in alleviating MIRI, we have summarized the potential drugs that can inhibit ferroptosis and/or regulate mitophagy . In path pathway , and pen pathway , providiIn present, although there are many studies on the mechanism of ferroptosis and mitophagy, the current drug studies on the treatment of MIRI mainly focus on inhibiting ferroptosis or regulating mitophagy alone. Damaged mitochondria will produce a large number of ROS, which promotes ferroptosis. Appropriate mitophagy can reduce mitochondria ROS production to alleviate MIRI. Our analysis showed that ferroptosis in MIRI may be mitochondrial ROS dependent. Since mitochondria are not the only source of ROS in cells, reducing ROS by regulating mitophagy can alleviate but not completely block ferroptosis. Therefore, compounds can simultaneously regulate mitophagy and inhibit ferroptosis have great potential to treat MIRI. Act as the “link” between ferroptosis and mitophagy, HIF, NLRP3 and mTOR can regulate both mitophagy and ferroptosis. Therefore, we analyzed the relationship between ferroptosis and mitophagy in MIRI based on the role of HIF, mTOR and NLRP3, summarized potential drugs that could treat MIRI by regulating mitophagy and/or ferroptosis, hoping to provide reference for the drug and methods development of MIRI therapy.


Herein, we describe the dynamics and spatial distribution of endothelial Ca2+ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca2+ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca2+ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca2+ signals. We further highlighted how distinct Ca2+ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca Consequently, endothelial Ca2+ signals regulate a wide range of cardiovascular functions, including vascular resistance and permeability, coagulation, leukocyte transmigration, neurovascular coupling, angiogenesis and vasculogenesis i i ,5,8. The vessels . Sproutigenesis) ,25,26,272+ signal.Herein, we provide an overview of how vascular endothelial cells regulate two representative examples of vascular functions, such as vasomotion and angiogenesis, by extracting the relevant information from the subcellular spatial profile and/or the temporal pattern of the underlying Ca2+]i can be elicited by neurohumoral agonists, e.g., acetylcholine i that invade the whole cytoplasm and sometimes propagate to neighboring endothelial cells i may rapidly return to the baseline due to the removal of cytosolic Ca2+ by Ca2+-clearing mechanisms, such as sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), plasma membrane Ca2+-ATPase (PMCA), Na+/Ca2+ exchanger (NCX) and mitochondria. In most cases, however, the initial elevation in [Ca2+]i may be followed by repetitive cycles of InsP3-induced ER Ca2+ release that occur as prolonged Ca2+ oscillations or by a sustained plateau level. In both cases, extracellular Ca2+ entry is required to refill the ER and prolong the Ca2+ response i; InsP3R1 supports irregular Ca2+ oscillations; and InsP3R3, which is the less sensitive isoform to both InsP3 and Ca2+, serves as an anti-oscillatory unit that generates monophasic Ca2+ signals. To illustrate how this concept also applies to the vascular endothelium, acetylcholine and glutamate trigger intracellular Ca2+ oscillations in mouse cerebrovascular endothelial cells expressing InsP3R1 and InsP3R2 [2+]i in their human counterparts expressing only InsP3R3 . En. En2+ pu <100 ms  [84]. Sury blips  [84]. Enal cells ,86,87.2+ activity has confirmed that, in native macro- and microvascular endothelial cells, the regenerative Ca2+ wave elicited by physiological autacoids, such as acetylcholine i that could spread to neighboring endothelial cells to ultimately coordinate the vascular function, as outlined in Direct imaging of endothelial Calcholine ,88,89,90lcholine ,92, is iterioles  and thenterioles  and rat 2+-permeable channels may also generate local Ca2+ microdomains that are located around the inner mouth of the open channel, do not generate cell-wide Ca2+ waves and are tightly coupled to specific Ca2+-dependent effectors. These local Ca2+ signals are known as Ca2+ pulsars, Ca2+ wavelets and Ca2+ sparklets  (2+ sparklets occur at the PM (2+ and InsP3) signals , wh, wh2+-pet the PM  and willt the PM  . Enactivity , but theeceptors . The mectivation . Neverth InsP3Rs . The inhe events . Recruitactivity .2+ pulsar sites can also be activated by sympathetic nerve stimulation of adjoining VSMCs via Ca2+ or InsP3 transfer through MEGJs in pressurized resistance arteries or arterioles . NM. NMARs) 2+ signals induced by SOCE activation in the endothelial lineage [2+ entry pathway that replenishes the ER Ca2+ store and sustains long-lasting oscillations in cultured endothelial cells [3-dependent reduction in ER Ca2+ concentration ([Ca2+]ER) and is mediated by the physical interaction between STIM1 , whereas STIM1 and TRPC1/TRPC4 channels mediate a current known as ISOC. In addition, a heteromeric complex comprising STIM1, Orai1 and TRPC1/TRPC4 has been described in some endothelial cells. This complex mediates an ICRAC-like current with intermediate biophysical features between ICRAC and ISOC i, which can spread to neighboring endothelial cells, thereby originating both intra-  composite Ca2+ events sustained by large clusters of InsP3Rs i superimposed on the spontaneous Ca2+ activity, e.g., spontaneous Ca2+ puffs, also termed Ca2+ noise i in ~90% of mouse aortic endothelial cells, while acetylcholine, substance P and bradykinin were significantly less effective . Al. Al2+ leing SOCE ,163.2+ signals in native endothelial cells is still lacking. A combination of high-speed TIRF microscopy i returns to the baseline after each transient i, but not biphasic Ca2+ signals of the same average amplitude, supported the nuclear translocation of NFAT2 in vascular endothelial cells i to induce migration in the leading tip cells. Interestingly, stromal-derived factor-1α (SDF-1α), a chemokine that is released following a drop in O2 tension to stimulate endothelial cell migration, elicits biphasic Ca2+ signals, but not intracellular Ca2+ oscillations, in several types of vascular endothelial cells . In. In2+ sincreases , whereas (NFAT2) . In acco of MLCK . Since tllations ,197. In llations . High-fr nucleus ,197. In esis see . For insal cells .2+ nanodomains i threshold that each Ca2+ spike must reach during an oscillatory train to induce the nuclear translocation of NF-κB in vascular endothelial cells is ~180 nM [2+]i threshold must also be exceeded in circulating ECFCs remains to be determined. This signaling pathway could be exploited for therapeutic purposes i. Zuccolo et al. showed that SDF-1α stimulates the Gi-protein-coupled receptor, CXCR4 to recruit PLCβ2, thereby inducing an initial increase in [Ca2+]i driven by ER Ca2+ release through InsP3Rs, followed by a sustained plateau that was mediated by SOCE i is still unclear. Similarly, it has long been known that VEGF only elicits biphasic Ca2+ signals in vascular endothelial cells, while it is now clear that it may also induce intracellular Ca2+ oscillations. Future work will have to scrutinize whether endothelial Ca2+ oscillations may also be triggered by other growth factors that are not known to elicit spiking Ca2+ activity, such as basic fibroblast growth factor, IGF-2 and angiopoietins [2+-permeable channels regulate crucial endothelium-dependent functions, including TRP Melastatin 2 (TRPM2), which can prevent endothelial dysfunction [2+ sparklets at specific membrane sites where they are tightly coupled with their downstream Ca2+-dependent effectors. However, these discrete Ca2+ events are yet to be measured. Similarly, local events of lysosomal Ca2+ release through TPCs or TRP mucolipin (TRPML) channels, which could either recruit juxtaposed InsP3Rs on the ER or regulate vesicular trafficking and autophagy, also deserve to be visualized in the endothelial lineage [2+ signals that are generated by the summation of multiple inputs, needs to be further explored [The spatio-temporal profile of the underlying Ca disease ,188,211. sulfide ,229. It oduction , but whepoietins ,231. Manfunction  and regufunction ,234; TRPfunction ,218 and function ; and finfunction ,236. The lineage ,237. Theexplored ,146.2+ signals fine-tune vascular function in health and can be targeted in disease [2+ machinery [2+ signals, ranging from discrete Ca2+ events to cell-wide Ca2+ waves, that can be severely attenuated by cardiovascular disorders [The combination of endothelial-cell-specific knockout mouse models and high-speed imaging  will be instrumental in assessing how endothelial Ca disease ,238. We achinery ,242,243.isorders .


G12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of Nras There is increasing evidence of a functional interaction between acute myeloid leukemia (AML) and immune cells, influencing disease outcome. Here the authors study how distinct oncogenes differentially affect the host immune response to leukemic cells in preclinical models of AML. AML is genetically heterogeneous with recurrent genetic abnormalities resulting in activation of signal transduction pathways, impaired function of lineage-specific transcription factors and dysregulation of epigenetic modifiers2. Survival and response to chemotherapy is dependent on the age and molecular profile of AML patients4. Despite chemotherapy, followed where possible by allogeneic hematopoietic stem cell transplantation , or low-intensity combination therapies for elderly patients5, long-term survival is <50% overall and is attributed to relapse or therapy resistance highlighting the importance of developing novel therapies.Acute myeloid leukemia (AML) is caused by the acquisition of genetic mutations in hematopoietic stem and progenitor cells (HSPCs) resulting in a block in myeloid differentiation and the expansion of immature myeloid blasts7. AML patients exhibit myeloid dysfunction, cytotoxic lymphocyte dysfunction of both NK and T cells, secretion of suppressive molecules and upregulation of immune suppressive ligands on AML cells13. Studies indicate that immune microenvironment composition is also important for response to chemotherapeutic treatments. For example, AML patients with abnormal NK cell function and downregulation of NK cytotoxicity surface receptors have defective NK clearance of leukemic blasts17. Lymphocyte recovery after chemotherapy is associated with improved survival and there are even rare cases of spontaneous remission after severe infections19. Early clinical trials suggest that combining hypomethylating agents and immune checkpoint inhibitors may have efficacy in AML, however these results have not yet been confirmed in randomized studies21. AML has a low somatic mutation burden and is predicted to have a low frequency of potential neoantigens23. This poses the question, what regulates immune responses in AML and can distinct genetic aberrations influence immunogenicity? Characterization of the immune microenvironment in specific types of AML, including the mechanisms of immune escape, may help to understand whether the endogenous immune response is capable of controlling, or eliminating AML.There is increasing evidence to support a functional interaction between AML and the immune system25. We find that distinct oncogenes alter the host immune response to the leukemia and that mutant Nras is a key determinant of this immunological selection27.Here, we investigate the immunogenicity of genetically distinct models of AML, representing common clinical and prognostic subsets of genetic alterations found in AML patientsAltogether, these data provide insights into endogenous anti-leukemia immune responses in AML and generate a path for the strategic use of immunotherapies for subsets of AML patients.G12D (AE/NrasG12D), representative of common genetic alterations found in patients with AML3  had similar HSPC baseline function assessed by colony formation in cytokine-enriched methylcellulose  AML developed in all recipients reconstituted with AML oncogene-expressing BM , MLL-AF9 (MA9) or AML1-ETO + NrasML3 Fig. . Rag2−/−o AMLs were transplanted into secondary (2o) non-irradiated immunodeficient Rag2−/−γc−/− or immunocompetent WT recipients  , Npm1fl-cA/+; Nrasfl-G12D/+), expressed from their respective endogenous promoters31. We expanded the AML by transplantation into immunodeficient mice and then transplanted this AML into tertiary (3o) non-irradiated immunodeficient Rag2−/−γc−/− or immunocompetent WT recipients. Npm1c/NrasG12D AML cells generated a rapid, fully penetrant AML when transplanted into Rag2−/−γc−/− recipients, however there was a marked delay in disease latency in immunocompetent WT recipients . Immune cell depletion was verified in the peripheral blood  increases T cell proliferation upon co-culture in comparison to non-transformed BM (Rag2−/−γc−/−) whereas MA9 AML did not , an expansion in CD4+ and CD8+ T effector memory (CD44+ CD62L−) and a decrease in CD4+ and CD8+ T central memory (CD44+ CD62L+) formation when compared to naïve controls , compared to AML passaged previously through immunodeficient Rag2−/−γc−/− mice   or immunodeficient  mice, suggesting that immunoediting is not observed in MA9 AML  Fig. . In contAML Fig.  and refl12D Fig. . This deG12D AML  control. Dual oncogene integration was confirmed by expression of both GFP (NrasG12D) and mCherry (Myc) fluorescent markers. Ectopic Myc expression resulted in only minor differences in MHC Class I surface expression (H2-Db and H2-Kb), but a marked increase in PD-L1 and CD86 expression  together with high expression of inhibitory immune checkpoint ligands PD-L1, GAL-9 and CD155 compared to MA9 and NrasG12D. This indicates that the non-immunogenic phenotype of BA/NH may be driven by inherent low antigen presentation and high expression of immune suppressive checkpoint molecules. In contrast, NrasG12D cells displayed a different cell surface phenotype with increased expression of antigen presentation machinery and immune-stimulatory ligands. Indeed, when we looked across a more extensive panel of molecular subtypes, RAS mutant human AML demonstrates high HLA Class II expression. It must be noted that MA9 AML exhibited an appreciable immunogenic phenotype despite exhibiting low levels of MHC Class I and II expression, suggesting a possible role for other immune cells such as NK cells in the control of this leukemia.Of the oncogenic drivers tested, the strongest immune response was induced by Nras37. It has been shown that HLA Class II expression in AML is in part dependent on the methylation status of the promoter of the transcriptional coactivator CIITA, and its expression, which is IFNγ responsive37. Interestingly, the expression of the co-stimulatory molecule CD80 is also IFNγ responsive38 and mutant Ras has recently been shown to drive a cell-intrinsic interferon response via increased expression of transposable elements39 providing a possible mechanism for the unique surface immunophenotype of the immunogenic NrasG12D AML. This also poses the interesting hypothesis as to whether AML immunogenicity can be enhanced by the administration of IFNγ, either alone or in combination with hypomethylating agents.Expression of the HLA Class II presentation machinery has recently been suggested as a determinant of immune evasion in hematological malignancies40. We identified distinct oncogene-dependent immune microenvironments in AML recipient immunocompetent mice. Expansion of CD8+ effector T cells in NrasG12D recipients indicates activation of the adaptive immune response that was observed to a much lesser extent in BA/NH or MA9 recipients. This was consistent with depletion experiments demonstrating a more dominant role for T cells in control of NrasG12D in comparison to MA9 AML. Furthermore, a higher frequency of CD8+ T cells in the NrasG12D AML microenvironment of moribund mice displayed expression of immunosuppressive receptors PD-1 and TIM-3. Consistent with this, examination of T cells from AML patients at diagnosis also revealed that a higher frequency of T cells in the RAS mutant AML tumor microenvironment display gene expression of PD-1 in comparison to MLL translocated AML, suggesting that RAS mutant AML can only progress in the context of inhibitory receptor expression on T cells.Consistent with the hypothesis that cell intrinsic differences in AML surface expression of immunomodulatory ligands dictate the potential for an anti-leukemia immune response, individual genetic aberrations also dictate the composition of the tumor microenvironment, including the degree of myeloid and lymphocyte cell infiltration and dysfunction27 and can be mediated by T cell-dependent selection against immunogenic neo-antigens and IFNγ associated genetic instability43. Here, the more immunogenic NrasG12D AML showed functional immunoediting in immunocompetent mice whereas the less immunogenic MA9 AML did not, possibly reflecting the difference in the efficacy of T cells in controlling these AMLs. Immunoediting has been described in mouse sarcoma models44, but has not been previously demonstrated in a syngeneic AML model. The different capacities for immunoediting presumably reflect the strength of the effector immune cell response and the degree of in vivo selective pressure. Immunoediting is also seen in highly selective clinical settings, specifically after the use of CD20-directed monoclonal antibodies and CD19-directed CAR-T cell therapy for ALL where CD20 and CD19 negative relapses are observed and facilitate escape from CAR-T cell killing46. Further evidence of immunoediting is seen in AML relapse after allogeneic BM transplantation which is frequently associated with downregulation of HLA Class II molecules47 or polymorphisms in the HLA region48.In addition to immune dysfunction, immune evasion may also be facilitated by immunoselection against the most immunogenic tumor cells, a dynamic process referred to as immunoeditingG12D AML to evade the immune system is likely to be multifaceted. While ICB clinical trials are ongoing in AML patients, preliminary data suggests that single agent ICB also has minimal activity in AML patients whereas ICB in combination with hypomethylating agent azacitidine has shown positive responses in a proportion of AML patients with higher pretherapy bone marrow CD3+ T cells and the presence of ASXL1 mutation, again suggesting that patient genetic profiles are important predeterminants of treatment efficacy49.Although immunoediting was characterized by the upregulation of the immunosuppressive ligand PD-L1, anti-PD1 therapy had limited efficacy in restoring the anti-leukemia immune response in this model, suggesting that the strategy employed in the immunoedited NrasG12D AML is driven by the coordinate downregulation of mutant Nras expression and upregulation of Myc-driven transcription. The down regulation of mutant Nras expression is consistent with the immunogenicity of this AML being in part driven by the presentation of immunogenic neo-antigens generated from mutant Nras. Our data are supported by previous studies in mouse models of sarcoma, that identified T cell-dependent immunoselection driving immunoediting against highly antigenic neo-epitopes derived from specific genetic mutations42. KRAS mutant-specific T cells have been found in melanoma patients showing that the immune system is capable of directly targeting mutant RAS derived neo-antigens50 and the presence of NRAS mutations may confer better response rates to immune checkpoint therapy51. RAS mutant AML has a relatively favorable prognosis after treatment with chemotherapy. It could be hypothesized that this may relate to the presence of a host immune response, and that this response can be unmasked after tumor de-bulking through chemotherapy.We hypothesize that immunoediting in Nras35. Consistent with this, we found that MYC transcriptional activity AML inversely correlates with the presence of a cytolytic immune infiltrate. We have demonstrated that the ectopic expression of Myc alone is able to reduce surface expression of both MHC Class I and II in non-immunoedited NrasG12D AML, in addition to driving the increased surface expression of PD-L1 and CD86. MYC activity has previously been implicated in regulating both transcription and translation of PD-L152. However, we believe that Myc activity has not previously been linked to the expression of antigen presentation machinery. Interestingly, recent studies have described a direct role for Myc in the repression of interferon response genes in NrasG12D driven models of pancreatic ductal adenocarcinoma and triple negative breast cancer54. It is tempting to speculate that MYC-mediated suppression of mutant RAS driven activation of interferon signaling may be an unappreciated aspect of this cooperative oncogenic relationship that is exploited by cancers of all cell lineages.There is increasing evidence showing that MYC is a critical modulator of an immunosuppressive tumor microenvironment and that MYC inactivation enables recruitment of lymphocytes into tumorsG12D, extrinsic immune pressure enables outgrowth of less immunogenic AML cells. NrasG12D shows transcriptional plasticity under immune selective pressure, and may undergo changes in gene and surface marker expression resulting in upregulation of an immunosuppressive phenotype. As immunotherapy approaches undergo clinical testing in myeloid blood cancers, these findings highlight that personalized and targeted treatment plans may be designed according to the genetics of AML patients.In summary, these data point to anti-leukemic immune responses being determined by specific oncogenic profiles. Critically, in the case of NrasAll animal experiments were approved by the QIMR Berghofer institutional ethics committee under protocols A11605M, A1212-619M and A1212-620M.−/−γc−/−(Rag2−/− Il2rg−/−) were back-crossed onto C57BL/6J. Pathogen-free mice were maintained with approval by QIMR Berghofer institutional ethics committee under protocol A11605M, A1212-619M and A1212-620M in a facility with an 8 to 8 light/dark cycle, temperature range of 19-21 degrees Celsius and 55-65% humidity. Npm1c/NrasG12D AML cells, generated as previously described31, were obtained from Prof. Wallace Langdon (UWA) and Prof. George Vassiliou (Wellcome Sanger Institute).Wild-type C57BL/6J mice were purchased from ARC Animal Resources Centre or Walter and Eliza Hall Institute for Medical Research. Rag257. Plasmids pMSCV-MLL-AF9-IRES-GFP (pMIG-MA9), pMSCV-NUP98-HOXA9-IRES-GFP (pMIG-NH9), pMSCV-BCR-ABL-IRES-GFP (pMIG-BA), pMSCV-AML1-ETO-IRES-GFP (pMIG-AE), pMSCV-IRES-GFP and the packaging plasmid pCL-Eco were a gift from Dr. D.G. Gilliland . pMSCV-GFP-IRES-NrasG12D (pMIG-NrasG12D) was a gift from A/Prof Ross Dickins . pMSCV-Myc-IRES-mCherry was a gift from Dr Gretchen Poortinga . Sanger sequencing was used to determine the exact sequence over the breakpoint of each fusion protein encoded in the listed constructs (Supplementary Table AML from primary hematopoietic stem and progenitor cells (HSPC) were generated as previously describedRag2−/−γc−/− mice (8-12 weeks of age) was depleted using Ter119, Gr1, and CD11b biotin-conjugated antibodies and Dynabeads (Invitrogen) according to the manufacturer’s instructions. Lineage-depleted BM cells at 2 × 106 cells/mL were cultured overnight  in RPMI supplemented with 10% FCS, 100 IU/mL Pencillin/Streptomycin, 10 ng/mL murine recombinant IL-3 (Peprotech), murine recombinant IL-6 (Peprotech) and 50 ng/mL murine recombinant stem cell factor (SCF) (Peprotech). Cells were re-suspended with 1 mL of unconcentrated retrovirus per well with polybrene (8 μg/mL) and HEPES (30 μL/mL) and mIL-3 10 ng/ml, mIL-6 10 ng/ml and mSCF 50 ng/ml (Peprotech). Transduction combinations included: MLL-AF9 alone; BCR-ABL and NUP98-HOXA9; or AML1-ETO and NrasG12D expressed using the retroviral backbone MSCV-IRES-GFP.Bone marrow (BM) from oC, 5% CO2. For primary (1o) transplants, BM cells were assessed for viability and GFP expression by flow cytometry prior to transplantation by lateral tail vein injection into primary Rag2−/−γc−/− recipients. For secondary (2o) and tertiary (3o) transplants, equal numbers of GFP+ AML cells were transplanted into non-irradiated wild-type C57BL/6J or Rag2−/−γc−/− recipients . Mice recruited to survival studies were euthanised when ethically required by a cumulative clinical score based on weight loss, posture, activity and white cell count.BM cells were transduced by two room temperature spin infections for 90 mins at 3000 rpm separated by three-hour incubation at 37o NrasG12D AML-bearing splenocytes were recovered and immediately transduced with either pMSCV-Myc-IRES-mCherry or the empty vector backbone retrovirus in short-term culture, as detailed above. Double positive GFP+, mCherry+ cells were sorted (BD FACSAriaTM) and expanded via transplantation in Rag2−/−γc−/− recipients.Cryopreserved 1+ cells using QuickExtractTM DNA Extraction Solution (Illumina) according to the manufacturer’s instruction. PCR was performed using the following primers: NUP98-HOXA9 , BCR-ABL , AML1-ETO , NRasG12D ORF .Genomic DNA was isolated from sorted GFPHistology samples were processed by the QIMR Berghofer Histology Facility. Briefly tissues were fixed in 10% neutral buffered formalin, embedded in paraffin prior to staining with haematoxylin Aarmstadt Hx Crystals (Merck) and Eosin Y (H&E). In addition, tissues were stained for peroxidase labeled Ki67  and phosphorylated histone H3 (p-H3)  with images captured on a Nikon Eclipse Ci, DS-Fi2 microscope or the Aperio Scanscope XT using Scanscope (version 102.0.7.5).In vivo antibody depletion in wild-type mice was performed using the following antibodies: anti-CD4 , anti-CD8β  and control IgG . Antibodies were injected into the intraperitoneal cavity on days −1 and 0 and then weekly for the duration of the experiment. Anti-PD-1  immune checkpoint inhibitor experiments commenced 7 days after transplant, with treatment every 3–4 days, with a total of 9 doses administered.G12D or MA9 AML previously expanded in Rag2−/−γc−/− mice. Whole splenocytes were harvested, labeled with Cell Trace Violet (CTV) and incubated for 72 h at a 5:1 ratio with and without irradiated (40 Gy) NrasG12D, MA9 (passaged through Rag2−/−γc−/− mice) or non-transformed Rag2−/−γc−/− bone marrow (BM) cells and 0.01μg/mL soluble CD3 . Dilution of CTV on CD4+ and CD8+ T cell populations was evaluated by flow cytometry. The proliferation indexes of CD4+ and CD8+ T cells were calculated by dividing the total number of divisions by the number of cells that underwent division.Non-irradiated wild-type C57BL/6J mice were transplanted with NrasBlood collected into EDTA-coated tubes was analyzed on a Hemavet 950 analyser (Drew Scientific) using Hemavet DMS Capture (version 1.0.0) software.TM or BD FACSAriaTM using FACS Diva Software (version 8.0.1).Spleens and livers were harvested into ice cold FACS buffer (PBS supplemented with 2% FCS v/v) and then emulsified through a 70 µM filter (BD Biosciences), centrifuged and washed in FACS buffer. Liver mononuclear cells were isolated using isotonic percoll gradient. Tissue samples were treated with Red Blood Cell Lysis Buffer . Samples were incubated in CD16/CD32 blocking antibody  before staining with appropriate fluorochrome-conjugated antibodies at the listed dilution  prior to snap freezing on dry ice. Total RNA was isolated from frozen cell pellets using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems). Samples were quantitated using a Qubit RNA HS Assay Kit (Molecular Probes), with integrity confirmed using the RNA 6000 PICO Kit (Agilent Technologies) and Agilent 2100 Bioanalyser (Agilent Technologies).GFP positive AML cells were isolated on BD FACSAria58 (version 2.5.2a) to the GRCm38, with assembly using the gene, transcript, and exon features of Ensembl (release 67). Expression was estimated using RSEM (version 1.2.30), with transcripts with zero read counts across all samples removed prior to analysis. Normalisation of read counts was performed by dividing by million reads mapped to generate counts per million (CPM), followed by the trimmed mean of M-values (TMM) method from the edgeR (version 3.28.1) package59. For the differential expression analysis, reads were filtered but not normalized, since edgeR performs normalisation (library size and RNA composition) internally. The glmFit function was used to fit a negative binomial generalised log-linear model to the read counts for each transcript. Transcript wise likelihood ratio tests were conducted for each comparison. Log2 transformed, normalized read counts were used for heatmaps and principle component analysis (PCA).Oligo d(T) captured mRNA (100 μg) was processed for Next Generation Sequencing (NGS) using the NEB Next Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). Quality was assessed using the High Sensitivity DNA Kit (Agilent) on the Agilent 2100 Bioanalyser with quantification using the Qubit DNA HS Assay Kit (Molecular Probes). Final libraries were sequenced using a high output single -end 75 bp flow cell (version 2) on the Illumina Nextseq 550 platform using NextSeq System Suite (version 2.1.2). Reads were trimmed for adapter sequences using Cutadapt (version 1.11) and aligned using STAR61. P-values were generated form 1000 gene set permutations, excluding gene sets with more than 3000 genes or <5 genes against custom made gene sets and Broads Hallmark and C2 database.Gene set enrichment analysis (GSEA) was performed using GSEA (version 4.1.0) from Broad Institute+ NrasG12D cells using Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) according to the manufacturer’s instructions. Reverse transcription was performed using Maxima H minus first strand cDNA synthesis kit with dsDNase according to the manufacturer (Thermofisher Scientific). Genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue kit according to the manufacturer’s instructions. RNA and DNA quantification concentration and purity were determined by Nanodrop spectrophometer (ThermoFisher Scientific). Q-PCR primers were designed using Primer3web (http://bioinfo.ut.ee/primer3/).DNase free RNA was extracted from sorted GFPPrimers used: Nras: Forward 5′ TGTTGGGAAAAGCGCCTTGA 3′ and reverse 5′ CCTGTCCTCATGTACTGGTCT 3′. Beta-actin (Actβ): Forward 5′ GACGATATCGCTGCGCTGGT 3′ and reverse 5′ CCACGATGGAGGGGAATA 3′. Primer specificity was determined by melt-curve analysis. Gene expression and copy number were quantified by Sybr green reaction (Life technologies) using ABI Viia7 qPCR machine with the QuantStudio Real-Time PCR System (version 1.3) according to standard protocols. Gene expression was determined by standard curve analysis and normalized to Actβ with relative gene expression and copy number expressed as a fold change.GSE6981 were downloaded, pre-processed and normalized as previously described29. Samples from AML patients with NRAS mutation (n = 42) and MLL-translocation (n = 10) were extracted for analysis. Using the GSVA (version 1.34.0) R package function ssgsea the relative enrichment of gene sets across was assessed for all genetic aberrations with more than 3 patients for genes encoding MHC Class II and for NRAS mutant samples was assessed for gene sets associated with cytolytic immune infiltration in AML37 or Myc gene target activation36.Microarray array data with accession GSE18538130. For MHC Class II gene expression comparison across oncogenes, CD33 expressing malignant annotated cells were extracted and B, T and NK cells excluded. For each sample MHC Class II gene expression was averaged. For comparison of the percentage of PDCD1 expressing cells oncogenes annotated T cells were extracted.Pre-processed and annotated single cell RNA-sequencing data of 5 AML patients with MLL-translocation, 6 patient with RAS mutations  and 7 healthy controls were obtained from GEO with accession t-test when normality and equal variance assumptions are met, Mann–Whitney test otherwise; unless otherwise described for more than two groups when normal distribution and equal variance assumption are met ordinary one-way ANOVA with post-Tukey multiple comparison test was performed, in case of violation of equal variance we performed Welch ANOVA or Kruskal–Wallis test with Dunn’s multiple comparisons test when normality is violated. Test for association between paired samples, using Pearson’s product moment correlation coefficient.Statistical analysis was performed using Prism (v7.02) as follows: Log-rank (Mantel-Cox) test for p values for all Kaplan–Meier survival analyses. To compare two groups unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Herein, we sought to examine the dynamic of intracellular release of Ca2+ and Zn2+ upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS) in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+ and Zn2+ ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+ channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+ or Zn2+. The source of EFS-induced rise in Ca2+ and Zn2+ seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+ and Zn2+ ions, whereby the removal of extracellular Ca2+ augmented the release of intracellular Ca2+ and Zn2+ and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+ is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potential in vitro.Recent pharmacological studies demonstrate a role for zinc (Zn In addition, a multitude of molecular components are involved in Ca2+ signaling pathways, working in combinations to produce specific Ca2+ signals of different spatial and temporal profiles  as a second messenger in certain cellular context was realized only recently  or buffered by specialized proteins in the cytosol  has been employed extensively neration . It is wneration . The eleneration . In addineration , ultimatneration .2+]i, with varying findings regarding the kinetic and source of release  containing 2 mg/ml Papain, followed by adding 1:1 HBSS media and centrifugation. The palleted tissue was resuspended in HBSS media, dissociated mechanically by pipetting and passed through 70 μM strainer, centrifuged and plated onto Poly-d-lysine coated coverslips in Neurobasal media  supplemented with 2% B27 , 1% GlutaMax , 200 μM Glucose solution  and 1% Pen/strep . Cultures were maintained at 37°C and 5% CO2. Typically, approximately 2.7 × 106 cells were seeded on 25 mm glass coverslips in 35 mm cell culture dishes. Medium was changed after 24 h to remove unattached cells. Experiments were conducted after 5 days post plating of the cortical cultures in order to avoid cultures being taken over by glial cells.The rat brain tissue dissociation protocols were approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre (RAC#: 2190006). Primary cortical cultures were obtained from brains of 6–8 weeks rats . Cortices were isolated and enzymatically digested in HBSS media  for 5 min and then blocked in 5% BSA in PBT (block buffer) for 60 min at room temperature. Samples were then incubated with primary antibody (diluted in the block buffer) overnight at 4°C. The following primary antibodies were used: mouse anti-MAP2ab , and rabbit anti-GFAP . Following three 5 min washes in PBT, secondary antibodies (1:1000 diluted in the block buffer) were applied for 1 h at room temperature. All samples were counterstained with 49,6-diamidino-2-phenylindole . Samples were then mounted onto glass slides with 5 μl of moviol aqueous mountant followed by viewing and image capturing under Zeiss florescence microscope using ZEN imaging software.in situ while recording a time lapse using Zeiss confocal imaging microscopy to monitor changes in the intensities of Ca2+ indicator Fluo-4 acetoxymethyl (AM) ester , Zn2+ indicator, Fluozin-1 acetoxymethyl (AM) ester , or membrane potential sensitive dye, bis--trimethine oxonol . The EFS parameters used in this study did not cause cell death as evaluated using trypan blue exclusion test, propidium iodide staining and absence of membrane blebbing.Primary cultures on coverslips were transferred into an EFS chamber . The chamber has two electrodes, separated by distance of 6.5 mm; each electrode has two ends, one end is immersed in the media and the other end extends outside of chamber . The exp2+ indicator Fluo-4 AM , Zn2+ indicator Fluozin-1 AM  or membrane potential sensitive dye DiBAC4(3)  in freshly prepared regular Krebs-HEPES buffer (pH 7.4) for 30 min at 37°C as previously described  water (Resistivity is 15 MΩ.cm at room temp) produced by the Milli-Q® Integral Water Purification Systems using M Quantum TIX filter . The system removes all trace ions including Zn2+). For imaging of organelles, cells were loaded in regular Krebs-HEPES buffer with ER-Tracker Blue-White DPX , LysoTracker Red DND-99 , or MitoTracker Red CMXRos . For experiments with the zinc chelator Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), cells were loaded in regular Krebs-HEPES buffer containing 50 μM TPEN. For experiments in Ca2+ free medium, cells were loaded with specific indicator in Ca2+ free Krebs-HEPES buffer in which the addition of CaCl2 was replaced with supplementation of 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). Image acquisition and analyses was performed using CLSM software (Zeiss META 510) or ZEN imaging software under Zeiss spinning wheel confocal microscope. Serial images were acquired at approximately 1 s intervals. A baseline recording was first taken then the stimulus was delivered at second 20, whereby cells were either stimulated with 25 mM KCl, 10 μM diazoxide or EFS as described in EFS method section for duration of 20 s. Imaging of cells exposed to EFS was performed in room temperature. For analysis, we performed background subtraction then we calculated ratio changes by normalizing intensity changes to the averaged intensity of the first three images before delivering the stimulus. Although our cultures contained many other cell types including glia, we targeted neurons only. This was made easier by using high resolution confocal fluorescence imaging which allowed us to identify neurons from other cells present in the isolated cultures.Cells were loaded with either Caescribed . Krebs-Ht-test was used for baseline comparative data and two-tailed student t-test was used for inter-group comparisons. A p ≤ 0.05 was considered significant.Paired samples ATP) activator diazoxide i and [Zn2+]i is unlikely to be due to the effect of transmembrane potential which itself is dependent on multiple factors including cellular orientation, direction and orientation of the electric field and activation or deactivation of voltage gated ion channels  the high fluorescence intensity of Fluozin-1 in the vesicles reflects high Zn2+ content and/or (ii) the high intensity reflects different intra-vesicular environment that changes the spectral characteristics of the dye independently of Zn2+. The latter would seem unlikely since Fluozin-1 AM intensity is highest in pH 7 and decrease at more acidic or basic pH  than Ca2+ and that a millimolar rise in intracellular levels of free Zn2+ inhibits the channel opening, thereby regulating Ca2+ release (2+ dependent Ca2+ influx in cortical neurons has been shown to contribute to neuronal cell death during oxidative stress (Accumulating evidence highlights the important roles for Znll death . Ca2+ det manner . Additior stores . The oth release . In addie stress .2+ and Zn2+ signaling in response to EFS. EFS in the absence of extracellular Ca2+ evoked significantly higher maximum release of intracellular Zn2+, indicating that extracellular Ca2+ may interfere with intracellular release of Zn2+. It is therefore tempting to speculate that, similar to the Zn2+ modulation of the cardiac RYR, Ca2+ entry through voltage sensitive Ca2+ channels may be also modulated and/or inhibited by binding of intracellular Zn2+ and that in the absence of extracellular Ca2+, Zn2+ will not bind to these channels leading to higher levels of free Zn2+ (2+ in response to EFS in the presence of extracellular Ca2+. Further investigations are required to decipher details of this interdependence relationship between Ca2+ and Zn2+ and to elucidate the downstream signaling and determine its consequence.Our study revealed a possible interaction between intracellular Caree Zn2+ . This ma2+ and Zn2+. Normalization to baseline assumes that the treatment does not affect baseline levels, which might not be always true. We therefore provided evidence of the effect of the various treatments on baseline values (In this study, we normalized fluorescence intensity to baseline in order to quantify the relative changes in intracellular Cae values . We showe values . Manipule values  however,e values . As for e values .2+ and Zn2+ from intracellular sources. Our study highlights an association between EFS-induced modification of membrane excitability and [Ca2+]i and [Zn2+]i signaling and supports the use of EFS as a tool to interrogate the dynamics of intracellular ions homeostasis.In summary, we showed that modifying membrane potential of cortical neurons by EFS resulted in hyperpolarization that is concurrently accompanied by release of CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre.AA and FAM: conception and design of the study, data analysis and interpretation, and manuscript writing. AA, SA, and FAM: collection and/or assembly of data. FAM: provision of study material and administrative and financial support. All authors contributed to final approval of the manuscript.


Chronological changes in the overactive bladder symptom score (OABSS) and the time-to-resolution of the OABSS were evaluated in 237 patients with prostate cancer who underwent low-dose-rate brachytherapy (LDR-BT) with iodine-125. The patients were divided into two groups: the OABSS resolution group and the resolution delay group. The OABSS in both groups worsened at 3 months following operation and gradually recovered at 9 months; however, the OABSS in the resolution delay group tended to worsen again after that. In the multivariate analysis, preoperative OABSS and the change from baseline to maximal OABSS were associated with OABSS resolution. To the best of our knowledge, this is the first study to evaluate the delayed resolution of storage symptoms.Urinary storage symptoms after low-dose-rate brachytherapy (LDR-BT) with iodine-125 have been noted to be less likely to improve to baseline compared to voiding symptoms. This study aimed to evaluate the chronological changes in the overactive bladder symptom score (OABSS) and the time-to-resolution of OABSS in patients undergoing LDR-BT. Patients with prostate cancer who underwent LDR-BT at Gifu University Hospital were enrolled. The OABSS was evaluated before and after LDR-BT. Patients were divided into the OABSS resolution and resolution delay groups, and the association between OABSS resolution delay and clinicopathological covariates was evaluated. In total, 237 patients were enrolled in this study, with a median follow-up of 88.3 months. The OABSS in both groups worsened at 3 months following operation and gradually recovered at 9 months; however, the OABSS in the resolution delay group tended to worsen again after that. In the multivariate analysis, preoperative OABSS and the change from baseline to maximal OABSS were associated with OABSS resolution. To our knowledge, this is the first study to evaluate the delayed resolution of OABSS after LDR-BT in patients with prostate cancer. A low baseline OABSS and significant changes in the OABSS from baseline were independent predictors of delayed OABSS resolution. Prostate cancer (PCa) has the highest incidence rate among men in Japan . AccordiRegarding urination in patients with PCa undergoing LDR-BT, the total International Prostate Symptom Score (IPSS) and quality of life due to urinary symptoms (IPSS-QOL) scores increased compared to baseline at 3 months following operation and returned to baseline by 36 months, whereas the overactive bladder symptom score (OABSS) did not return to baseline even at 36 months after LDR-BT . In our Here, we aimed to investigate the predictive factors influencing the delayed resolution of OABSS in patients with PCa who underwent LDR-BT.We conducted a retrospective cohort study of consecutive patients with clinical T1c/T2/T3a PCa, according to the 7th American Joint Committee on Cancer Staging Manual , who undhttps://www.med.gifu-u.ac.jp/visitors/disclosure/docs/2020-210.pdf (accessed on 6 June 2023).An institutional review board of Gifu University authorized the present study . Since this is a retrospective study, informed consent was waived. According to the regulations of the Japanese Ethics Committee and ethical guidelines, retrospective and observational studies using existing data and other materials do not require written consent because the results of these studies have already been released to the public. Details of this study, which are available only in Japanese, can be found at ® delivery system  or loose 125I radioactive seeds , which were implanted by real-time transrectal ultrasound (TRUS)-guided transperineal technique [The enrolled patients received either a linked seed using the ProLinkechnique .The latest American Association of Physicists in Medicine Task Group 43 formalism and Variseed version 7.1  were used for treatment planning and post-implant dosimetry. One month after LDR-BT, post-implant dosimetry was performed using computed tomography (CT) and magnetic resonance imaging (MRI). CT was performed using a CT scanner  with a 16 or 64 detector array. MRI was performed using a 5-channel SENSE cardiac coil with easy breathing, a slice thickness of 3 mm, and no cross-gap . The following dose parameters were used and analyzed in this study: the minimum dose received by 90% of the prostate gland (D90), percentage of the target volume receiving a minimum of 100% of the prescribed dose (V100), minimum dose received by 30% of the urethral volume (UD30), rectal volume receiving 100% of the prescribed dose (RV100), and rectal volume receiving 150% of the prescribed dose (RV150).IPSS, IPSS-QOL, OABSS, uroflowmetry (UFM), and post-voiding residual volume (PVR) were evaluated before LDR-BT; 1, 3, 6, 9, 12, 18, and 24 months after LDR-BT; and annually thereafter.p-values were two-tailed, with a p-value < 0.05 considered statistically significant. The primary endpoints were the chronological changes and time-to-resolution of the OABSS after LDR-BT. The secondary endpoint was to analyze the relationship between delayed resolution of OABSS and clinicopathological covariates. Patients were divided into OABSS resolution and resolution delay groups. Resolution of the OABSS was defined as the return of the OABSS to baseline scores after LDR-BT. Chronological changes in OABSS were represented by line graphs with median and interquartile range (IQR) values. Data analysis was performed using JMP Pro 16 software . Continuous variables were analyzed using the Wilcoxon signed-rank test, and categorical variables were analyzed using Fisher’s exact test. The Kaplan–Meier method was used for estimating the resolution of OABSS and assessing the proportion of OABSS resolution. The resolution of OABSS in each group was analyzed using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model. All Patients with PCa and lymph node metastases, distant metastases, prior transurethral resection of the prostate, maximal flow velocity (Qmax) <10 mL/s, or missing OABSS values up to 36 months after LDR-BT were excluded from the study. Finally, 237 patients met the inclusion criteria. The chronological changes from baseline in the OABSS after LDR-BT are shown in p = 0.018; p < 0.001; The results of the multivariate Cox proportional hazards regression analysis for OABSS resolution are shown in LDR-BT, EBRT, or RP is a standard primary curative radical treatment for localized PCa ,14. The A relatively large number of previous studies examining chronological changes regarding LUTS after LDR-BT have been reported ,23,24,25Previous reports have shown an association between GU toxicity after LDR-BT and the delayed resolution of OABSS, and combination therapy with LDR-BT and EBRT has been associated with late GU toxicity and LUTS ,21,23,24This study had several limitations. First, potential bias may be inherent because it was a retrospective, single-center, non-randomized study. Secondly, the number of enrolled patients was relatively small; therefore, caution must be exercised when interpreting the findings of this study. Third, because the decision to resolve the OABSS was based on the judgment of the physician or individual patient, the interpretation of the results may not have been based on certain criteria, suggesting that the results may have differed from those obtained when the diagnosis was based on clear criteria. In addition, the IPSS and OABSS results were obtained from individual patients, suggesting that the assessment of LUTS may not be consistent . Fourth,To the best of our knowledge, this is the longest follow-up study to evaluate chronological changes in OABSS in patients with PCa after LDR-BT. In addition, this study identified independent factors influencing OABSS resolution. A low OABSS before LDR-BT and a large change in OABSS from baseline were independent predictors of delayed OABSS resolution.