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Despite high remission rates after therapy,60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC),which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs,we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34(+)CD38(-) hematopoietic stem cells (HSC),CD34(+)CD38(-) LSCs,and CD34(+)CD38(+) LPs,all derived from the same patients' bone marrow (BM) specimens. In this manner,we identified multiple differentially expressed miRNAs,in particular miR-126,which was highly expressed in HSCs and increased in LSCs compared with LPs,consistent with a stem-like cell function. High miR-126 expression in AML was associated with poor survival,higher chance of relapse,and expression of genes present in LSC/HSC signatures. Notably,attenuating miR-126 expression in AML cells reduced in vitro cell growth by inducing apoptosis,but did not affect the survival of normal BM in which it instead enhanced expansion of HSCs. Furthermore,targeting miR-126 in LSCs and LPs reduced their clonogenic capacity and eliminated leukemic cells,again in the absence of similar inhibitory effects on normal BM cells. Our results define miR-126 as a therapeutic focus to specifically eradicate LSCs and improve AML outcome. View Publication

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks,making them attractive candidate HSC regulators. We report that miR-126,a miRNA expressed in HSC and early progenitors,plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion,resulting in expansion of mouse and human long-term repopulating HSC. Conversely,enforced miR-126 expression impaired cell-cycle entry,leading to progressively reduced hematopoietic contribution. In HSC/early progenitors,miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway,attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size,demonstrating the importance of miRNA in the control of HSC function. View Publication

Natural killer (NK) cells play a crucial role in immune surveillance by recognizing and eliminating tumor cells. However,tumors employ various mechanisms to evade NK cell-mediated immunity. NKp30 is a potent activating receptor on NK cells,but its function can be inhibited by specific ligands secreted by cancer cells. Here,we identified dipeptidase 1 (DPEP1) as a novel ligand for NKp30 in KM12C colon cancer cells,using co-immunoprecipitation,confocal microscopy,and flow cytometry. We examined how the DPEP1–NKp30 interaction affects NK cell activity and found that NK cytotoxicity increased in KM12C cells with DPEP1 knockdown but was significantly reduced in HCT116 cells overexpressing DPEP1. We further demonstrated that DPEP1 is secreted via extracellular vesicles and that its interaction with NKp30 suppressed the expression and secretion of perforin 1,granzyme B,CD107a,and interferon-γ in NK92 cells. In a xenograft mouse model treated with NK92 cells,tumors derived from HCT116/DPEP1 cells were significantly larger than those from HCT116/mock cells. Using peripheral blood-derived human NK cells,we confirmed that DPEP1 inhibited both cytotoxicity and granzyme B secretion. These findings suggest that disrupting the DPEP1–NKp30 interaction may enhance NK cell-mediated cytotoxicity and represent a novel therapeutic strategy for cancer immunotherapy. The online version contains supplementary material available at 10.1038/s41598-025-18475-z. View Publication

SummaryKCNQ1/Kv7,a low-voltage-gated K+ channel,regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes,its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s),we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation,but affected channel function by increasing spike frequency and Ca2+ flux,leading to insulin hypersecretion. With prolonged culturing,the mutant islets decreased their secretion and gradually deteriorated,modeling a diabetic state,which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and ?-cell survival in hereditary diabetes pathology. Graphical abstract Highlights•A permanent neonatal diabetes melitus patient carries a homozygous KCNQ1 mutation•KCNQ1R397W is loss of function and shows atypical electrophysiology in hESC-islets•Under high glucose,elevated Ca2+ flux leads to insulin hypersecretion•Mutant cells gradually switch phenotype,deteriorate,accelerated by high glucose Biological sciences; Endocrinology; Endocrinology; Health sciences; Internal medicine; Medical specialty; Medicine; Natural sciences; Physiology View Publication

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination. View Publication

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However,the role of PKCiota in lung tumor development has not been investigated. To address this question,we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene,Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore,the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus,Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore,aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. View Publication

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity,a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs,with the former currently undergoing phase I clinical trials. We show here that ST1926,CD437,and active congeners cause DNA damage. Cellular and subcellular COMET assays,H2AX phosphorylation (gamma-H2AX),and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4,an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts,which are selectively resistant to RRMs,do not show any sign of DNA damage after treatment with ST1926,CD437,and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs,increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated,ataxia telangectasia RAD3-related,and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926,indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. View Publication

PURPOSE The radiosensitivity of the normal intestinal epithelium is the major limiting factor for definitive radiotherapy against abdominal malignancies. Radiosensitizers,which can be used without augmenting radiation toxicity to normal tissue,are still an unmet need. Inhibition of proteosomal degradation is being developed as a major therapeutic strategy for anticancer therapy as cancer cells are more susceptible to proteasomal inhibition-induced cytotoxicity compared with normal cells. Auranofin,a gold-containing antirheumatoid drug,blocks proteosomal degradation by inhibiting deubiquitinase inhibitors. In this study,we have examined whether auranofin selectively radiosensitizes colon tumors without promoting radiation toxicity in normal intestine. EXPERIMENTAL DESIGN The effect of auranofin (10 mg/kg i.p.) on the radiation response of subcutaneous CT26 colon tumors and the normal gastrointestinal epithelium was determined using a mouse model of abdominal radiation. The effect of auranofin was also examined in a paired human colonic organoid system using malignant and nonmalignant tissues from the same patient. RESULTS Both in the mouse model of intestinal injury and in the human nonmalignant colon organoid culture,auranofin pretreatment prevented radiation toxicity and improved survival with the activation of p53/p21-mediated reversible cell-cycle arrest. However,in a mouse model of abdominal tumor and in human malignant colonic organoids,auranofin inhibited malignant tissue growth with inhibition of proteosomal degradation,induction of endoplasmic reticulum stress/unfolded protein response,and apoptosis. CONCLUSIONS Our data suggest that auranofin is a potential candidate to be considered as a combination therapy with radiation to improve therapeutic efficacy against abdominal malignancies. View Publication

Accumulating data have shown that targeting breast cancer stem cells (CSCs) is an auspicious way for anticancer therapies. This study demonstrated that the antirheumatic drug auranofin is a potent CSC inhibitor with anti-CSC action on breast cancer. This research focused on investigating the effect of auranofin on breast cancer and CSCs and its cellular mechanism. Mammosphere formation,colony formation,levels of CD44 high /CD24 low,and aldehyde dehydrogenase 1 expression in the cells were evaluated after auranofin treatment. The anti-CSC properties of auranofin were further examined by gel shift assay and cytokine detection. Auranofin suppressed cell growth,colony formation,migration,and mammosphere formation and triggered apoptosis in breast cancer. Auranofin decreased the CD44 high /CD24 low - and aldehyde dehydrogenase-expressed subpopulations,as well as the Stat3-DNA interaction and phosphorylated Stat3 level. Auranofin also decreased the extracellular levels of interleukin-8 (IL-8) in the mammosphere media. Auranofin suppressed the Stat3/IL-8 signal and killed CSCs; therefore,it may be a potential target for CSCs. View Publication

Pediatric high-grade astrocytomas (HGAs) account for 15-20% of all pediatric central nervous system tumors. These neoplasms predominantly involve the supratentorial hemispheres or the pons--diffuse intrinsic pontine gliomas (DIPG). Assumptions that pediatric HGAs are biologically similar to adult HGAs have recently been challenged,and the development of effective therapeutic modalities for DIPG and supratentorial HGA hinges on a better understanding of their biological properties. Here,20 pediatric HGAs (9 DIPGs and 11 supratentorial HGAs) were subject to gene expression profiling following approval by the research ethics board at our institution. Many of these tumors showed expression signatures composed of genes that promote G1/S and G2/M cell cycle progression. In particular,Aurora kinase B (AURKB) was consistently and highly overexpressed in 6/9 DIPGs and 8/11 HGAs. Array data were validated using quantitative real-time PCR and immunohistochemistry,as well as cross-validation of our data set with previously published series. Inhibition of Aurora B activity in DIPG and in pediatric HGA cell lines resulted in growth arrest accompanied by morphological changes,cell cycle aberrations,nuclear fractionation and polyploidy as well as a reduction in colony formation. Our data highlight Aurora B as a potential therapeutic target in DIPG. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis,the animal model for MS,myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby,the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently,B cells were found to participate in the pathogenesis of CNS autoimmunity,with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore,myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue. View Publication