第11卷，第1期

With morphological features resembling hepatocellular carcinoma, hepatoid adenocarcinoma of the lung (HAL) is a rare and aggressive subtype of lung cancer with a 5-year survival of only 8%. Most HAL patients present increased serum alpha-fetoprotein (AFP) levels that are commonly related to poor prognosis. Most insights into HAL have come from case series or reports, and TP53 is frequently mutated in HAL. The heterogeneity of HAL complicates the diagnosis. With no established standard, the management of HAL is modeled on that of classical lung adenocarcinoma, although with dismal outcomes. Herein, we reported the effective management of a stage IIIb HAL patient with perioperative chemoimmunotherapy and curative-intent surgery. The patient remained disease-free and minimal residual disease (MRD)-negative for more than one year after surgery.

1317

Acute myeloid leukemia (AML) is a rapidly lethal blood cancer characterized by aberrant proliferation and differentiation of myeloid progenitors in the hematopoietic tissue. In recent decades, AML patients are significantly increased. In 2019 alone, 21,450 new AML cases were diagnosed in the United States, with an estimated 10,920 deaths due to AML. What’s worse, AML occurs with an increasing incidence with advanced age, and the survival rate remains dismal with a median overall survival of only 5-10 months. Notwithstanding great advances in allogenic transplant and chemotherapy, the therapeutic outcomes are unfavorable due to the disease relapse and mortality rates. More importantly, the development of more specific and effective therapies was hampered in part by the lack of proper animal models for AML. Thus, management and elucidation of the pathological mechanism of AML remain an ongoing challenge, and the development of effective AML mouse models and treatment options are urgently needed.

1364

Hirschsprung’s disease (HSCR) is the most common type of developmental gastrointestinal malformation causing intestinal obstruction in children, with an incidence of 1/5000 in live births. It is characterized by the absence of intrinsic ganglion cells in the myenteric and submucosal plexuses of the distal gastrointestinal tract, leading to the dysregulation of smooth muscle contraction/relaxation and resulting in intestinal obstruction. Depending on the length of the aganglionic intestine, HSCR can be categorized into short-segmental HSCR (S-HSCR), which affects up to the sigmoid colon from the anus, and long-segmental HSCR (LHSCR), which affects beyond the sigmoid colon. In some rare conditions, aganglionic lesions can affect the full length of the colon or even the whole intestine.

1511

There are no approved therapeutic drugs for nonalcoholic steatohepatitis (NASH) due to the many bottlenecks, including the lack of preclinical animal models that can perfectly mimic human NASH features, such as systemic metabolic disorders, hepatic steatosis, intense liver inflammation and fibrosis, liver tumorigenesis, and cardiovascular complications. Pigment epitheliumderived factor (PEDF) is a lipid metabolism regulator with high expression in the liver, and its reduced expression in the liver is closely related to the development of nonalcoholic fatty liver disease (NAFLD). Besides, high-fat diet (HFD)-fed low-density lipoprotein receptor knockout (LDLR knockout, LKO) mice are often used as animal models to study atherosclerosis and fatty liver.5 Here, we attempt to further knock out PEDF in HFD-fed LKO mice to induce premature development of inflammation and fibrosis, leading to a better NASH model. The results showed that HFD-fed LDLR/PEDF double knockout (LDLR/PEDF DKO, L/P DKO) mice can ideally mimic the disease progression and pathological characteristics of human NASH, not only show systemic changes but also experience disease progression from mild to severe NASH accompanied by serious liver inflammation, tumorigenesis, and cardiovascular injury. The multiomics analysis and related validation results suggest that PEDF deficiency is involved in the occurrence and development of NASH by affecting lipid and glutathione metabolism.

1288

Genetic mutations cause aberrant splicing, one of the important molecular mechanisms in human diseases. IVS2-654, a mutation causing aberrant splicing of b-globin premRNA and contributing to β-globin deficiency, is one of the most common diseases in Southeast Asia and China. In our previous work, we found a TTTV protospacer adjacent motif (PAM) by the Cas12a system and the editing efficiency of IVS2-654 C > T achieving 76.7%. Here, we present that the efficiency and persistence of IVS2-654 C > T can be rescued through electroporation of Cas9 ribonucleoprotein (RNPs) with multiple single guide RNAs (sgRNAs) into hematopoietic stem and progenitor cells (HSPCs), introducing high-efficiency indels of pre-mRNA with the aberrant splice sites disrupted and restoring normal β-globin expression. Notably, we found those multiple adjacent sgRNAs could induce large fragment deletion rather than regular small indels, leading to normal coding sequence (CDS) region and directly restoring the β-globin gene function of IVS2-654 C > T without off-target effects. Our strategies could directly rescue the β-globin gene function of IVS2-654 C > T with a higher restoration by multiple sgRNAs co-delivered.

2104

Etoposide is widely used for cancer chemotherapy in the clinic. However, long-term etoposide treatment can lead to adverse effects or drug resistance. To improve the situation, we evaluated the therapeutic efficiency of etoposide combined with inhibitors of bromodomain and extraterminal (BET) family proteins, which have recently emerged as novel anti-cancer targets due to their critical roles in cancer development. Firstly, we showed BRD4, one of the main targets of BET inhibitors, was involved in DNA damage response (DDR) via the homologous recombination (HR) repair pathway. Then we found BRD4 deficiency and inhibition reduced ataxia-telangiectasia mutated (ATM) recruitment and activation by down-regulating the expression of MRN complex (MRE11-RAD50-NBS1) proteins. Subsequently, our data showed that BET inhibitors could sensitize cancer cells to etoposide treatment both in vitro and in vivo. In summary, BRD4 inhibitors enhance the anticancer effect of etoposide by suppressing the MRN-ATM axis in DDR, thus indicating the important value of these inhibitors as candidates for combination with etoposide in chemotherapy.

1289

One of the major obstacles for nucleic acid-based medicine is cellular uptake due to the concern of efficiency and potential toxicity of the vehicle. In this work, we developed a novel approach to eliminate the need for a vehicle to allow for the cellular uptake of siRNA-based therapy with a unique modification strategy that also further enhances therapeutic efficacy. This discovery may be of great importance for the development of siRNA-based therapeutics as cellular uptake remains a critical challenge. In this study, we have tested the potential of this modification strategy for siRNA using a siRNA that targets B-cell lymphoma-2 (BCL-2). BCL-2 is an important anti-apoptotic gene and is overexpressed in many different cancers. As a result, venetoclax, a BCL-2 inhibitor, has been developed and is currently used either alone or in combination with obinutuzumab (Gazyva) or rituximab (Rituxan) to treat certain types of chronic lymphocytic leukemia (CLL) or certain types of small lymphocytic lymphoma (SLL). It is also used in combination with either azacitidine (Vidaza), decitabine (Dacogen), or cytarabine as a treatment for acute myeloid leukemia (AML). In this study, we generated a modified siRNA against BCL-2 in which all uracil residues in the sense and antisense strands are replaced with 5-fluorouracil (5-FU). 5-FU is a uracil analog that blocks de novo pyrimidine biosynthesis by forming a suicide complex with its target protein thymidylate synthase (TYMS, TS) and its metabolite, fluorodeoxyuridine monophosphate (FdUMP). Based on this, 5-FU is one of the major chemotherapeutic agents used to treat various tumor types.

1237

Sox2 is a key transfer factor for maintaining pluripotency and self-renewal in embryonic stem cells (ESCs), though the mechanism of its transcriptional regulation in ESCs has not been fully addressed. Distal enhancer-promoter interactions are vital for Sox2 transcription activity in mammals. However, how these diverse interactions individually influence Sox2 gene regulation in mouse ESCs remains unclear. Previous studies found that three distal enhancers (termed E1, E2, and E3) interact with Sox2 promoter. E1 forms an mESC-specific interaction, while E2 functions in mouse neural progenitor cells (mNPCs) and has not been shown to directly interact with Sox2 in mESCs. E3 can interact with Sox2 in mouse ESCs (mESCs), mouse neural stem cells (mNSCs), and mNPCs. Therefore, there are three classes of E-P associations in mESCs: specific, indirect, and common. In mESCs, E1 and E3 display enhancer activity, while E2 shows weak activity as measured by fluorescence reporter assays.

1185

As a bone-confined chronic degenerative disorder, the progression of osteoporosis is characterized by abnormal crosstalk between osteoblasts and osteoclasts, leading to an imbalance of bone remodeling in adults. Several studies reported that arachidonic acid ester 12 lipoxygenase (ALOX12) acts as a regulator in bone genesis by participating in the activation of the peroxisome proliferatoractivated receptor g (PPARG) pathway through its reaction product. A positive correlation between ALOX12 gene polymorphism and bone mineral density (BMD) has also been verified, indicating that serum Se deficiency was accompanied by some ALOX12 variation, contributing to the peak BMD and the development of osteoporosis. Another multidomain enzyme involved in DNA methylation, cystathionine beta-synthase (CBS), regulates the conversion of homocysteine into glutathione. Mutations in CBS lead to the production of more sulfur end-products from the methylation cycle. The newborn CBS-knockout (KO) mice who received treatment with recombinant poly ethylene glycol human truncated CBS (PEG-CBS) were rescued from osteoporosis-like symptoms. In patients with osteoporosis, the expression of CBS was found to be downregulated in femur tissues, leading to a lower BMD. These findings support the idea that DNA methylation-related enzymes, including ALOX12 and CBS, are important for the occurrence of osteoporosis. However, the specificities in these 2 genes of osteoporosis human samples are largely unclear. Thus, we aimed to explore the impact of the DNA methylation level on select candidate genes in osteoporosis. These results will be of great help in optimizing treatments and for the early diagnosis of osteoporosis, complying with the principles of precision medicine.

1288

Persistent gastritis induced by Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric cancer (GC). H. pylori is prevalent in about 50% world’s population, while it causes cancer in less than 2% of exposed individuals. Our studies found that H. pylori infection can induce oncogenic properties in AGS cells by deregulating multiple factors associated with the cell cycle, apoptosis, and other important events associated with cancer progression. Another study, in which we isolated H. pylori from the gastric biopsy and juice sample of suspected gastritis patients, found differential growth patterns of these bacteria. Previous studies have reported the difference between the microbiota in gastric juice and mucosal lining (biopsy). However, none of the reports analyzed the variation between the H. pylori residing in biopsy and juice samples and their morphological, physiological, and pathogenic differences. In this study, we characterized the H. pylori isolates from the two different physiological locations (gastric epithelium and gastric juice) of the same subjects. We assessed the morphological features of the biopsy and juice isolates. The expression profile of GC marker genes, inflammatory genes, metalloproteinases, and the expression of regulatory genes NFκB and β-catenin at 6, 12, and 24 h post-H. pylori infection was also evaluated along with oncogenic property resistance to anoikis.

1180

During a sudden viral pandemic, where the pathogenesis and nature of the infection are unclear, early and accurate identification of the patients infected with the pandemiccausing pathogen is crucial to prevent excess mortality. During the coronavirus disease 2019 (COVID-19) pandemic, early diagnosis of severe acute respiratory syndrome virus-2 (SARS-CoV-2) is limited by the incidence of the symptoms that are shared with acute respiratory illnesses (ARI) caused by other pathogens, including bacteria and viruses. Although pathogen-specific diagnosis methods like reverse transcription-quantitative polymerase chain reaction (RTPCR) remain gold-standard for SARS-COV-2 diagnosis, they also have several disadvantages, such as false negatives due to low or fluctuating viral loads and long processing time with the expertise needed for handling. The host immune response to different pathogen infections can be utilized for pathogen-specific diagnosis. This emerging approach has been shown to differentiate between bacterial and viral infections accurately. Identifying SARS-CoV-2 specific host transcriptome gene classifiers that can differentiate ARI caused by SARS-CoV-2 and other pathogens can aid in the early diagnosis of COVID-19 patients.

1238

Renal cell carcinoma, which consists of three main histological subtypes, namely, clear cell renal cell carcinoma (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC), accounts for most kidney cancer cases. ccRCC accounts for approximately 85% of all RCC cases, and it is a malignant tumor with multiple molecular features and a poor prognosis. Studies have shown that ccRCC is one of the most immune and vaso-invasive cancer types. Considering that ccRCC is not sensitive to either radiotherapy or chemotherapy, targeted therapies, such as vascular endothelial growth factor inhibitors/tyrosine kinase inhibitors and immunotherapy, are now the mainstay first-line therapies that are used in the clinic. However, a significant proportion of patients do not respond to the two treatments mentioned above or have high recurrence rates, which are related to the high degree of tumor heterogeneity and the tumor microenvironment (TME) of ccRCC. Single-cell transcriptome sequencing provides accurate gene expression data at the single-cell level. Integrated single-cell and large transcriptome data from ccRCC samples provide comprehensive information, such as information about prognosis, metastasis, and response to sorafenib and anti-PD-1 treatment. In the present study, we aimed to integrate bulk and single-cell transcriptome data to reveal the clinically relevant cell types in renal cell carcinoma, aiming to reveal the underlying molecular mechanism.

1168

1332

Pure red cell aplasia (PRCA) is a bone marrow failure syndrome characterized by severe anemia, reticulocytopenia, selective reduction, and even the absence of bone marrow erythroid precursors, while leukocyte and platelet counts are normal. Acquired PRCA can be divided into primary and secondary. Both are mediated by immune responses. Reduction in the quantity and dysfunction of committed erythroid progenitors (EPs), including burst forming uniterythroid (BFU-E) and colony forming unit-erythroid (CFU-E), may be the source of reduced erythropoiesis and needs to be elucidated. High throughput sequencing is of great significance for the in-depth study of reduced or absent EPs. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyze EPs from the bone marrow of PRCA patients, to explore the pathogenesis of PRCA and guide treatments.

1290

Gliomas originating from anatomically and developmentally distinct brain regions have different clinical outcomes. However, the molecular landscape underlying this difference remains largely unknown. We analyzed key molecular mutations via sequencing and correlated them with clinical characteristics in 180 adult patients with gliomas originating from the neocortex, mesocortex, and cerebellum. Cases of cerebellar origin had significantly longer survival than those of supratentorial origin, consistent with higher rates of mutation in key genes associated with supratentorial gliomas. In high-grade gliomas (HGGs), shorter survival was found in cases of neocortex origin compared to those of mesocortex or cerebellum origin, consistent with a higher rate of mutation in the hTERT promotor. In lowgrade gliomas (LGGs), cases of supratentorial origin also exhibited higher mutation rates in specific genes. Different driver genes that potentially underlie the progression from LGG to HGG were identified for glioma per origin. In summary, our data shed light on the molecular mechanisms for the different clinical characteristics of gliomas of different origins. These findings highlight the heterogeneity of the molecular landscape of gliomas originating from different brain regions and can provide a critical foundation for targeted therapy against gliomas.

1257

Hepatocellular carcinoma (HCC) is a key cause of morbidity and mortality, which is a global health problem. Elucidating the molecular and cellular mechanisms of HCC progression is helpful to find either early markers for diagnosis or novel drug targets for drug development. Some evidence showed that methyltransferase like 3 (METTL3), a central methyltransferase of the epi-transcriptomic complex that catalyzes m⁶A mRNA modifications, is associated with HCC. Based on the data collected from HCC cell lines and nude mouse models in which METTL3 was modulated, researchers concluded that METTL3 promoted the pathogenesis of HCC, and indicated that inhibition of METTL3 might be an effective approach for HCC treatment. However, the HCC cell lines and nude mouse models are not the ideal tools because they do not really reflect the function of METTL3 in the pathogenesis of HCC in vivo. The in vivo function of METTL3 in the pathogenesis of HCC by using a hepatocyte-specific Mettl3 knockout (Mettl3-HKO) mouse model is largely unknown.

1325

SP100 is an antiviral protein that restricts the productive stage of human papillomavirus (HPV) and multiple other viruses, and viruses in turn block SUMO-1-mediated stabilization of SP100 and promotes its degradation. Interferon (IFN) signaling could still produce more SP100 through transcription to counteract viruses. Viruses also disable the transcriptional up-regulation of SP100 to achieve persistent infection in hosts. Chromosome-19 miRNA cluster (C19MC) miRNAs confer variable levels of resistance to different types of viral infections and here we use SP100 mRNA as our target for understanding the tumor context in which it is expressed or suppressed, and its relationship with C19MC-directed antiviral response miRNAs in human skin cutaneous melanoma (SKCM-TCGA). We show that, high SP100 mRNA expression reflects better survival in melanoma patients and that, the genomic landscape of the SP100 gene is subjected to copy number alteration in SP100^Low melanomas with recurrent breakpoints in chromosome-2q between SP100 and SP110 gene loci and centromere. Besides, the C19MC miRNA-520G promotes SP100 mRNA expression and impedes melanin biosynthesis with down-regulated SLC45A2 and increased HTR2B mRNAs which are known indirect regulators of the tyrosine pool and melanin biosynthesis.

1303

Fractures have an extraordinarily negative impact on individuals’ quality of life and functional status. Nonunion or disability of fracture is a major health issue with important clinical, social, and economic implications. Mesenchymal stem cells (MSCs) play an indispensable role in the initiation of the fracture repair process including the formation of a callus which is replaced by new bone. The use of MSCs in the treatment of fractures is very attractive as they can reduce the time of healing and occurrence of nonunion. However, the effects of MSCs are often hindered by the harsh ischemic micro-environment at the fracture sites, such as low cell survival rate and differentiation in vivo. Histone modifications are one of the most important epigenetic regulations with the ability to control the fate of stem cells. Lysine demethylase 5C (Kdm5c) is frequently mutated in patients with X-linked intellectual disabilities, many of whom exhibit physical and behavioral abnormalities, including epilepsy, short stature, etc. In our previous study, we investigated the de-differentiated MSCs with enhanced osteogenic differentiation capacity and found that Kdm5c might be involved in regulating the properties of dedifferentiated osteogenic MSCs by PCR array. However, it is still unclear whether Kdm5c plays a role in osteogenesis, bone formation, and fracture repair.

2847

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease of genetic origin characterized by abnormally dilated capillaries and a wide spectrum of symptoms, including headaches, seizures, neurological deficits, and intracerebral hemorrhage. Its unpredictable clinical course and the current lack of therapies make the identification of prognostic and predictive biomarkers an imperative research challenge. Herein, we provide evidence that galectin-3 (Gal-3), a major tissue and circulating biomarker of oxidative stress and inflammation, is significantly up-regulated both in CCM patients and experimental models. Specifically, whole transcriptome sequencing, qRT-PCR, and Western blotting studies demonstrated a significant up-regulation of Gal-3 expression levels both in surgical CCM specimens and in blood samples of CCM patients. Moreover, immunohistochemical analyses showed strong Gal-3 immunoreactivity in CCM lesion endothelial cells and infiltrating leukocytes. Furthermore, using cellular and animal models, we found that Gal-3 expression levels are inversely correlated with those of KRIT1, the major causative gene for CCM disease, implying a functional relationship. Overall, our findings demonstrate for the first time that Gal-3 up-regulation occurs in CCM disease and is linked to a causative gene, suggesting that it may serve as a useful biomarker of prognostic and predictive value for risk stratification and treatment of CCM patients.

1301

Autosomal dominant polycystic liver disease (ADPLD) refers to a condition characterized by the presence of numerous cholangiocytes-lined and fluid-filled cysts in the liver and the absence of polycystic kidney disease. Although patients with ADPLD may be asymptomatic, some patients suffer from abdominal pain, gastroesophageal reflux, and nausea, because of hepatomegaly. These symptoms compromise the patient’s quality of life. In severe cases, complications such as intracystic hemorrhage, infection, and rupture may occur. This disease exhibits high genetic heterogeneity. Six genes, including PRKCSH, SEC63, ALG8, SEC61B, GANAB and LRP5, have been identified to have pathogenic variants causing ADPLD. However, about 55%e 70% of cases cannot be explained by the known loci. Hepatic cysts in ADPLD are derived from cholangiocytes. The development and progressive expansion of cysts are associated with a process called ductal plate malformation (DPM), which manifests as hyperproliferation and perturbed polarization of cholangiocytes as well as the abnormal structure and function of primary cilia in cholangiocytes. Most of the proteins encoded by genes associated with ADPLD are localized in the endoplasmic reticulum. So far, the role of ciliary-associated genes’ mutations in the pathogenesis of ADPLD has not been reported. Bardet-Biedl syndrome 4 (BBS4) is associated with the formation of the primary cilium and its mutation causes Bardet-Biedl syndrome, a rare autosomal recessive disorder. However, the role of BBS4 in ADPLD has not yet been reported.

1278

MET gene alterations in lung cancer patients mainly include exon 14 skipping and gene amplification, which are the key therapeutic targets and drive resistance to tyrosine kinase inhibitors (TKIs). However, the structural variants of MET, such as MET fusions, are much rarer (0.26%), as reported in a Chinese non-small cell lung cancer (NSCLC) cohort. Several recurrent MET fusions, such as KIF5B-MET and HLA-DRB1-MET, were reported as oncogenic drivers and showed favorable responses to crizotinib. In addition, MET fusions have been found to mediate resistance to EGFR-TKIs. With the application of comprehensive genomic analyses in clinical samples, an increasing number of MET fusion partners have been identified; however, the clinical and molecular characteristics of patients harboring such MET fusions remain to be investigated in large cohorts.

1248

Diffuse large B cell lymphoma (DLBCL) is one of the most prevalent lymphoid malignancies. The current standard of care can cure about two-thirds of DLBCL patients. Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare but highly aggressive form of mature B-cell lymphoma that accounts for approximately 1%-9% of testicular malignancies. Different from nodal DLBCL, PT-DLBCL has a markedly worse prognosis because of inferior response to the current treatment regimens and significant extranodal tropism. Three main questions remained unresolved in the field of PT-DLBCL research. Previously, two prognostically important categories of DLBCL have been defined: activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL. Most PT-DLBCLs were classified as ABC-DLBCL. However, the exact cells of origin (COO) of PT-DLBCL were still under debate. Secondly, the lack of transcriptomics profiling of PT-DLBCL at the single-cell level hindered the discovery of intra-tumor heterogeneity. Finally, the role of the testis microenvironment in PT-DLBCL was unclear. Herein, we performed single-cell RNA sequencing (scRNASeq) and bulk whole exome sequencing (WES) to reveal the molecular landscape of PT-DLBCL. We defined predictive signatures based on gene profiles of the pivotal clusters. Our study could be a benefit for the clinical classification of PTDLBCL and provide valuable therapeutic targets.

2842

Dilated cardiomyopathy (DCM) is a frequently inherited heart condition. A familial transmission rate of 20%-35% has been documented, and most of these cases are autosomal dominant. This condition has a high rate of morbidity and mortality, which can lead to cardiac arrest and persistent heart failure. Several biological processes are regulated by the constitutively active serine/threonine protein kinase glycogen synthase kinase-3β (GSK-3β), constitutively active. It is a negative regulator of glucose homeostasis and is involved in energy metabolism, inflammation, endoplasmic reticulum stress, mitochondrial dysfunction, and apoptotic pathways. E-cadherin is repressed in epithelialemesenchymal transition (EMT), allowing the passage of attached epithelial cells to the mesenchymal state. B-Catenin represses E-cadherin expression to promote EMT. In the WNT pathway, GSK-3β regulates E-cadherin transcription by phosphorylating bcatenin to trigger proteasomal degradation. Although numerous mechanisms remain unknown, GSK-3β has demonstrated a significant role in heart cardiomyocyte differentiation, hypertrophy, and fibrosis. The ability of GSK-3β to control cardiac hypertrophy, which can both cause and treat disease, may have a dosage-dependent impact. Abnormalities in the cardiac Z-disc were found in GSK-3β conditional knockout mice, which affected myocardial contraction. Few people with primary GSK-3β mutations have been identified, and prior research has mostly focused on gene-edited mice or cells. A potentially pathogenic GSK-3β variant was identified in the current study in a patient with DCM, which has significant ramifications for future genetic research on both DCM and GSK-3β.

1429

Chronic myelogenous leukemia (CML) is a malignancy from bone marrow myeloid stem cells mainly driven by the fusion gene BCR-ABL. In addition to BCR-ABL, other genes including RNF6 are also dysregulated in CML cells. RNF6, a ubiquitin ligase of the RING family, promotes various cancer cell proliferation, chemoresistance, and tumor growth in vivo by targeting various proteins for ubiquitination and degradation, including SHP1, TLE3, FOXA1, and MAD1. However, its specific mechanism in CML is not known.

1253

Most basal-like breast cancers (BLBCs) have a poor prognosis and high crossover with triple-negative breast cancers (TNBCs). However, approximately 50% of TNBC patients develop chemoresistance. Dexamethasone reportedly induces Krüppel-like factor 5 (KLF5) and can cause docetaxel and cisplatin resistance in TNBC. A super-enhancer can maintain the transcription of KLF5. Bromodomain-containing 4 (BRD4) is a transcriptional coactivator in superenhancers, and the BRD4 inhibitor compound 870 could strongly inhibit KLF5 transcription. Forkhead box class O 1 (FOXO1) promotes chemoresistance to doxorubicin in breast cancer. FOXO1 has been shown to promote SOX2 transcription, which plays a critical role in cancer stemness. In addition, FOXO1 promoted KLF5 transcription in diabetic cardiomyopathy. However, the roles of FOXO1 and KLF5 in chemotherapy remain exclusive. In the present study, we examined the function of FOXO1 and the relationship between FOXO1 and KLF5 in BLBCs. We used epirubicin (EPI) and cisplatin (DDP) to treat BLBCs. The expression of FOXO1 and KLF5 was down-regulated, whereas KLF5 overexpression decreased the sensitivity of BLBCs to these drugs. FOXO1 regulated KLF5 in the BLBC chemotherapy response, and a novel therapeutic strategy targeting KLF5 and FOXO1 was proposed.

1225

Increasing evidence supports the hypothesis of autologous immune attack in severe aplastic anemia (SAA): the predominant role of activated cytotoxic T cells (CTL) expressing g-interferon in inhibiting the growth of bone marrow (BM) cells, putative autoantigens, and oligoclonal expansion of CD8+ T cells. For SAA patients, the definitive therapies are immunosuppressive therapy (IST) or hematopoietic stem transplantation (HSCT); IST is most widely applied in the clinic because of the lack of HLAmatched sibling or unrelated donors, patients’ age, and the cost of HSCT. However, only about 60% of SAA patients are responders after receiving IST, and less than 10% achieve complete remission (CR); effective biomarkers for the efficacy prediction of IST in SAA patients are lacking. Our previous publications have demonstrated that T cell receptor (TCR) repertoire profiling has been identified as a biomarker for predicting the clinical outcomes and efficacy of patients. However, systematic evaluation of the predictive value of the TCR repertoire for SAA patients during IST is still little known.

1262

Non-alcoholic fatty liver disease (NAFLD) is a liver condition that is widely prevalent across the world. A considerable number of people with NAFLD have the potential to progress to a more severe form of the condition known as nonalcoholic steatohepatitis (NASH), accompanied by bridging fibrosis. This advancement is more likely if the patient has metabolic risk factors such as obesity or type 2 diabetes that deteriorate over time. Additionally, even slight inflammation or fibrosis in NAFLD can significantly increase the likelihood of progression compared to steatosis alone. This underscores the importance of revising the present methods of monitoring NAFLD patients to ensure early detection and effective management of the disease. The pattern of DNA methylation varies with the onset of a wide range of NAFLD since it is extremely susceptible to internal and external environmental stressors; hepatic DNA methyltransferase has been connected to the NAFLD activity score (NAS) and has been demonstrated to have higher activity in NASH than simple steatosis. Thus, the current work aimed to clarify the role of DNA methylated genes, i.e., sphingomyelin phosphodiesterase 3, Smpd3 in liver tissue and adipose tissue of NASH and mild fibrosis, for identifying biomarkers for differentiating different stages of NAFLD and providing precision medicine approaches to NAFLD diagnosis and management.

1294

Wnt signaling plays a major role in regulating cell proliferation and differentiation. The Wnt ligands are a family of 19 secreted glycoproteins that mediate their signaling effects via binding to Frizzled receptors and LRP5/6 coreceptors and transducing the signal either through b-catenin in the canonical pathway or through a series of other proteins in the noncanonical pathway. Many of the individual components of both canonical and noncanonical Wnt signaling have additional functions throughout the body, establishing the complex interplay between Wnt signaling and other signaling pathways. This crosstalk between Wnt signaling and other pathways gives Wnt signaling a vital role in many cellular and organ processes. Dysregulation of this system has been implicated in many diseases affecting a wide array of organ systems, including cancer and embryological defects, and can even cause embryonic lethality. The complexity of this system and its interacting proteins have made Wnt signaling a target for many therapeutic treatments. However, both stimulatory and inhibitory treatments come with potential risks that need to be addressed. This review synthesized much of the current knowledge on the Wnt signaling pathway, beginning with the history of Wnt signaling. It thoroughly described the different variants of Wnt signaling, including canonical, noncanonical Wnt/PCP, and the noncanonical Wnt/Ca²⁺ pathway. Further description involved each of its components and their involvement in other cellular processes. Finally, this review explained the various other pathways and processes that crosstalk with Wnt signaling.

1372

Mesenchymal stem cells (MSCs), the most well-studied cell type in the field of stem cell therapy, have multi-lineage differentiation and self-renewal potential. MSC-based therapies have been used to treat diverse diseases because of their ability to potently repair tissue and locally restore function. An increasing body of evidence demonstrates that paracrine function is central to the effects of MSC-based therapy. Growth factors, cytokines, chemokines, extracellular matrix components, and extracellular vehicles all contribute to the beneficial effects of MSCs on tissue regeneration and repair. The paracrine substances secreted by MSCs change depending on the tissue microenvironment and biological behavior. In this review, we discuss the bioactive substances secreted by MSCs depending on the microenvironment and biological behavior and their regulatory mechanisms, which explain their potential to treat human diseases, to provide new ideas for further research and clinical cell-free therapy.

1434

Chemotherapy is an effortless and frequently used approach in cancer therapy. However, in most cases, it can only prolong life expectancy and does not guarantee a complete cure. Furthermore, chemotherapy is associated with severe adverse effects, one of the major complications of effective cancer therapy. In addition, newly published research outputs show that cancer stem cells are involved in cancer disease progression, drug resistance, metastasis, and recurrence and that they are functional in the trans-differentiation capacity of cancer stem cells to cancer cells in response to treatments. Novel strategies are therefore required for better management of cancer therapy. The prime approach would be to synthesize and develop novel drugs that need extensive resources, time, and endurance to be brought into therapeutic use. The subsequent approach would be to screen the anti-cancer activity of available non-cancerous drugs. This concept of repurposing non-cancer drugs as an alternative to current cancer therapy has become popular in recent years because using existing anticancer drugs has several adverse effects. Micronutrients have also been investigated for cancer therapy due to their significant anti-cancer effects with negligible or no side effects and availability in food sources. In this paper, we discuss an ideal hypothesis for screening available noncancerous drugs with anticancer activity, with a focus on cancer stem cells and their clinical application for cancer treatment. Further, drug repurposing and the combination of micronutrients that can target both cancers and cancer stem cells may result in a better therapeutic approach leading to maximum tumor growth control.

1485

A pre-metastatic niche (PMN) is a protective microenvironment that facilitates the colonization of disseminating tumor cells in future metastatic organs. Extracellular vesicles (EVs) play a role in intercellular communication by delivering cargoes, such as noncoding RNAs (ncRNAs). The pivotal role of extracellular vesicle-derived noncoding RNAs (EV-ncRNAs) in the PMN has attracted increasing attention. In this review, we summarized the effects of EV-ncRNAs on the PMN in terms of immunosuppression, vascular permeability and angiogenesis, inflammation, metabolic reprogramming, and fibroblast alterations. In particular, we provided a comprehensive overview of the effects of EV-ncRNAs on the PMN in different cancers. Finally, we discussed the promising clinical applications of EV-ncRNAs, including their potential as diagnostic and prognostic markers and therapeutic targets.

1445

Intra-tumor heterogeneity is now arguably one of the most-studied topics in tumor biology, as it represents a major obstacle to effective cancer treatment. Since tumor cells are highly diverse at genetic, epigenetic, and phenotypic levels, intra-tumor heterogeneity can be assumed as an important contributing factor to the nullification of chemotherapeutic effects, and recurrence of the tumor. Based on the role of heterogeneous subpopulations of cancer cells with varying cell-cycle dynamics and behavior during cancer progression and treatment; herein, we aim to establish a comprehensive definition for adaptation of neoplastic cells against therapy. We discuss two parallel and yet distinct subpopulations of tumor cells that play pivotal roles in reducing the effects of chemotherapy: "resistant" and "tolerant" populations. Furthermore, this review also highlights the impact of the quiescent phase of the cell cycle as a survival mechanism for cancer cells. Beyond understanding the mechanisms underlying the quiescence, it provides an insightful perspective on cancer stem cells (CSCs) and their dual and intertwined functions based on their cell cycle state in response to treatment. Moreover, CSCs, epithelialemesenchymal transformed cells, circulating tumor cells (CTCs), and disseminated tumor cells (DTCs), which are mostly in a quiescent state of the cell cycle are proved to have multiple biological links and can be implicated in our viewpoint of cell cycle heterogeneity in tumors. Overall, increasing our knowledge of cell cycle heterogeneity is a key to identifying new therapeutic solutions, and this emerging concept may provide us with new opportunities to prevent the dreadful cancer recurrence.

3034

The interaction between cluster of differentiation 47 (CD47) and signal regulatory protein a (SIRPa) protects healthy cells from macrophage attack, which is crucial for maintaining immune homeostasis. Overexpression of CD47 occurs widely across various tumor cell types and transmits the "don’t eat me" signal to macrophages to avoid phagocytosis through binding to SIRPa. Blockade of the CD47-SIRPa axis is therefore a promising approach for cancer treatment. Lymphoma is the most common hematological malignancy and is an area of unmet clinical need. This review mainly described the current strategies targeting the CD47-SIRPα axis, including antibodies, SIRPa Fc fusion proteins, small molecule inhibitors, and peptides both in preclinical studies and clinical trials with Hodgkin lymphoma and non-Hodgkin lymphoma.

1345

Ribonucleotide reductase M2 (RRM2) is a small subunit in ribonucleotide reductases, which participate in nucleotide metabolism and catalyze the conversion of nucleotides to deoxynucleotides, maintaining the dNTP pools for DNA biosynthesis, repair, and replication. RRM2 performs a critical role in the malignant biological behaviors of cancers. The structure, regulation, and function of RRM2 and its inhibitors were discussed. RRM2 gene can produce two transcripts encoding the same ORF. RRM2 expression is regulated at multiple levels during the processes from transcription to translation. Moreover, this gene is associated with resistance, regulated cell death, and tumor immunity. In order to develop and design inhibitors of RRM2, appropriate strategies can be adopted based on different mechanisms. Thus, a greater appreciation of the characteristics of RRM2 is a benefit for understanding tumorigenesis, resistance in cancer, and tumor microenvironment. Moreover, RRM2-targeted therapy will be more attention in future therapeutic approaches for enhancement of treatment effects and amelioration of the dismal prognosis.

1361

The hypoxic microenvironment is an essential characteristic of most malignant tumors. Notably, hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulatory factor of cellular adaptation to hypoxia, and many critical pathways are correlated with the biological activity of organisms via HIF-1α. In the intra-tumoral hypoxic environment, HIF-1α is highly expressed and contributes to the malignant progression of tumors, which in turn results in a poor prognosis in patients. Recently, it has been indicated that HIF-1α involves in various critical processes of life events and tumor development via regulating the expression of HIF-1α target genes, such as cell proliferation and apoptosis, angiogenesis, glucose metabolism, immune response, therapeutic resistance, etc. Apart from solid tumors, accumulating evidence has revealed that HIF-1α is also closely associated with the development and progression of hematological malignancies, such as leukemia, lymphoma, and multiple myeloma. Targeted inhibition of HIF-1α can facilitate an increased sensitivity of patients with malignancies to relevant therapeutic agents. In the review, we elaborated on the basic structure and biological functions of HIF-1α and summarized their current role in various malignancies. It is expected that they will have future potential for targeted therapy.

1366

N6-methyladenosine (m⁶A) modifications, as one of the most common forms of internal RNA chemical modifications in eukaryotic cells, have gained increasing attention in recent years. The m⁶A RNA modifications exert various crucial roles in various biological processes, such as embryonic development, neurogenesis, circadian rhythms, and tumorigenesis. Recent advances have highlighted that m⁶A RNA modification plays an important role in immune response, especially in the initiation and progression of autoimmune diseases. In this review, we summarized the regulatory mechanisms of m⁶A methylation and its biological functions in the immune system and mainly focused on recent progress in research on the potential role of m⁶A RNA methylation in the pathogenesis of autoimmune diseases, thus providing possible biomarkers and potential targets for the prevention and treatment of autoimmune diseases.

1358

CRISPR/Cas9 is an effective gene editing tool with broad applications for the prevention or treatment of numerous diseases. It depends on CRISPR (clustered regularly interspaced short palindromic repeats) as a bacterial immune system and plays as a gene editing tool. Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing approaches, it has been broadly investigated to treat numerous hereditary and acquired illnesses, including cancers, hemolytic diseases, immunodeficiency disorders, cardiovascular diseases, visual maladies, neurodegenerative conditions, and a few X-linked disorders. CRISPR/Cas9 system has been used to treat cancers through a variety of approaches, with stable gene editing techniques. Here, the applications and clinical trials of CRISPR/Cas9 in various illnesses are described. Due to its high precision and efficiency, CRISPR/Cas9 strategies may treat gene-related illnesses by deleting, inserting, modifying, or blocking the expression of specific genes. The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed. The use of transfection vehicles for CRISPR/Cas9, including viral vectors (such as an Adeno-associated virus (AAV)), and the development of non-viral vectors is also considered.

1943

In recent years, significant breakthroughs have been made in the field of gene therapy. Adeno-associated virus (AAV) is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of interest. Among the AAV vectors, AAV serotype 8 (AAV8) has attracted much attention for its efficient and stable gene transfection into specific tissues. Currently, recombinant AAV8 has been widely used in gene therapy research on a variety of diseases, including genetic diseases, cancers, autoimmune diseases, and viral diseases. This paper reviewed the applications and challenges of using AAV8 as a vector for gene therapy, with the aim of providing a valuable resource for those pursuing the application of viral vectors in gene therapy.

1333

Adhesion G protein-coupled receptors (aGPCRs) are the second largest diverse group within the GPCR superfamily, which play critical roles in many physiological and pathological processes through cellecell and celleextracellular matrix interactions. The adhesion GPCR Adgrg6, also known as GPR126, is one of the better-characterized aGPCRs. GPR126 was previously found to have critical developmental roles in Schwann cell maturation and its mediated myelination in the peripheral nervous system in both zebrafish and mammals. Current studies have extended our understanding of GPR126-mediated roles during development and in human diseases. In this review, we highlighted these recent advances in GPR126 in expression profile, molecular structure, ligandereceptor interactions, and associated physiological and pathological functions in development and diseases.

1384

PARP inhibitors (PARPi) are a kind of cancer therapy that targets poly (ADP-ribose) polymerase. PARPi is the first clinically approved drug to exert synthetic lethality by obstructing the DNA single-strand break repair process. Despite the significant therapeutic effect in patients with homologous recombination (HR) repair deficiency, innate and acquired resistance to PARPi is a main challenge in the clinic. In this review, we mainly discussed the underlying mechanisms of PARPi resistance and summarized the promising solutions to overcome PARPi resistance, aiming at extending PARPi application and improving patient outcomes.

1304

Exosomes are small membrane vesicles containing microRNA, RNA, DNA fragments, and proteins that are transferred from donor cells to recipient cells. Tumor cells release exosomes to reprogram the factors associated with the tumor microenvironment (TME) causing tumor metastasis and immune escape. Emerging evidence revealed that cancer cell-derived exosomes carry immune inhibitory molecule program death ligand 1 (PD-L1) that binds with receptor program death protein 1 (PD-1) and promote tumor progression by escaping immune response. Currently, some FDA-approved monoclonal antibodies are clinically used for cancer treatment by blocking PD-1/PD-L1 interaction. Despite notable treatment outcomes, some patients show poor drug response. Exosomal PD-L1 plays a vital role in lowering the treatment response, showing resistance to PD-1/PD-L1 blockage therapy through recapitulating the effect of cell surface PD-L1. To enhance therapeutic response, inhibition of exosomal PD-L1 is required. Calcium signaling is the central regulator of tumorigenesis and can regulate exosome biogenesis and secretion by modulating Rab GTPase family and membrane fusion factors. Immune checkpoints are also connected with calcium signaling and calcium channel blockers like amlodipine, nifedipine, lercanidipine, diltiazem, and verapamil were also reported to suppress cellular PD-L1 expression. Therefore, to enhance the PD-1/PD-L1 blockage therapy response, the reduction of exosomal PD-L1 secretion from cancer cells is in our therapeutic consideration. In this review, we proposed a therapeutic strategy by targeting calcium signaling to inhibit the expression of PD-L1-containing exosome levels that could reduce the anti-PD-1/PD-L1 therapy resistance and increase the patient’s drug response rate.

1347

N6-methyladenosine (m⁶A) is the most prevalent modification in the eukaryotic transcriptome and has a wide range of functions in coding and noncoding RNAs. It affects the fate of the modified RNA, including its stability, splicing, and translation, and plays an important role in post-transcriptional regulation. Bones play a key role in supporting and protecting muscles and other organs, facilitating the movement of the organism, ensuring blood production, etc. Bone diseases such as osteoarthritis, osteoporosis, and bone tumors are serious public health problems. The processes of bone development and osteogenic differentiation require the precise regulation of gene expression through epigenetic mechanisms including histone, DNA, and RNA modifications. As a reversible dynamic epigenetic mark, m⁶A modifications affect nearly every important biological process, cellular component, and molecular function, including skeletal development and homeostasis. In recent years, studies have shown that m⁶A modification is involved in osteogenesis and bone-related diseases. In this review, we summarized the proteins involved in RNA m⁶A modification and the latest progress in elucidating the regulatory role of m⁶A modification in bone formation and stem cell directional differentiation. We also discussed the pathological roles and potential molecular mechanisms of m⁶A modification in bone-related diseases like osteoporosis and osteosarcoma and suggested potential areas for new strategies that could be used to prevent or treat bone defects and bone diseases.

1350

Extracellular vesicles (EVs) are nano-size vesicles secreted naturally by all cells into the extracellular space and have been recognized as important cellecell mediators in multicellular organisms. EVs contain nucleic acids, proteins, lipids, and other cellular components, regulating many basic biological processes and playing an important role in regenerative medicine and diseases. EVs can be traced to their cells of origin and exhibit a similar function. Moreover, EVs demonstrate low immunogenicity, good biocompatibility, and fewer side effects, compared to their parent cells. Mesenchymal stem cells (MSCs) are one of the most important resource cells for EVs, with a great capacity for self-renewal and multipotent differentiation, and play an essential role in stem cell therapy. The mechanism of MSC therapy was thought to be attributed to the differentiation of MSCs after targeted migration, as previously noted. However, emerging evidence shows the previously unknown role of MSC-derived paracrine factors in stem cell therapy. Especially EVs derived from oral tissue MSCs (OMSC-EVs), show more advantages than those of all other MSCs in tissue repair and regeneration, due to their lower invasiveness and easier accessibility for sample collection. Here, we systematically review the biogenesis and biological characteristics of OMSC-EVs, as well as the role of OMSC-EVs in intercellular communication. Furthermore, we discuss the potential therapeutic roles of OMSC-EVs in oral and systemic diseases. We highlight the current challenges and future directions of OMSC-EVs to focus more attention on clinical translation. We aim to provide valuable insights for the explorative clinical application of OMSC-EVs.

1318

Ferroptosis is a novel form of regulated cell death characterized by iron-dependent excessive lipid peroxidation. The core organelle involved in ferroptosis is mitochondria. Mitochondria undergoing ferroptosis are distinct from normal mitochondria in terms of morphology, biochemistry, gene expression, and energy metabolism. An increasing number of studies have shown that mitochondria and their associated metabolic pathways mediate ferroptosis in the development and progression of breast cancer. In this review, we discuss the relevant research about ferroptosis in breast cancer and provide a comprehensive summary of mitochondrial regulation in ferroptosis from the perspective of lipid metabolism, oxidative phosphorylation, ion metabolism, glycometabolism, and nucleotide metabolism. We also summarize the application of mitochondrial metabolism-related pathways as ferroptosis treatment targets. Here we provide new insights into the relationship between mitochondria, ferroptosis, and breast cancer treatment.

1370

The incidence and mortality rates of cancer are increasing every year worldwide but the survival rate of cancer patients is still unsatisfactory. Therefore, it is necessary to further elucidate the molecular mechanisms involved in tumor development and drug resistance to improve cancer cure or survival rates. In recent years, autophagy has become a hot topic in the field of oncology research, which plays a double-edged role in tumorigenesis, progression, and drug resistance. Meanwhile, long non-coding RNA (lncRNA) has also been shown to regulate autophagy, and the two-sided nature of autophagy determines the dual regulatory role of autophagy-related lncRNAs (ARlncRNAs). Therefore, ARlncRNAs can be effective therapeutic targets for various cancers. Furthermore, the high abundance and stability of ARlncRNAs in tumor tissues make them promising biomarkers. In this review, we summarized the roles and mechanisms of ARlncRNAs in tumor cell proliferation, apoptosis, migration, invasion, drug resistance, angiogenesis, radiation resistance, and immune regulation. In addition, we described the clinical significance of these ARlncRNAs, including as biomarkers/therapeutic targets and their association with clinical drugs.

1341

As the most common internal modification of mRNA, N⁶-methyladenosine (m⁶A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m⁶A methyltransferase (writer), methyltransferase-like 3 (METTL3) addsm⁶A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whetherm6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m⁶A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126’s downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m⁶A plays a role in pre-miRNA processing that contributes to tumorigenesis.

1492

Therapeutic targeting FOXO3A (a forkhead transcription factor) represents a promising strategy to suppress acute myeloid leukemia (AML). However, the effective inhibitors that target FOXO3A are lacking and the adaptive response signaling weakens the cytotoxic effect of FOXO3A depletion on AML cells. Here, we show that FOXO3A deficiency induces a compensatory response involved in the reactive activation of mTOR that leads to signaling rebound and adaptive resistance. Mitochondrial metabolism acts downstream of mTOR to provoke activation of JNK/c-JUN via reactive oxygen species (ROS). At the molecular level, FOXO3A directly binds to the promoter of G protein gamma subunit 7 (GNG7) and preserves its expression, while GNG7 interacts with mTOR and restricts phosphorylated activation of mTOR. Consequently, combinatorial inhibition of FOXO3A and mTOR show a synergistic cytotoxic effect on AML cells and prolongs survival in a mouse model of AML. Through a structure-based virtual screening, we report one potent small-molecule FOXO3A inhibitor (Gardenoside) that exhibits a strong effect of anti-FOXO3A DNA binding. Gardenoside synergizes with rapamycin to substantially reduce tumor burden and extend survival in AML patient-derived xenograft model. These results demonstrate that mTOR can mediate adaptive resistance to FOXO3A inhibition and validate a combinatorial approach for treating AML.

1351

CDC42 controls intestinal epithelial (IEC) stem cell (IESC) division. How aberrant CDC42 initiates intestinal inflammation or neoplasia is unclear. We utilized models of inflammatory bowel diseases (IBD), colorectal cancer, aging, and IESC injury to determine the loss of intestinal Cdc42 upon inflammation and neoplasia. Intestinal specimens were collected to determine the levels of CDC42 in IBD or colorectal cancer. Cdc42 floxed mice were crossed with Villin-Cre, Villin-CreER^T2 and/or Lgr5-eGFP-IRES-CreER^T2, or Bmi1-CreER^T2 mice to generate Cdc42 deficient mice. Irradiation, colitis, aging, and intestinal organoid were used to evaluate CDC42 upon mucosal inflammation, IESC/progenitor regenerative capacity, and IEC repair. Our studies revealed that increased CDC42 in colorectal cancer correlated with lower survival; in contrast, lower levels of CDC42 were found in the inflamed IBD colon. Colonic Cdc42 depletion significantly reduced Lgr5⁺ IESCs, increased progenitors’ hyperplasia, and induced mucosal inflammation, which led to crypt dysplasia. Colonic Cdc42 depletion markedly enhanced irradiation-or chemical-induced colitis. Depletion or inhibition of Cdc42 reduced colonic Lgr5⁺ IESC regeneration. In conclusion, depletion of Cdc42 reduces the IESC regeneration and IEC repair, leading to prolonged mucosal inflammation. Constitutive monogenic loss of Cdc42 induces mucosal inflammation, which could result in intestinal neoplasia in the context of aging.

1340

Emerging evidence suggested that zinc finger protein 831 (ZNF831) was associated with immune activity and stem cell regulation in breast cancer. Whereas, the roles and molecular mechanisms of ZNF831 in oncogenesis remain unclear. ZNF831 expression was significantly diminished in breast cancer which was associated with promoter CpG methylation but not mutation. Ectopic over-expression of ZNF831 suppressed breast cancer cell proliferation and colony formation and promoted apoptosis in vitro, while knockdown of ZNF831 resulted in an opposite phenotype. Anti-proliferation effect of ZNF831 was verified in vivo. Bioinformatic analysis of public databases and transcriptome sequencing both showed that ZNF831 could enhance apoptosis through transcriptional regulation of the JAK/STAT pathway. ChIP and luciferase report assays demonstrated that ZNF831 could directly bind to one specific region of STAT3 promoter and induce the transcriptional inhibition of STAT3. As a result, the attenuation of STAT3 led to a restraint of the transcription of Bcl2 and thus accelerated the apoptotic progression. Augmentation of STAT3 diminished the apoptosis-promoting effect of ZNF831 in breast cancer cell lines. Furthermore, ZNF831 could ameliorate the anti-proliferation effect of capecitabine and gemcitabine in breast cancer cell lines. Our findings demonstrate for the first time that ZNF831 is a novel transcriptional suppressor through inhibiting the expression of STAT3/Bcl2 and promoting the apoptosis process in breast cancer, suggesting ZNF831 as a novel biomarker and potential therapeutic target for breast cancer patients.

1328

Reactive oxidative species (ROS) production-driven ferroptosis plays a role in acute kidney injury (AKI). However, its exact molecular mechanism is poorly understood. Scavenger receptor CD36 has important roles in oxidizing lipids, lipid accumulation, metabolic syndrome, and insulin resistance in chronic kidney disease, but its roles remain unexplored in AKI. The present study investigated the role and mechanism of CD36 in regulating proximal tubular cell ferroptosis and AKI. The expression of CD36 was found to be significantly up-regulated in AKI renal tissues and correlated with renal function, which might serve as an independent biomarker for AKI patients. Moreover, in adult mice subjected to AKI, deletion of CD36 (CD36^-/-) induced tubular cell ROS accumulation, ferroptosis activation, and renal injury. Mechanistically, combining LC-MS/MS, co-IP, and ubiquitination analyses revealed that CD36 could specifically bind to ferroptosis suppressor protein 1 (FSP1) and regulate its ubiquitination at sites K16 and K24, leading to FSP1 degradation and progression of ferroptosis in AKI. The present results emphasize a novel mechanism of CD36 in cisplatin-induced AKI. The discovery of the special CD36 roles in promoting ferroptosis and AKI development by regulating the ubiquitination of FSP1 in proximal tubular cells may be potential therapeutic targets for AKI. Moreover, CD36 may play a key role in the progression of AKI. Therefore, targeting CD36 may provide a promising treatment option for AKI.

1515

Cartilage development is controlled by the highly synergistic proliferation and differentiation of growth plate chondrocytes, in which the Indian hedgehog (IHH) and parathyroid hormone-related protein-parathyroid hormone-1 receptor (PTHrP-PTH1R) feedback loop is crucial. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) branch of the unfolded protein response (UPR) is essential for normal cartilage development. However, the precise role of ER stress effector IRE1α, encoded by endoplasmic reticulum to nucleus signaling 1 (ERN1), in skeletal development remains unknown. Herein, we reported that loss of IRE1α accelerates chondrocyte hypertrophy and promotes endochondral bone growth. ERN1 acts as a negative regulator of chondrocyte proliferation and differentiation in postnatal growth plates. Its deficiency interrupted PTHrP/PTH1R and IHH homeostasis leading to impaired chondrocyte hypertrophy and differentiation. XBP1s, produced by p-IRE1α-mediated splicing, binds and up-regulates PTH1R and IHH, which coordinate cartilage development. Meanwhile, ER stress cannot be activated normally in ERN1-deficient chondrocytes. In conclusion, ERN1 deficiency accelerates chondrocyte hypertrophy and cartilage mineralization by impairing the homeostasis of the IHH and PTHrP/PTH1R feedback loop and ER stress. ERN1 may have a potential role as a new target for cartilage growth and maturation.

1727

Glioblastoma (GBM) is a malignant brain tumor that grows quickly, spreads widely, and is resistant to treatment. Fibroblast growth factor receptor (FGFR)1 is a receptor tyrosine kinase that regulates cellular processes, including proliferation, survival, migration, and differentiation. FGFR1 was predominantly expressed in GBM tissues, and FGFR1 expression was negatively correlated with overall survival. We rationally designed a novel small molecule CYY292, which exhibited a strong affinity for the FGFR1 protein in GBM cell lines in vitro. CYY292 also exerted an effect on the conserved Ser777 residue of FGFR1. CYY292 dose-dependently inhibited cell proliferation, epithelialemesenchymal transition, stemness, invasion, and migration in vitro by specifically targeting the FGFR1/AKT/Snail pathways in GBM cells, and this effect was prevented by pharmacological inhibitors and critical gene knockdown. In vivo experiments revealed that CYY292 inhibited U87MG tumor growth more effectively than AZD4547. CYY292 also efficiently reduced GBM cell proliferation and increased survival in orthotopic GBM models. This study further elucidates the function of FGFR1 in the GBM and reveals the effect of CYY292, which targets FGFR1, on downstream signaling pathways directly reducing GBM cell growth, invasion, and metastasis and thus impairing the recruitment, activation, and function of immune cells.

1365

Cancer stem cells (CSCs) are considered tumor-initiating cells and the main drivers of disease progression. Targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the up-regulation of IL-13RA2 expression in colorectal cancer (CRC) tissues and spheroid cells. The expression of IL-13RA2 was positively correlated with canonical stemness markers in CRC. We further demonstrated that the level of IL-13 was up-regulated in the serum of CRC patients. Biologically, recombinant IL-13 (rIL-13) stimulation promoted the sphere formation, proliferation, and migration of CRC cells in vitro and enhanced tumorigenesis in vivo. This phenotype could be reversed by knocking down IL-13RA2. Mechanistically, IL-13 activated autophagy by inducing LC3I/LC3II transformation in CRC-CSCs, which was crucial for the biological functions of IL-13. We further demonstrated that IL-13RA2 acted as a modular link of the E3 ligase UBE3C and the substrate p53 protein, enhancing the interaction of UBE3C and p53, thereby inducing the K48-linked ubiquitination of p53. In conclusion, the IL-13/IL-13RA2 signaling cascade promotes CRC-CSC self-renewal and tumorigenesis by inducing p53 ubiquitination, adding an important layer to the connection between IL-13 and p53, which can be translated into novel targeted therapies.

1739