Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

Background: Lung cancer is a malignant tumor with metastatic potential. Chemokine ligand 14 (CXCL14) has been reported to be associated with different cancer cell migration and invasion. However, few studies have explored the function of CXCL14 and its specific receptor in lung cancer metastasis. This study aims to determine the mechanism of CXCL14-promoted cancer metastasis. Methods: The expression of CXCL14, atypical chemokine receptor 2 (ACKR2), and epithelial mesenchymal transition (EMT) markers was evaluated by the public database of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and immunofluorescence (IF). Migration and wound healing assays were used to observe the motility of cancer cells. A luciferase reporter assay was performed to analyze transcription factor activity. The metastasis of lung cancer cells was evaluated in an orthotopic model. Results: We have presented that overexpression of CXCL14 and ACKR2 was observed in lung cancer datasets, human lung tumor sections, and lung cancer cells. Furthermore, the migration of CXCL14-promoted lung cancer cells was determined in vitro and in vivo. In particular, ACKR2 knockdown abolished CXCL14-induced cancer cell motility. Additionally, ACKR2 was involved in CXCL14-triggered phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src signaling pathway and subsequently upregulated nuclear factor κB (NF-κB) transcription activity leading to EMT and migration of lung cancer cells. These results indicated that the CXCL14/ACKR2 axis played an important role in lung cancer metastasis. Conclusion: This study is the first to reveal the function of CXCL14 in promoting EMT and metastasis in lung cancer. As a specific receptor for CXCL14 in lung cancer, ACKR2 mediates CXCL14-induced signaling that leads to cell motility. Our findings can be used as a prognostic biomarker of lung cancer metastasis.

Sepsis-associated encephalopathy (SAE), as shown as acute and long-term cognitive impairment, is associated with increased mortality of sepsis. The causative factors of SAE are diverse and the underlying pathological mechanisms of SAE remain to be fully elucidated. Multiple studies have demonstrated a crucial role of microglia in the development of SAE, but the role of neutrophils and neutrophil extracellular traps (NETs) in SAE is still unclear. Here, we firstly show that in murine sepsis model, neutrophils and NETs promote blood-brain barrier (BBB) disruption, neuronal apoptosis and microglia activation in hippocampus and induce hippocampus-dependent memory impairment. Anti-Gr-1 antibody or DNase I treatment attenuates these sepsis-induced changes. Then, we find that genetic deletion of neutrophil GSDMD or PD-L1 reduces NET release and improves SAE in murine sepsis model. Finally, in human septic neutrophils, p-Y705-Stat3 binds to PD-L1, promotes PD-L1 nuclear translocation and enhances transcription of the gasdermin D (GSDMD) gene. In summary, our findings firstly identify a novel function of PD-L1 in maintaining transcriptional activity of p-Y705-Stat3 to promote GSDMD-dependent NET release in septic neutrophils, which plays a critical role in the development of SAE.

Induced tumor-suppressing cells (iTSCs) can be generated from cancer and non-cancer cells. Here, three paradoxical maxims for the action of iTSCs are reviewed: the secretion of tumor-suppressing proteins, their role as a "double-edged" sword, and the elimination of lesser-fit cancer cells. "Super-fit" cancer cells secrete an array of proteins, most of which contribute to enhancing their growth and removing "lesser-fit" cancer cells. These maxims explain the potential dilemma with therapeutic agents since the inhibitory agents tend to promote the synthesis of tumor-promoting proteins. The maxims suggest the possibility of a novel treatment option using cancer-guided evolutionary-fit iTSCs.

Exosomes, as therapeutically relevant cell-secreted extracellular vesicles, have attracted enormous interest because they participate in intercellular communication and facilitate wound healing. Stem cell-derived exosomes exhibit similar biological effects to source cells with the exception of low immunogenicity and no tumorigenicity, as well as superior efficacy in promoting wound healing. Exosomes accelerate wound healing by promoting angiogenesis and cell proliferation, as well as balancing inflammatory responses. Particularly, when exosomes are genetically modified or used in combination with materials, they can exhibit better comprehensive therapeutic properties, such as enriching active ingredients, targeted delivery, and physiological barrier to penetration, which are not available in traditional single products. Besides, exosomes have also been considered for diagnostic and therapeutic uses related to wounds, such as repairing complex wounds, enhancing graft success, treating related complications, and serving as diagnostic biomarkers. However, their clinical applications still face challenges, as reliable commercial products are not yet available. This review will focus on recent research advances that describe the characteristics and isolation of exosomes, introduce the sources of exosomes suitable for wound repair and related complications, illustrate the value of engineered exosomes and their development directions in the future, and provide evidence for the potential therapeutic application of exosomes in wound healing, as well as discuss potential risks, challenges, and solutions for future applications.

IFNγ has long been recognised as a key mediator of tumour immunity and angiostasis. However, IFNγ modulation for cancer therapy is still unsuccessful due to its complex effects on various host cells. In this study, we found that treatment of Lewis lung carcinoma transplants with cisplatin often caused IFNγ-dependent tumour vascular damage. IFNγ induced endothelial glycolysis and lactate production, leading to enhanced endocytosis of vascular endothelial (VE)-cadherin and vessel leakage. We have also developed anti-IFNγ nanoparticles coated with a clot-binding peptide CREKA (CREKA-lipo-anti-IFNγ), which targets the fibrin-fibronectin complex that appears in the leaky site of damaged tumour blood vessels. Blocking IFNγ activity in the leakage site of capillaries using nanoparticles rescued VE-cadherin distribution on the endothelial cellular surface, promoted blood vessel integrity, and improved drug delivery. In conclusion, IFNγ blockade in capillary leak site protected tumour blood vessels from lactate-dependent VE-cadherin loss and enhanced drug delivery during chemotherapy, which provides a basis for tissue-specific IFNγ blockade for tumour therapy.

Radiotherapy is the most predominant treatment strategy for lung squamous cell carcinoma (LUSC) patients, but radioresistance is the major obstacle to therapy effectiveness. The mechanisms and regulators of LUSC radioresistance remain unclear. Here, lactotransferrin (LTF) is found to be significantly upregulated in radioresistant LUSC cell lines (H226R and H1703R) and clinical samples and promotes radioresistance of LUSC both in vitro and in vivo. Comprehensive enrichment analyses suggested that LTF potentially modulates autophagy in LUSC. Interestingly, the level of autophagy was raised in the radioresistant cells, and suppression of autophagy sensitized LUSC to irradiation. Functional experiments showed that LTF deficiency inhibits cellular autophagy through the AMPK pathway, ultimately leading to radiosensitization. Mechanistically, LTF can directly interact with AMPK to facilitate its phosphorylation and activate autophagy signaling. Moreover, NEAT1 functions as a ceRNA that targets miR-214-5p resulting in an increased LTF expression. Intriguingly, SP2, a transcription factor regulated by AMPK, induced NEAT1 expression by directly binding to its promoter region and thus forming a LTF/AMPK/SP2/NEAT1/miR-214-5p feedback loop. Our work reveals for the first time that LTF induces radioresistance by promoting autophagy and enhancing its self-expression via forming a positive feedback loop, suggesting that LTF is an appealing radiosensitization target for treating LUSC.

Translation machinery associated 7 homolog (TMA7) is closely related to proliferation-related diseases. However, the function and regulatory mechanism of TMA7 in laryngeal squamous cell carcinoma (LSCC) remain unclear. The present study aimed to investigate the effect of TMA7 on the occurrence and development of LSCC and to study the mechanism of TMA7. TMA7 is upregulated in LSCC tissues and associated with poor prognosis. After TMA7 downregulation, the autophagy level was increased, and the proliferation, migration, and invasion of LSCC cells were inhibited. The m6A methylated reader IGF2BP3 enhanced the stability of TMA7 and reduced the level of autophagy. TMA7 interacted directly with UBA2. Furthermore, the activation of the IGF2BP3-regulated TMA7-UBA2-PI3K pathway is the primary mechanism by which TMA7 inhibits autophagy and promotes the progression of LSCC. The current study revealed that IGF2BP3-mediated TMA7 m6A modification promotes LSCC progression and cisplatin-resistance through UBA2-PI3K pathway, providing new insights into the autophagy-related mechanism, potential biomarkers, and therapeutic targets for LSCC.

Alcohol use disorder (AUD) is one of the most prevalent neuropsychological disorders worldwide, and its pathogenesis is convoluted and poorly understood. There is considerable evidence demonstrating significant associations between multiple heritable factors and the onset and progression of AUD. In recent years, a substantial body of research conducted by emerging biotechnologies has increasingly highlighted the crucial roles of noncoding RNAs (ncRNAs) in the pathophysiology of mental diseases. As in-depth understanding of ncRNAs and their mechanisms of action, they have emerged as prospective diagnostic indicators and preclinical therapeutic targets for a variety of psychiatric illness, including AUD. Of note, dysregulated expression of ncRNAs such as circRNAs, lncRNAs and miRNAs was routinely found in AUD individuals, and besides, exogenous regulation of partial ncRNAs has also been shown to be effective in ameliorating alcohol preference and excessive alcohol consumption. However, the exact molecular mechanism still remains elusive. Herein, we systematically summarized current knowledge regarding alterations in the expression of certain ncRNAs as well as their-mediated regulatory mechanisms in individuals with AUD. And finally, we detailedly reviewed the potential theranostics applications of gene therapy agents targeting ncRNAs in AUD mice. Overall, a deeper comprehension of functional roles and biological mechanisms of ncRNAs may make significant contributions to the accurate diagnosis and effective treatment of AUD.

In gastric cancer, lymph node metastasis (LNM) is the major metastasis route, and lymphatic invasion is the precursor of LNM. Tumor-associated neutrophils (TANs) promote LNM. However, the molecular mechanisms underlying TANs-mediated lymphatic invasion and/or LNM remain unclear. Herein, we revealed that high level of TANs was the independent risk factor for lymphatic invasion and LNM respectively, and lymphatic tumor cell-neutrophil clusters were positively correlated with LNM. Crosstalk between neutrophils and tumor cells was required for enhanced tumor cell invasiveness, endowing neutrophils to boost epithelial-to-mesenchymal transition (EMT) of tumor cells and in turn promoting LNM. Mechanically, tumor cells educated neutrophils via TGFβ1 to produce more FAM3C through Smad2/3 signaling activation, and FAM3C promoted tumor cell EMT through JNK-ZEB1/Snail signaling pathway. The crosstalk enhanced the affinity of neutrophils with tumor cells through interaction of integrins α6β1 and α6β4 with CD151. Furthermore, studies using tumor-bearing mice demonstrated that neutrophils were the important driver for gastric cancer tumorigenesis and invasiveness. The study clearly identifies the functional roles of TANs in promoting tumor invasion, and facilitates a better understanding of novel mechanisms responsible for LNM of gastric cancer, which provides potential targets for developing new strategies to prevent or treat LNM in gastric cancer.

Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy and associated with poor prognosis. Lack of therapeutic methods for CCA and insensitivity of targeted therapy and immunotherapy make its treatment challenging. NUF2, a component of Ndc80 kinetochore complex, is implicated in the initiation and development of multiple cancers. However, the role and mechanism of NUF2 in CCA is still unclear. In this research, we investigated the biological processes and underlying mechanisms of NUF2 in CCA. We discovered that the expression of NUF2 was upregulated in CCA and negatively correlated with prognosis. Changes in NUF2 levels had an impact on cell proliferation and migration. Moreover, NUF2 functioned as an oncogene to promote the progression of CCA through p38/MAPK signaling by inhibiting p62 binding of TFR1 and affecting its autophagic degradation. In addition, TFR1 promoted CCA progression and Kaplan-Meier analyses uncovered patients with high expression of TFR1 was associated with the poor survival. In conclusion, our study demonstrated that NUF2 promoted CCA progression by regulating TFR1 protein degradation, and the NUF2/TFR1/MAPK axis could be an excellent therapeutic target for CCA.

Serine/threonine kinases (STK3) is a core component of the Hippo pathway and modulates oxidative stress and inflammatory responses in cardiovascular diseases. However, its potential role in septic cardiomyopathy remains undefined. STK3-mediated phosphorylation of Kelch-like ECH-associated protein 1 (KEAP1) was shown to suppress antioxidant gene transcription controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages. To explore whether STK3 induces KEAP1-mediated suppression of Nrf2 in septic cardiomyopathy, wild-type and global STK3 knockout (STK3 -/- ) mice were treated with LPS. LPS treatment upregulated cardiac STK3 expression. STK3 deletion attenuated myocardial inflammation and cardiomyocyte death, and improved myocardial structure and function. In LPS-challenged HL-1 cardiomyocytes, shRNA-mediated STK3 knockdown normalized mitochondrial membrane potential and ATP production, attenuated apoptosis, and rescued antioxidant gene expression by preventing Nrf2 downregulation. Co-IP, docking analysis, western blotting, and immunofluorescence assays further showed that STK3 binds and phosphorylates KEAP1, promoting Nrf2 downregulation. Accordingly, transfection of phosphodefective KEAP1 mutant protein in cardiomyocyte restored Nrf2 expression and mitochondrial performance upon LPS, while expression of a phosphomimetic KEAP1 mutant abolished the mitochondria-protective and pro-survival effects of STK3 deletion. These findings suggest that STK3 upregulation contributes to septic cardiomyopathy by phosphorylating KEAP1 to promote Nrf2 degradation and suppression of the antioxidant response.

Cisplatin is widely recommended in combination for the treatment of tumors, thus inevitably increasing the incidence of cisplatin-induced acute kidney injury. Mitophagy is a type of mitochondrial quality control mechanism that degrades damaged mitochondria and maintains cellular homeostasis. Ferroptosis, a new modality of programmed cell death, is characterized by iron-dependent phospholipid peroxidation and oxidative membrane damage. However, the role of mitophagy in ferroptosis in kidney disease is unclear. Here, we investigated the mechanism underlying both BNIP3-mediated and PINK1-PARK2-mediated mitophagy-induced attenuation of ferroptosis in cisplatin-induced acute kidney injury. The results showed that cisplatin induced mitochondrial injury, ROS release, intracellular iron accumulation, lipid peroxidation and ferroptosis in the kidney, which were aggravated in Bnip3 knockout, Pink1 knockout or Park2 knockout cisplatin-treated mice. Ferrstatin-1, a synthetic antioxidative ferroptosis inhibitor, rescued iron accumulation, lipid peroxidation and ferroptosis caused by inhibition of mitophagy. Thus, the present study elucidated a novel mechanism by which both BNIP3-mediated and PINK1-PARK2-mediated mitophagy protects against cisplatin-induced renal tubular epithelial cell ferroptosis through the ROS/HO1/GPX4 axis.

Renal cell carcinoma (RCC) is a serious threat to people's health due to its rapid progression, and patients easily develop resistance to targeted therapy. The absent in melanoma 2 (AIM2) is a receptor protein that has recently been proposed to play an important role in various diseases. In this study, AIM2 was identified as a new biomarker of RCC and promoted RCC progression and sunitinib resistance in an inflammasome-independent manner. Mechanistically, AIM2 promoted FOXO3a phosphorylation and proteasome degradation, thereby reducing its transcriptional effect on ACSL4 and inhibiting ferroptosis. In summary, AIM2 promoted RCC progression and sunitinib resistance through FOXO3a-ACSL4 axis-regulated ferroptosis, which could provide new ideas and therapeutic targets for RCC diagnosis and treatment.

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

tRNA is one of the most conserved and abundant RNA species, which plays a key role during protein translation. tRNA molecules are post-transcriptionally modified by tRNA modifying enzymes. Since high-throughput sequencing technology has developed rapidly, tRNA modification types have been discovered in many research fields. In tRNA, numerous types of tRNA modifications and modifying enzymes have been implicated in biological functions and human diseases. In our review, we talk about the relevant biological functions of tRNA modifications, including tRNA stability, protein translation, cell cycle, oxidative stress, and immunity. We also explore how tRNA modifications contribute to the progression of human diseases. Based on previous studies, we discuss some emerging techniques for assessing tRNA modifications to aid in discovering different types of tRNA modifications.

Gut microbiota was only considered as a commensal organism that aids in digestion, but recent studies revealed that the microbiome play a critical role in both physiological and pathological immune system. The gut microbiome composition is altered by environmental factors such as diet and hygiene, and the alteration affects immune cells, especially T cells. Advanced genomic techniques in microbiome research defined that specific microbes regulate T cell responses and the pathogenesis of immune-mediated disorders. Here, we review features of specific microbes-T cell crosstalk and relationship between the microbes and immunopathogenesis of diseases including in cancers, autoimmune disorders and allergic inflammations. We also discuss the limitations of current experimental animal models, cutting-edge developments and current challenges to overcome in the field, and the possibility of considering gut microbiome in the development of new drug.

Curcumin is a natural polyphenol phytochemical derived from turmeric with antioxidant, anti-inflammatory, and anticancer properties but is concerned about poor solubility in water, absorption, and metabolic stability. Potent metastatic osteosarcoma is the most common primary bone cancer in children, adolescents, and young adults. It is responsible for low survival rates because of its high rate of metastasis to the lungs. To improve poor bioavailability, numerous curcumin analogs were developed to possess anticancer characteristics through a variety of biological pathways involved in cytotoxicity, proliferation, autophagy, sensitizing chemotherapy, and metastases. This review provides an overview of their various pharmacological functions, molecular mechanisms, and therapeutic potential as a remedy for human osteosarcoma. To enhance therapeutic efficacy, several liposomal nanoparticles, nanocarriers, multifunctional micelles, and three-dimensional printed scaffolds have also been developed for the controlled delivery of curcumin targeting human osteosarcoma cells. Consequently, curcumin and several potential analogs and delivery formulations are optimistic candidates to improve the currently available strategy for human osteosarcoma. However, further insight into the mechanism of action of promising curcumin analogs and the development of carriers in clinical trials of osteosarcoma needs to be investigated to improve their overall potency and clinical utility, in particular the anti-metastatic effect.

Myocardial ischemia-reperfusion (I/R) damage is characterized by mitochondrial damage in cardiomyocytes. Transmembrane BAX inhibitor motif containing 6 (TMBIM6) and presenilin-2 (PS2) participate in multiple mitochondrial pathways; thus, we investigated the impact of these proteins on mitochondrial homeostasis during an acute reperfusion injury. Myocardial post-ischemic reperfusion stress impaired myocardial function, induced structural abnormalities and promoted cardiomyocyte death by disrupting the mitochondrial integrity in wild-type mice, but not in TMBIM6 transgenic mice. We found that TMBIM6 bound directly to PS2 and promoted its post-transcriptional degradation. Knocking out PS2 in mice reduced I/R injury-induced cardiac dysfunction, inflammatory responses, myocardial swelling and cardiomyocyte death by improving the mitochondrial integrity. These findings demonstrate that sufficient TMBIM6 expression can prevent PS2 accumulation during cardiac I/R injury, thus suppressing reperfusion-induced mitochondrial damage. Therefore, TMBIM6 and PS2 are promising therapeutic targets for the treatment of cardiac reperfusion damage.

Cardiac fibroblasts are crucial for scar formation and cardiac repair after myocardial infarction (MI). Collagen triple helix repeat containing 1 (CTHRC1), an extracellular matrix protein, is involved in the pathogenesis of vascular remodeling, bone formation, and tumor progression. However, the role and underlying mechanism of CTHRC1 in post-MI wound repair are not fully clear. Bioinformatics analysis demonstrated CTHRC1 up-regulation in cardiac fibroblasts after ischemic cardiac injury. Serum levels of CTHRC1 were increased in MI mice and CTHRC1 expression was up-regulated in cardiac fibroblasts after MI. In vitro results showed that the induction of CTHRC1 expression in cardiac fibroblasts was mediated by canonical TGFβ1-Smad2/3 signaling axis. Moreover, CTHRC1 improved wound healing and boosted cardiac fibroblast activation in vitro. Cthrc1 deficiency aggravated cardiac function and reduced collagen deposition as well as increased mortality attributable to cardiac rupture after MI. Consistent with above phenotypes, reduced the levels of myocardial CD31, α-smooth muscle actin, collagen I, and collagen III was observed, whereas myocardial expression of matrix metalloproteinase 2 and matrix metalloproteinase 9 were increased in Cthrc1 knockout mice post-MI. Above effects could be partly reversed by rCTHRC1 protein or rWNT5A protein. Our study indicates that cardiac fibroblast-derived, canonical TGFβ1-Smad2/3-dependent CTHRC1 could improve wound repair and prevent cardiac rupture after MI via selectively activating non-canonical WNT5A-PCP signaling pathway.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.