Search

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Engaging in activity has proven to have beneficial effects on different facets of well-being, such as conditions related to the deterioration of the nervous system. Non-coding RNAs (ncRNAs) and exosomal ncRNAs associated with vesicles have been recognized as influencers of gene expression and cell signaling, potentially contributing to the positive impact of physical activity on neurodegenerative conditions. It is hypothesized that exercise-induced changes in ncRNA expression may regulate key processes involved in neuroprotection, including neuroinflammation, oxidative stress, protein aggregation, and synaptic function. Exercise has shown promise in preventing neurodegenerative diseases (NDs), and ncRNAs and exosomal ncRNAs are emerging as potential mediators of these benefits. In review, we explored how ncRNAs and exosomal ncRNAs play a role in enhancing the impacts of activity on neurodegenerative disorders for future treatments. Research studies, both preclinical and clinical, that have documented the use of various exercises and their effects on ncRNAs and exosomal ncRNAs for the treatment of NDs have been compiled and enlisted from the PubMed database, spanning the time period from the year 2000 up to the current time. Studies show that manipulating specific ncRNAs or harnessing exercise-induced changes in ncRNA expression and exosomal cargo could potentially be utilized as therapeutic strategies for preventing or treating NDs. In conclusion, studies suggest that various exercise modalities, including aerobic, resistance, and high-intensity interval training, can modulate the expression of ncRNAs and exosomal ncRNAs in the context of NDs. The altered ncRNA profiles may contribute to the neuroprotective and therapeutic effects observed with exercise interventions. However, more research is needed to fully understand the underlying mechanisms and to further explore the potential of exercise-induced ncRNA signatures as biomarkers and therapeutic targets for neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Melatonin released from the pineal gland plays an important role in maintaining the light/dark cycle. Melatonin exerts its effects on various organs through receptor and nonreceptor pathways. Recently, the role of melatonin in various metabolic disorders has been investigated. This review focuses on the molecular pathways associated with melatonin and its role in metabolic disorders. In humans, melatonin acts through two G protein-coupled receptors (MT1 and MT2). Melatonin modulates insulin release, such as elevated insulin levels in the evening compared to morning hours, exerts cardioprotective effects through the cGMP pathway and nitric oxide production in endothelial cells, and controls oxidative stress and apoptosis in myocardial tissue. Melatonin through MT2 receptors increases lipolysis and thermogenesis, which have a positive effect on weight reduction in obese individuals. Currently, most drugs that target melatonin receptors are primarily used to treat neurological disorders. A detailed investigation to explore the role of melatonin and its signalling pathway in peripheral organs is essential to develop therapeutic molecules for managing metabolic disorders. [ABSTRACT FROM AUTHOR]

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given this small number of signaling pathways, the existence of multiple related ligands for many of these pathways represents a key evolutionary innovation for encoding complexity into cell–cell signaling. Relatedly, crosstalk between pathways is another critical feature, which allows a modest number of pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells that express a given signaling ligand but also those cells that specifically coexpress pairs of signaling ligands. We present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor β (TGF β), Janus kinase/signal transducer and activator of transcription (JAK/STAT), Jun kinase (JNK), and platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-related receptor (PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets and that they can be used to identify cells and tissues that coexpress pairs of ligands. As a proof of principle, we demonstrate that the 4th chromosome TGF β ligands myoglianin and maverick are broadly coexpressed in muscles and other tissues of both larva and adults and that the JAK/STAT ligands upd2 and upd3 are partially coexpressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the 7 Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways. [ABSTRACT FROM AUTHOR]

Introduction: Anemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO). Pegmolesatide (by Hansoh Pharma) is currently the only marketed long-acting EPO mimetic peptide (EMP) for the treatment of anemia in both dialysis and non-dialysis CKD patients. This paper aimed to explore the long-acting erythropoiesis stimulating molecular mechanism of Pegmolesatide. Methods: In vitro assays were utilized to assess Pegmolesatide erythropoietin receptor (EPOR) affinity, competitive binding, cell proliferation/survival, apoptosis, cell surface receptor expression, and signal transduction. Pharmacokinetics (PK) and Pharmacodynamics (PD) parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following repeated administration. Results: In vitro Pegmolesatide demonstrated enhanced binding stability and prolonged residency at EPOR, surpassing erythropoiesis-stimulating agents (ESAs) rHuEPO and Darbepoetin. This sustained EPOR binding facilitated heightened endogenous EPOR expression post-drug withdrawal, maintaining downstream signal transduction pathways (JAK2/STAT5, ERK1/2 MAPK) for erythropoiesis. Pegmolesatide promoted UT-7 cell proliferation & survival and suppressed apoptosis. Following a single 0.08 mg/kg dose of Pegmolesatide in BALB/c mice, reticulocyte count, red blood cells, hemoglobin, and hematocrit persisted at elevated levels 4-6 days after administration. In the two clinical Phase II studies dose-dependent increases in hemoglobin and prolonged response duration were independently observed. Pegmolesatide showed significant PK-PD dual prolongation effects and was well tolerated. Adverse events were mild and manageable, with no reports of severe anaphylaxis. Discussion: Preclinical and clinical evidence signifies that Pegmolesatide is a unique, potent PEGylated EPO-memetic peptide (EMP) with a prolonged PD efficacy and PK half-life and a good safety-tolerability profile. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play. Trial registration: Phase IIa clinical study of Pegmolesatide on renal anemia, CTR20140533. Initial Public Notice 14 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. Phase II clinical study of Pegmolesatide on renal anemia, CTR20140539. Initial Public Notice 26 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. [ABSTRACT FROM AUTHOR]

Extracellular membrane vesicles (EVs) offer promising values in various medical fields, e.g., as biomarkers in liquid biopsies or as native (or bioengineered) biological nanocarriers in tissue engineering, regenerative medicine and cancer therapy. Based on their cellular origin EVs can vary considerably in composition and diameter. Cell biological studies on mammalian prominin-1, a cholesterol-binding membrane glycoprotein, have helped to reveal new donor membranes as sources of EVs. For instance, small EVs can originate from microvilli and primary cilia, while large EVs might be produced by transient structures such as retracting cellular extremities of cancer cells during the mitotic rounding process, and the midbody at the end of cytokinesis. Here, we will highlight the various subcellular origins of prominin-1+ EVs, also called prominosomes, and the potential mechanism(s) regulating their formation. We will further discuss the molecular and cellular characteristics of prominin-1, notably those that have a direct effect on the release of prominin-1+ EVs, a process that might be directly implicated in donor cell reprogramming of stem and cancer stem cells. Prominin-1+ EVs also mediate intercellular communication during embryonic development and adult homeostasis in healthy individuals, while disseminating biological information during diseases. [ABSTRACT FROM AUTHOR]

Extracellular vesicles, or exosomes, are produced by every type of cell and contain metabolites, proteins, lipids, and nucleic acids. Their role in health and disease is to influence different aspects of cell biology and to act as intermediaries between cells. Follicular fluid exosomes or extracellular vesicles (FF-EVs) secreted by ovarian granulosa cells are critical mediators of ovary growth and maturation. The movement and proteins of these exosomes are crucial in the regulation of cellular communication and the aging of cells, a process termed inflammaging. Menopause, a natural progression in the aging of females, is often accompanied by numerous negative symptoms and health issues. It can also act as a precursor to more severe health problems, including neurological, cardiovascular, and metabolic diseases, as well as gynecological cancers. Researchers have discovered pathways that reveal the diverse effects of exosome-driven cellular communication and oocyte development in the follicular fluid. It also explores the complex functions of FF exosomal proteins in the pathologies associated with menopause. [ABSTRACT FROM AUTHOR]

TGFβ family ligands are synthesized as precursors consisting of an N-terminal prodomain and C-terminal growth factor (GF) signaling domain. After proteolytic processing, the prodomain typically remains noncovalently associated with the GF, sometimes forming a high-affinity latent procomplex that requires activation. For the TGFβ family ligand anti-Müllerian hormone (AMH), the prodomain maintains a high-affinity interaction with its GF that does not render it latent. While the prodomain can be displaced by the type II receptor, AMHR2, the nature of the GF:prodomain interaction and the mechanism of prodomain displacement by AMHR2 are currently unknown. We show here that the AMH prodomain exhibits an atypical two-domain structure, containing a dimerizing and a GF-binding domain connected through a flexible linker. Cryo-EM and genomic analyses show that the distinctive GF-binding domain, the result of an exon insertion 450 Mya, comprises a helical bundle and a belt-like structure which interact with the GF at the type II and I receptor binding sites, respectively. The dimerizing domain, which adopts a TGFβ-like propeptide fold, covalently connects two prodomains through intermolecular disulfide bonds. Disease mutations map to both the GF-binding and dimerization domains. Our results support a model where AMHR2 displaces the helical bundle and induces a conformational change in the GF, followed by release of the prodomain and engagement of the type I receptor. Collectively, this study shows that the AMH prodomain has evolved an atypical binding interaction with the GF that favors, without disrupting signaling, the maintenance of a noncovalent complex until receptors are engaged. [ABSTRACT FROM AUTHOR]

The Neural Crest cells are multipotent progenitor cells formed at the neural plate border that differentiate and give rise to a wide range of cell types and organs. Directional migration of NC cells and their correct positioning at target sites are essential during embryonic development, and defects in these processes results in congenital diseases. The NC migration begins with the epithelial-mesenchymal transition and extracellular matrix remodeling. The main cellular mechanisms that sustain this migration include contact inhibition of locomotion, co-attraction, chemotaxis and mechanical cues from the surrounding environment, all regulated by proteins that orchestrate cell polarity and motility. In this review we highlight the molecular mechanisms involved in neural crest cell migration and polarity, focusing on the role of small GTPases, Heterotrimeric G proteins and planar cell polarity complex. Here, we also discuss different congenital diseases caused by altered NC cell migration. [ABSTRACT FROM AUTHOR]

CXXC type zinc finger protein 5 (CXXC5) is a member of the ZF-CXXC family and plays a pivotal role in signal integration and information transfer within cell signaling network. CXXC5 acts as a regulator in various physiological processes, and abnormalities in its protein structure or function have been linked to multiple pathological processes. In this article, we correspondingly describe the composition of the ZF-CXXC family, emphatically introducing the features of the CXXC5 gene and protein, review the role of CXXC5 in cellular signaling networks, the physiological and pathological processes associated with CXXC5 dysregulation, and particularly focus on the correlation between CXXC5 and cancers. Finally, we summarize the current therapies targeting CXXC5 and their potential applications, and discuss the intriguing findings from current studies, and the opportunities and challenges in future. [ABSTRACT FROM AUTHOR]

S6 Kinase 2 (S6K2) is a key regulator of cellular signaling and is crucial for cell growth, proliferation, and survival. This review is divided into two parts: the first focuses on the complex network of upstream effectors, downstream modulators, and post-translational modifications (PTMs) that regulate S6K2 activity. We emphasize the dynamic nature of S6K2 regulation, highlighting its critical role in cellular homeostasis and its potential as a therapeutic target in diseases like cancer. The second part utilizes in silico analyses, employing computational tools to model S6K2's three-dimensional structure and predict its interaction networks. Molecular dynamics simulations and docking studies reveal potential binding sites and interactions with novel known inhibitors. We also examine the effects of environmental contaminants that potentially disrupt S6K2 function and provide insights into the role of external factors that could impact its regulatory mechanisms. These computational findings provide a deeper understanding of the conformational dynamics of S6K2 and its interactions with its inhibitors. Together, this integrated biochemical and computational approach enhances our understanding of S6K2 regulation and identifies potential new therapeutic strategies targeting S6K2 in the oncology setting. [ABSTRACT FROM AUTHOR]

Simple Summary: In animals, the proteins encoded by genes of the tumor necrosis factor superfamily (TNFSF) play crucial roles in cell signaling. They regulate cell differentiation, survival, and programmed cell death in multiple organs and tissues, with particularly significant functions in the immune system. This study characterizes the TNFSF genes present in all major vertebrate lineages and proposes a model for the evolution of this superfamily since the emergence of vertebrates. The model can serve as a tool to interpret functional data and to generate new hypotheses regarding the roles of TNFSF genes in multiple species. This study characterizes the evolution of the tumor necrosis factor superfamily (TNFSF) across vertebrate lineages, both cyclostomes and gnathostomes, by combining sequence similarity and synteny data for the genes from 23 model species. The available evidence supports a simple model in which most of the diversity found in living species can be attributed to the expansion of four genes found in an ancestor of all vertebrates before the first of the genome duplications that occurred in the vertebrate lineages. It is inferred that the ancestor of all cyclostomes possessed only six TNFSF genes. A cyclostome-specific genome triplication had little effect on the total number of these genes. The ancestor of all gnathostomes, due to the effect of a second genome duplication plus additional single-gene duplications, already had 21 TNFSF genes. In several gnathostome lineages, particularly in some tetrapods, the TNF superfamily has significantly contracted due to numerous gene losses. This evolutionary model provides a framework for exploring functional data, showing that the descendants of different ancestral genes have acquired distinct roles, most prominently in the innate and adaptive immune systems, which led to a species-specific refinement of which TNFSF genes were conserved or lost. Several data hitherto difficult to interpret (the interactions of very different TNFSF ligands with the same receptors; the ability of the same ligands to bind alternative receptors, with or without death domains; and the cooperation of different ligands in specific functions) can be explained as consequences of the evolutionary history of the TNF superfamily. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute members of the dual-specificity family of protein phosphatases that dephosphorylate the MAPKs. MKP-5 dephosphorylates the stress-responsive MAPKs, p38 MAPK and JNK, and has been shown to promote tissue fibrosis. Here, we provide insight into how MKP-5 regulates the transforming growth factor-β (TGF-β) pathway, a well-established driver of fibrosis. We show that MKP-5-deficient fibroblasts in response to TGF-β are impaired in SMAD2 phosphorylation at canonical and non-canonical sites, nuclear translocation, and transcriptional activation of fibrogenic genes. Consistent with this, pharmacological inhibition of MKP-5 is sufficient to block TGF-β signaling, and that this regulation occurs through a JNK-dependent pathway. By utilizing RNA sequencing and transcriptomic analysis, we identify TGF-β signaling activators regulated by MKP-5 in a JNK-dependent manner, providing mechanistic insight into how MKP-5 promotes TGF-β signaling. This study elucidates a novel mechanism whereby MKP-5-mediated JNK inactivation is required for TGF-β signaling and provides insight into the role of MKP-5 in tissue fibrosis. [ABSTRACT FROM AUTHOR]

Immunomechanobiology, the study of how physical forces influence the behavior and function of immune cells, is a rapidly growing area of research. It is becoming increasingly recognized that mechanical stimuli, such as fluid shear forces, are a critical determinant of immune cell regulation. In this review, we discuss the principles and significance of various mechanical forces present within the human body, with a focus on fluid shear flow and its impact on immune cell activation and function. Moreover, we discuss engineering approaches used to study immune cell mechanobiology, and their implications in health and diseases such as cancer, autoimmune disorders, and infectious disease. [ABSTRACT FROM AUTHOR]

Purpose of the Review: Preclinical and clinical evidence support the notion that neurodevelopmental disorders (NDDs) are synaptic disorders, characterized by excitatory-inhibitory imbalance. Despite this, NDD drug development programs targeting glutamate or gamma-aminobutyric acid (GABA) receptors have been largely unsuccessful. Nonetheless, recent drug trials in Rett syndrome (RTT), fragile X syndrome (FXS), and other NDDs targeting other mechanisms have met their endpoints. The purpose of this review is to identify the basis of these successful studies. Recent Findings: Despite increasing evidence of disruption in synaptic homeostasis, most genetic variants associated with NDDs implicate proteins involved in cell regulation and not in neurotransmission. Metabolic processes, in particular mitochondrial function, appear to play a role in NDD pathophysiology. NDDs are also characterized by distinctive cell signaling abnormalities, which link cellular and synaptic homeostasis. Recent successful trials in NDDs, including those of trofinetide, the first drug specifically approved for one of these disorders (i.e., RTT), implicate the targeting of downstream processes (i.e., signaling pathways) rather than neurotransmitter receptors. Summary: Recent positive drug studies in NDDs and their underlying mechanisms, in conjunction with new knowledge on the pathophysiology of these disorders, support the concept that targeting signaling and cellular and synaptic homeostasis may be a preferred approach for ameliorating synaptic abnormalities in many NDDs. [ABSTRACT FROM AUTHOR]

ABSTRACT αβT cells protect vertebrates against many diseases, optimizing surveillance using mechanical force to distinguish between pathophysiologic cellular alterations and normal self‐constituents. The multi‐subunit αβT‐cell receptor (TCR) operates outside of thermal equilibrium, harvesting energy via physical forces generated by T‐cell motility and actin‐myosin machinery. When a peptide‐bound major histocompatibility complex molecule (pMHC) on an antigen presenting cell is ligated, the αβTCR on the T cell leverages force to form a catch bond, prolonging bond lifetime, and enhancing antigen discrimination. Under load, the αβTCR undergoes reversible structural transitions involving partial unfolding of its clonotypic immunoglobulin‐like (Ig) domains and coupled rearrangements of associated CD3 subunits and structural elements. We postulate that transitions provide critical energy to initiate the signaling cascade via induction of αβTCR quaternary structural rearrangements, associated membrane perturbations, exposure of CD3 ITAMs to phosphorylation by non‐receptor tyrosine kinases, and phase separation of signaling molecules. Understanding force‐mediated signaling by the αβTCR clarifies long‐standing questions regarding αβTCR antigen recognition, specificity and affinity, providing a basis for continued investigation. Future directions include examining atomistic mechanisms of αβTCR signal initiation, performance quality, tissue compliance adaptability, and T‐cell memory fate. The mechanotransduction paradigm will foster improved rational design of T‐cell based vaccines, CAR‐Ts, and adoptive therapies. [ABSTRACT FROM AUTHOR]

Combinatorial networks of cell adhesion molecules and cell surface receptors drive fundamental aspects of neural circuit establishment and function. However, the intracellular signals orchestrated by these cell surface complexes remain less understood. Here, we report that the Ga12/13 pathway lies downstream of several GPCRs with critical synaptic functions. Impairment of the Ga12/13 pathway in postnatal hippocampal neurons diminishes inhibitory inputs without altering neuronal morphology or excitatory transmission. Ga12/13 signaling in hippocampal CA1 neurons in vivo selectively regulates PV interneuron synaptic connectivity, supporting an inhibitory synapse subtype-specific function of this pathway. Our studies establish Ga12/13 as a signaling node that shapes inhibitory hippocampal circuitry. [ABSTRACT FROM AUTHOR]

In the realms of bioengineering and biopharmaceuticals, there exists a critical demand for advanced genetic tools that can interact with specific cell signaling pathways to accurately identify and target various cell types. This research introduces the innovative CRISPR‐ADAReader system, which enables precise manipulation of cell activity through sensing target RNA. Featuring both positive and negative feedback loops, the system allows for tailored regulation across different cell types in response to various internal signals, showcasing exceptional programmability, specificity, and sensitivity. By choosing distinct RNAs as activation signals, the CRISPR‐ADAReader efficiently monitors and alters targeted cell behaviors. In a case study focusing on retinoblastoma treatment, the system distinctively initiates positive feedback and self‐silencing actions by detecting MCYN and Rb transcripts, thus safeguarding normal retinal pigment epithelial cells while promoting apoptosis in cancer cells. Moreover, the CRISPR‐ADAReader demonstrates significant anti‐tumor effectiveness in live models, markedly reducing retinoblastoma cell proliferation through the activation of several cancer‐suppression pathways, outperforming the capabilities of the ADAR‐sensor. Notably, the system also shows an excellent in vivo safety profile. In conclusion, the CRISPR‐ADAReader system represents a groundbreaking method for the detection and editing of RNA, offering a potent instrument for the customized and precise governance of cell behavior. [ABSTRACT FROM AUTHOR]

Sevenless (Sev) is a Drosophila receptor tyrosine kinase (RTK) required for the specification of the R7 photoreceptor. It is cleaved into α and β subunits and binds the ectodomain of the G-protein-coupled receptor bride of sevenless (Boss). Previous work showed that the Boss ectodomain could bind but not activate Sev; rather, the whole seven-pass transmembrane Boss was required. Here, we show that Sev does not need to be cleaved to function and that a single-pass transmembrane form of Boss activates Sev. We use cryo-electron microscopy and biophysical methods to determine the structural basis of ligand binding and pH-dependent dimerization of Sev, and we discuss the implications in the process of Sev activation. The Sev human homolog, receptor oncogene from sarcoma 1 (ROS1), is associated with oncogenic transformations, and we discuss their structural similarities. [Display omitted] • Cryo-EM structures of ligand-free and Boss-bound sevenless • Sevenless receptor tyrosine kinase shows pH-dependent dimerization at low pH • A single-pass transmembrane form of Boss activates sevenless • Sevenless and its human homolog ROS1 have similar structures Cerutti et al. explore the biology of the sevenless receptor tyrosine kinase and its ligand, bride of sevenless, that specify the Drosophila R7 photoreceptor. They identify unique pH-dependent dimerization properties of sevenless and structural similarities with its human homolog ROS1, which is associated with oncogenic transformations. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models.We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses.Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect.In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse. [ABSTRACT FROM AUTHOR]