Inhibition Of Hedgehog Signaling Research Articles

Inhibition of Hedgehog Signaling Ameliorates Severity of Chronic Pancreatitis in Experimental Mouse Models.

Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas with no specific cure. Research highlighting the pathogenesis and especially the therapeutic aspect remains limited. Aberrant activation of developmental pathways in adults have been implicated in several diseases. Hedgehog pathway is a notable embryonic signaling pathway, known to promote fibrosis of various organs when over-activated. The aim of this study is to explore the role of hedgehog pathway in the progression of CP and evaluate its inhibition as a novel therapeutic strategy against CP. CP was induced in mice by repeated injections of L-arginine or Caerulein in two separate models. Mice were administered with the FDA approved pharmacological hedgehog pathway inhibitor, Vismodegib during or after establishing the disease condition to inhibit hedgehog signaling. Various parameters of CP were analyzed to determine the effect of hedgehog pathway inhibition on the severity and progression of the disease. Our study shows that hedgehog signaling was over-activated during CP and its inhibition was effective in improving the histopathological parameters associated with CP. Vismodegib administration not only halted the progression of CP but was also able to resolve already established fibrosis. Additionally, inhibition of hedgehog signaling resulted in reversal of pancreatic stellate cell activation ex vivo. Conclusions: Findings from our study justify conducting clinical trials using Vismodegib against CP and thus, could lead to development of novel therapeutic strategy for the treatment of CP.

The Nociceptor Primary Cilium Contributes to Mechanical Nociceptive Threshold and Inflammatory and Neuropathic Pain.

The primary cilium, a single microtubule-based organelle protruding from the cell surface and critical for neural development, also functions in adult neurons. While some dorsal root ganglion neurons elaborate a primary cilium, whether it is expressed by and functional in nociceptors is unknown. Recent studies have shown a role of Hedgehog, whose canonical signaling is primary cilium dependent, in nociceptor sensitization. We establish the presence of primary cilia in soma of rat nociceptors, where they contribute to mechanical threshold, prostaglandin E2 (PGE2)-induced hyperalgesia, and chemotherapy-induced neuropathic pain (CIPN). Intrathecal administration of siRNA targeting Ift88, a primary cilium-specific intra-flagellar transport (IFT) protein required for ciliary integrity, resulted in attenuation of Ift88 mRNA and nociceptor primary cilia. Attenuation of primary cilia was associated with an increase in mechanical nociceptive threshold in vivo, and decrease in nociceptor excitability in vitro, abrogation of hyperalgesia, and nociceptor sensitization induced by both a prototypical pronociceptive inflammatory mediator PGE2 and paclitaxel CIPN, in a sex-specific fashion. siRNA targeting Ift52, another IFT protein, and knockdown of NompB, the Drosophila Ift88 ortholog, also abrogated CIPN and reduced baseline mechanosensitivity, respectively, providing independent confirmation for primary cilia control of nociceptor function. Hedgehog-induced hyperalgesia is attenuated by Ift88 siRNA, supporting a role for primary cilia in Hedgehog-induced hyperalgesia. Attenuation of CIPN by cyclopamine (intradermal and intra-ganglion), which inhibits Hedgehog signaling, supports a role of Hedgehog in CIPN. Our findings support a role of the nociceptor primary cilium in control of mechanical nociceptive threshold and inflammatory and neuropathic pain, the latter Hedgehog-dependent.Significance statement Many neurons have a tiny antenna-like structure, the primary cilium, protruding from their cell body, which processes information about the extracellular environment as well as regulates intracellular signaling. We report experiments aimed at understanding the role of the primary cilium in pain sensory neurons (nociceptors), including in the setting of inflammatory and neuropathic pain. We establish that nociceptors bear a primary cilium and show that this organelle regulates detection of noxious stimuli and contributes to nociceptor sensitization, using both the rat and the fruit fly as model organisms to manipulate primary cilia in pain states. We also identify primary cilium dependence of the contribution of Hedgehog to pain states.

The senescence-associated secretome of Hedgehog-deficient hepatocytes drives MASLD progression.

The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA-Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid-restricted high-fat diet (CDA-HFD) to induce MASLD, Smo- mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo- mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo- hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD-fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence.

Pharmacologic Hedgehog inhibition modulates the cytokine profile of osteolytic breast cancer cells

The establishment and progression of bone metastatic breast cancer is supported by immunosuppressive myeloid populations that enable tumor growth by dampening the innate and adaptive immune response. Much work remains to understand how to target these tumor-myeloid interactions to improve treatment outcomes. Noncanonical Hedgehog signaling is an essential component of bone metastatic tumor progression, and prior literature suggests a potential role for Hedgehog signaling and its downstream effector Gli2 in modulating immune responses. In this work, we sought to identify if inhibition of noncanonical Hedgehog signaling alters the cytokine profile of osteolytic breast cancer cells and the subsequent communication between the tumor cells and myeloid cells. Examination of large patient databases revealed significant relationships between Gli2 expression and expression of markers of myeloid maturation and activation as well as cytokine expression. We found that treatment with HPI-1 reduced tumor cell expression of numerous cytokine genes, including CSF1, CSF2, and CSF3, as well as CCL2 and IL6. Secreted CSF-1 (M−CSF) was also reduced by treatment. Changes in tumor-secreted factors resulted in polarization of THP-1 monocytes toward a proinflammatory phenotype, characterized by increased CD14 and CD40 surface marker expression. We therefore propose M−CSF as a novel target of Hedgehog inhibition with potential future applications in altering the immune microenvironment in addition to its known roles in reducing tumor-induced bone disease.

Blocking smoothened cholesterylation: A strategy for overcoming drug resistance in cancer

In this issue of Cell Chemical Biology, Liu etal.1 report the identification of Q29, a synthetic diterpenoid that blocks covalent cholesterol modification of smoothened (SMO) and inhibits hedgehog signaling. Q29 is capable of suppressing tumor cell growth, both invitro and invivo, and overcoming resistance to SMO inhibitors.

IC Regimen: Delaying Resistance to Lorlatinib in ALK Driven Cancers by Adding Repurposed Itraconazole and Cilostazol.

Lorlatinib is a pharmaceutical ALK kinase inhibitor used to treat ALK driven non-small cell lung cancers. This paper analyses the intersection of past published data on the physiological consequences of two unrelated drugs from general medical practice-itraconazole and cilostazol-with the pathophysiology of ALK positive non-small cell lung cancer. A conclusion from that data analysis is that adding itraconazole and cilostazol may make lorlatinib more effective. Itraconazole, although marketed worldwide as a generic antifungal drug, also inhibits Hedgehog signaling, Wnt signaling, hepatic CYP3A4, and the p-gp efflux pump. Cilostazol, marketed worldwide as a generic thrombosis preventative drug, acts by inhibiting phosphodiesterase 3, and, by so doing, lowers platelets' adhesion, thereby partially depriving malignant cells of the many tumor trophic growth factors supplied by platelets. Itraconazole may enhance lorlatinib effectiveness by (i) reducing or stopping a Hedgehog-ALK amplifying feedback loop, by (ii) increasing lorlatinib's brain levels by p-gp inhibition, and by (iii) inhibiting growth drive from Wnt signaling. Cilostazol, surprisingly, carries minimal bleeding risk, lower than that of aspirin. Risk/benefit assessment of the combination of metastatic ALK positive lung cancer being a low-survival disease with the predicted safety of itraconazole-cilostazol augmentation of lorlatinib favors a trial of this drug trio in ALK positive lung cancer.

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway

BackgroundTrans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown.Methods4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling.ResultsDistraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force.ConclusionsOur study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

Molecular Profiling and the Interaction of Somatic Mutations with Transcriptomic Profiles in Non-Melanoma Skin Cancer (NMSC) in a Population Exposed to Arsenic

Exposure to inorganic arsenic (As) is recognized as a risk factor for non-melanoma skin cancer (NMSC). We followed up with 7000 adults for 6 years who were exposed to As. During follow-up, 2.2% of the males and 1.3% of the females developed basal cell carcinoma (BCC), while 0.4% of the male and 0.2% of the female participants developed squamous cell carcinoma (SCC). Using a panel of more than 400 cancer-related genes, we detected somatic mutations (SMs) in the first 32 NMSC samples (BCC = 26 and SCC = 6) by comparing paired (tissue–blood) samples from the same individual and then comparing them to the SM in healthy skin tissue from 16 participants. We identified (a) a list of NMSC-associated SMs, (b) SMs present in both NMSC and healthy skin, and (c) SMs found only in healthy skin. We also demonstrate that the presence of non-synonymous SMs in the top mutated genes (like PTCH1, NOTCH1, SYNE1, PKHD1 in BCC and TP53 in SCC) significantly affects the magnitude of differential expressions of major genes and gene pathways (basal cell carcinoma pathways, NOTCH signaling, IL-17 signaling, p53 signaling, Wnt signaling pathway). These findings may help select groups of patients for targeted therapy, like hedgehog signaling inhibitors, IL17 inhibitors, etc., in the future.

P-EKKE Alleviates Myocardial Infarction (MI) in MI Rats by Inhibiting Hedgehog Signaling Pathway-mediated Inflammation and Inhibiting α-actin Mediated Myocardial Fibrosis

Background: Myocardial infarction (MI) is an ischemic heart disorder that causes apoptosis or necrosis of myocardial cells. Objective: The study aimed to evaluate the effect of P-EKKE on myocardial infarction and explore the associated mechanisms in MI rats. Methods: The MI in rats was established by ligating the left coronary artery of rats; the rats were divided into the MI group (without treatment) and the P-EKKE group (treated with P-EKKE). Normal rats were assigned to the NC group (without treatment) and the sham group (under LAD without ligation). Cardiac function was evaluated using echocardiography. The MI area was measured with TTC staining. Histological analysis was performed to evaluate inflammation (HE staining) and myocardial fibrosis (Masson and immunofluorescence staining). RT-PCR and Western blotting were used to determine Gli-1/SHH expression in myocardial tissues. Results: P-EKKE clearly improved the cardiac function of MI rats. The area of myocardial infarction in MI rats undergoing P-EKKE treatment (P-EKKE group) was found to be predominantly decreased compared to MI rats without treatment (p < 0.05). P-EKKE treatment clearly inhibited apoptosis and increased H3S10ph expression in the area of myocardial infarction of MI rats compared to MI rats without treatment (p < 0.05). P-EKKE treatment significantly alleviated inflammation and decreased myocardial fibrosis in the area of myocardial infarction in MI rats compared to MI rats without treatment (p < 0.05). P-EKKE significantly increased the expression of Gil-1 and SHH in myocardial infarction of MI rats compared to MI rats without treatment (p < 0.05). Conclusion: P-EKKE inhibited myocardial infarction and played an anti-inflammatory and myocardial protective role in MI rats. P-EKKE inhibited myocardial inflammation by activating the hedgehog signaling pathway and inhibited myocardial fibrosis by decreasing α-actin expression.

Abstract 3586: A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors

Abstract Background: Neuroendocrine tumors (NETs) represent heterogenous malignancies whose origins and etiology remain poorly understood. Men1-driven reprogramming of neural crest-derived glial cells was recently implicated in NET development. In these studies, aberrant Sonic hedgehog (SHH) pathway activation known to pattern neural cell fate coincided with the neuroendocrine phenotype in mice. Here, we investigated the hypothesis that loss of menin encoded by the MEN1 gene drives SHH-dependent oncogenic reprogramming of NETs by modulating the neural crest cell fate. Methods: Glial cell specific Men1 deletion was accomplished by expressing Cre recombinase downstream of the human glial fibrillary acidic protein promoter (GFAPΔMen1). Hedgehog (HH) activation of Men1-deficient glial cells was blocked by deleting the gene encoding primary ciliary protein Kif3a required for transducing SHH signaling. The resulting GFAPΔMen1 mice were evaluated for NET development and dysregulated hormone profiles. To identify aberrant SHH activation leading to neuroendocrine reprogramming, we performed single cell RNA- and Assay for Transposase Accessible Chromatin sequencing on GFAPΔMen1 NETs and primary glial cultures. Hyperactivation of SHH in human gastroenteropancreatic (GEP)-NETs was confirmed by immunofluorescent staining and western blot. Lastly, primary mouse and human NET organoids were treated with an agonist and inhibitors of HH signaling and evaluated for ERK/AKT activation, proliferation, and transcript fluctuations indicative of neural crest cell reprogramming. Results: GFAPΔMen1 mice developed NETs in the pancreas, pituitary, and small intestine. Astonishingly, impaired SHH activation in GFAP+/Men1−/− cells abolished the development of NETs and restored hormone levels to that of wild type mice. GFAPΔMen1 NETs and glial cultures showed increased SHH signaling and chromatin accessibility of HH pathway genes, whereas additive Kif3a deletion blocked HH activation. Moreover, menin negative glial cells demonstrated a phenotypic shift in neuroglial lineages that suggested SHH-mediated reprogramming of neural crest-derived cells following the loss of menin. Consistent with hyperactive SHH activation in the GFAPΔMen1 mice, human GEP-NETs overexpressed SHH and downstream signaling targets. Functionally, SHH induced ERK/AKT activation, cell proliferation, and neuroendocrine transcript expression in human and mouse NET organoids, whereas pharmacological inhibition of HH signaling reversed these effects. Conclusions: Our observations implicate neural crest-derived glial cells as potential neuroendocrine cell precursors that are susceptible to transformation through increased HH signaling. These studies warrant future investigation into the delivery of Hedgehog inhibitors in the adjuvant setting for NET treatment. Citation Format: Suzann Duan, Ricky A. Sontz, Martin Deymier, Juanita L. Merchant. A Hedgehog-dependent signaling axis drives neural plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3586.

Synthesis and molecular docking simulations of novel azepines based on quinazolinone moiety as prospective antimicrobial and antitumor hedgehog signaling inhibitors

A series of novel azepine derivatives based on quinazolinone moiety was synthesized through the reaction of quinazolinone chalcones (2a–d) either with 2-amino aniline in acidic medium to give diazepines (3a–d) or with 2-aminophenol to offer oxazepine (4a–d). The structure of the synthesized compounds was confirmed via melting points, elemental analyses, and different spectroscopic techniques. Moreover, these newly compounds mode of action was investigated in-silico using molecular docking against the outer membrane protein A (OMPA), exo-1,3-beta-glucanase for their antimicrobial activity, and against Smoothened (SMO), transcription factor glioma-associated homology (SUFU/GLI-1), the main proteins of Hedgehog signaling pathway to inspect their anticancer potential. Our results showed that, diazepine (3a) and oxazepine (4a) offered the highest binding energy against the target OMPA/ exo-1,3-beta-glucanase proteins and exhibited the potent antimicrobial activities against E. coli, P. aeruginosa, S. aureus, B. subtilis, C. Albicans and A. flavus. As well, diazepine (3a) and oxazepine (4a) achieved the best results among the other compounds, in their binding energy against the target SMO, SUFU/GLI-1 proteins. The in-vitro cytotoxic study was done for them on panel of cancer cell lines HCT-116, HepG2, and MCF-7 and normal cell line WI-38. Conclusively, it was revealed that molecular docking in-silico simulations and the in-vitro experiments were agreed. As a result, our findings elucidated that diazepine (3a) and oxazepine (4a), have the potential to be used as antimicrobial agents and as possible cancer treatment medications.

Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms

Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.

Pharmacology of Veratrum californicum Alkaloids as Hedgehog Pathway Antagonists.

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each.

Vicious Cycle-Breaking Lipid Nanoparticles Remodeling Multicellular Crosstalk to Reverse Liver Fibrosis.

During liver fibrogenesis, the reciprocal crosstalk among capillarized liver sinusoidal endothelial cells (LSECs), activated hepatic stellate cells (HSCs), and dysfunctional hepatocytes constructs a self-amplifying vicious cycle, greatly exacerbating the disease condition and weakening therapeutic effect. Limited by the malignant cellular interactions, the previous single-cell centric treatment approaches show unsatisfactory efficacy and fail to meet clinical demand. Herein, a vicious cycle-breaking strategy is proposed to target and repair pathological cells separately to terminate the malignant progression of liver fibrosis. Chondroitin sulfate-modified and vismodegib-loaded nanoparticles (CS-NPs/VDG) are designed to efficiently normalize the fenestrae phenotype of LSECs and restore HSCs to quiescent state by inhibiting Hedgehog signaling pathway. In addition, glycyrrhetinic acid-modified and silybin-loaded nanoparticles (GA-NPs/SIB) are prepared to restore hepatocytes function by relieving oxidative stress. The results show successful interruption of vicious cycle as well as distinct fibrosis resolution in two animal models through multiregulation of the pathological cells. This work not only highlights the significance of modulating cellular crosstalk but also provides a promising avenue for developing antifibrotic regimens.

Current Status of Hedgehog Signaling Inhibitors.

The Hedgehog (Hh) signaling pathway plays a crucial role in diverse biological processes such as cell differentiation, proliferation, senescence, tumorigenesis, malignant transformation, and drug resistance. Aberrant Hh signaling, resulting from mutations and excessive activation, can contribute to the development of various diseases during different stages of biogenesis and development. Moreover, it has been linked to unfavorable outcomes in several human cancers, including basal cell carcinoma (BCC), multiple myeloma (MM), melanoma, and breast cancer. Hence, the presence of mutations and excessive activation of the Hh pathway presents obstacles and constraints in the realm of cancer treatment. Extant research has demonstrated that small molecule inhibitors are regarded as the most effective therapeutic approaches for targeting the Hh pathway in contrast to traditional chemotherapy and radiotherapy. Consequently, this review focuses on the present repertoire of small molecule inhibitors that target various components of the Hh pathway, including Hh ligands, Ptch receptors, Smo transmembrane proteins, and Gli nuclear transcription factors. This study provides a comprehensive analysis of small molecules' structural and functional aspects in the preclinical and clinical management of cancer. Additionally, it elucidates the obstacles encountered in targeting the Hh pathway for human cancer therapy and proposes potential therapeutic approaches.

Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG invivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

The deubiquitinating enzyme USP44 suppresses hepatocellular carcinoma progression by inhibiting Hedgehog signaling and PDL1 expression

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. Research into the key genes that maintain the malignant behavior of cancer cells is crucial for the treatment of HCC. Here, we identified ubiquitin‐specific peptidase 44 (USP44), a member of the deubiquitinase family, as a novel regulator of HCC progression. The tumor suppressive function of USP44 was evaluated in a series of in vitro and in vivo experiments. Through quantitative proteomics examination, we demonstrated that USP44 inhibits HCC PDL1 expression by downregulating the Hedgehog (Hh) signaling pathway. Mechanistically, we found that USP44 directly interacts with Itch, an E3 ligase involved in Hh signaling, and promotes the deubiquitination and stabilization of Itch. These events result in the proteasomal degradation of Gli1 and subsequent inactivation of Hh signaling, which ultimately suppresses PDL1 expression and the progression of HCC. Furthermore, the HCC tissue microarray was analyzed by immunohistochemistry to evaluate the pathological relevance of the USP44/Itch/Gli1/PDL1 axis. Finally, the Gli1 inhibitor GANT61 was found to act in synergy with anti-PDL1 therapy. Overall, USP44 can act as a suppressive gene in HCC by modulating Hh signaling, and co-inhibition of Gli1 and PDL1 might be an effective novel combination strategy for treating HCC patients.

The HEDGEHOG-GLI1 pathway is important for fibroproliferative properties in keloids and as a candidate therapeutic target

Keloids are benign fibroproliferative skin tumors caused by aberrant wound healing that can negatively impact patient quality of life. The lack of animal models has limited research on pathogenesis or developing effective treatments, and the etiology of keloids remains unknown. Here, we found that the characteristics of stem-like cells from keloid lesions and the surrounding dermis differ from those of normal skin. Furthermore, the HEDGEHOG (HH) signal and its downstream transcription factor GLI1 were upregulated in keloid patient–derived stem-like cells. Inhibition of the HH-GLI1 pathway reduced the expression of genes involved in keloids and fibrosis-inducing cytokines, including osteopontin. Moreover, the HH signal inhibitor vismodegib reduced keloid reconstituted tumor size and keloid-related gene expression in nude mice and the collagen bundle and expression of cytokines characteristic for keloids in ex vivo culture of keloid tissues. These results implicate the HH-GLI1 pathway in keloid pathogenesis and suggest therapeutic targets of keloids.

The Challenge of Treating Relapsed Myeloid Leukemia in Children with Down Syndrome - a Targeted Analysis Using Patient-Derived Xenograft Models

Background. Myeloid leukemia occurs with a 150-fold increased incidence in children with Down syndrome (ML-DS) and is characterized by distinct disease mechanism and response to treatment. In the context of trisomy 21, somatic GATA1 mutations first initiate preleukemic transient abnormal myelopoiesis, a disorder of fetal liver hematopoiesis. Co-operating events, which target cohesin complex components, epigenetic modifiers or signal transducers including RAS pathway genes (Yoshida 2013, Labuhn M 2019), then drive transformation to ML-DS in 20% of infants with TAM, typically by age 4 years. Abnormal sonic hedgehog signaling was recently associated with trisomy 21 (Galati 2019). Its role in the development of ML-DS is unknown. Hypersensitivity of ML-DS blasts to chemotherapy with cytarabine and other agents accounts at least in part for the more favorable response of ML-DS compared to acute myeloid leukemia of children without DS. Contemporary treatment protocols for ML-DS achieve event-free and overall survival of approx. 89% and 90% at 5 years (Taub 2017). In marked contrast, patients who develop a relapse of ML-DS face a significantly lower probability of survival (EFS 20.9% and OS 22% at 3 years, Raghuram 2023), compared to children with relapsed AML who do not have DS, and appear to be resistant against conventional chemotherapy. Since the few survivors with a relapsed ML-DS are almost exclusively found among patients who first achieve a second remission and then undergo hematopoietic stem cell transplantation, successful identification of agents with efficacy against the blasts of relapsed ML-DS is essential for survival. We tested if blasts of relapsed ML-DS had lost their sensitivity to cytarabine and whether new drugs could be identified that target pathways active in the blasts of ML-DS. Patients, Materials and Methods . We used patient-derived xenografts (PDX) of human ML-DS and relapsed ML-DS blasts in immunodeficient mice (NSG and NSG-W41) to determine in vivo responses to standard chemotherapeutic agents (cytarabine, vincristine) and novel approaches such as demethylating agents (azacytidine), inhibition of histone deacetylation (panobinostat), mTOR (rapamycin), PARP (olaparib), and sonic hedgehog signaling (glasdegib) alone or in combination (glasdegib + cytarabine; panobinostast + azacytidine). Injection of leukemic cells was performed intrafemorally. Endpoint was inhibition of leukemic engraftment compared to control recipients receiving vehicle only. Results.Responses of primary and relapsed ML-DS samples to drugs tested in PDX models showed marked heterogeneity. While some ML-DS samples showed resistance to cytarabine at the time of relapse this was not the universal cause of failure of primary treatment of ML-DS and some blasts of relapsed ML-DS retained sensitivity to cytarabine. Some primary and relapsed ML-DS blasts showed unexpected sensitivity to vincristine, a drug more commonly used to treat acute lymphoblastic leukemia. Despite intriguing rationales we observed no responses of primary and relapsed ML-DS blasts to the PARP inhibitor olaparib, mTOR inhibition by rapamycin and sonic hedgehog inhibition by glasdegib. Conclusions. Loss of hypersensitivity to cytarabine was not universal at the time of ML-DS relapse. Some agents not typically used to treat AML, such as vincristine, showed unexpected efficacy whereas others lacked responses despite a plausible mechanistic rationale. Patient-specific molecular mechanisms underlying relapsed ML-DS are likely to have an impact on drug sensitivity. They should be determined for all patients with relapsed ML-DS to assist identification of targets and selection of drugs in order to establish urgently needed but currently still lacking efficacious treatment for relapsed ML-DS.

Inhibition of Hedgehog signaling ameliorates foam cell formation by promoting autophagy in early atherosclerosis

Macrophages are the origin of most foam cells in the early stage of atherosclerotic plaques. However, the mechanism involved in the formation of macrophage-derived foam cell formation remains unclear. Here, we revealed that the hedgehog (Hh) signaling is critical in autophagy-lysosome pathway regulation and macrophage-derived foam cell formation. Inhibition of Hh signaling by vismodegib ameliorated lipid deposition and oxidative stress level in atherosclerotic plaques in high-fat diet-fed apoE−/− mice. For mechanistic study, how the Hh signaling modulate the process of foam cell formation were accessed afterward. Unexpectedly, we found that suppression of Hh signaling in apoE−/− mice had no significant impact on circulating cholesterol levels, indicating that Hh pathway modulate the procession of atherosclerotic plaque not through a traditional lipid-lowing mechanism. Instead, vismodegib was found to accelerate autophagosomes maturation as well as cholesterol efflux in macrophage-derived foam cell and in turn improve foam cell formation, while autophagy inhibitors (LY294002 or CQ) administration significantly attenuated vismodegib-induced cholesterol efflux and reversed the effect on foam cell formation. Therefore, our result demonstrated that inhibition of the Hh signaling pathway increases cholesterol efflux and ameliorates macrophage-derived foam cell formation by promoting autophagy in vitro. Our data thus suggested a novel therapeutic target of atherosclerosis and indicated the potential of vismodegib to treat atherosclerosis.