Activation Of JAK-STAT Pathway Research Articles

Extracellular nicotinamide phosphoribosyltransferase visfatin activates JAK2-STAT3 pathway in cancer-associated fibroblasts to promote colorectal cancer metastasis.

Metastasis is one of the major challenges in the treatment of colorectal cancer (CRC), during which cancer-associated fibroblasts (CAFs) in the tumor microenvironment are critically involved. In this study, we aim to explore the regulatory role of extracellular nicotinamide phosphoribosyltransferase Visfatin and its impact on CRC metastasis. To examine the effect of visfatin on CAFs, human CRC tissue-derived CAFs were exposed to visfatin, and the expression of inflammatory factors, activation of JAK-STAT pathway and production of ROS in CAFs were assessed. To examine the effect of visfatin-treated CAFs on CRC metastasis, human CRC cell line SW480 or SW620 were cultured with the conditioned medium derived from visfatin-treated CAFs, and the invasion and migration ability of SW480 or SW620 cells were evaluated by transwell migration and matrigel invasion assays. Our previous study found that visfatin, a secreted form of nicotinamide phosphoribosyltransferase that governs the rate-limiting step of NAD synthesis, promoted CRC metastasis. However, little is known about the effect of visfatin on CAFs. The conditioned medium derived from visfatin- treated CAFs promotes the migratory and invasive capability of CRC cells, and enhance lung metastasis in mouse model. Visfatin treatment stimulated the expression of a couple of inflammatory factors in CAFs, which was mediated by visfatin-induced activation of JAK- STAT pathway and accumulation of ROS. Inhibition of JAK-STAT pathway or neutralization of cellular ROS attenuated visfatin-mediated migration and invasion of CRC cells. The present work highlights a critical role of visfatin in the crosstalk between CRC cells and CAFs, which moonlight as a non-metabolic extracellular signal molecule to hijacks JAK-STAT pathway in CAFs to promote CRC metastasis.

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse post-translational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of a complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple co-localized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Transcriptomics analysis of differential gene expression and immune and inflammatory response mechanisms in patients with typical and non-criteria obstetric antiphospholipid syndrome (OAPS and NC-OAPS)

In this study, we investigated the molecular differences between patients with typical obstetric antiphospholipid syndrome (OAPS) and patients with non-criteria obstetric antiphospholipid syndrome (NC-OAPS) patients through transcriptome sequencing of peripheral blood samples from ten OAPS patients and ten NC-OAPS patients. Differentially expressed genes (DEGs) were identified, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, protein-protein interaction (PPI) analysis, and competitive endogenous RNA (ceRNA) network construction to identify hub genes. Verification was performed via Quantitative Real-time PCR (qPCR) in OAPS (n=9) and NC-OAPS (n=12) samples. We identified 240 DEGs in two groups. GO and KEGG analyses reviewed upregulated in pathways related to the inflammatory response; immune response; antigen processing and presentation; Th1, Th2, and Th17 cell differentiation; and NK cell-mediated cytotoxicity in OAPS patients. PPI and ceRNA network analyses identified key genes, with significant upregulation of CXCR2, JAK2, and MPO found in the OAPS group, which correlated with severe inflammation, JAK-STAT pathway activation, and increased NET activity in neutrophils. Other genes such as CD4, IL2RB, and NKG7, are involved in T-cell and NK cell regulation. Our results indicate enhanced inflammatory and immune responses in OAPS patients, suggesting more severe immune activity than in NC-OAPS patients, providing a basis for precise diagnostic and therapeutic strategies.

Recombinant leptins affect lipid metabolism in hepatocytes of Cynoglossus semilaevis

Leptin is a peptide hormone primarily produced by adipose tissue, which plays a crucial role in regulating energy balance by controlling appetite and energy expenditure. To better understand the intricate physiological functions of leptin in hepatocytes in tongue sole (Cynoglossus semilaevis), this study presents an integrated transcriptomic and metabolomic analysis of tongue sole hepatocytes following stimulation with lepA and lepB. Comparative analysis identified 2971 and 2900 upregulated differentially expressed genes (DEGs) and 1895 and 1821 downregulated DEGs in response to lepA and lepB stimulation, respectively. Notably, 5706 genes were commonly regulated by both stimulations, suggesting overlapping functional roles of these two leptin proteins. GO and KEGG enrichment analysis indicated significant enrichment in immune response (JAK-STAT signaling pathway, NF-κB signaling pathway, etc.) and lipid metabolism pathways, such as fatty acid synthesis, with similar findings for lepB. Metabolomic analysis demonstrated clear separation between treatment and control groups, with 34 medium- to long-chain fatty acids and numerous differentially expressed metabolites (DEMs) identified. KEGG analysis of the metabolome paralleled the transcriptomic findings, with shared pathways in lipid metabolism and immune function. Finally, joint analysis highlighted co-enrichment in linoleic acid metabolism and leishmaniasis pathways. Detailed mechanistic insights revealed the activation of JAK-STAT and NF-κB signaling pathways, modulation of arachidonic acid metabolism, and the influence on the MAPK signaling pathway. Additionally, lepA uniquely co-enriched in the fatty acid synthesis pathway, with specific gene regulation affecting the synthesis of various fatty acids. The study concluded that lepA and lepB exerted significant regulatory effects on lipid metabolism and immune responses in tongue sole hepatocytes through complex molecular networks. To our current knowledge, this represents the first direct evidence of leptin's role in immune function within teleost fish and offers valuable insights into the molecular mechanisms of lipid metabolism underlying the actions of lepA and lepB.

Up-regulated Lnc BTU promotes the production of duck plague virus DNA polymerase and inhibits the activation of JAK-STAT pathway to facilitate duck plague virus replication

Duck plague virus (DPV) is the only herpes virus known to be transmissible among aquatic animals, leading to immunosuppression in ducks, geese and swans. Long noncoding RNAs (LncRNA) are known to participate in viral infections, acting as either immune defenders or viral targets to evade the host response, but their precise roles in waterfowl virus infections are yet to be fully understood. This study aimed to investigate the role of LncRNA in DPV-induced innate immune responses. Results showed that DPV infection greatly upregulated Lnc BTU expression in duck embryo fibroblasts (DEF) and Lnc BTU promoted DPV replication. Mechanically, 4 DPV proteins, namely UL46, UL42, VP22 and US10, interacted with Lnc BTU, leading to its upregulation. Specifically, Lnc BTU facilitated the production of DNA polymerase by enhancing UL42 expression, thereby promoting DPV replication. Additionally, Lnc BTU suppressed STAT1 expression by targeting the DNA binding domain (DBD) and promoting STAT1 degradation through the proteasome pathway. Furthermore, Lnc BTU inhibited the production of key antiviral factors such as IFN-α, IFN-β, MX and OASL during DPV infection. Treatment with 2 JAK-STAT pathway activators in DEFs resulted in the inhibition of Lnc BTU expression and DPV replication. Interestingly, DPV infection led to a decrease in STAT1 levels, which was reversed by Si-Lnc BTU. These findings suggest that DPV relies on Lnc BTU to inhibit the activation of the JAK-STAT pathway and limit the production of type 1 interferons (IFN) to complete immune evasion. Our study highlights the novel role of DPV proteins UL46, UL42, VP22, US10 as RNA-binding proteins in modulating the innate antiviral immune response, and discover the role of a new host factor, Lnc BTU, in DPV immune evasion, Lnc BTU and STAT1 can be used as a potential therapeutic target for DPV infection and immune evasion.

Siglec7 functions as an inhibitory receptor of non-specific cytotoxic cells and can regulate the innate immune responses in a primitive vertebrate (Oreochromis niloticus)

In mammals, siglec7, an integral component of the siglecs, is principally found on the surface of natural killer (NK) cells, macrophages, and monocytes, where it interacts with various pathogens to perform immunological regulatory activities. Nonetheless, the immune defense and mechanism of siglec7 in early vertebrates remain unknown. In this study, we identified siglec7 from Oreochromis niloticus (OnSiglec7) and revealed its immune functions. Specifically, OnSiglec7 was abundantly expressed in immune-related tissues of healthy tilapia and its transcription level was strongly activated after being challenged with A. hydrophila, S. agalactiae, and Poly: IC. Meanwhile, OnSiglec7 protein was purified and analyzed, which could recognize multiple pathogens through binding and agglutinating activity. Moreover, OnSiglec7-positive cells were mainly distributed in non-specific cytotoxic cells (NCC) of tilapia HKLs and showed cell membrane localization. Furthermore, OnSiglec7 blockage affected multiple innate immune responses (inflammation, apoptosis, and pyroptosis process) by regulating the activation of MAPK, NF-κB, TLR, and JAK-STAT pathways. Finally, OnSiglec7 blockage also greatly enhanced the cytotoxic effect of tilapia NCC. Summarily, this study uncovers immune functions and mechanisms of siglec7 in primitive vertebrates, thereby enhancing our understanding of the systemic evolution and ancient functions of other siglecs within the host's innate immune system (to our knowledge).

Host JAK-STAT activity is a target of parasitoid wasp virulence strategies.

Innate immune responses that allow hosts to survive infection depend on the action of multiple conserved signaling pathways. Pathogens and parasites in turn have evolved virulence factors to target these immune signaling pathways in an attempt to overcome host immunity. Consequently, the interactions between host immune molecules and pathogen virulence factors play an important role in determining the outcome of an infection. The immune responses of Drosophila melanogaster provide a valuable model to understand immune signaling and host-pathogen interactions. Flies are commonly infected by parasitoid wasps and mount a coordinated cellular immune response following infection. This response is characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in the host hemocoel. The conserved JAK-STAT signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Here we show that activity of Stat92E, the D. melanogaster STAT ortholog, is induced in immune tissues following parasitoid infection. Virulent wasp species are able to suppress Stat92E activity during infection, suggesting they target JAK-STAT pathway activation as a virulence strategy. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with a gain-of-function mutation in hopscotch, the D. melanogaster JAK ortholog, indicating that they inhibit the activity of the core signaling components of the JAK-STAT pathway. Our data suggest that parasitoid wasp virulence factors block JAK-STAT signaling to overcome fly immune defenses.

Perilipin1 inhibits Nosema bombycis proliferation by promoting Domeless- and Hop-mediated JAK-STAT pathway activation in Bombyx mori.

Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.

BIA-ALCL and BIA-SCC: Updates on Clinical Features and Genetic Mutations for Latest Recommendations

Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) and Breast Implant-Associated Squamous Cell Carcinoma (BIA-SCC) are emerging neoplastic complications related to breast implants. While BIA-ALCL is often linked to macrotextured implants, current evidence does not suggest an implant-type association for BIA-SCC. Chronic inflammation and genetics have been hypothesized as key pathogenetic players, although for both conditions, the exact mechanisms and specific risks related to breast implants are yet to be established. While the genetic alterations in BIA-SCC are still unknown, JAK-STAT pathway activation has been outlined as a dominant signature of BIA-ALCL. Recent genetic investigation has uncovered various molecular players, including MEK-ERK, PI3K/AKT, CDK4-6, and PDL1. The clinical presentation of BIA-ALCL and BIA-SCC overlaps, including most commonly late seroma and breast swelling, warranting ultrasound and cytological examinations, which are the first recommended steps as part of the diagnostic work-up. While the role of mammography is still limited, MRI and CT-PET are recommended according to the clinical presentation and for disease staging. To date, the mainstay of treatment for BIA-ALCL and BIA-SCC is implant removal with en-bloc capsulectomy. Chemotherapy and radiation therapy have also been used for advanced-stage BIA-ALCL and BIA-SCC. In-depth characterization of the tumor genetics is key for the development of novel therapeutic strategies, especially for advanced stage BIA-ALCL and BIA-SCC, which show a more aggressive course and poor prognosis.

Mechanisms and therapeutic prospect of the JAK-STAT signaling pathway in liver cancer.

Liver cancer (LC) poses a significant global health challenge due to its high incidence and poor prognosis. Current systemic treatment options, such as surgery, chemotherapy, radiofrequency ablation, and immunotherapy, have shown limited effectiveness for advanced LC patients. Moreover, owing to the heterogeneous nature of LC, it is crucial to uncover more in-depth pathogenic mechanisms and develop effective treatments to address the limitations of the existing therapeutic modalities. Increasing evidence has revealed the crucial role of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in the pathogenesis of LC. The specific mechanisms driving the JAK-STAT pathway activation in LC, participate in a variety of malignant biological processes, including cell differentiation, evasion, anti-apoptosis, immune escape, and treatment resistance. Both preclinical and clinical investigations on the JAK-STAT pathway inhibitors have exhibited potential in LC treatment, thereby opening up avenues for the development of more targeted therapeutic strategies for LC. In this study, we provide an overview of the JAK-STAT pathway, delving into the composition, activation, and dynamic interplay within the pathway. Additionally, we focus on the molecular mechanisms driving the aberrant activation of the JAK-STAT pathway in LC. Furthermore, we summarize the latest advancements in targeting the JAK-STAT pathway for LC treatment. The insights presented in this review aim to underscore the necessity of research into the JAK-STAT signaling pathway as a promising avenue for LC therapy.

SOCS3 inhibiting JAK-STAT pathway enhances oncolytic adenovirus efficacy by potentiating viral replication and T-cell activation.

Oncolytic viruses (OVs) are emerging as a potentially useful treatment for malignancies due to the capabilities of direct oncolysis and immune induction. Improving the replication of OVs is an effective approach to enhance the oncolytic effects. Here, we observed that cancer cells with deficiencies in JAK-STAT pathway showed greater sensitivity to oncolytic adenovirus (oAd), and JAK inhibitor could enhance the replication of oAd. Therefore, we constructed a novel oAd expressing SOCS3, a major negative regulator of JAK-STAT pathway, and confirmed that oAd-SOCS3 exhibited a more significant antitumor effect than oAd-Ctrl both in vitro and in vivo. Mechanistically, SOCS3 inhibited the activation of JAK-STAT pathway, resulting in stronger tumor selective replication of oAd and downregulated expression of PD-L1 on cancer cells as well. Both benefits could collectively awaken antitumor immunity. This study highlights the importance of JAK-STAT pathway in viral replication and confirms the treatment of oAd-SOCS3 in potential clinical applications.

Ruxolitinib ameliorated coxsackievirus B3-induced acute viral myocarditis by suppressing the JAK-STAT pathway

BackgroundAccumulating evidences have demonstrated that overwhelming inflammation occurs in the process of Coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVM). No specific therapy is available. More than an effective Janus-associated kinase (JAK) inhibiter, ruxolitinib exerts a critical role in the inflammatory diseases. In this study, we investigated the potential effect of ruxolitinib on CVB3-induced acute viral myocarditis. MethodIn vivo, BALB/c mice were intraperitoneally injected of CVB3, treated of a successive gavage of ruxolitinib for seven days, and subjected to a series of analysis. In vitro, primary bone marrow-derived macrophages (BMDMs) and cardiac fibroblasts were isolated, cultured, treated, harvested and finally detected. ResultsIn vivo, acute viral myocarditis was successfully induced by the injection of CVB3 characterized by impaired cardiac function, predominant infiltration of inflammatory cells, necroptosis of myocardium, great increase of cardiac troponin I (cTnI) and cytokine levels, replication of CVB3, and excessive activation of JAK-STAT pathways. Oral administration of ruxolitinib suppressed the activation of JAK-STAT pathway in a dosage-dependent way, lessened the infiltration of inflammatory cells and necroptosis of myocardium, reduced the levels of cTnI and cytokines, and finally alleviated CVB3-induced cardiac dysfunction, with the reduced production of type I interferon and no promising effect on the replication of CVB3. In vitro, the treatment of ruxolitinib inhibited the activation of JAK-STAT pathway and increase of multiple cytokines mRNA levels in BMDMs and had no protective effect against CVB3 replication in cardiac fibroblasts. ConclusionsOur study suggested that ruxolitinib ameliorated CVB3-induced AVM by inhibiting the activation of JAK-STAT pathway, infiltration of inflammatory cells and necroptosis of myocardium, which may provide a novel strategy for AVM therapy.

CXCL5 promotes tumorigenesis and angiogenesis of glioblastoma via JAK-STAT/NF-κb signaling pathways.

The tumor microenvironment contains chemokines that play a crucial role in various processes, such as tumorigenesis, inflammation, and therapy resistance, in different types of cancer. CXCL5 is a significant chemokine that has been shown to promote tumor proliferation, invasion, angiogenesis, and therapy resistance when overexpressed in various types of cancer. This research aims to investigate the impact of CXCL5 on the biological functions of glioblastoma (GBM). The TCGA GBM and GEO databases were utilized to perform transcriptome microarray analysis and oncogenic signaling pathway analysis of CXCL5 in GBM. Validation of CXCL5 expression was performed using RT-qPCR and Western Blot. The impact of CXCL5 on cell proliferation, tumorigenesis, and angiogenesis in GBM was assessed through various methods, including cell proliferation assay, cloning assay, intracranial xenograft tumor models, and tube formation assay. Clinical prognosis was evaluated in 59 samples of gliomas with varying degrees of malignancy (grades 2, 3, and 4) and the TCGA GBM database, based on CXCL5 expression levels. The activities of the JAK-STAT and NF-κB signaling pathways were detected using Western Blot. The expression of CXCL5 was highly enriched in GBM. Moreover, the inhibition of CXCL5 showed a significant efficacy in suppressing cellular proliferation and angiogenesis, resulting in extended survival rates in xenograft mouse models in comparison to the control group. Notably, pretreatment with dapsone exhibited a reversal of the impact of CXCL5 on the formation of colonies and tubes in GBM cells. Elevated expression of CXCL5 was correlated with poor outcomes in GBM patients. Furthermore, the overexpression of CXCL5 has been associated with the activation of JAK-STAT and NF-κB signaling pathways. CXCL5 plays an important role in tumorigenesis and angiogenesis, indicating the potential for novel therapies targeting CXCL5 in GBM.

EBF1-JAK2 inhibits the PAX5 function through physical interaction with PAX5 and kinase activity.

Gene aberrations of B-cell regulators and growth signal components such as the JAK-STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1-JAK2 (E-J). E-J caused constitutive activation of JAK-STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E-J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E-J with PAX5 and kinase activity of E-J were required for E-J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E-J-positive ALL cells, which suggests that E-J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins.

Inflammation perturbs NK cell phenotype and function in chronic myeloid leukemia (CML)

Abstract Natural killer (NK) cells have the capacity to recognize and clear cancer cells by releasing cytotoxic granules. In fact, NK cells’ mature phenotype and abundance are associated with prolonged treatment-free remission in chronic myeloid leukemia (CML). However, NK cell anti-leukemic activity is suppressed during the disease, and the environmental triggers of this impairment are not fully characterized. Given the role of inflammatory cytokines in the progression of myeloid malignancies, we define their impact on NK cell function in a BCR-ABL1 +CML chimeric mouse model. Consistent with clinical observations, NK cells are reduced in counts in leukemic mice, display immature phenotype, low degranulation rate, and altered expression of activating and inhibitory receptors. Single-cell RNAseq analysis confirmed these observations at the transcriptional level, and revealed enrichment in genes and pathways associated with inflammatory cytokine response (e.g., GM-CSF, LTα/β, XCL1, IL-6st, IL-2Ra, SOCS1/2/3, CIS; IL-6/STAT3 and TNFa/NF-kB pathways) in CML-exposed NK cells. To validate the clinical relevance of our findings, we characterized NK cells from CML patients. As expected, NK cell frequencies are reduced in our cohort and display diminished K562-targeted degranulation. Mirroring mouse scRNAseq data, patient NK cells possess a pro-inflammatory gene signature with the activation of JAK-STAT, TNFa/NF-kB, and PI3K-Akt signaling pathways. As this is likely triggered by leukemic cytokines, we found that leukemic serum dampens ex vivo NK cell degranulation capacity. Thus, we speculate that NK cells are sensitive to CML-associated cytokines and that inflammation represents an optimal target for NK-boosting immunotherapies.

NOTCH pathway inactivation reprograms stem-like oral cancer cells to JAK-STAT dependent state and provides the opportunity of synthetic lethality

BackgroundWe have recently provided the evidence of interconvertible cellular states, driving non-genetic heterogeneity among stem-like oral cancer cells (oral-SLCCs). Here, NOTCH pathway-activity status is explored as one of the possible mechanisms behind this stochastic plasticity. MethodsOral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivo effects were studied by xenograft growth in zebrafish embryo. ResultsWe have observed stochastic plasticity in oral-SLCCs, spontaneously maintaining both NOTCH-active and inactive states. While cisplatin refraction was associated with post-treatment adaptation to the active-state of NOTCH pathway, oral-SLCCs with inactive NOTCH pathway status showed aggressive tumor growth and poor prognosis. RNAseq analysis clearly suggested the upregulation of JAK-STAT pathway in NOTCH pathway-inactive subset. The 3D-spheroids with lower NOTCH-activity status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib or siRNA mediated downregulation of tested partners STAT3/4. Oral-SLCCs were programmed to adapt the inactive status of NOTCH pathway by exposing to γ-secretase inhibitors, LY411575 or RO4929097, followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This approach resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft initiation in Zebrafish embryos. ConclusionStudy revealed for the first time that NOTCH pathway-inactive state exhibit activation of JAK-STAT pathways, as synthetic lethal pair. Therefore, co-inhibition of these pathway may serve as novel therapeutic strategy against aggressive oral cancer.

Abstract 6548: Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis

Abstract Although the development of single-cell RNAseq (scRNAseq) has improved the knowledge of the tumor microenvironment (TME), there is not a clear understanding of specific cell subtypes, markers that define these cell populations, nor the interaction between them. Previously, we showed that inflammasome activation is correlated with antitumor responses triggered by immune checkpoint blockade (ICB). This highlights a central role for TMEM176B, mostly expressed in myeloid cells, that impairs the recruitment of cytotoxic immune cells in the TME by inhibiting NLRP3 inflammasome activation. Still, the roles of the different subsets of myeloid and T cells in the TME and to what extent they are connected remain controversial. Here, we used scRNAseq data from human tumors to identify and characterize cDC2, Th17 and exhausted CD8+ T cell subpopulations in cancer. Five scRNAseq data sets of melanoma, basal cell carcinoma, breast, lung, colorectal, and ovary cancer patients were integrated, accounting for 382,019 immune cells in total. We applied and compared different bioinformatic tools for scRNASeq data normalization and data sets integration to find conserved immune cell populations across samples, data sets, and cancer types. Then, we evaluated different parameters using well-established methods for clustering and cell type classification to identify subtypes of DCs and T cells in the integrated data. We combined automatic methods with manual analysis of cluster markers to improve robustness and confidence in the cell populations identified. Three clusters of cDC2 were defined and characterized using different scRNAseq analysis tools for pathway enrichment analysis and transcription factors (TF) activation prediction. We found a subset of inflammatory cDC2 with high gene expression of IL1B and NLRP3, in contrast with another cluster characterized by high expression of TMEM176B and CD14. A third cluster showed an intermediate transcriptomic profile. The TMEM176B+ CD14+ cluster was characterized by the activation of JAK-STAT and hypoxia pathways, and the TF FOXL2, ETS2, and ESR2. The NLRP3+ IL1B+ cluster showed more activation of MAPK, Trail, and PI3K pathways, and the TF CEBPE. Moreover, the activation of TNFα and NFkB signaling pathways was found in both clusters. In addition, a conserved population of Th17 cells in the integrated data was observed. Th17 cells were heterogeneous, making the subpopulations and transcriptome signature difficult to define. Further analysis is being performed to identify Th17 subsets and their potential interactions with the cDC2 subclusters. A robust identification and characterization of these immune cell subsets will build towards our understanding of the interactions between these subpopulations triggered by inflammasome activation within the TME and the mechanisms behind ICB response. Citation Format: Yamil D. Mahmoud, Natalia Rego, Marcela Vilarino, Florencia Veigas, Maria R. Girotti, Juan M. Perez Saez, Gabriel A. Rabinovich, Marcelo Hill. Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6548.

Mesenchymal stromal cells in tumor microenvironment remodeling of BCR-ABL negative myeloproliferative diseases.

Chronic myeloproliferative neoplasms encompass the BCR-ABL1-negative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are characterized by calreticulin (CALR), myeloproliferative leukemia virus proto-oncogene (MPL) and the tyrosine kinase Janus kinase 2 (JAK2) mutations, eventually establishing a hyperinflammatory tumor microenvironment (TME). Several reports have come to describe how constitutive activation of JAK-STAT and NFκB signaling pathways lead to uncontrolled myeloproliferation and pro-inflammatory cytokines secretion. In such a highly oxidative TME, the balance between Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs) has a crucial role in MPN development. For this reason, we sought to review the current literature concerning the interplay between HSCs and MSCs. The latter have been reported to play an outstanding role in establishing of the typical bone marrow (BM) fibrotic TME as a consequence of the upregulation of different fibrosis-associated genes including PDGF- β upon their exposure to the hyperoxidative TME characterizing MPNs. Therefore, MSCs might turn to be valuable candidates for niche-targeted targeting the synthesis of cytokines and oxidative stress in association with drugs eradicating the hematopoietic clone.

Oncogenic role of microRNA-19b-3p-mediated SOCS3 in glioma through activation of JAK-STAT pathway.

The altered expression of microRNA (miRNA) has been implicated in glioma. Here, the current study aimed to clarify the oncogenic effects of miR-19b-3p on cellular processes of glioma and to elucidate the underlying mechanism associated with SOCS3 and the JAK-STAT signaling pathway. Differentially expressed genes related to glioma were initially identified via microarray analysis. Twenty-five glioma patients were selected for clinical data collection, while additional 12 patients with traumatic brain injuries were selected as controls. Cell senescence was assessed by β-galactosidase staining, proliferation by MTT assay and apoptosis by flow cytometry following gain- and/or loss-of-function of miR-19b-3p or SOCS3. Glioma xenograft mouse model was developed through subcutaneous injection to nude mice to provide evidence in vivo. The glioma patients exhibited overexpressed miR-19b-3p and poorly-expressed SOCS3. SOCS3 was identified as a target gene of miR-19b-3p through dual-luciferase reporter gene assay. miR-19b-3p repressed SOCS3 expression and activated the JAK-STAT signaling pathway. Furthermore, miR-19b-3p inhibition promoted apoptosis and senescence, and suppressed cell proliferation through inactivation of the JAK-STAT signaling pathway and up-regulation of SOCS3. The reported regulatory axis was validated in nude mice as evidenced by suppressed tumor growth. Taken together, this study demonstrates that miR-19b-3p facilitates glioma progression via activation of the JAK-STAT signaling pathway by targeting SOCS3, highlighting a novel therapeutic target for glioma treatment.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.