TNFα-induced NF-κB Activation Research Articles

Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Arbitrary Ca2+ regulation for endothelial nitric oxide, NFAT and NF-κB activities by an optogenetic approach.

Modern western dietary habits and low physical activity cause metabolic abnormalities and abnormally elevated levels of metabolites such as low-density lipoprotein, which can lead to immune cell activation, and inflammatory reactions, and atherosclerosis. Appropriate stimulation of vascular endothelial cells can confer protective responses against inflammatory reactions and atherosclerotic conditions. This study aims to determine whether a designed optogenetic approach is capable of affecting functional changes in vascular endothelial cells and to evaluate its potential for therapeutic regulation of vascular inflammatory responses in vitro. We employed a genetically engineered, blue light-activated Ca2+ channel switch molecule that utilizes an endogenous store-operated calcium entry system and induces intracellular Ca2+ influx through blue light irradiation and observed an increase in intracellular Ca2+ in vascular endothelial cells. Ca2+-dependent activation of the nuclear factor of activated T cells and nitric oxide production were also detected. Microarray analysis of Ca2+-induced changes in vascular endothelial cells explored several genes involved in cellular contractility and inflammatory responses. Indeed, there was an increase in the gene expression of molecules related to anti-inflammatory and vasorelaxant effects. Thus, a combination of human blue light-activated Ca2+ channel switch 2 (hBACCS2) and blue light possibly attenuates TNFα-induced inflammatory NF-κB activity. We propose that extrinsic cellular Ca2+ regulation could be a novel approach against vascular inflammation.

Clinical and Biological Effects of Canakinumab in Lower-Risk Myelodysplastic Syndromes (MDS): Results from a Phase 2 Clinical Trial

Interleukin 1 beta (IL-1B) induced inflammasome activation is a mechanism of cancer initiation and progression (Schroder K, Cell, 2010). Our previous studies (Ganan-Gomez I, Blood 2021) showed that IL-1B was one of the most overexpressed pro-inflammatory cytokines in the hematopoietic stem and progenitor cells (HSPCs) of patients with clonal cytopenia of undetermined significance (CCUS) carrying DNMT3a and TET2 mutations. Given that IL-1B upregulation induces aberrant myeloid differentiation of HSPCs (Pietras EM, Nat Cell Biol., 2016), we hypothesize that IL-1B plays a critical role in MDS pathogenesis, and that targeting IL-1B signaling overcomes aberrant MDS HSPCs' myeloid skewing. To test this hypothesis, we designed a clinical trial to evaluate the IL-1B inhibitor canakinumab's (NCT04239157) safety profile and its clinical and biological effects in patients with lower-risk MDS. Patients and Methods: Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS, normal renal and hepatic functions, and performance status, who had previously received at least 1 line of other therapy were included in the study (Table1). Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. Bone marrow (BM) samples were obtained before therapy and after 1, 2, and 4 cycles of therapy during the trial and after every 3 or 4 cycles of therapy thereafter. Single-cell RNA sequencing (scRNA-seq; 10X Genomics) analyses were performed in lineage negative (Lin-)CD34+ HSPCs and mononuclear cells (MNCs) isolated at different therapy timepoints from the BM of 1 patient who achieved hematological improvement after canakinumab treatment, 2 patients who achieved stable disease, and 2 patients who did not respond to this therapy. The clinical characteristics of the 5 patients whose samples were analyzed by scRNA-seq analyses are included in Table 1. Results: Twenty-six patients have been enrolled in the clinical trial. Patients' clinical characteristics are included in Table 1. Twenty-three patients were evaluable for response. Among the 22 out of the 23 patients who were transfusion dependent at baseline, one achieved transfusion independence, and another had platelet count improvement. Three out of the 23 had progressive disease, and 18 had stable disease. Treatment was well tolerated with no severe adverse events. One patient died of sepsis unrelated to therapy. Overall, we did not observe any improvement in the blast counts and cytogenetic and/or mutational burden. To evaluate the biological effects of canakinumab, we performed scRNA-seq of Lin-CD34+ HSPCs (Figure 1) and MNCs at therapy baseline and after 2, 4, 5, 8, and 11 cycles of treatment. Consistent with our initial hypotheses, these analyses revealed that canakinumab decreased the frequencies of hematopoietic stem cells (HSCs) and multipotent myeloid progenitors (MPPs). Differential analysis between the samples showed that HSCs and MPPs significantly downregulated the expression of genes involved in cytokine signaling (e.g., IL-1B, CXCL2, and CXCL8), TNFα-induced activation of NF-κB, and hypoxia pathways, which demonstrated efficient target engagement of canakinumab on MDS cells. Similar results were also observed in downstream monocytes and B cells. In contrast, we did not observe any significant change in the HSPC architecture in the BM of patients with stable disease or whose disease did not respond to canakinumab. Consistent with our previous results in early stage MDS, the patient who achieved hematological improvement after canakinumab therapy was the only one among the cohort who carried a single mutation affecting DNMT3A (Table1). Conclusion. This study demonstrates that canakinumab is safe in MDS and efficiently targets IL-1B signaling. However, the biological effect of canakinumab has not yet translated in clinical responses in the subset of patients enrolled in this clinical trial. The study will accrue transfusion independent patients and patients with early stage MDS as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 8: Celastrol targets multiple pathways and suppress cell invasion in lung cancer growth and metastasis

Abstract Lung cancer remains the leading cause of cancer-related deaths in the United States and worldwide. Non-small cell-lung cancer (NSCLC) accounts for 85% of all lung cancer cases. Plant-derived compounds have historically led to some of our most useful cancer drugs (e.g., paclitaxel, vincristine, etc). Celastrol (CEL), a triterpenoid isolated from Tripterygium wilfordii, has attracted significant attention for its strong anticancer properties. CEL demonstrate time- and dose-dependent anti-proliferative activity against H1299 and A549 cells with IC50 0.63 and 1.8 µM, respectively and shows dose dependent inhibition of A549 colony formation. CEL has minimal effect against the normal bronchial epithelium Beas2b. We have also demonstrated that CEL loaded onto bovine milk-derived exosomes (ExoCEL) enhanced therapeutic response vs. the free CEL in tumor xenograft model and lacks systemic and immunotoxicity. Our data indicate that the wound healing and cell migration rate was significantly reduced in the CEL treatment. In fact, the effect was time and dose dependent as analyzed by Wimasis software. CEL is an inhibitor of TAK1 and has shown to suppress basal TGF-β1 and TNF-α induced NF-κB activity, however, the precise mechanisms remain to be identified. Here, we systemically explored the mechanism of CEL by which it inhibits the growth and metastasis of NSCLC using RNAseq analysis. RNAseq followed by differential expression of genes demonstrate a total of over 600 genes modulated by the CEL treatment of which 287 genes were upregulated while 314 genes were downregulated. Gene Ontology analysis, genome analysis along with reactome pathway enrichment analyses indicated that CEL-treated genes are involved in cell cycle, EMT, PI3K-AKT and p53 signaling and pathways related to lung cancer and its metastasis. The molecules of major pathways involved in the RNAseq analysis were analyzed by western blot. Our findings indicated significant decrease in TGFβ-induced migratory behavior of cancer cells and reversal of TGFβ-mediated changes in EMT proteins such as ZO1, Vimentin, β-catenin and claudin by CEL in H1299 lung cancer cells. Cancer cells exhibited up-regulated N-cadherin with loss of E-cadherin called the "cadherin switch" and downregulation of E-cadherin is one of the hallmarks of EMT. We also confirmed that CEL inhibit TGFβ-induced cMyc expression by decreasing PI3K and AKT phosphorylation via TGF-β/non-Smad pathway and show dose- dependent reduction of pAKT, PI3K, cMyc and Smad4 in the absence and presence of TGF-β. In summary, the analysis of biological functions of the most differentially expressed genes revealed possible mechanisms of CEL for its strong activity against lung cancer. Pathways identification provide information about the potential use of CEL to inhibit tumorigenesis, progression, and metastasis of lung cancer. (Supported by Research and Development Office, KSA Grant# RDO-2003). Citation Format: Farrukh Aqil, Raghuram Kandimalla, Neha Tyagi, Mohd Saeed, Ramesh Gupta. Celastrol targets multiple pathways and suppress cell invasion in lung cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 8.

Abstract 2988: Proteasomal deubiquitinases represent an attractive therapeutic target in head and neck squamous cell carcinomas (HNSCC)

Abstract Despite recent advances in treatment options for Human Papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC), the overall survival (OS) rate for HNSCC is low, demonstrating the need for novel therapies for these cancers. Targeting the ubiquitin-proteasome system (UPS) has emerged as a potential target for the development of novel anti-cancer therapies. Bortezomib, a first generation proteasome inhibitor clinically approved for the treatment of Multiple Myeloma, has previously been demonstrated to induce tumor regression in a subset of HNSCC patients through the inhibition of canonical NFκB activity; however, the failure of Bortezomib to inhibit other pro-survival pathways such as MAPK and STAT3 signalling resulted in heterologous responses. A recently developed alternative to proteasome inhibitors is the small molecule b-AP15, which inhibits the proteasomal deubiquitinases USP14 and UCHL5, thus allowing more specificity and less toxicity than proteasome inhibitors. Here, we show b-AP15 inhibits proliferation and colony formation in multiple HPV- and HPV+ HNSCC cell lines, with minimal effects in normal oral keratinocytes. Furthermore, b-AP15 induces significant caspase-dependent apoptosis. Using siRNA targetting USP14 and UCHL5, only USP14 depletion significantly reduced cell proliferation and colony formation. Mechanistically, b-AP15 treatment reduces TNFα-induced NFκB activity and the expression of pro-survival proteins such as cIAP2 and TRAF2, promoting the formation of the TNFR complex II and sensitizing cells to TNFα-induced cell death. Finally, TCGA data demonstrates that USP14 is highly expressed in HNSCC when compared with normal tissue and is significantly correlated with worse overall survival. Together, we have identified that inhibition of proteasomal deubiquitinases inhibits the proliferation and survival of HNSCC cells and enhanced TNFα-induced cell death via the inhibition of NFκB activity. Our data suggest that combination therapies with b-AP15 could potentially offer a clinical benefit in HNSCC patients by promoting TNFα-induced cytotoxicity. Further studies will focus on the mechanism by which USP14 regulates NFκB signalling in HNSCC cells and the effect of b-AP15 activity in combination with radiation treatment in in vivo mouse xenograft models. Citation Format: Ethan L. Morgan, Tiffany Toni, Xinping Yang, Hui Cheng, Ramya Viswanathan, Zhong Chen, Carter Van Waes. Proteasomal deubiquitinases represent an attractive therapeutic target in head and neck squamous cell carcinomas (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2988.

An L-theanine derivative targets against SARS-CoV-2 and its Delta and Omicron variants

Recent research efforts have shown that tea has activities against SARS-CoV-2. However, the active compounds and the action mechanisms are largely unknown. Here we study the inhibitory potential of L-theanine from tea and its semi-synthesized derivative, a small-molecule fluorescent compound, ethyl 6-bromocoumarin-3-carboxylyl L-theanine (TBrC) against infection and replication of SARS-CoV-2 and the underlying mechanisms of action. We reveal that TBrC has potential activities against SARS-CoV-2 in addition to its activity against lung cancer. TBrC showed extracellular inhibition of SARS-CoV-2 Mpro/3CL and the host cell receptor ACE2 while interacting with the viral spike glycoproteins (wild-type, Delta, and Omicron mutants). Moreover, TBrC and L-theanine significantly suppressed growth and TNFα-induced nuclear transcriptional activation of NF-κB in human lung cancer cells without affecting the viability of normal lung cells, suggesting a potential protection of TBrC and L-theanine from pulmonary damages in SARS-CoV-2 infected patients, especially for lung cancer patients with SARS-CoV-2 infection.

Altersolanol B, a fungal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adenocarcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways

The present study focused on the apoptosis-inducing effects and cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line, MCF-7. AB demonstrated approximately 4-fold greater antiproliferative activity in ER+ MCF-7 cells (IC50 5.5 μM) compared to the ER-negative (triple-negative) MDA-MB-231 (IC50 21.3 μM). The viability of normal breast fibrocystic epithelial cells, MCF-10A, was unaffected. AB induced intrinsic apoptosis in MCF-7 cells; it triggered the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP), upregulated the expression of pro-apoptotic Bax, and downregulated the expression of anti-apoptotic Bcl-2. AB induced cell cycle arrest at G0/G1, as indicated by the downregulation of key checkpoint proteins operating at the G0/G1 phase of the cell cycle (cyclin D1, CDK4 and CDK2). The observed increase in p21Waf1/Cip1 and p53 expression may facilitate cell cycle arrest, and the subsequent induction of apoptosis. AB lacked significant effects on intracellular ROS levels, while it down-regulated nuclear factor erythroid 2-related factor 2 (Nrf2), and the Nrf2-dependent antioxidant enzyme, heme oxygenase-1. The compound disrupted AKT signaling through the downregulation of phospho-AKT and phospho-FOXO1, and the upregulation of PTEN, a phosphatase and tumor suppressor that negatively regulates the PI3K/AKT pathway. AB also disrupted the phosphorylation of AKT-controlled eukaryotic translation initiation factor, 4E-BP1, and GSK-3β, both of which are aberrantly regulated in human cancer. The AB-dependent downregulation of NF-κB was corroborated by the inhibition of TNFα-induced NF-κB activity as monitored in a luciferase reporter. The NF-κB inhibitory activity of AB was 3-fold more potent than that of the standard inhibitor, N-p-Tosyl-l-phenylalanine chloromethyl ketone. In addition to reducing the pro-survival effects of NF-кB, the inhibition of AKT phosphorylation by AB may also lead to FOXO1-mediated growth arrest and apoptosis. AB upregulated the expression of phospho-MKK4 and phospho-p38, and downregulated the expression of phospho-MEK1/2 and phospho-ERK1/2 indicating opposing effects on the two important oncogenic signaling cascades that are aberrantly activated in many cancers. AB disrupted both the AKT and ERK1/2 signaling pathways leading to apoptosis in ER+ MCF-7 cells through mitochondria-associated mechanisms coupled with the potent inhibition of NF-кB activation. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. Our preliminary study suggested that the THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthraquinone that possess intrinsic redox activity and DNA interacting ability. This study supports the continued investigation of THAQs as lead molecules in anticancer drug discovery and development.

Analgesic, anti-inflammatory and NF-\u03baB inhibitory activity of aerial parts of Cestrum diurnum

BackgroundCestrum diurnum L. (Solanaceae), locally known as buno-Hasnahena, is widely used in different traditional medicinal practices to treat pain, burn, swelling and related disorders. Adequate evidence is not available to support its medicinal properties for further use and drug development. Present study was designed to evaluate its traditional use in pain and inflammation with further characterisation of its chemical constituents through liquid chromatography-mass spectroscopic (LC-MS) analysis.MethodsAntinociceptive and analgesic potential of methanol extract of the aerial parts of C. diurnum was carried out using carrageenan induced paw oedema and formalin induced paw licking test in mice at the oral doses of 150 and 300 mg/kg body weight. Inhibition of the inflammatory mediator nuclear factor kappa B (NF-κB) was evaluated by tumour necrosis factor α (TNF-α) induced NF-κB activation assay in macrophage cells at the concentration of 100 μg/ml. LC-MS analysis of the extract was performed to characterise the active component responsible for bioactivities.ResultsThe extract significantly inhibited (p < 0.05) carrageenan induced paw oedema at both doses tested and the effect persisted throughout the entire experimental period of 3 h with the highest activity (50% inhibition) observed at 3rd h. Further, the extract significantly inhibited (p < 0.05) formalin induced paw licking both in the early and late phase at the aforementioned dose levels. The extract also downregulated the expression of NF-κB p65 protein in the TNF-α induced NF-κB activation assay. LC-MS analysis of the extract indicated the presence of some important secondary metabolites including nicotine, nornicotine, ursolic acid, vitamin D3 and its derivatives.ConclusionsThe results of this study supported the folkloric uses of the plant in pain and inflammations. The insights and observations suggest the action might involve downregulation of NF-κB p65 protein expression and/or inhibition of autacoids (histamine, serotonin, prostaglandin).

TRIM67 Suppresses TNFalpha-Triggered NF-kB Activation by Competitively Binding Beta-TrCP to IkBa.

The transcription factor NF-κB plays an important role in modulation of inflammatory pathways, which are associated with inflammatory diseases, neurodegeneration, apoptosis, immune responses, and cancer. Increasing evidence indicates that TRIM proteins are crucial role in the regulation of NF-κB signaling pathways. In this study, we identified TRIM67 as a negative regulator of TNFα-triggered NF-κB activation. Ectopic expression of TRIM67 significantly represses TNFα-induced NF-κB activation and the expression of pro-inflammatory cytokines TNFα and IL-6. In contrast, Trim67 depletion promotes TNFα-induced expression of TNFα, IL-6, and Mcp-1 in primary mouse embryonic fibroblasts. Mechanistically, we found that TRIM67 competitively binding β-transducin repeat-containing protein (β-TrCP) to IκBα results inhibition of β-TrCP-mediated degradation of IκBα, which finally caused inhibition of TNFα-triggered NF-κB activation. In summary, our findings revealed that TRIM67 function as a novel negative regulator of NF-κB signaling pathway, implying TRIM67 might exert an important role in regulation of inflammation disease and pathogen infection caused inflammation.

Stapling proteins in the RELA complex inhibits TNFα-induced nuclear translocation of RELA.

Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1β-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

The Transcriptional and Epigenetic Reprogramming of Aged Hematopoietic Stem Cells Drives Myeloid Rewiring in Clonal Hematopoiesis-Associated Cytopenias

Patients (pts) with myelodysplastic syndromes (MDS) have few therapy options. Interventions to improve outcomes should consider strategies that arrest MDS in its early phases, when symptoms are minimal and prolonged survival is expected.To develop prevention strategies that arrest MDS before the disease outcomes become irreversibly dismal, we dissected the molecular and biological mechanisms that maintain MDS in one of its premalignant phases, clonal cytopenia of undetermined significance (CCUS). Recognizing that CCUS is an aging-related disease, we first sought to determine, at the single-cell level, how CCUS affects the transcriptional and epigenetic profile of the aging hematopoietic stem and progenitor cell (HSPC) compartment and overcomes aging-induced degenerative phenotypes. We performed single-cell RNA sequencing (scRNA-seq) analysis of Lin -CD34 + HSPCs isolated from the bone marrow (BM) of 3 young healthy donors (yHDs), 4 elderly HDs (eHDs), and 3 elderly pts with CCUS carrying mutations in common MDS driver genes.We found that the frequencies of hematopoietic stem cells (HSCs) and megakaryocytic (Mk)/erythroid (Er) progenitors were increased at the expense of myeloid (My) progenitors in eHDs as compared with yHDs (Fig. a). In contrast, CCUS pts had a predominant My-biased HSPC distribution (Fig. a). However, immunophenotypic quantification in large cohorts of eHDs and CCUS pts revealed that CCUS pts' BM had significantly fewer CD34 +CD38 - HSC populations and CD34 +CD38 + My progenitors, suggesting that My bias in CCUS results from the aberrant My differentiation of HSCs rather than My cell expansion. Further differential expression analysis among the scRNA-seq clusters showed that, compared with yHD HSCs, eHD HSCs were characterized by a significant upregulation of genes involved in the TNFα-induced activation of NF-κB (e.g., MCL1; Fig. b), which is consistent with previous findings that aged HSCs undergo transcriptional reprogramming to maintain their survival in response to changes in the systemic environment (He et al. Blood 2020). In contrast, CCUS HSCs, compared with eHD HSCs, overexpressed regulators of translation, respiratory electron transport, and mitochondrial translation initiation (Fig. c), which underscores these cells' state of proliferation and metabolic activation and their ability to overcome aging-induced phenotypic alterations.To evaluate whether the aberrant lineage differentiation in eHD and CCUS HSPCs arose from the altered fate determination of HSCs, we performed single-cell assays for transposase-accessible chromatin sequencing to profile chromatin accessibility in sorted HSCs or Lin -CD34 + HSPCs from yHDs, eHDs, and CCUS pts. Consistent with our transcriptomic data, compared with yHD HSCs, eHD HSCs were mostly poised for Mk differentiation, whereas CCUS HSPCs were poised for lymphoid/My differentiation. Indeed, eHD HSCs had an increased activity of transcriptional factors belonging to the NF-κB family and open peaks at the distal elements of genes involved in hemostasis (Fig. d). In contrast, CCUS HSCs were poised to downregulate the expression of genes involved in NF-κB signaling and Mk/Er differentiation (Fig. e).These results suggested that CCUS HSCs are highly metabolically active to maintain My differentiation. Indeed, metabolomic analyses confirmed that intermediates of oxidative phosphorylation were significantly upregulated in CCUS CD34 + cells as compared with eHD CD34 + cells (Fig. f). Further, scRNA-seq analysis of mononuclear cells isolated from the BM of 3 CCUS and 3 eHD samples revealed the widespread upregulation of genes involved in protein processing and mitochondrial metabolism. This analysis also revealed impaired terminal My differentiation despite the HSPC My bias, with decreased frequencies of monocytic cells, and an intriguing expansion of cytotoxic cell subsets in the BM of CCUS pts.In conclusion, our results demonstrate that CCUS HSCs carrying MDS driver mutations evade aging-induced phenotypic degeneration, become metabolically active, and have aberrant My skewing. Our study clarifies the molecular mechanisms underlying MDS initiation and offers an opportunity for early therapeutic intervention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

St. John's Wort alleviates dextran sodium sulfate-induced colitis through pregnane X receptor-dependent NFκB antagonism.

St. John's wort (SJW), from traditional herbs, activates the pregnane X receptor (PXR), a potential drug target for treating inflammatory bowel disease (IBD). However, how SJW alleviates dextran sodium sulfate (DSS)-induced experimental IBD by activating PXR is unknown. To test this, PXR-humanized, wild-type (WT) and Pxr-null mice, primary intestinal organoids cultures, and the luciferase reporter gene assays were employed. In vivo, a diet supplemented with SJW was found to activate intestinal PXR both in WT and PXR-humanized mice, but not in Pxr-null mice. SJW prevented DSS-induced IBD in PXR-humanized and WT mice, but not in Pxr-null mice. In vitro, hyperforin, a major component of SJW, activated PXR and suppressed tumor necrosis factor (TNF)α-induced nuclear factor (NF) κB translocation in primary intestinal organoids from PXR-humanized mice, but not Pxr-null mice. Luciferase reporter gene assays showed that hyperforin dose-dependently alleviated TNFα-induced NFκB transactivation by activating human PXR in Caco2 cells. Furthermore, SJW therapeutically attenuated DSS-induced IBD in PXR-humanized mice. These data indicate the therapeutic potential of SJW in alleviating DSS-induced IBD in vivo, and TNFα-induced NFκB activation in vitro, dependent on PXR activation, which may have clinical implications for using SJW as a herbal drug anti-IBD treatment.

Structure activity relationship (SAR) study identifies a quinoxaline urea analog that modulates IKKβ phosphorylation for pancreatic cancer therapy.

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKβ) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S177, S181) in IKKβ is a key event that drives tumor necrosis factor (TNF) α induced NF-κB mediated gene expression. Here we conducted structure activity relationship (SAR) study to improve potency and oral bioavailability of a quinoxaline analog 13-197 that was previously reported as a NFκB inhibitor for pancreatic cancer therapy. The SAR led to the identification of a novel quinoxaline urea analog 84 that reduced the levels of p-IKKβ in dose- and time-dependent studies. When compared to 13-197, analog 84 was ∼2.5-fold more potent in TNFα-induced NFκB inhibition and ∼4-fold more potent in inhibiting pancreatic cancer cell growth. Analog 84 exhibited ∼4.3-fold greater exposure (AUC0-∞) resulting in ∼5.7-fold increase in oral bioavailability (%F) when compared to 13-197. Importantly, oral administration of 84 by itself and in combination of gemcitabine reduced p-IKKβ levels and inhibited pancreatic tumor growth in a xenograft model.

Dammarane triterpenes and phytosterols from Dysoxylum tpongense Pierre and their anti-inflammatory activity against liver X receptors and NF-κB activation

Dysoxylum tpongense Pierre (local name ‘Huynh Dan Bap’) belonging to family Meliaceae, is a tree (3–10 m height), distributed in the mountainous areas (ca. 1000 m a.s.l.) in North Vietnam. From the dichloromethane fraction of the methanol extract of the leaves and stems of this plant, six dammarane triterpenes, one furanoid diterpene together with three sterols were isolated. Evaluation of biological activities of isolated compounds showed that cabraleahydroxylactone (5), cabraleahydroxylactone 3-acetate (6), and stigmast-4-en-3-one (10) possessed an anti-inflammatory effect against Liver X receptor (LXR) activation in HepG2 cell line model with IC50 values of 20.29 ± 3.69, 24.32 ± 2.99, and 7.09 ± 0.97 (μM), respectively. While three other triterpenoid compounds aglinin C 3- acetate (1), aglinin C (2), and 24-epi-cabraleadiol (4) presented the most significant inhibitory effect against TNF-α induced NF-κB activation in HepG2 cell line in a dose-dependent manner with IC50 values of 12.45 ± 2.37, 23.32 ± 3.25, and 13.95 ± 1.57 μM, respectively. As stigmast-4-en-3-one (10), with structure closely similar to cholesterol, acted selectively on LXRs but not on NF-kB activation pathway, this suggests that stigmast-4-en-3-one (10) can be potentially applied as an agonist on LXR signaling pathway. Pathways LXRs-NF-κB-iNOS expression have a close relationship and play a crucial role in proceeding metabolic abnormalities like atherosclerosis, obesity, inflammation, etc. Thus, the findings showed that dammarane-type triterpenoids from D. tpongense are worthy of further investigation for potential LXR agonists and potent anti-atherogenic agents against atherosclerotic lesion progression.

Purple corn extract alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like phenotypes in BALB/c mice

ABSTRACT Zea mays L. (Poaceae), also known as purple corn, is an annual herbaceous plant that is grown as food for human consumption in a variety of forms, including cooking oils and sweeteners in processed food and beverage products. The purpose of this study was to determine whether a novel purple corn extract, FB801, might have an anti-atopic dermatitis (AD) effect on AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Topical sensitization (1%) and challenge (0.3%) by DNCB were performed on the dorsal skin and right ear of BALB/c mice to induce AD. Following FB801 and dexamethasone administered orally, the severity of skin lesions was examined macroscopically and histologically. Serum levels of immunoglobulin E (IgE) and various cytokines were determined by enzyme-linked immunosorbent assay. Oral administration of FB801 significantly reduced typical symptoms of AD (erythema/bleeding, swelling, molting/erosion and scaling/drying), scratching frequencies, and the recruitment of inflammatory and mast cells. In addition, FB801 suppressed serum levels of IgE and T helper (Th)2 type cytokines such as interleukin (IL)-4 and IL-10 in DNCB-treated BALB/c mice. Furthermore, FB801 reduced the degradation of inhibitor of nuclear factor-κB proteins (NF-κB) in tumor necrosis factor (TNF)-α-stimulated human keratinocyte (HaCaT) cells. These results suggest that FB801 inhibited the development of the AD-like skin symptoms by regulating Th1 and Th2 responses in the skin lesions in mice and suppressing TNF-α induced NF-κB activation in HaCaT cells, suggesting that FB801 has potential application as an effective alternative therapy for the prevention and management of AD.

MyD88-Dependent Signaling Is Required for HOIP Deficiency–Induced Autoinflammation

The linear ubiquitin chain assembly complex (LUBAC) plays pivotal roles in regulating lymphocyte activation, inflammation, and cell death. This is highlighted by the fact that patients with mutations in LUBAC catalytic subunit HOIP suffer from autoinflammation combined with immunodeficiency. Although defective development of T and B cells resulting from HOIP deficiency in adaptive immunity can explain immunodeficiency, the pathogenesis of autoinflammation is not clear. In this study, we found that dendritic cell (DC)-specific deletion of HOIP resulted in spontaneous inflammation, indicating the essential role of HOIP in maintaining DC homeostasis. Although HOIP deficiency in DCs did not affect TNF-α-induced NF-κB activation, it enhanced TNF-α-induced apoptosis and necroptosis. However, crossing HoipDC KO mice with TNFR1-knockout mice surprisingly could not rescue the systematic inflammation, suggesting that the autoinflammation is not due to the effect of HOIP on TNF-α signaling. In contrast, treatment of HoipDC KO mice with antibiotics reduced the inflammation, implying that TLR signaling may contribute to the inflammatory phenotype found in HoipDC KO mice. Consistently, we found that LPS induced more cell death and significantly higher levels of IL-1α and IL-1β in HoipDC KO cells. Importantly, MyD88 deficiency rescued the inflammatory phenotype in HoipDC KO mice. Together, these findings reveal the indispensable function of HOIP in maintaining DC homeostasis, and MyD88-dependent proinflammatory signal plays a substantial role in the pathogenesis of human autoinflammation associated with HOIP mutations.

Inhibition of p62-ZZ Domain-Mediated Signaling Overcomes Bortezomib Resistance in Multiple Myeloma Cells Independent of Their p53 Status

Multiple myeloma (MM) cells depend on autophagy as well as proteasome activity for their survival. In MM cells, autophagy is essential to reduce the extraordinary ubiquitinated protein system stress derived from high levels of immunoglobulin synthesis. Thus, proteasome inhibitors (PIs) decrease tumor burden in the majority of MM patients but eventually a PI resistant MM clone(s) arises. Bortezomib (BTZ) resistance has been reported to correlate with p53 status of MM cells. Previous studies showed that MM patients harboring p53 genetic abnormalities are commonly resistant to standard therapies, increasing the need for new druggable targets.A possible mechanism of BTZ resistance involves the cargo receptor for ubiquitin-mediated autophagy SQSTM1/p62, that enhances MM cells survival by increasing their autophagic flux. We found that MM cells with wild type p53, are more sensitive to PI treatment, while MM cells harbouring mutant p53 or lacking p53 are more resistant to BTZ-mediated cytotoxicity. BTZ treatment induced a modest level of apoptosis in MM1.S cells (p53 wild type) at high doses (>5nM) by triggering the cleavage of caspase 3 and PARP-1. As expected BTZ treatment induced a significant compensatory increase in SQSTM1/p62 at the mRNA and protein levels in these cells. Conversely, BTZ treatment of p53 mutant MM cells failed to induce apoptosis, exhibiting only a mild effect on viability. This occurred in spite of a similar increase in SQSTM1/p62 expression as seen in MM.1S cells.To determine the role of p62 in the BTZ resistance of p53 mutant MM cells, we tested a novel small-molecule p62-ZZ domain inhibitor, XRK3F2 we recently identified. XRK3F2 treatment of mice with established MM, dramatically increased bone formation in MM-bearing bones, although it did not affect tumour burden in vivo . In vitro studies showed that high concentrations (10μM) of XRK3F2 were required to decrease the viability of primary and MM cell lines and induce a time-dependent activation of caspases 3, 7 and 9. XRK3F2 treatment dose-dependently increased levels of the lipidated form of LC3, LC3II, suggesting activation of pro-survival autophagy. Surprisingly, the expression levels of SQSTM1/p62 were either unchanged or increased following XRK3F2 treatment despite the increase of autophagosome formation, indicating that XRK3F2 inhibited the autophagic flux in MM cells. Defective autophagy occurs in tumor cells and leads to accumulation of p62, further promoting tumorigenesis by altering NFκB regulation and gene expression. We found that XRK3F2 treatment also significantly inhibited TNFα-induced NFκB activation, by inhibiting p65 nuclear translocation. These data suggested that XRK3F2 may sensitize MM cells to BTZ-induced cytotoxicity and possibly overcome BTZ resistance.To test this hypothesis, human MM cell lines MM1.S (p53 wild type), U266 (p53 mutant) and KMS-11 (p53 null) were exposed to different doses of a BTZ-XRK3F2 combination comprised of varying concentrations of BTZ combined with XRK3F2 at 5μM. We found treatment with the BTZ-XRK3F2 combination significantly reduced cell viability compared with either drug alone at the same doses, and increased MM cells sensitivity to BTZ, independent of their p53 status (IC50 BTZ vs BTZ-XRK3F2 for MM1.S 3 vs 1.5nM of BTZ; for U266 10 vs 1.7nM of BTZ; for KMS-11 7 vs 1nM BTZ).Mechanistic studies revealed that in p53 wild type MM cells, the combination (Bortezomib 3nM - XRK3F2 5 μM) significantly increased annexin V positive cells after 6h of treatment. This was followed by a reduction in expression of the anti-apoptotic Bcl-2 protein (mRNA and protein levels) and extensive activation of caspase 3 and cleaved PARP1 after 24h, demonstrating apoptosis-mediated cell death. Importantly, p62 expression was significantly reduced by the BTZ-XRK3F2 combination. The decrease in p62 level may result as a consequence of its caspase 3-dependent degradation. In p53 mutant cells, the combination significantly induced PARP1 cleavage, although only mildly increased caspase 3 activation. These results imply that the BTZ-XRK3F2 combination activates multiple parallel death pathways to enhance both apoptosis and necrosis.Taken together, these data suggest that p62-ZZ antagonists, alone or in combination with PIs, may have both anti-tumor and bone anabolic effects, and may overcome PI resistance in MM cells, making p62 an attractive target for the treatment of MM. DisclosuresXie:Oxis Biotech: Consultancy. Roodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Herpes Simplex Virus Type 2 Glycoprotein D Inhibits NF-κB Activation by Interacting with p65.

NF-κB plays a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. HSV type 2 (HSV-2) is one of the most predominant sexually transmitted pathogens worldwide, and its infection increases the risk of HIV type 1 (HIV-1) acquisition and transmission. HSV-2 glycoprotein D (gD), highly homologous to HSV-1 gD, is essential for viral adhesion, fusion, entry, and spread. It is known that HSV-1 gD can bind herpesvirus entry mediator (HVEM) to trigger NF-κB activation and thereby facilitate viral replication at the early stage of infection. In this study, we found that purified HSV-2 gD triggered NF-κB activation at the early stage of infection, whereas ectopic expression of HSV-2 gD significantly downregulated TNF-α-induced NF-κB activity as well as TNF-α-induced IL-6 and IL-8 expression. Mechanistically, HSV-2 gD inhibited NF-κB, but not IFN-regulatory factor 3 (IRF3), activation and suppressed NF-κB activation mediated by overexpression of TNFR-associated factor 2 (TRAF2), IκB kinase α (IKKα), IKKβ, or p65. Coimmunoprecipitation and binding kinetic analyses demonstrated that HSV-2 gD directly bound to the NF-κB subunit p65 and abolished the nuclear translocation of p65 upon TNF-α stimulation. Mutational analyses further revealed that HSV-2 gD interacted with the region spanning aa 19-187 of p65. Findings in this study together demonstrate that HSV-2 gD interacts with p65 to regulate p65 subcellular localization and thereby prevents NF-κB-dependent gene expression, which may contribute to HSV-2 immune evasion and pathogenesis.