NF-κB Target Genes Research Articles

Knockout of LRRC8A Anion Channels in Vascular Smooth Muscle Cells Impairs NF-κB and Hif1α-mediated Signaling

Vascular smooth muscle cell (VSMC) inflammation associated with the development of vascular disease, including hypertension. We have demonstrated that knockout (KO) or inhibition of LRRC8A anion channels in VSMCs attenuates reactive oxygen species (ROS) production by NADPH oxidase 1, impairs TNFα-induced inflammation, and promotes vascular relaxation in murine mesenteric arteries. To explore the mechanism of these effects we performed RNA sequencing (RNAseq) on LRRC8A wild-type (WT) and knockout (KO) VSMCs (N = 3 pairs) under control conditions and 6hrs following TNFα treatment (10ng/ml). The data were normalized by Median ratio (DESeq2) and differential expression was determined by DESeq2 method using Partek software and pathway enrichment analysis was done by KEGG pathway. Differential expression of genes of interest was confirmed by quantitative PCR (qPCR) in both cultured cells and in whole aortic tissues (endothelium-denuded, N = 3) from LRRC8A WT and KO mice. HIF1α transcriptional activity was quantified by luciferase reporter assays in cells exposed to hypoxia (1% oxygen) or cobalt chloride (300μM) for 6 hrs. RNAseq demonstrated broad impairment of pro-inflammatory signaling in KO cells. This was consistent with previous observations of impaired nuclear factor kappa B (NF-κB) reporter activation by TNFα in the absence of LRRC8A. Compared to WT VSMCs, resting LRRC8A KO VSMCs exhibited statistically significant downregulation of 33 NF-κB target genes including the IL-6 (Il6) and IL-1β (Il1r1) receptors. TNFα treatment increased the expression of 37 NF-κB target genes in WT cells. Most of these genes (34 genes) were not significantly activated by TNFα in KO cells, while 3 genes (chemokine (C-C motif) ligand 20; CCL20, prostaglandin E synthase; PTGES and vascular endothelial growth factor C; VEGFC) were significantly downregulated. Pathway analysis data revealed that Hif1α signaling appear in the top downregulated pathway and it was impaired as 14 Hif1α target genes were downregulated in unstimulated KO VSMCs. In addition, expression of Rps4i, a long non-coding mRNA associated with inhibition of HIF1α signaling was significantly upregulated. Luciferase reporter assays following exposure to hypoxia or cobalt chloride confirmed impaired Hif1α activation in LRRC8A KO cells. Important target genes for HIF1α, including HK2, LDHB, ACE, BMP4 and PFKP, were less activated, while RPS4i remained upregulated compared to WT cells. These findings demonstrate disruption of both NF-κB and HIF1α signaling in VSMCs lacking LRRC8A. This anion channel represents a novel target for anti-inflammatory therapy in vascular disease. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

Abstract 3995: A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells

Abstract Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable success in hematological malignancies but only modest efficacy in solid tumors. Apoptosis induced by Fas receptor signaling can be a substantial barrier to CAR T-cell antitumor activity, as many solid tumors express high levels of Fas ligand (FasL). Here we describe a novel synthetic construct where the ectodomain of Fas receptor is fused to the signaling endodomain of a stimulatory receptor, creating a Fas signal redirect receptor (Fas-SRR). This unique design not only prevents Fas-dependent cell death, but also augments the long-term killing potential of induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) cells. Fas-SRR constructs with unique T-cell activation signaling endodomains were designed and compared to two control arms: a dominant negative receptor (Fas-DNR), which blocks Fas signal but does not promote activation; and a Fas-41BB fusion receptor, where the 41BB endodomain is fused to FasR ectodomain to direct activation upon FasL:Fas engagement. Fas-SRR designs were validated in Jurkat reporter cells treated with Fas agonist to measure signal redirection via Fas-SRR. To conduct a head-to-head comparison, CAR-iT cells were engineered to express a CAR under the regulation of the TRAC locus and to express either a Fas-DNR, a Fas-41BB, or FAS-SRR constructs at high purity (>90% transgene expression). No major differences were seen in maturation phenotype (>95% TRAC-CAR expression) of these cells, indicating compatibility of the FAS-SRR constructs with CAR-iT cell generation. Functional testing of the Fas-SRR+ CAR-iT cells was performed in a long-term restimulation assay where effector cells were repeatedly exposed to target cells over multiple rounds of co-culture. Complete loss of target cell lysis was observed by round six in the co-cultures with FAS-DNR cells, where loss of CAR-iT cell persistence led to accumulation of target cells. After eight rounds of co-culture, the Fas-41BB cells and the majority of Fas-SSR cell constructs also began to exhibit minimal activity toward target cells. However, the top Fas-SRR cell constructs continued to show durable antitumor activity (>97% cytolysis) through the end of the 9-round rechallenge assay. Of note, GSEA analysis identified significant upregulation of NFkB and RELA target genes in cells having the top FAS-SRR construct versus the less durable designs. These results reinforce the advantage of attenuating Fas-FasL interactions to prevent apoptosis and, more importantly, demonstrate the unique therapeutic value in redirecting Fas signaling to improve T-cell potency over an extended duration. Additional studies are underway to further investigate the implications of Fas signal redirection in CAR T and CAR-iT cells and to further understand the module of genes involved in supporting serial stimulation performance. Citation Format: Christopher Ecker, Eigen Peralta, Hui-Yi Chu, Lorraine Loter, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells [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 3995.

Abstract 52: CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells

Abstract Background: Common failures of chimeric antigen receptor (CAR)-T cell therapy regimens stem from immune escape of target antigen low tumors. Strategies to improve CAR-T cell therapy efficacy include enhancing intrinsic effector function while preventing T cell exhaustion. The ubiquitin-modifying enzyme A20, is a key negative regulator of the NF-kB pathway downstream of T cell receptor activation in T cells. Dysfunctional CD8 T cells in tumors have high expression of A20, and deletion of A20 enhances antitumor activity of CD8 T cells in murine tumor models. We tested whether CRISPR-mediated knock-out (KO) of tumor necrosis factor α-induced protein 3 (TNFAIP3), the gene for A20, increases the effector function and alters the metabolic state of CAR-T cells. Results: A20 deletion led to upregulation of NF-κB target genes and resulted in higher levels of activation marker expression and effector cytokine secretion compared to unarmored CAR-T upon incubation with target cells. In addition, A20 KO CAR-T cells maintained prolonged expression of activation markers upon antigen clearance in the absence of enhanced differentiation, and were more resistant to dysfunction in a repeat challenge serial kill assay. In vivo, A20 deficient CAR-T exhibited increased anti-tumor efficacy against low, medium, and high target expressing tumor models. Consistent with NF-κB upregulation, A20 KO cells significantly upregulated key glycolytic intermediates suggesting that amino acid anabolism was fueled by glycolysis and the pentose phosphate pathway. Moreover, the preferential increase of glycolysis over oxidative phosphorylation confirmed that A20 KO CAR-T cells were skewed towards an effector-like phenotype. Notably, glutathione and its cysteine-glycine bi-products were upregulated upon activation to a higher level in A20 KO CAR-T suggesting enhanced resistance to oxidative stress, in line with their prolonged persistence. Conclusions: CRISPR-mediated knock-out of A20 lowers the activation threshold of CAR-T cells producing enhanced in vitro and in vivo anti-tumor efficacy particularly against tumors with very low levels of target expression. A20 KO induces metabolic rewiring of CAR-T cells by promoting glycolysis and amino acid anabolism to sustain their superior effector functions. This intrinsic armoring strategy can be applied to any CAR-T in any indication to increase tumor control and reduce immune escape. Citation Format: Nina Jia Chu, Carina Nava-Barbero, Ashley Merlino, Ping-Hsien Lee, David Baker, Ben Taylor, Gordon Moody, Letizia Giardino. CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells [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 52.

WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-κB termination.

Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of the RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we find that two structurally similar substrate-recognizing components of Cullin-RING E3 ligases, WSB1 and WSB2, can recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. We showed that WSB1/2 negatively regulated a subset of NF-κB target genes via associating with chromatin where they targeted methylated RelA for ubiquitination, facilitating the termination of NF-κB-dependent transcription. WSB1/2 specifically interacted with methylated lysines (K) 314 and 315 of RelA via their N-terminal WD-40 repeat (WDR) domains, thereby promoting ubiquitination of RelA. Computational modeling further revealed that a conserved aspartic acid (D) at position 158 within the WDR domain of WSB2 coordinates K314/K315 of RelA, with a higher affinity when either of the lysines is methylated. Mutation of D158 abolished WSB2's ability to bind to and promote ubiquitination of methylated RelA. Together, our study identifies a novel function and the underlying mechanism for WSB1/2 in degrading chromatin-bound methylated RelA and preventing sustained NF-κB activation, providing potential new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

Hepatoprotective effects of Elaeagnus latifolia fruit extract against acetaminophen-induced hepatotoxicity in mice: Mechanistic insights

Elaeagnus latifolia fruit extract (EE) has been reported to contain antioxidant and anti-inflammatory phytochemicals. The present study aimed to examine the anti-hepatotoxic effects of EE in acetaminophen (APAP)-injected mice. Pretreatment with EE for 7 days in APAP mice showed significant reductions in the levels of AST and ALT and necrotic area, accompanied by decreases in liver oxidative stress, nitric oxide, and infiltrating inflammatory cells, compared to the APAP-only group. EE markedly down-regulated expression of CYP2E1, JNK, Bax, NOX subunit, and NF-κB target genes, and up-regulated expression of Bcl-2 and Nrf2 target genes in the livers. Furthermore, EE effectively lowered serum and hepatic lipids, and down-regulated expression of SREBP-1c/2 genes. EE was found to contain phenolic compounds, flavonoids, and proanthocyanidins. Therefore, mechanistic insights into EE's anti-hepatotoxic effects may be associated with preventing the abnormal expression of genes involved in toxic metabolite generation, hepatotoxic signaling, apoptosis, oxidative stress, inflammation, and lipid synthesis.

The pomegranate-derived peptide Pug-4 alleviates nontypeable Haemophilus influenzae-induced inflammation by suppressing NF-kB signaling and NLRP3 inflammasome activation.

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is the most common cause of exacerbation of chronic obstructive pulmonary disease (COPD), of which an excessive inflammatory response is a hallmark. With the limited success of current medicines there is an urgent need for the development of novel therapeutics that are both safe and effective. In this study, we explored the regulatory potential of pomegranate-derived peptides Pug-1, Pug-2, Pug-3, and Pug-4 on NTHi-induced inflammation. Our results clearly showed that to varying degrees the Pug peptides inhibited NTHi-induced production of IL-1β, a pivotal cytokine in COPD, and showed that these effects were not related to cytotoxicity. Pug-4 peptide exhibited the most potent inhibitory activity. This was demonstrated in all studied cell types including murine (RAW264.7) and human (differentiated THP-1) macrophages as well as human lung epithelial cells (A549). Substantial reduction by Pug-4 of TNF-α, NO and PGE2 in NTHi-infected A549 cells was also observed. In addition, Pug-4 strongly inhibited the expression of nuclear-NF-κB p65 protein and the NF-κB target genes (determined by IL-1β, TNF-α, iNOS and COX-2 mRNA expression) in NTHi-infected A549 cells. Pug-4 suppressed the expression of NLRP3 and pro-IL-1β proteins and inhibited NTHi-mediated cleavage of caspase-1 and mature IL-1β. These results demonstrated that Pug-4 inhibited NTHi-induced inflammation through the NF-κB signaling and NLRP3 inflammasome activation. Our findings herein highlight the significant anti-inflammatory activity of Pug-4, a newly identified peptide from pomegranate, against NTHi-induced inflammation. We therefore strongly suggest the potential of the Pug-4 peptide as an anti-inflammatory medicine candidate for treatment of NTHi-mediated inflammation.

Lysine-36 of Drosophila histone H3.3 supports adult longevity.

Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several post-translational histone modifications have been linked to the maintenance of longevity. Here, we focus on lysine-36 of the replication-independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine-to-arginine mutant that blocks the activity of its cognate-modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in nondividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposons and by suppressing aberrant immune signaling.

Poly-(ADP-ribose)polymerases inhibitionby olaparib attenuates activities of the NLRP3 inflammasome and of NF-κB in THP-1 monocytes.

Poly-(ADP-ribose) polymerases (PARPs) are a protein family that make ADP-ribose modifications on target genes and proteins. PARP family members contribute to the pathogenesis of chronic inflammatory diseases, including atherosclerosis, in which monocytes/macrophages play important roles. PARP inhibition is protective against atherosclerosis. However, the mechanisms by which PARP inhibition exerts this beneficial effect are not well understood. Here we show that in THP-1 monocytes, inhibition of PARP by olaparib attenuated oxidized low-density lipoprotein (oxLDL)-induced protein expressions of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing-3 (NLRP3) inflammasome components: NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. Consistent with this effect, olaparib decreased oxLDL-enhanced interleukin (IL)-1β and IL-18 protein expression. Olaparib also decreased the oxLDL-mediated increase in mitochondrial reactive oxygen species. Similar to the effects of the NLRP3 inhibitor, MCC950, olaparib attenuated oxLDL-induced adhesion of monocytes to cultured human umbilical vein endothelial cells and reduced foam cell formation. Furthermore, olaparib attenuated the oxLDL-mediated activation of nuclear factor (NF)-κB through the oxLDL-mediated increase in IκBα phosphorylation and assembly of NF-κB subunits, demonstrated by co-immunoprecipitation of IκBα with RelA/p50 and RelB/p52 subunits. Moreover, PARP inhibition decreased oxLDL-mediated protein expression of a NF-κB target gene, VCAM1, encoding vascular cell adhesion molecule-1. This finding indicates an important role for NF-κB activity in PARP-mediated activation of the NLRP3 inflammasome. Thus, PARP inhibition by olaparib attenuates NF-κB and NLRP3 inflammasome activities, lessening monocyte cell adhesion and macrophage foam cell formation. These inhibitory effects of olaparib on NLRP3 activity potentially protect against atherosclerosis.

Lawsonia intracellularis regulates nuclear factor-κB signalling pathway during infection.

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells.

E3 ubiquitin ligase BCA2 promotes breast cancer stemness by up-regulation of SOX9 by LPS.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. Breast cancer stem cells (BCSCs) are believed to play a crucial role in the carcinogenesis, therapy resistance, and metastasis of TNBC. It is well known that inflammation promotes stemness. Several studies have identified breast cancer-associated gene 2 (BCA2) as a potential risk factor for breast cancer incidence and prognosis. However, whether and how BCA2 promotes BCSCs has not been elucidated. Here, we demonstrated that BCA2 specifically promotes lipopolysaccharide (LPS)-induced BCSCs through LPS induced SOX9 expression. BCA2 enhances the interaction between myeloid differentiation primary response protein 88 (MyD88) and Toll-like receptor 4 (TLR4) and inhibits the interaction of MyD88 with deubiquitinase OTUD4 in the LPS-mediated NF-κB signaling pathway. And SOX9, an NF-κB target gene, mediates BCA2's pro-stemness function in TNBC. Our findings provide new insights into the molecular mechanisms by which BCA2 promotes breast cancer and potential therapeutic targets for the treatment of breast cancer.

Balance between Pro- and Antifibrotic Proteins in Mesenchymal Stromal Cell Secretome Fractions Revealed by Proteome and Cell Subpopulation Analysis.

Multipotent mesenchymal stromal cells (MSCs) regulate tissue repair through paracrine activity, with secreted proteins being significant contributors. Human tissue repair commonly results in fibrosis, where fibroblast differentiation into myofibroblasts is a major cellular mechanism. MSCs' paracrine activity can inhibit fibrosis development. We previously demonstrated that the separation of MSC secretome, represented by conditioned medium (CM), into subfractions enriched with extracellular vesicles (EV) or soluble factors (SF) boosts EV and SF antifibrotic effect. This effect is realized through the inhibition of fibroblast-to-myofibroblast differentiation in vitro. To unravel the mechanisms of MSC paracrine effects on fibroblast differentiation, we performed a comparative proteomic analysis of MSC secretome fractions. We found that CM was enriched in NF-κB activators and confirmed via qPCR that CM, but not EV or SF, upregulated NF-κB target genes (COX2, IL6, etc.) in human dermal fibroblasts. Furthermore, we revealed that EV and SF were enriched in TGF-β, Notch, IGF, and Wnt pathway regulators. According to scRNAseq, 11 out of 13 corresponding genes were upregulated in a minor MSC subpopulation disappearing in profibrotic conditions. Thus, protein enrichment of MSC secretome fractions and cellular subpopulation patterns shift the balance in fibroblast-to-myofibroblast differentiation, which should be considered in studies of MSC paracrine effects and the therapeutic use of MSC secretome.

Retraction: Loss of Metallothionein Predisposes Mice to Diethylnitrosamine-Induced Hepatocarcinogenesis by Activating NF-κB Target Genes.

Retraction: Loss of Metallothionein Predisposes Mice to Diethylnitrosamine-Induced Hepatocarcinogenesis by Activating NF-κB Target Genes.

Liver receptor homolog-1 (NR5A2) orchestrates hepatic inflammation and TNF-induced cell death

The nuclear receptor liver receptor homolog-1 (LRH-1) has been shown to promote apoptosis resistance in various tissues and disease contexts; however, its role in liver cell death remains unexplored. Hepatocyte-specific deletion of LRH-1 causes mild steatosis and inflammation but unexpectedly shields female mice from tumor necrosis factor (TNF)-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes show markedly attenuated estrogen receptor alpha and elevated nuclear factor κB (NF-κB) activity, while LRH-1 overexpression inhibits NF-κB activity. This inhibition relies on direct physical interaction of LRH-1's ligand-binding domain and the Rel homology domain of NF-κB subunit RelA. Mechanistically, increased transcription of anti-apoptotic NF-κB target genes and the proteasomal degradation of pro-apoptotic BCL-2 interacting mediator of cell death prevent mitochondrial apoptosis and ultimately protect mice from TNF-induced liver damage. Collectively, our study emphasizes LRH-1 as a critical, sex-dependent regulator of cell death and inflammation in the healthy and diseased liver.

MALT1 Protease Inhibition Overcomes BTK Inhibitor Resistance and Shows Synergistic Activity with Venetoclax in Models of B Cell Lymphoma and Leukemia

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Anti-Inflammatory Activity of the Constituents from the Leaves of Perilla frutescens var. acuta.

Perilla frutense var. acuta (Lamiaceae) has been used to treat indigestion, asthma, and allergies in traditional medicine. In this study, luteolin 7-O-diglucuronide (1), apigenin 7-O-diglucuronide (2), and rosmarinic acid (3) were isolated from the leaves of P. frutescens var. acuta through various chromatographic purification techniques. Several approaches were used to investigate the anti-inflammatory activity of the constituents (1-3) and their working mechanisms. In silico docking simulation demonstrated that 1-3 would work as a PPAR-α/δ/γ agonist, and in vitro PPAR-α/δ/γ transcriptional assay showed that the Perilla water extract (PWE) and 3 increased PPAR-α luciferase activity (1.71 and 1.61 times of the control (PPAR-α + PPRE, p < 0.001)). In the NF-κB luciferase assay, 1 suppressed NF-κB activity the most (56.83% at 5 µM; 74.96% at 10 µM; 79.86% at 50 µM). In addition, 1 and 2 inhibited the mRNA expression of NF-κB target genes, including Il6, Mcp1, and Tnfa, at 50 µM, and 3 suppressed the genes at the mRNA level in a dose-dependent manner. We report that 1 and 2 exert anti-inflammatory effects through NF-κB inhibition, and the PPAR-α/NF-κB signaling pathway is related to the anti-inflammatory activity of 3.

Targeting PPARγ/ NF-κB Signaling Pathway by Britannin, a Sesquiterpene Lactone from Inula aucheriana DC., in Gastric Cancer.

Gastric cancer is one of the most common and deadliest malignancies in the world. Therefore, there is an urgent need to develop new and effective agents to reduce mortality. The plants of genus Inula have gained the attention of researchers worldwide as a rich source of potent medicinal compounds. This study explores the anti-cancer activity of Britannin, a sesquiterpene lactone isolated from Inula aucheriana DC., and its molecular mechanism in gastric cancer cells, AGS and MKN45. Cytotoxicity was evaluated through the MTT assay following 24 h, 48 h, and 72 h treatment with different concentrations of Britannin. Apoptosis rate and caspase-3 activity were measured 24 h after treatment by Britannin. . Western blotting was performed to determine the expression of the NF-κB, IκBα, and PPARγ proteins. Moreover, quantitative RT-PCR was applied to measure the expression of NF-κB target genes. We showed that Britannin induced cell growth inhibition and apoptosis in gastric cancer cells. Britannin caused an elevation in mRNA and protein levels of PPARγ. The involvement of PPARγ was more confirmed using GW9662, a PPARγ inhibitor. Suppression of NF-κB was demonstrated by western blot analysis. Down-regulation of MMP-9, TWIST-1, COX-2, and Bcl-2 and up-regulation of Bax were also observed in gastric cancer cells. These results imply that activation of the PPARγ signaling pathway through suppression of NF-κB underlies the anti-cancer properties of Britannin in gastric cancer. Therefore, Britannin could be considered as a promising anti-cancer candidate for further evaluation.

Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma.

The overactivation of NF-κB signaling is a key hallmark for the pathogenesis of extranodal natural killer/T cell lymphoma (ENKTL), a very aggressive subtype of non-Hodgkin's lymphoma yet with rather limited control strategies. Previously, we found that the dysregulated exportin-1 (also known as CRM1) is mainly responsible for tumor cells to evade apoptosis and promote tumor-associated pathways such as NF-κB signaling. Herein we reported the discovery and biological evaluation of a potent small molecule CRM1 inhibitor, LFS-1107. We validated that CRM1 is a major cellular target of LFS-1107 by biolayer interferometry assay (BLI) and the knockdown of CRM1 conferred tumor cells with resistance to LFS-1107. We found that LFS-1107 can strongly suppresses the growth of ENKTL cells at low-range nanomolar concentration yet with minimal effects on human platelets and healthy peripheral blood mononuclear cells. Treatment of ENKTL cells with LFS-1107 resulted in the nuclear retention of IkBα and consequent strong suppression of NF-κB transcriptional activities, NF-κB target genes downregulation and attenuated tumor cell growth and proliferation. Furthermore, LFS-1107 exhibited potent activities when administered to immunodeficient mice engrafted with human ENKTL cells. Therefore, LFS-1107 holds great promise for the treatment of ENKTL and may warrant translation for use in clinical trials. Yang's laboratory was supported by the National Natural Science Foundation of China (Grant: 81874301), the Fundamental Research Funds for Central University (Grant: DUT22YG122) and the Key Research project of 'be Recruited and be in Command' in Liaoning Province (Personal Target Discovery for Metabolic Diseases).

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes’ clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

KDM2A Deficiency in the Liver Promotes Abnormal Liver Function and Potential Liver Damage.

Dysregulation of metabolic functions in the liver impacts the development of diabetes and metabolic disorders. Normal liver function can be compromised by increased inflammation via the activation of signaling such as nuclear factor (NF)-κB signaling. Notably, we have previously identified lysine demethylase 2A (KDM2A)-as a critical negative regulator of NF-κB. However, there are no studies demonstrating the effect of KDM2A on liver function. Here, we established a novel liver-specific Kdm2a knockout mouse model to evaluate KDM2A's role in liver functions. An inducible hepatic deletion of Kdm2a, Alb-Cre-Kdm2afl/fl (Kdm2a KO), was generated by crossing the Kdm2a floxed mice (Kdm2afl/fl) we established with commercial albumin-Cre transgenic mice (B6.Cg-Tg(Alb-cre)21Mgn/J). We show that under a normal diet, Kdm2a KO mice exhibited increased serum alanine aminotransferase (ALT) activity, L-type triglycerides (TG) levels, and liver glycogen levels vs. WT (Kdm2afl/fl) animals. These changes were further enhanced in Kdm2a liver KO mice in high-fat diet (HFD) conditions. We also observed a significant increase in NF-κB target gene expression in Kdm2a liver KO mice under HFD conditions. Similarly, the KO mice exhibited increased immune cell infiltration. Collectively, these data suggest liver-specific KDM2A deficiency may enhance inflammation in the liver, potentially through NF-κB activation, and lead to liver dysfunction. Our study also suggests that the established Kdm2afl/fl mouse model may serve as a powerful tool for studying liver-related metabolic diseases.