Target Genes In Cells Research Articles

4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study.

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4β,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4β,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Abstract PO4-14-01: Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy

Abstract Breast cancer (BC) is a complex disease with distinct subtypes, including triple-negative breast cancer (TNBC), which lacks targeted therapies. CDK8 and CDK19, components of the mediator complex involved in transcriptional regulation, have emerged as potential therapeutic targets in BC. Transcriptomic analysis revealed that elevated CDK8 expression was associated with poor prognosis across all BC subtypes. Furthermore, previous studies demonstrated that sensitivity to CDK8/CDK19 inhibition correlated with high STAT3 phosphorylation and enrichment of TNF/NF-kB and STAT target genes in responder cells. In this study, we evaluated the responsiveness of TNBC cell lines to RVU120, a highly selective and potent CDK8/CDK19 inhibitor currently undergoing clinical trials for acute myeloid leukemia (AML), high-risk myelodysplastic syndrome (HR-MDS), and solid tumors. Notably, the sensitivity of TNBC cell lines to RVU120 varied depending on the culture conditions, with the presence of epidermal growth factor (EGF) identified as a major determinant of response variability. Considering the role of EGFR in activating downstream signaling pathways, including MAPK and STAT, and previous observations of synergy between small molecule CDK8 inhibitors and MEK inhibitors in neuroblastoma, we investigated MEK inhibitors as potential combination partners in hormone-negative BC. Among the 15 tested BC cell lines, a synergy between CDK8/CDK19 inhibitors and MEK inhibitors was observed in 5 cell lines, antagonism in another 5 cell lines, and no synergy in the remaining 5 cell lines, based on the analysis with Combenefit software (Loewe additivity model). Importantly, cell lines where synergy of the combination therapy has been observed, exhibited EGFR amplification and markers of activated RAS pathway. This pattern of response was consistent across different MEK and MEK/RAF inhibitors, including selumetinib, trametinib, and avutometinib. These findings highlight the synergistic potential of combining RVU120 with MEK inhibitors in hormone-negative BC, particularly in TNBC with EGFR amplification and an active RAS pathway. Ongoing studies involving RNAseq analysis and organoid screening aim to further elucidate the underlying mechanisms and confirm the efficacy of this combination therapy. Furthermore, in vivo efficacy experiments are necessary to validate in vitro activity of both compounds. Successful development of this therapeutic concept relies on the validation and implementation of selected partner drugs targeting RAS pathway, as well as the identification of suitable biomarkers such as EGFRamp or RAS-score. Promising findings of this study provide a rationale for exploring the combination of CDK8/CDK19 inhibitor RVU120 and MEK inhibitors as a potential targeted therapy strategy for hormone-negative BC. Citation Format: Urszula Pakulska, Adrianna Moszynska, Justyna Martyka, Jerzy Woznicki, Elzbieta Adamczyk, Marta Obacz, Kinga Keska Izworska, Katarzyna Wiklik, Agata Stachowicz, Hendrik Nogai, Milena Mazan, Tomasz Rzymski. Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-01.

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.

A statistical approach for identifying single nucleotide variants that affect transcription factor binding

Non-coding variants located within regulatory elements may alter gene expression by modifying transcription factor (TF) binding sites, thereby leading to functional consequences. Different TF models are being used to assess the effect of DNA sequence variants, such as single nucleotide variants (SNVs). Often existing methods are slow and do not assess statistical significance of results. We investigated the distribution of absolute maximal differential TF binding scores for general computational models that affect TF binding. We find that a modified Laplace distribution can adequately approximate the empirical distributions. A benchmark on invitro and invivo datasets showed that our approach improves upon an existing method in terms of performance and speed. Applications on eQTLs and on a genome-wide association study illustrate the usefulness of our statistics by highlighting cell type-specific regulators and target genes. An implementation of our approach is freely available on GitHub and as bioconda package.

Abstract LB033: Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells

Abstract Because of the Hippo pathway’s unique ability to restrict TAZ/YAP-TEAD in promoting regeneration, any abnormality of its core components, may promote the migration, invasion, and malignancy of cancer cells. Aberrant overexpression of YAP/TAZ-TEAD promotes tumorigenesis and is therefore considered oncogenes in many solid tumors. Drug resistance is a major factor undermining the efficacy of cancer drugs. As YAP/TAZ-TEAD complex plays a role in intrinsic and acquired resistance to various chemotherapeutic and targeted therapy drugs, there is increasing interest in combining a TEAD inhibitor with various cancer therapies. Since elevated YAP/TAZ-TEAD activity has been implicated in multiple cancer types at various stages of cancer progression and cancer types, Drugging the TEAD hydrophobic pocket, which was discovered in 2015, remains as an attractive and proven strategy to modulate the activity. Despite the progresses, only 3 small molecule TEAD inhibitors are currently being tested in Phase I clinical trials which includes: VT3989b NCT04665206 from Vivacare, IK-930b NCT05228015 from Ikena Oncology and IAG-933, NCT0485737 from the Novartis Oncology. As such, there is a need to uncover alternative TEAD inhibitors for cancer therapeutics. We report novel small molecule inhibitors that form a covalent complex with the conserved cysteine of the TEAD palmitoylation site. Compounds inhibited TEAD transcriptional target genes in cancer cells. Cellular proliferation and colony forming assay data strongly displayed cellular sensitivity in Hippo signaling altered cancer cell lines. TEAD1 dependency of gastric cancer cell lines enhance cellular sensitivity in response to small molecule inhibitors. Transcriptional profiling and RNA seq data indicate that the covalent inhibitors specifically downregulate down-stream transcriptional target genes. With our exciting key data, we are exploring multiple other oncology targets to use our Pan-TEAD inhibitors either as a monotherapy or combination therapy. We expect to complete our Technology Disclosure on additional targets before the meeting and to add exciting data for the Cancer drug development community. Citation Format: Ramesh Kumar, Joel Toh, Joanne Thian, Noorul Farzana Mohideen, Jialin Sun, Sayan Chakraborty, Jayantha Gunaratne, Wanjin Hong. Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB033.

Targeting histone demethylases JMJD3 and UTX: selenium as a potential therapeutic agent for cervical cancer

BackgroundThe intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease.ResultSelenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets.ConclusionsOur study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

Dietary phytochemicals as regulators of gut inflammation in the context of type 2 diabetes

Numerous disorders, including type 2 diabetes and even COVID-19, are linked to poor gut health and inflammation(1,2). In addition to impacting food digestion and absorption, gut inflammation worsens diabetes outcomes by causing gut microbial dysbiosis, disrupting tight junctions (allowing microbial metabolites to freely enter into circulation), and altering glucose absorption(3). ACE2 is a crucial regulator of gut health and has received much attention during the COVID-19 pandemic due to its role as a major viral entry protease. Studies have shown that the ACE2/Ang-(1-7)/Mas axis is important in managing inflammation and maintaining normal glucose metabolism(3). Dietary phytochemicals are plant bioactive compounds, with promising anti-inflammatory and anti-diabetic properties, and may affect these processes. In this work, we aimed to look at the link between inflammation, ACE2 and the glucose transporters, SGLT1 and GLUT2, and how phytochemicals could be used to normalise the changes brought about by inflammation in Caco-2/TC7 human intestinal epithelium cells. We first examined how gut inflammation, ACE2 and glucose transporters are related and proceeded to look at the effect of some chosen phytochemicals on regulating glucose transport via modulation of the ACE2/Ang(1-7)/Mas axis. This included genistein (an isoflavone from soyabeans), sulforaphane (an isothiocyanate found in Brassica, especially broccoli), apigenin (a flavone found in vegetables and herbs), and artemisinin (a sesquiterpene lactone used as a drug). The impact of phytochemicals on the SARS-CoV-2 viral entry receptors, ACE2 and TMPRSS2, was also examined as a secondary outcome. To induce inflammation, the Caco2/TC7 cells were co-stimulated with IL-1β (25 ng/mL) and TNF-α (50 ng/mL) for varying durations (24 h, 48 h, 72 h, 168 h) and changes in target gene expression (ACE2, SGLT1, GLUT2, TMPRSS2) were assessed by droplet digital PCR. IL-6 and IL-8 were assessed as markers of inflammation in the cell culture media by multiplex ELISA. Inflammation increased ACE2, TMPRSS2 and SGLT1 mRNA. ACE2 increased with cytokine exposure duration, coupled with an obvious decrease in IL-8, SGLT1 and TMPRSS2. Pearson correlation analysis revealed that the increase in ACE2 was strongly associated with decreases in SGLT1 (r = −0.99, p<0.01) and IL-8 (r = −0.959, p<0.05), implying ACE2 to play a crucial role in gut inflammation and postprandial glycaemia. After establishing the gut cell inflammation model, we compared the effect of the phytochemicals on our target genes in cells cultured in normal and pro-inflammatory environments. None of the tested phytochemicals were effective in reducing IL-8 secretion, while phytochemicals showed varying effects on the target genes. Genistein normalised the effects of inflammation on the target genes with less effect from the other tested phytochemicals. However, further research is required to assess the importance of genistein in vivo in the context of gut inflammation and type 2 diabetes.

Extracellular Vesicles Contribute to Viral-Induced Hepatocellular Carcinoma: Understanding Their Involvement in Viral Hepatitis and Their Potential as Biomarkers for Early Hepatocellular Carcinoma Detection.

The high global prevalence of hepatitis B and hepatitis C and the poor prognosis of hepatitis B and hepatitis C-associated hepatocellular carcinoma (HCC), necessitates the early diagnosis and treatment of the disease. Recent studies show that cell-to-cell communication via extracellular vesicles (EVs) is involved in the HCC progression. The objective of the following study was to explore the role of EVs in the progression of viral-induced HCC and investigate their potential for the early diagnosis of cancer. First, the mRNA derived from EVs of HCC patients was compared to the mRNA derived from EVs from the healthy controls. Expression analysis of ANGPTL3, SH3BGRL3, and IFITM3 genes from the EVs was done. Afterward, to confirm whether hepatocytes can uptake EVs, HuH7 cells were exposed to EVs, and the expression analysis of downstream target genes (AKT, TNF-α, and MMP-9) in Huh7 cells was done. Transcriptional analysis showed that in the EVs from HCC patients, the expression levels of ANGPTL3, SH3BGRL3, and IFITM3 were significantly increased by 2.62-, 4.3-, and 9.03-folds, respectively. The downstream targets, AKT, TNF-α, and MMP-9, also showed a considerable change of 4.1-, 1.46-, and 5.05-folds, respectively, in Huh7 cells exposed to HCC EVs. In conclusion, the following study corroborates the role of EVs in HCC progression. Furthermore, the significant alteration in mRNA levels of the selected genes demonstrates their potential to be used as possible biomarkers for the early diagnosis of HCC.

Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

USP40 promotes hepatocellular carcinoma progression through a YAP/USP40 positive feedback loop

Yes-associated protein (YAP) is an essential driver of hepatocellular carcinoma (HCC) progression and the ubiquitin-proteasome system controls its abundance. However, the role of ubiquitin-specific protease 40 (USP40) in YAP stability remains unclear. Here, USP40 was first identified as a novel regulator of YAP abundance and its target genes in HCC cells. USP40 interacted with YAP to remove the lysine 48 (K48)-linked polyubiquitination of YAP at K252 and K315 sites, thereby maintaining YAP stability. USP40 facilitated the proliferation, colony formation, migration and spheroid formation of HCC cells in vitro and promoted HCC growth in vivo in a YAP-dependent manner. In turn, YAP transcriptionally activated USP40 expression in HCC cells. RNA sequencing analysis showed that about 37% of USP40-regulated genes overlapped with YAP-regulated genes. Interestingly, stiffness-induced USP40 upregulation was abolished by YAP knockdown, and USP40 knockdown attenuated stiffness-induced YAP accumulation in HCC cells. Clinical data demonstrated that USP40 was positively associated with YAP expression in HCC tissues and its high expression indicated a poor prognosis. In conclusion, the USP40/YAP positive feedback loop contributes to HCC progression, suggesting that USP40 may be a promising drug target for anti-HCC.

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 5777: Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs)

Abstract The purpose of this study was to determine if EWS/FLI1 (E/F), the oncogenic fusion protein that drives Ewing Sarcoma (ES), can be rewired to enhance expression of BCL6-controlled genes using Transcriptional Chemical Inducers of Proximity (TCIPs), a novel bivalent small molecule platform. TCIP molecules recruit a transcriptional regulator, such as BRD4, to the transcriptional repressor BCL6. Upon binding, a ternary complex is formed on chromatin at BCL6-bound loci, causing transcriptional upregulation of BCL6 target genes. Since E/F does not have a bona fide ligand, we have used an exogenously expressed N terminal FKBPF36V-EWS/FLI1 (N-FK-E/F) fusion system to study how modulation of E/F by TCIPs impacts the transcriptome of ES cells. To eliminate background signal from endogenous E/F, we used CRISPR/Cas9 to knockout (KO) endogenous E/F. Our exogenously expressed N-FK-E/F has mutated PAM motifs and is resistant to KO. In our proof-of-concept study we used TCIP1 consisting of ortho-AP1867 (oAP), a synthetic ligand for FKBPF36V, linked to the BCL6 inhibitor BI3812 (BI) to rewire N-FK-E/F. We have also tested a negative control TCIP (TCIP NEG) that links oAP to a BI derivative that abolishes BCL6 binding. TCIP NEG has similar physiochemical properties to our active TCIP1; however, it cannot bind BCL6, serving as a negative control for ternary complex formation. TCIP1 rapidly and dose-dependently increases the RNA and protein expression of several BCL6 target genes, like p21, in multiple ES models that stably express N-FK-E/F. TCIP NEG does not increase the expression of BCL6 target genes in ES cells. TCIP1 induces ternary complex formation in ES cell lysates, and ternary complex formation is necessary for activity in ES cells. TCIP1 is specific and does not increase BCL6 target gene levels to the same extent in ES cells expressing a FKBP-GFP construct or in parental ES cells lacking N-FK-E/F. RNA sequencing of EWS502 N-FK-E/F expressing cells shows that TCIP1 treatment induces a larger increase in expression of BCL6 target gene RNA than BCL6 inhibition. Moreover, CUT&RUN experiments in EWS502 N-FK-E/F expressing cells suggest TCIP1 leads to an accumulation of N-FK-E/F at the promoters of BCL6 target genes. Motif analysis showed that TCIP1 increases the likelihood of N-FK-E/F binding to BCL6 consensus sequences as wells as increases the likelihood of BCL6 binding to E/F recognition sequences. In future studies we will conduct RNA sequencing, CUT&RUN, and other ‘omics’ approaches (e.g. DisP-seq) to compare TCIP1 to TCIP NEG in multiple cell lines at multiple concentrations and time points. Our data suggests that N-FK-E/F can be rewired to induce the expression of BCL6 target genes in ES. Since ES is driven solely by E/F, TCIPs may be promising next generation therapeutics. Our study provides a proof-of-concept that will be the foundation for the development of future TCIP molecules that recruit endogenous E/F once suitable ligands are developed. Citation Format: Michael J. Bond, Ryan P. Golden, Roman C. Sarott, Giulia DiGiovanni, Basel A. Karim, Briana Howard, Kenneth Ross, Nathanael S. Gray, Kimberly Stegmaier. Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs) [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 5777.

Integrating network pharmacology with microRNA microarray analysis to identify the role of miRNAs in thrombosis treated by the Dahuang Zhechong pill

Background:Thrombotic diseases are the leading causes of death worldwide, urging for improvements in treatment strategies. Dahuang Zhechong pill (DHZCP) is a traditional Chinese medicine widely used for treating thrombotic diseases; however, the underlying mechanisms remain unclear. This study aimed to explore the potential mechanisms of DHZCP in treating thrombosis with a focus on bioinformatics and miRNAs. Methods:We used network pharmacology to explore the targets of thrombosis treated with DHZCP and performed microarray analysis to acquire miRNA profiles and predict the target genes in thrombin-stimulated MEG-01 cells treated with DHZCP. Based on the overlapping of targets, we carried out a component-target-miRNA network and enrichment analysis and validated the selected miRNAs and mRNAs using quantitative reverse transcription-polymerase chain reaction. Results:Our data showed 850 targets of 230 active ingredients of DHZCP and 1214 thrombosis-related genes; 235 targets were common. We identified 32 miRNAs that were regulated by thrombin stimulation but regulated reversely by DHZCP treatment in MEG-01 cells, and predicted 1846 targets with function annotation. We analyzed conjointly 23 integrating targets from network pharmacology and microarray. HIF1A, PIK3CA, MAPK1 and BCL2L1 emerged as key nodes in the network diagrams. We confirmed the differential expression of seven miRNAs, one mRNA (BCL2L1) and platelet surface protein. Conclusions:This study showed that miRNAs and their targets, such as BCL2L1, played crucial roles in platelet activation during DHZCP intervention in thrombosis, highlighting their potential to alleviate platelet activation and increase cell apoptosis. The study’s findings could help develop new strategies for improving thrombosis treatment.

Salidroside inhibited the proliferation of gastric cancer cells through up-regulating tumor suppressor miR-1343-3p and down-regulating MAP3K6/MMP24 signal molecules

ABSTRACT Salidroside inhibited the proliferation of cancer cell. Nevertheless, the mechanism has not been completely clarified. The purpose of the study is to explore the mechanisms of salidroside against gastric cancer. To analyze the changes of microRNA (miRNA) in gastric cancer cells under the treatment of salidroside, the miRNA expression was analyzed by using RNA-seq in cancer cells for 24 h after salidroside treatment. The differentially expressed miRNAs were clustered and their target genes were analyzed. Selected miRNA and target mRNA genes were further verified by q-PCR. The expressions of target genes in cancer cells were detected by immunohistochemistry. Cancer cell apoptotic index was significantly increased after salidroside treatment. The proliferation of gastric cancer cells were blocked at S-phase cell cycle. The expression of 44 miRNAs changed differentially after salidroside treatment in cancer cells. Bioinformatic analysis showed that there were 1384 target mRNAs corresponding to the differentially expressed miRNAs. Surprisingly, salidroside significantly up-regulated the expression of tumor suppressor miR-1343-3p, and down-regulated the expression of MAP3K6, STAT3 and MMP24-related genes. Salidroside suppressed the growth of gastric cancer by inducing the cancer cell apoptosis, arresting the cancer cell cycle and down-regulating the related signal transduction pathways. miRNAs are expressed differentially in gastric cancer cells after salidroside treatment, playing important roles in regulating proliferation and metastasis. Salidroside may suppress the growth of gastric cancer by up-regulating the expression of the tumor suppressor miR-1343-3p and down-regulating the expression of MAP3K6 and MMP24 signal molecules.

Baicalein Alleviates Arsenic-induced Oxidative Stress through Activation of the Keap1/Nrf2 Signalling Pathway in Normal Human Liver Cells.

Oxidative stress is a key mechanism underlying arsenicinduced liver injury, the Kelch-like epichlorohydrin-related protein 1 (Keap1)/nuclear factor E2 related factor 2 (Nrf2) pathway is the main regulatory pathway involved in antioxidant protein and phase II detoxification enzyme expression. The aim of the present study was to investigate the role and mechanism of baicalein in the alleviation of arsenic-induced oxidative stress in normal human liver cells. Normal human liver cells (MIHA cells) were treated with NaAsO2 (0, 5, 10, 20 μM) to observe the effect of different doses of NaAsO2 on MIHA cells. In addition, the cells were treated with DMSO (0.1%), NaAsO2 (20 μM), or a combination of NaAsO2 (20 μM) and Baicalein (25, 50 or 100 μM) for 24 h to observe the antagonistic effect of Baicalein on NaAsO2. Cell viability was determined using a Cell Counting Kit- 8 (CCK-8 kit). The intervention doses of baicalein in subsequent experiments were determined to be 25, 50 and 100μM. The intracellular content of reactive oxygen species (ROS) was assessed using a 2',7'-dichlorodihydrofluorescein diacetate (DCFHDA) probe kit. The malonaldehyde (MDA), Cu-Zn superoxide dismutase (Cu-Zn SOD) and glutathione peroxidase (GSH-Px) activities were determined by a test kit. The expression levels of key genes and proteins were determined by real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blotting. Baicalein upregulated the protein expression levels of phosphorylated Nrf2 (p-Nrf2) and nuclear Nrf2, inhibited the downregulation of Nrf2 target genes induced by arsenic, and decreased the production of ROS and MDA. These results demonstrate that baicalein promotes Nrf2 nuclear translocation by upregulating p-Nrf2 and inhibiting the downregulation of Nrf2 target genes in arsenic-treated MIHA cells, thereby enhancing the antioxidant capacity of cells and reducing oxidative stress. Baicalein alleviated arsenic-induced oxidative stress through activation of the Keap1/Nrf2 signalling pathway in normal human liver cells.

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.

Discovery of anticancer compound possessing potential to bind γ-secretase catalytic subunit and inhibit notch promoter activity

Gamma secretase (GS) is an important therapeutic target in anticancer drug discovery. Increased GS activity activates notch signaling pathway which is associated with cancer stemness and drug resistance in cancer cells. A total of 69,075 natural and their derivative compounds were screened to identify the lead compound on the basis of in silico GS catalytic domain binding potential and in vitro selective anticancer efficacy. STOCK1N-23234 showed higher dock score (−11.82) compared to DAPT (−9.2) in molecular docking experiment and formed hydrogen bond with the key amino acid (Asp385) involve in catalysis process. Molecular dynamics (MD) simulation parameters (RMSD, RMSF, Rg, SASA and hydrogen bond formation) revealed that the STOTCK1N-23234 formed structurally and energetically stable complex with the GS catalytic domain with lower binding energy (−22.79 kcal/mol) compared to DAPT (−16.22 kcal/mol). STOCK1N-23234 showed better toxicity (up to 60%) against colon and breast cancer cells (HCT-116 and MDA-MB-453) at 1–70 µM concentration. Interestingly, STOCK1N-23234 did not showed cytotoxicity against human normal breast cells (MCF-10A). STOCK1N-23234 treatment significantly decreased sphere formation, notch promoter activity, and transcription of notch target genes (Hes-1 and Hey-1) in HCT-116 cells derived colonosphere. Confocal microscopy revealed that STOTCK1N-23234 treatment at test concentration induced apoptosis related morphological changes, reduced mitochondria membrane potential and increased reactive oxygen species production in HCT-116 cells compared to non-treated cells. In conclusion, STOCK1N-23234 is a novel lead natural anticancer compound which requires in depth validation in cancer preclinical models. Communicated by Ramaswamy H. Sarma

Abstract A080: Target genes in pancreatic cancer cells of the pan-quadruplex clinical candidate compound QN-302 revealed by comparative transcriptome profiling

Abstract QN-302 is a tetra-substituted naphthalene derivative designed to have high affinity to quadruplexes in cancer-related genes, which have multiple quadruplex occurrence in their promoter sequences. QN-302 has ca 1 nM in vitro activity against a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines and shows high activity against four distinct PDAC in vivo models, including the KPC genetic model. QN-302 is currently being developed for clinical trials in solid tumors including PDAC. It has also recently been granted orphan drug drug (ODD) status by the FDA. We have previously reported on whole-genome transcriptome studies for QN-302, which show that it selectively targets quadruplex-rich genes in PDAC-related pathways, and thus acts as a pan-quadruplex agent. We have recently extended our analysis to include two closely related analogs of QN-302 as well as data from a gemcitabine-resistant line. QN-302 is consistently ca 10-fold more potent in PDAC cells and in vivo than the second-best analog. RNA-seq whole-cell and comparative bioinformatics analyses have been used to probe these differences. This has resulted in the identification of those target genes that the naphthalene diimide family have in common, many of which are involved in PDAC pathways such as hedgehog, axon guidance, WNT/β-catenin and hippo. It has also been possible to identify a small number of quadruplex target genes that are highly and selectively down-regulated by QN-302, and which are also highly up-regulated in human PDAC. We suggest that these contribute to the exceptional in vivo potency of this compound. Citation Format: Ahmed Ahmed, Tariq Arshad, Maria Roman-Escorza, Dan M. Neidle, Stephen Neidle. Target genes in pancreatic cancer cells of the pan-quadruplex clinical candidate compound QN-302 revealed by comparative transcriptome profiling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A080.

FOXM1 derived from Triple negative breast cancer exosomes promotes cancer progression by activating IDO1 transcription in macrophages to suppress ferroptosis and induce M2 polarization of Tumor-associated macrophages.

To explore the oncogenic mechanism of FOXM1 in the tumor microenvironment (TME) regarding triple negative breast cancer (TNBC) promotion. The mRNA and protein levels of target genes in TNBC cells and their exosomes were detected by RT-qPCR and western blot. Co-culture models of TNBC cells and THP-1/M0 macrophages was established to detect the impact of co-culture on FOXM1 expression and macrophage polarization direction. The bioinformatics website was used to predict the binding sites between the FOXM1 and IDO1 promoter, which were further validated using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. Finally, after erastin-induced ferroptosis, Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and other experiments were conducted to investigate whether the FOXM1/IDO1 axis regulates M2 macrophage polarization through ferroptosis. It was found that FOXM1 was highly expressed in exosomes derived from TNBC cells, and TNBC cells upregulated FOXM1 expression in THP-1 cells through exosomes to promote M2 macrophage polarization. Furthermore, FOXM1 upregulated IDO1 in M2-type TAMs by regulating transcription. Lastly, FOXM1/IDO1 inhibited ferroptosis, promoting M2 macrophage polarization, thereby advancing TNBC progression. In conclusions, FOXM1 derived from TNBC cell-derived exosomes activated IDO1 transcription in TAMs to inhibit ferroptosis, promoting TAMs' M2 polarization and exerting carcinogenic effects.