Activation Of Target Genes Research Articles

Notch Signaling: An Emerging Paradigm in the Pathogenesis of Reproductive Disorders and Diverse Pathological Conditions.

The highly conserved Notch pathway, a pillar of juxtacrine signaling, orchestrates intricate intercellular communication, governing diverse developmental and homeostatic processes through a tightly regulated cascade of proteolytic cleavages. This pathway, culminating in the migration of the Notch intracellular domain (NICD) to the nucleus and the subsequent activation of downstream target genes, exerts a profound influence on a plethora of molecular processes, including cell cycle progression, lineage specification, cell-cell adhesion, and fate determination. Accumulating evidence underscores the pivotal role of Notch dysregulation, encompassing both gain and loss-of-function mutations, in the pathogenesis of numerous human diseases. This review delves deep into the multifaceted roles of Notch signaling in cellular dynamics, encompassing proliferation, differentiation, polarity maintenance, epithelial-mesenchymal transition (EMT), tissue regeneration/remodeling, and its intricate interplay with other signaling pathways. We then focus on the emerging landscape of Notch aberrations in gynecological pathologies predisposing individuals to infertility. By highlighting the exquisite conservation of Notch signaling in Drosophila and its power as a model organism, we pave the way for further dissection of disease mechanisms and potential therapeutic interventions through targeted modulation of this master regulatory pathway.

The diverging roles of insulin-like growth factor binding proteins in pulmonary arterial hypertension

Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH.

Systematic investigation of Radix Salviae for treating diabetic peripheral neuropathy disease based on network Pharmacology.

Diabetic peripheral neuropathy (DPN) is a debilitating complication of diabetes mellitus with limited available treatment options. Radix Salviae, a traditional Chinese herb, has shown promise in treating DPN, but its therapeutic mech-anisms have not been systematically investigated. Radix Salviae (Danshen in pinin), a traditional Chinese medicine (TCM), is widely used to treat DPN in China. However, the mechanism through which Radix Salviae treats DPN remains unclear. Therefore, we aimed to explore the mechanism of action of Radix Salviae against DPN using network pharmacology. The active ingredients and target genes of Radix Salviae were screened using the TCM pharmacology database and analysis platform. The genes associated with DPN were obtained from the Gene Cards and OMIM databases, a drug-com-position-target-disease network was constructed, and a protein-protein inter-action network was subsequently constructed to screen the main targets. Gene Ontology (GO) functional annotation and pathway enrichment analysis were performed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) using Bioconductor. A total of 56 effective components, 108 targets and 4581 DPN-related target genes of Radix Salviae were screened. Intervention with Radix Salviae for DPN mainly involved 81 target genes. The top 30 major targets were selected for enrichment analysis of GO and KEGG pathways. These results suggested that Radix Salviae could treat DPN by regulating the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway. Therefore, Danshen may affect DPN by regulating inflammation and apoptosis.

Mechanism of action of Coptidis Rhizome in treating periodontitis based on network pharmacology and in vitro validation

ObjectiveExplore the therapeutic mechanism of Coptidis Rhizome (CR) in periodontitis using network pharmacology, and validate it through molecular docking and in vitro experiments.MethodsScreened potential active components and target genes of CR from TCMSP and Swiss databases. Identified periodontitis-related target genes using GeneCards. Found common target genes using Venny. Conducted GO and KEGG pathway analysis. Performed molecular docking and in vitro experiments using Berberine, the main active component of CR, on lymphocytes from healthy and periodontitis patients. Assessed effects on inflammatory factors using CCK-8, flow cytometry, and ELISA.ResultsFourteen active components and 291 targets of CR were identified. 30 intersecting target genes with periodontitis were found. GO and KEGG analysis revealed oxidative stress response and IL-17 signaling pathway as key mechanisms. Molecular docking showed strong binding of Berberine with ALOX5, AKT1, NOS2, and TNF. In vitro experiments have demonstrated the ability of berberine to inhibit the expression of Th17 + and other immune related cells in LPS stimulated lymphocytes, and reduce the secretion of IL-6, IL-8, and IL-17.ConclusionCR treats periodontitis through a multi-component, multi-target, and multi-pathway approach. Berberine, its key component, acts through the IL-17 signaling pathway to exert anti-inflammatory effects.

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity.

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.

MiR-29 and MiR-140 regulate TRAIL-induced drug tolerance in lung cancer

ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has chemotherapeutic potential as a regulator of an extrinsic apoptotic ligand, but its effect as a drug is limited by innate and acquired resistance. Recent findings suggest that an intermediate drug tolerance could mediate acquired resistance, which has made the main obstacle for limited utility of TRAIL as an anti-cancer therapeutics. We propose miRNA-dependent epigenetic modification drives the drug tolerant state in TRAIL-induced drug tolerant (TDT). Transcriptomic analysis revealed miR-29 target gene activation in TDT cells, showing oncogenic signature in lung cancer. Also, the restored TRAIL-sensitivity was associated with miR-29ac and 140-5p expressions, which is known as tumor suppressor by suppressing oncogenic protein RSK2 (p90 ribosomal S6 kinase), further confirmed in patient samples. Moreover, we extended this finding into 119 lung cancer cell lines from public data set, suggesting a significant correlation between TRAIL-sensitivity and RSK2 mRNA expression. Finally, we found that increased RSK2 mRNA is responsible for NF-κB activation, which we previously showed as a key determinant in both innate and acquired TRAIL-resistance. Our findings support further investigation of miR-29ac and -140-5p inhibition to maintain TRAIL-sensitivity and improve the durability of response to TRAIL in lung cancer.

Total ginsenosides extend healthspan of aging Drosophila by suppressing imbalances in intestinal stem cells and microbiota

BackgroundDisruption of stem cell and microbial homeostasis accelerates the aging process. Hence, maintaining these balances effectively delays aging and alleviates the symptoms of age-related diseases. Recent research indicates that targeting endoplasmic reticulum (ER) stress and immune deficiency (IMD) signalling may play a positive role in maintaining homeostasis in aging intestinal stem cells (ISC) and microbial equilibrium. Previous research has suggested that total ginsenosides (TG) derived from Panax ginseng C. A. Meyer may exhibit potential anti-aging properties by mitigating ER stress and mediating the IMD pathway. Nevertheless, it remains unclear whether TG improve ISC and microbial homeostasis by modulating ER stress and the IMD pathway to promote healthy aging. PurposeTo elucidate whether TG promotes healthspan in Drosophila and its underlying molecular mechanisms, focusing on its role in regulating ER stress and the IMD pathway to maintain ISC and intestinal microbiota homeostasis. MethodsHigh performance liquid chromatography was performed to detect the main saponin monomer in TG. Survival rate, gut length, barrier function, and feeding/excretion behaviour assays were used to evaluate the effects of TG on the lifespan and gut health of Drosophila. At the stem cell level, “esg-luciferase” reporter system, esg-GFP/delta stem cell fluorescent labelling, and phospho-histone H3+ mitotic activity assays were employed to determine whether TG prevented natural aging or oxidative stress-associated ISC over-proliferation in Drosophila. Immunofluorescence staining was used to detect the effects of TG on ER stress during aging. Overexpression or interference of ER stress target genes and their related c-Jun N-terminal kinase (JNK) gene was manipulated using gene editing technology to verify the molecular mechanism by which TG maintains age-related ISC proliferation homeostasis. Molecular docking and isothermal titration calorimetry were used to verify the direct interactions between TG and ER stress target genes. In addition, at the intestinal flora level, 16S rDNA sequencing was used to analyse the effect of TG on the diversity and abundance of Drosophila intestinal flora and the possible functional pathways involved. RT-qPCR was performed to determine whether TG mediated the expression of target genes in the IMD pathway. A dominant bacterial species-specific mono-association analysis were performed to verify whether the effects of TG on IMD target genes and ISC proliferation depended on the direct control of the dominant bacterial species. ResultsOur results suggest that administration of TG delays the decline in gut morphology and function in aging Drosophila. TG prevents age-associated ISC hyperproliferation by inhibiting ER stress IRE1-mediated JNK signaling. Furthermore, oral TG prevented aging-associated ISC and gut microbiota dysbiosis by remodelling the gut microbiota and inhibiting Acetobacter-mediated activation of IMD target genes. ConclusionTG promotes healthy aging by inhibiting the excessive proliferation of ISC and alleviating intestinal microbial imbalance, thereby providing new insights for the research and development of anti-aging TG products.

Abstract P10: p44, the Amino-terminal Lacking Isoform of p53, is Dispensable for Tumor Suppression but Regulates Male Fertility

Abstract p53 is the most studied tumor-suppressor, by virtue of its functional inactivation in most human cancers. One aspect that has not received sufficient attention is the function of the various forms of p53, such as p47, that lacks the amino-terminal transactivation domain. Hence, the goal of this work has been to systematically characterize p47, which was found to retain the ability to selective transactivate apoptotic target gene activation (and thus the ability to induce apoptosis), but lacks the ability to induce the expression of genes involved in cell cycle arrest. Consistently, patients with tumors that have p53 mutations that lead to p47 expression have a better prognosis. We have uncovered the mechanistic basis of the selective transcriptional activity of p47, which is associated with selective co-factor binding. In addition, we have generated mice that express only p47 (without p53), and show that they are susceptible to spontaneous tumors, like human patients who have germ-line mutations that lead to only p47 expression. Thus, p47 appears to be a null allele in mice with respect to tumor formation, and is incapable of inducing selective target gene activation nor apoptosis, highlighting species-specific differences in its functionality. Further characterization of these mice will be reported. Citation Format: RH Chia, Beng Hooi Phang, Fanny Xueting Teo, Hannah Lau, Kanaga Sabapathy. p44, the Amino-terminal Lacking Isoform of p53, is Dispensable for Tumor Suppression but Regulates Male Fertility [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P10.

The Experimental and In Silico-Based Evaluation of NRF2 Modulators, Sulforaphane and Brusatol, on the Transcriptome of Immortalized Bovine Mammary Alveolar Cells.

Changes during the production cycle of dairy cattle can leave these animals susceptible to oxidative stress and reduced antioxidant health. In particular, the periparturient period, when dairy cows must rapidly adapt to the sudden metabolic demands of lactation, is a period when the production of damaging free radicals can overwhelm the natural antioxidant systems, potentially leading to tissue damage and reduced milk production. Central to the protection against free radical damage and antioxidant defense is the transcription factor NRF2, which activates an array of genes associated with antioxidant functions and cell survival. The objective of this study was to evaluate the effect that two natural NRF2 modulators, the NRF2 agonist sulforaphane (SFN) and the antagonist brusatol (BRU), have on the transcriptome of immortalized bovine mammary alveolar cells (MACT) using both the RT-qPCR of putative NRF2 target genes, as well as RNA sequencing approaches. The treatment of cells with SFN resulted in the activation of many putative NRF2 target genes and the upregulation of genes associated with pathways involved in cell survival, metabolism, and antioxidant function while suppressing the expression of genes related to cellular senescence and DNA repair. In contrast, the treatment of cells with BRU resulted in the upregulation of genes associated with inflammation, cellular stress, and apoptosis while suppressing the transcription of genes involved in various metabolic processes. The analysis also revealed several novel putative NRF2 target genes in bovine. In conclusion, these data indicate that the treatment of cells with SFN and BRU may be effective at modulating the NRF2 transcriptional network, but additional effects associated with cellular stress and metabolism may complicate the effectiveness of these compounds to improve antioxidant health in dairy cattle via nutrigenomic approaches.

A Network Pharmacological Analysis of Celabenzine Protecting Central Nervous System from Damage Through RhoA Gene Modulation

Central nervous system diseases pose a significant threat to human health. Clinical observations have demonstrated that celabenzine, the primary active component of the natural plant ginseng, can potentially alleviate cognitive dysfunction in patients with central nervous system disorders. However, the specific mechanisms of its action remain unclear. The PubMed database was used to identify the targets of celabenzine. Subsequently, a Venn diagram of intersecting targets was constructed by utilizing the identified central nervous system injury targets. Additionally, Cytoscape software (version 3.7.2) was used to construct active ingredient-target and Protein-protein interaction networks. The Metascape database was used to perform Gene Ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Molecular docking was performed on active ingredients and target genes. Network pharmacology analysis revealed that celabenzine shares 175 intersecting targets with central nervous system damage, manifesting its effects through PI3K-Akt and MAPK signaling pathways and proteoglycans in the cancer pathway. Celabenzine demonstrates a strong binding affinity with its target gene, RhoA, indicating stable binding. This study was accomplished using network pharmacology and bioinformatics. Our findings suggested that celabenzine shows its protective effects against central nervous system damage by modulating the RhoA gene.

Endocrine Resistance in Breast Cancer: The Role of mTOR Signaling in Mediating Resistance to Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators (SERMs) have been widely prescribed and effective as a first-line endocrine therapy to treat ER+ breast cancer. Tamoxifen, the most used SERM in the treatment of breast cancer, has been shown to be effectively anti-proliferative in breast tissue and has made a tremendous contribution to reducing breast cancer mortality. Vast experimental evidence from seven sources supports tamoxifen’s ability in repressing the expression of estrogen-responsive genes involved in cancer growth. The binding of tamoxifen to the estrogen receptor prevents the recruitment of coactivators to the complex and instead promotes the recruitment of corepressors and histone deacetylases, thus inhibiting transcriptional activation of target genes. However, the issue of endocrine resistance remains a predominant problem with this therapy. Sources have found that endocrine resistance can arise due to dysregulations in the mTOR signaling pathway. Experiments have revealed some hope regarding a mechanism by which we can re-sensitize the breast cancer cells to the therapy, notably by knocking down YAP/TAZ or PSAT1 in the mTOR pathway. Despite this discovery, endocrine resistance prevails due to irregularities in additional pathways. Therefore, subsequent research is crucial to identify more targets that, when knocked down, enable re-sensitization of resistant cells, restoring full therapeutic ability of SERMs in afflicted women.

The impact of canonical Wnt transcriptional repressors TLE3 and TLE4 on postsynaptic transcription at the neuromuscular junction.

Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [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 6441.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer [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 6271.

Abstract 2067: QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators

Abstract Integrative analyses of CRISPR/Cas9 screening datasets from different sources revealed - besides known oncogenes - a series of transcriptional master regulators as most prominent context-specific dependencies in cancer that remained largely inaccessible to conventional drug discovery approaches. New chemical entities (NCEs) that specifically interfere with a transcription factor (TF) at any level should induce and be detectable based on selective effects on their primary biochemical activity - i.e., the transcriptional activation of specific target genes. However, the definition of direct targets of a TF and, more generally, the unbiased detection of direct transcriptional effects has remained challenging due to the limited time resolution of conventional techniques for gene perturbation and transcriptional profiling. To overcome this limitation, QUANTRO Therapeutics relies on SLAMseq technology that, by metabolic labeling de novo-synthesized RNA immediately after intervention and conserving the information for later readout, provides unprecedent time-resolution in transcriptomic analysis. Aiming to target previously undruggable targets, we set out to first build definitive knowledge around their direct effect on transcription, inducing their quick protein degradation through the auxin-inducible degron (AID) technology followed by immediate transcriptional readout though SLAMseq -before any secondary, confounding effect could appear in the cell. The targets´ mRNA fingerprints produced are then used as a reference and cross-compared with existing and new chemical entities to: (i) benchmark the real mode of action of existing drugs; (ii) identify new drug candidates that specifically interfere with disease-causing transcriptional programs in cancer; (iii) interrogate the efficiency, selectivity, and potency of drug candidates during Hit-validation and Hit-to-Lead activities. Proof of concept data demonstrated that time-resolved transcriptional profiling can reveal drugs´ mode of action with unprecedent precision and sensitivity, when drugs act on Targets that have a direct effect on transcriptional regulation - such as TFs and signaling pathways. Building on this knowledge, QUANTRO Therapeutics unleashed the unique potential of mRNA fingerprinting technology for HTS-compatible drug discovery by (1) establishing scalable and automated workflows for metabolic RNA labeling, treatment of cells, and RNA extraction; (2) developing algorithms for combinatorial mRNA fingerprint-readout that allow for the simultaneous assessment of TF signatures by multiplexed targeted RNA sequencing (QUANTROseq), thus transforming the precision and scope of cell-based compound screens. Citation Format: Arianna Sabò, Nina Fasching Griesmayr, Tobias Neumann, Raphael Manzenreither, Maja Ivankovic, Rodrigo Pacheco Valamatos Costa, Sarah Rieser, Sara Scinicariello, Petr Triska, Anna Stingeder, Astrid Gruss, Ryan Cubero, Olga Frank, Ivica Sowemimo, Paul Kirchgatterer, Adriana Cantoran, Stefan Ameres, Johannes Zuber, Michael Bauer. QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators [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 2067.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p<0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [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 6799.

Abstract 5940: JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator

Abstract Background: As a key tumor suppressor, p53 precisely regulates cellular events such as cell cycle arrest, apoptosis, senescence, and DNA repair under physiological conditions. In 50-60% of human cancers, TP53 gene is mutated. The TP53 Y220C is a hotspot loss-of-function mutation occurring in around 1% of solid tumors. Application of p53-Y220C reactivator for restoration of p53 function represents a promising treatment strategy for patients with this mutation. Methods: The Induced Allosteric Drug Discovery Platform (IADDP), which integrates medicinal chemistry, biochemical and biophysical studies, and computational techniques, was applied for the development of JAB-30355. The proportion of p53-Y220C with wild-type protein-like structure after JAB-30355 treatment was determined by immunoprecipitation. Thermal shift assay was applied to assess the thermal stability of JAB-30355-reactivated p53-Y220C protein. DNA binding assay was developed to evaluate the in vitro DNA binding activity. p53-luciferase reporter system was developed to evaluate the cellular transcriptional activity. qPCR and Western blotting were performed to evaluate the transcriptional and translational levels of p53 downstream targets, respectively. CellTiter-Glo assay was performed to evaluate the inhibitory activity of JAB-30355 on the viability of tumor cell lines harboring wild-type or Y220C mutated TP53. In vivo PK-PD study was conducted to evaluate the relationship between JAB-30355 concentration and expression of p53 target genes. Multiple CDX and PDX mouse models harboring TP53 Y220C were used to evaluate the antitumor activity of JAB-30355. Results: Immunoprecipitation assay and thermal shift assay results demonstrated that JAB-30355 efficiently restored the wild-type p53-like structure and enhanced protein stability of p53-Y220C in a dose-dependent manner. In addition, JAB-30355 significantly enhanced the DNA binding activity of p53-Y220C and subsequently increased the expression of p53 target genes, such as CDKN1A, MDM2 and PUMA. JAB-30355 inhibited cell viability of multiple TP53 Y220C-mutated cancer cell lines with IC50s ranging from 0.2 to 0.7 μM, and exhibited good selectivity against TP53 wild-type cells. Overall, the in vitro data demonstrated that JAB-30355 is a potent and selective p53-Y220C reactivator. In vivo PK-PD study showed good correlation between JAB-30355 exposure and activation of p53 target genes. Furthermore, JAB-30355 exhibited dose-dependent anti-tumor activity, inducing tumor stasis or regression in multiple CDX and PDX models of ovarian cancer, pancreatic cancer, gastric cancer, and small cell lung cancer, with overall good tolerability. Conclusions: JAB-30355 is a highly potent, selective, and orally bioavailable p53-Y220C reactivator that will be tested in clinical space soon. Citation Format: Qian Zheng, Peng Wang, Chen Liang, Yao Li, Xin Sun, Amin Li, Wei Zhang, Wei Long, Yanping Wang. JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator [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 5940.

Abstract 1734: Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC

Abstract Background: Approximately 75% of Human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors carry genetic and expression alterations in the family of protein methyltransferases and demethylases, underscoring the importance of these enzymes in the pathogenesis of HNSCC. SMYD3 a lysine methyltransferase that is overexpressed in HPV-negative HNSCC. We recently reported that nuclear SMYD3 plays dual role in immune-related gene regulation through H3K4me3 or H4K20me3, leading to gene activation or silencing, respectively in HPV-negative HNSCC. While SMYD3 has been reported to write and bind to H3K4me3 activating target gene expression, its function in gene silencing is understudied. This study aims to determine the mechanism(s) through which SMYD3 functions both as a transcriptional activator and a repressor within the same cell context, specifically in HPV-negative HNSCC. Methods: immunoprecipitation (IP) of SMYD3 was performed from nuclear extracts isolated from HN-6 cells to identify candidate interacting partners of SMYD3. The nuclear immunoprecipitates were sent for MS analysis to identify candidate nuclear interacting partners of SMYD3. CUT&RUN assays for selected candidate interacting proteins and SMYD3 will be performed in control HN-6 cells versus SMYD3 KO cells to assess genomic co-occupancy with SMYD3. Further, CUT&RUN assays for repressive marks H4K20me3, H3K27me3 and H3K9me3 have been performed in HN-6 and SMYD3 KO cells to assess co-occupancy with SMYD3 as well as its effect of SMYD3 on the genome-wide mapping of these marks. Finally, CUT&RUN assays for SMYD3 followed by re-CUT&RUN of repressive histone marks are ongoing to identify co-occupied loci. Results: Preliminary analysis of our nuclear SMYD3 IP results has identified a chromatin modifier with known repressive function. Co-immunoprecipitation experiments to validate this interaction are planned. CUT&RUN assays for repressive histone marks in HN-6 versus SMYD3 KO cells have revealed impact on H4K20me3 and H3K27me3. Conclusions: Our preliminary results suggest that SMYD3 may exert its silencing function by regulating the genome-wide deposition of H4K20me3.Our nuclear SMYD3 IP analysis has identified candidate interacting partners that may assist SMYD3 in promoting the deposition of these repressive marks. CUT&RUN assays for SMYD3 followed by re-CUT&RUN for H4K20me3 and H3K27me3 bound chromatin may provide further insight about whether SMYD3 co-occupies specific genomic regions together with these repressive marks. This project is expected to provide mechanistic insights into the bifaceted functions of SMYD3 within the same cell context using HPV-negative HNSCC cell lines as an experimental model system.. Citation Format: Jawad Akhtar, Sohyoung Kim, Vassiliki Saloura. Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC [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 1734.

Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

Integration of meta-analysis and network pharmacology analysis to investigate the pharmacological mechanisms of traditional Chinese medicine in the treatment of hepatocellular carcinoma.

Background: This study will explore the therapeutic value of traditional Chinese medicine (TCM) in Hepatocellular Carcinoma (HCC) through meta-analysis, combined with network pharmacology analysis. Methods: The results of randomized controlled trials on TCM and HCC were retrieved and summarized from multiple databases. The effective active com-pounds and target genes of the high-frequency TCM were obtained using the TCMSP database, and disease targets of HCC were acquired through the public disease database. The network pharmacology analysis was used to get the core genes and investigate the potential oncogenic molecular mechanism. Results: A total of 14 meta-analysis studies with 1,831 patients suggested that therapy combined TCM is associated with better clinical efficacy and survival prognosis, as well as avoiding many adverse events. A total of 156 compounds, 247 herbal target genes and 36 core genes were identified. The function analysis suggested above genes may participate development in HCC through regulating some pathways, such as HIF-1 pathway and PD-L1 immune-related pathway. Conclusion: TCM, as a novel, safe, and effective multi-mechanism therapy, holds greater value in the treatment of HCC.