Transcriptional Downregulation Research Articles

Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L

BackgroundCerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) – a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer.MethodsOvarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins.ResultsFor each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level.ConclusionsOur study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress

Mechanisms of functional cross-talk between global transcriptional repression and efficient DNA damage repair during genotoxic stress are poorly known. In this study, using human AF9 as representative of Super Elongation Complex (SEC) components, we delineate detailed mechanisms of these processes. Mechanistically, we describe that Poly-Serine domain-mediated oligomerization is pre-requisite for AF9 YEATS domain-mediated TFIID interaction-dependent SEC recruitment at the promoter-proximal region for release of paused RNA polymerase II. Interestingly, during genotoxic stress, CaMKII-mediated phosphorylation-dependent nuclear export of AF9-specific deacetylase HDAC5 enhances concomitant PCAF-mediated acetylation of K339 residue. This causes monomerization of AF9 and reduces TFIID interaction for transcriptional downregulation. Furthermore, the K339 acetylation-dependent enhanced AF9-DNA-PKc interaction leads to phosphorylation at S395 residue which reduces AF9-SEC interaction resulting in transcriptional downregulation and efficient repair of DNA damage. After repair, nuclear re-entry of HDAC5 reduces AF9 acetylation and restores its TFIID and SEC interaction to restart transcription.

Tribbles pseudokinase 3 promoted renal fibrosis by regulating the expression of DNA damage-inducible transcript 3 in diabetic nephropathy.

Diabetic nephropathy (DN) is a severe complication of prolonged diabetes, impacting millions worldwide with an increasing incidence. This study investigates the role of tribbles pseudokinase 3 (TRIB3), a protein implicated in the progression of DN, focusing on its mechanisms underlying glomerular damage. Through analysis of the Gene Expression Omnibus (GEO) database, we identified TRIB3 among differentially expressed genes in streptozotocin (STZ)-treated C57BL/6J mice. Both in vitro and in vivo experiments were conducted to examine the effects of TRIB3 inhibition on high glucose (HG)-induced damage in podocytes and DN mouse models. The results demonstrated that TRIB3 inhibition reduced inflammatory responses and extracellular matrix (ECM) production in MPC5 cells, mediated by the downregulation of DNA damage-inducible transcript 3 (DDIT3) - a critical regulator of proinflammatory cytokine secretion and ECM synthesis. Inhibiting TRIB3 decreased inflammatory factors and ECM deposition in diabetic mice in vivo, confirming its pivotal role in DN pathogenesis. These findings indicate that TRIB3 and its interaction with DDIT3 contribute significantly to DN by promoting inflammatory cascades and ECM accumulation, presenting potential therapeutic targets for managing the disease.

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells.

Defects in the FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone composed of SSRP1 and SUPT16H, are implicated in intellectual disability. Here, we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain. We show that the FACT complex binds to the promoter region of the estrogen-related receptor (ERR) gene and positively regulates ERR expression. ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis. Dysfunction of the FACT complex leads to the downregulation of ERR transcription, resulting in a decreased ratio of glycolysis to oxidative phosphorylation (G/O) in neuroblasts. Consequently, neuroblasts exhibit smaller cell sizes, lower proliferation potential, and altered cell fates. Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex. Thus, the G/O ratio, mediated by the FACT complex, plays a crucial role in neuroblast cell fate maintenance. Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of β-AR-ROS signaling.

Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of β-adrenergic receptor (β-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and β-MHC), β-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of β-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-β-AR-ROS and NPR-A signaling.

Fabrication of low-viscosity injectable mineralized hydrogel sensor with redox-activated hybrid nanoparticle structure for cancer detection

A redox-responsive low-viscosity, injectable hydrogel (LVI-gel) sensor with capabilities involving bimodal cancer detection via the incorporation of glutathione (GSH) and reactive oxygen species (ROS)-activated nanoparticles (PD-Cu2+) is proposed and made injectable with varying degrees of mineralization. The PD-Cu2+ complex, in the presence of overexpressed GSH and ROS, undergoes a structural change that causes a regain of fluorescence and a shift in conductivity, accompanied by the variations in the mechanical, and adhesive properties for cancer detection. The release of catechol moieties brought about by the structural change of the PD-Cu2+ complex influences the shift in hydrogen bonding, resulting in increased water uptake, further reducing viscosity (η decreases by 95.14 %) and increasing adhesiveness (360.28 % increase) in the presence of GSH 10 mM. The alterations in mechanical and electrical properties further affect the bending response via LCR (control: ΔR/R0 ∼ 0.55 and GSH 10 mM: ΔR/R0 ∼ 0.89), which was confirmed in vitro via wireless response with HeLa, PC-3, and B16F10 as cancer cells. The effect of Cu2+-catalyzed oxidation of GSH was observed in vivo from the immunohistochemical studies and the downregulation of transcription for GPx1/Rn18s. Additionally, increased superoxide dismutase (SOD2) and catalase (CAT) activity along with the downregulation of the oncogenic NFκβ and TNF gene are observed. Finally, the ex situ sensing activity was monitored by the shift in resistance and the wireless response, demonstrating the ease with which the sensor could diagnose.

LINC00887 Acts as an Enhancer RNA to Promote Medullary Thyroid Carcinoma Progression by Binding with FOXQ1.

Medullary thyroid carcinoma (MTC) is a rare but aggressive endocrine malignancy that originates from the parafollicular C cells of the thyroid gland. Enhancer RNAs (eRNAs) are non-coding RNAs transcribed from enhancer regions, which are critical regulators of tumorigenesis. However, the roles and regulatory mechanisms of eRNAs in MTC remain poorly understood. This study aims to identify key eRNAs regulating the malignant phenotype of MTC and to uncover transcription factors involved in the regulation of key eRNAs. GSE32662 and GSE114068 were used for the identification of differentially expressed genes, eRNAs, enhancers and enhancer-regulated genes in MTC. Metascape and the transcription factor affinity prediction method were used for gene function enrichment and transcription factor prediction, respectively. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to assess the binding of histone H3 lysine 27 acetylation (H3K27ac)-enriched regions to anti- H3K27ac. RIP-qPCR was used to detect the binding between FOXQ1 and LINC00887. CCK8 and Transwell were performed to measure the proliferation and invasion of MTC cells, respectively. Intracellular reactive oxygen species (ROS) levels were quantified using a ROS assay kit. Four eRNAs (H1FX-AS1, LINC00887, MCM3AP-AS1 and A1BG-AS1) were screened, among which LINC00887 was the key eRNA promoting the proliferation and invasion of MTC cells. A total of 135 genes controlled by LINC00887-regulated enhancers were identified; among them, BCL2, PRDX1, SFTPD, TPO, GSS, RAD52, ZNF580, and ZFP36L1 were significantly enriched in the "ROS metabolic process" term. As a transcription factor regulating genes enriched in the "ROS metabolic process" term, FOXQ1 could recruit LINC00887. Overexpression of FOXQ1 restored LINC00887 knockdown-induced downregulation of GSS and ZFP36L1 transcription in MTC cells. Additionally, FOXQ1 overexpression counteracted the inhibitory effects of LINC00887 knockdown on the proliferation and invasion of MTC cells and the promotion of intracellular ROS accumulation induced by LINC00887 knockdown. LINC00887 was identified as a key eRNA promoting the malignant phenotype of MTC cells. The involvement of FOXQ1 was essential for LINC00887 to play a pro-tumorigenic role in MTC. Our findings suggest that the FOXQ1/LINC00887 axis is a potential therapeutic target for MTC.

Role of PKCδ in Acute HFD-induced Increased Tonic GABA Current in Cardiac-projecting DMV Neurons

High fat diet (HFD) is associated with reduced cardiac vagal motor output, a major contributor to progression of cardiovascular diseases. In cardiac projecting neurons in the dorsal motor nucleus of the vagus (CVNDMV), 15-day HFD exposure increases tonic GABAA receptor (GABAAR) current, which contributes to dampening cardiac parasympathetic output. However, the mechanism underlying this increase inhibition is unknown. In this study, we aimed to decipher the mechanism(s) underlying this acute HFD-induced tonic GABAAR current in CVNDMV. We hypothesized that increased activity of protein kinase C δ isoform (PKCδ) enhances tonic GABAAR current in CVNDMV after HFD since PKCδ activity is upregulated and this type of PKCδ activation promotes GABAAR membrane stabilization as well as increases tonic GABAAR current in neurons. Initial experiments using cardiac retrograde tracing paired with whole-cell patch-clamp electrophysiology in male C57BL/6Js confirmed that general inhibition of PKC activity with GFX (10 μM) eliminated HFD-induced increases in tonic GABAAR current in CVNDMV after 15D HFD (HFDGFX = 0.55 ± 0.34 pA/pF; N=3 cells / 2 mice vs NFDGFX = 0.87 ± 0.42 pA/pF; N=3 cells / 2 mice). To investigate the specific role of PKCδ, we first determined that PKCδ was expressed in all putative cholinergic neuronal subpopulations of DMV by mining a publicly available single nucleus RNAseq dataset (The Broad Institute Single Cell Portal) followed by confirmation that PKCδ was present in CVNDMV from normal mice (Ct value= 14.6 ± 0.74, N=8 cells/ 4 mice). PKCδ selective inhibition with rottlerin (10μM), similar to GFX, diminished the increase in tonic GABAAR current after 15D HFD (HFDno inhibitor vs rottlerin= 1.32 ± 0.14 vs 0.46 ± 0.06 pA/pF, P=0.0004; NFDrottlerin vs HFDrottlerin = 0.41 ±0.08 vs 0.46 ± 0.06 pA/pF, P=0.68; NNFD rottlerin = 3 cells /2 mice, NHFD rottlerin = 5 cells /3 mice). There was no effect of GFX or rottlerin on tonic current in NFD animals (NFDno inhibitor vs GFX: P=0.95; NFDno inhibitor vs rottlerin: P=0.42), suggesting that inhibition of PKCδ did not alter tonic current in normal conditions. Importantly, the impact of inhibition of PKCδ was specific to tonic GABAAR currents since rottlerin had no effect on phasic GABAAR neurotransmission (amplitude: P= 0.07; frequency: P=0.09; decay time: P=0.25). To begin elucidating mechanisms for an increase in PKCd activity, we examined single cell transcriptional expression in CVNDMV after 15D HFD and we revealed a strong trend for lower fold expression of PKCδ mRNA in HFD (2ΔΔCtNFD vs HFD = 1.41 ± 0.42 vs 0.51 ± 0.09, P=0.058, NNFD = 8 cells / 4 mice, NHFD = 8 cells / 4 mice). Altogether, these preliminary results suggest that PKCδ mediates HFD-induced increases in tonic GABAAR current. Given the differences between increase PKCδ functional expression and putative transcriptional downregulation, it is possible that HFD-induced increases of PKCδ activity occur at post-transcriptional level. Further experiments will be done to investigate PKCδ protein expression and its colocalization with GABAARs in CVNDMV. The outcome of this study, therefore, will provide understanding on mechanism to develop treatments targeting PKCδ for cardiovascular diseases associated with HFD. R01HL157366 NIHLB to CRB. 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.

N-acetylcysteine alleviates arsenic trioxide-induced reductions in hepatic catalase gene expression both in vitro and in vivo

Arsenic trioxide (ATO), one of the oldest and most frequently used poisons, is well-known in forensic science for inducing hepatotoxicity. The regulation of peroxisomal antioxidative enzyme catalase (CAT) involves intricate mechanisms at both transcriptional and post-transcriptional levels. However, the molecular mechanisms underlying the regulation of CAT gene expression in hepatic cells remain elusive. Furthermore, the regulation of CAT gene expression evident in animals administered with ATO in vivo is not well-explored, although several studies have revealed ATO-induced reductions in CAT enzymatic activity in rat livers. In this study, we revealed ATO-dependent reductions in CAT gene expression in both rat liver and Huh-7 human hepatoma cells. Our results indicate that the decline in CAT enzymatic activity can be attributed, at least in part, to the downregulation of its gene expression. The ATO-induced reduction in CAT expression was concurrent with the reduction in peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator (PGC)-1α and inactivation of PPARγ, both considered as positive regulators of CAT gene expression. Moreover, antioxidant N-acetylcysteine (NAC) demonstrated the capability to alleviate the downregulation of CAT gene expression both in vivo and in vitro. Additionally, NAC played a role in alleviating ATO-induced hepatotoxicity, potentially by mitigating the transcriptional downregulation of the CAT gene. Altogether, these results indicate that ATO exerts toxicity by inhibiting the antioxidant defense mechanism, which may be useful for forensic diagnosis of arsenic poisoning and clinical treatment of mitigating ATO-induced hepatotoxicity.

Involvement of E3 ubiquitin ligase NEDD4-mediated YY1 ubiquitination in alleviating idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and lethal lung disease characterized by progressive lung scarring. This study aims to elucidate the role of the E3 ubiquitin ligase NEDD4 in the ubiquitination of YY1 and its subsequent impact on TAB1 transcription, revealing a possible molecular mechanism in the development of IPF. Through bioinformatics analysis and both in vitro and in vivo experiments, we observed differential expression levels of NEDD4 and YY1 between normal and IPF samples, identifying NEDD4 as an upstream E3 ubiquitin ligase of YY1. Furthermore, binding sites for the transcription factor YY1 on the promoter region of TAB1 were discovered, indicating a direct interaction. In vitro experiments using HEPF cells showed that NEDD4 mediates the ubiquitination and degradation of YY1, leading to suppressed TAB1 transcription, thereby inhibiting cell proliferation and fibrogenesis. These findings were corroborated by in vivo experiments in an IPF mouse model, where the ubiquitination pathway facilitated by NEDD4 attenuated IPF progression through the downregulation of YY1 and TAB1 transcription. These results suggest that NEDD4 plays a crucial role in the development of IPF by modulating YY1 ubiquitination and TAB1 transcription, providing new insights into potential therapeutic targets for treating IPF.

SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF.

Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1) in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that TGFβ functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the anti-fibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis.

MicroRNA164e suppresses NAC100 transcription factor-mediated synthesis of seed storage proteins in chickpea.

Development of protein-enriched chickpea varieties necessitates an understanding of specific genes and key regulatory circuits that govern the synthesis of seed storage proteins (SSPs). Here, we demonstrated the novel involvement of Ca-miR164e-CaNAC100 in regulating SSP synthesis in chickpea. Ca-miRNA164e was significantly decreased during seed maturation, especially in high-protein accessions. The miRNA was found to directly target the transactivation conferring C-terminal region of a nuclear-localized transcription factor, CaNAC100 as revealed using RNA ligase-mediated-rapid amplification of cDNA ends and target mimic assays. The functional role of CaNAC100 was demonstrated through seed-specific overexpression (NACOE) resulting in significantly augmented seed protein content (SPC) consequential to increased SSP transcription. Further, NACOE lines displayed conspicuously enhanced seed weight but reduced numbers and yield. Conversely, a downregulation of CaNAC100 and SSP transcripts was evident in seed-specific overexpression lines of Ca-miR164e that culminated in significantly lowered SPC. CaNAC100 was additionally demonstrated to transactivate the SSP-encoding genes by directly binding to their promoters as demonstrated using electrophoretic mobility shift and dual-luciferase reporter assays. Taken together, our study for the first time established a distinct role of CaNAC100 in positively influencing SSP synthesis and its critical regulation by CamiR164e, thereby serving as an understanding that can be utilized for developing SPC-rich chickpea varieties.

Combining WO3@AuNPs with Poly(amidoamine) Allows Sensitive Electrochemical Detection of DR1 Based on Dual Signal Amplification.

Down-regulator of transcription 1 (DR1) is considered as a biomarker of hashimoto's thyroiditis(HT), which is a risk factor for thyroid cancer. Here, a label-free electrochemical biosensor for DR1 detection was constructed based on polyamidoamine(PAMAM)polymer and the nanocomposite (WO3@AuNPs) composed of tungsten trioxide (WO3) and gold nanoparticles (AuNPs). WO3@AuNPs was obtained by combining monolayer WO3nanosheets, which has high conductivity, and AuNPs. The modification of WO3@AuNPs can not only increase the conductivity of the electrode but also provide more active sites for signaling units, thus greatly improve the sensitivity of the sensor. The polymer PAMAM is biocompatible and non-immunogenic, and its end functional group can bind to the target molecules, providing them with more binding sites and thus improving the sensitivity of the sensor. Under optimal conditions, the label-free biosensor showed a good linear relationship between the logarithm of DR1 concentration and the impedance in the range of 10 fg·mL-1to 100 ng·mL-1, with a detection limit as low as 0.3 fg·mL-1. Besides, this label-free electrochemical platform exhibited satisfactory selectivity and anti-interference capability in human serum samples. Therefore, this method has considerable potential in clinical detection of DR1.

Abstract CT105: Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug

Abstract The human genome contains G-rich regions that are over-represented in many cancer gene promoters. These can form higher-order G-quadruplex arrangements under the influence of an appropriate and selective small molecule compound. The resulting ligand-G-quadruplex complex may be resistant to polymerase read-through and thus results in transcriptional down-regulation of target genes. QN-302, based on a tetra-substituted naphthalene diimide chemotype, is a potent and selective G-quadruplex-binding compound that has been designed and initially developed at University College London. It displays single-digit cell growth inhibition in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. RNA-seq analysis shows that it is a pan-quadruplex agent, down-regulating the expression of multiple G-quadruplex-promoter cancer genes and pathways in PDAC. It also shows in vivo anti-tumor activity in several animal models for PDAC, with low toxicity observed at therapeutic doses. QN-302 was licensed to Qualigen Therapeutics Inc in January 2022. It was granted Orphan Drug designation by the FDA in December 2022 for PDAC. This was followed by extensive regulatory toxicological and pharmacological studies together with successful scale-up synthesis, purification and formulation. QN-302 was granted IND clearance in August 2023 by the FDA for phase 1 clinical trials in advanced or metastatic solid tumors. This Phase 1a clinical trial is being conducted in two USA cancer centers (START Midwest, Grand Rapids, MI and Honor Health, Scottsdale, AZ) using a protocol based on 28-day once-weekly dose escalation and evaluation of three potential biomarkers, two of which have been suggested by transcriptomic analysis as being indicative of G-quadruplex-induced drug response and accompanying decrease of anti-tumor activity. So far 1 Cohort (3 patients) has been dosed at the lowest dose of 1.62 mg of QN-302 as part of this Phase 1a clinical trial. Patient #1, a male patient of metastatic pancreatic cancer, is currently in Cycle 4 of therapy showing Stable Disease and no AEs or SAEs reported. Patient #2, a female patient of metastatic colorectal cancer, completed Cycle 2 after which she showed progression upon CT Scan before entering Cycle 3 and was taken off study. Patient #3, a male patient of metastatic PDAC, is currently ending Cycle 2, about to enter Cycle 3, showing Stable Disease upon CT scan. All patients dosed showed no AEs, SAEs or side effects. They were also clinically observed to have improved QOL compared to prior SOC while on therapy with QN-302, This presentation will outline this unique approach and summarize safety, tolerability, PK/PD data and any early clinical data as described above including preliminary biomarker data now that clinical evaluation has commenced. The advantages and challenges of QN-302 will also be discussed. Citation Format: Tariq Arshad, Stephen Neidle, Sreenivasa Chandana, Erkut Borazanci. Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug [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 CT105.

Co-targeting BET, CBP, and p300 inhibits neuroendocrine signalling in androgen receptor-null prostate cancer.

There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

RNA expression changes driven by altered epigenetics status related to NASH etiology

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem due to the increased obesity rates, among other factors. In its more severe stage (NASH), inflammation, hepatocellular ballooning and fibrosis are present in the liver, which can further evolve to total liver dysfunction or even hepatocarcinoma. As a metabolic disease, is associated to environmental factors such as diet and lifestyle conditions, which in turn can influence the epigenetic landscape of the cells, affecting to the gene expression profile and chromatin organization. In this study we performed ATAC-sequencing and RNA-sequencing to interrogate the chromatin status of liver biopsies in subjects with and without NASH and its effects on RNA transcription and NASH etiology. NASH subjects showed transcriptional downregulation for lipid and glucose metabolic pathways (e.g., ABC transporters, AMPK, FoxO or insulin pathways). A total of 229 genes were differentially enriched (ATAC and mRNA) in NASH, which were mainly related to lipid transport activity, nuclear receptor-binding, dicarboxylic acid transporter, and PPARA lipid regulation. Interpolation of ATAC data with known liver enhancer regions showed differential openness at 8 enhancers, some linked to genes involved in lipid metabolism, (i.e., FASN) and glucose homeostasis (i.e., GCGR). In conclusion, the chromatin landscape is altered in NASH patients compared to patients without this liver condition. This alteration might cause mRNA changes explaining, at least partially, the etiology and pathophysiology of the disease.

From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease.

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM_024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD.

Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Type IV Collagen Promotes Adipogenic Differentiation of Adipose Stem Cells.

Type IV collagen is a major component of the extracellular matrix in adipose tissue. It is secreted during the lipogenic differentiation of mesenchymal stem cells, but its direct impact and mechanism on the differentiation of adipose-derived stem cells (ASCs) into lipids are unclear. In this study, ASCs were obtained from human liposuction samples and cultured. Lipogenic induction of ASCs was achieved using lipogenic induction medium. Immunofluorescence analysis revealed differential expression of type IV collagen during the early and late stages of adipogenic induction, displaying a distinct morphological encapsulation of ASCs. Silencing of type IV collagen using siRNA resulted in a significant decrease in adipogenic capacity, as indicated by reduced lipid droplet formation and downregulation of adipogenic-related gene transcription. Conversely, supplementation of the culture medium with synthetic type IV collagen demonstrated enhanced adipogenic induction efficiency, accompanied by upregulation of YAP/TAZ protein expression and its downstream target gene transcription. Furthermore, inhibition of the YAP/TAZ pathway using the inhibitor Blebbistatin attenuated the functionality of type IV collagen, leading to decreased lipid droplet formation and downregulation of adipocyte maturation-related gene expression. These findings highlight the crucial role of type IV collagen in promoting adipogenic differentiation of ASCs and suggest its involvement in the YAP/TAZ-mediated Hippo pathway.No Level Assigned This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .