Cell Cycle Regulation Pathways Research Articles

Identification and evaluation of candidate COVID-19 critical genes and medicinal drugs related to plasma cells

The ongoing COVID-19 pandemic, caused by the SARS-CoV-2 virus, represents one of the most significant global health crises in recent history. Despite extensive research into the immune mechanisms and therapeutic options for COVID-19, there remains a paucity of studies focusing on plasma cells. In this study, we utilized the DESeq2 package to identify differentially expressed genes (DEGs) between COVID-19 patients and controls using datasets GSE157103 and GSE152641. We employed the xCell algorithm to perform immune infiltration analyses, revealing notably elevated levels of plasma cells in COVID-19 patients compared to healthy individuals. Subsequently, we applied the Weighted Gene Co-expression Network Analysis (WGCNA) algorithm to identify COVID-19 related plasma cell module genes. Further, positive cluster biomarker genes for plasma cells were extracted from single-cell RNA sequencing data (GSE171524), leading to the identification of 122 shared genes implicated in critical biological processes such as cell cycle regulation and viral infection pathways. We constructed a robust protein-protein interaction (PPI) network comprising 89 genes using Cytoscape, and identified 20 hub genes through cytoHubba. These genes were validated in external datasets (GSE152418 and GSE179627). Additionally, we identified three potential small molecules (GSK-1070916, BRD-K89997465, and idarubicin) that target key hub genes in the network, suggesting a novel therapeutic approach. These compounds were characterized by their ability to down-regulate AURKB, KIF11, and TOP2A effectively, as evidenced by their low free binding energies determined through computational analyses using cMAP and AutoDock. This study marks the first comprehensive exploration of plasma cells’ role in COVID-19, offering new insights and potential therapeutic targets. It underscores the importance of a systematic approach to understanding and treating COVID-19, expanding the current body of knowledge and providing a foundation for future research.

Transcriptomic landscape of quiescent and proliferating human corneal stromal fibroblasts

This study analyzed the transcriptional changes in primary human corneal stromal fibroblasts (hCSFs) grown under quiescent (serum-free) and proliferating (serum-supplemented) culture conditions to identify genes, pathways, and protein‒protein interaction networks influencing corneal repair and regeneration. Primary hCSFs were isolated from donor human corneas and maintained in serum-free or serum-laden conditions. RNA was extracted from confluent cultures using Qiagen kit and subjected to RNA sequencing (RNAseq) analysis. Differential gene expression (DGE) and pathway enrichment analyses were conducted using DESeq2 and Gene Set Enrichment Analysis (GSEA), respectively. Protein‒protein interaction (PPI) networks were created exploiting the STRING database and analyzed with Cytoscape and the cytoHubba plugin. RNA-seq revealed 5,181 genes that were significantly differentially expressed/changed among the 18,812 annotated genes (p value ˂0.05). A cutoff value of a log2-fold change of ±1.5 or greater was used to identify 674 significantly upregulated and 771 downregulated genes between quiescent and proliferating hCSFs. Pathway enrichment analysis revealed significant changes in genes linked to cell cycle regulation, inflammatory, and oxidative stress response pathways, such as E2F Targets, G2M Checkpoint, and MYC Targets, TNFA signaling via NF-kB, and oxidative phosphorylation. Protein-protein interaction network analysis highlighted critical hub genes. The FGF22, CD34, ASPN, DPT, LUM, FGF10, PDGFRB, ECM2, DCN, VEGFD, OMD, OGN, ANGPT1, CDH5, and PRELP were upregulated, whereas genes linked to cell cycle regulation and mitotic progression, such as BUB1, TTK, KIF23, KIF11, BUB1B, DLGAP5, NUSAP1, CCNA2, CCNB1, BIRC5, CDK1, KIF20A, AURKB, KIF2C, and CDCA8, were downregulated. The RNA sequences and gene count files have been submitted to the Gene Expression Omnibus (accession # GSE260476). Our study provides a comprehensive information on the transcriptional and molecular changes in hCSFs under quiescent and proliferative conditions and highlights key pathways and hub genes.

Exploring the mechanism of genistein in treating hepatocellular carcinoma through network pharmacology and molecular docking

Abstract Objectives Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, with treatment options limited and outcomes often poor, especially in advanced stages. This study explores the therapeutic potential of genistein, a soybean-derived isoflavone, on HCC using network pharmacology to uncover its multi-targeted anti-cancer mechanisms. Methods Potential targets of genistein were predicted using databases such as Super-PRED, PharmMapper, and SwissTargetPrediction. Abnormally expressed genes in HCC tissues were analyzed from TCGA and GEO datasets, with genes linked to the prognosis of HCC patients selected as potential therapeutic targets. GO and KEGG pathway enrichment analyses were conducted for both genistein’s targets and the HCC-related gene set. Key targets were identified through network analysis using Cytoscape software. Molecular docking was performed with Autodock to assess the binding affinity between genistein and these key targets. The therapeutic effects of genistein on HCC were validated through animal experiments and cell line studies. Results This study identified 343 potential targets for genistein in treating hepatocellular carcinoma (HCC). Analyses revealed enrichment in cell cycle regulation pathways through GO and KEGG assessments. Transcriptomic data from HCC datasets unveiled 184 potential therapeutic targets, emphasizing cell cycle regulation. Notably, 12 proteins were identified as targets of both genistein and HCC treatment. Molecular docking studies demonstrated genistein’s strong binding affinity with CDC25C and MELK. In vitro and in vivo validations affirmed genistein’s role in inhibiting HCC proliferation by inducing G2/M phase arrest. This study elucidates genistein’s multi-target mechanism in suppressing HCC cell proliferation, supporting its potential clinical application in HCC treatment. Conclusions This study demonstrates that genistein effectively suppresses the malignant growth of HCC by interfering with the transition from the G2 to M phase, revealing a multifaceted mechanism of action.

LncRNA LOC730101 Promotes Darolutamide Resistance in Prostate Cancer by Suppressing miR-1-3p.

Antiandrogen is part of the standard-of-care treatment option for metastatic prostate cancer. However, prostate cancers frequently relapse, and the underlying resistance mechanism remains incompletely understood. This study seeks to investigate whether long non-coding RNAs (lncRNAs) contribute to the resistance against the latest antiandrogen drug, darolutamide. Our RNA sequencing analysis revealed significant overexpression of LOC730101 in darolutamide-resistant cancer cells compared to the parental cells. Elevated LOC730101 levels were also observed in clinical samples of metastatic castration-resistant prostate cancer (CRPC) compared to primary prostate cancer samples. Silencing LOC730101 with siRNA significantly impaired the growth of darolutamide-resistant cells. Additional RNA sequencing analysis identified a set of genes regulated by LOC730101, including key players in the cell cycle regulatory pathway. We further demonstrated that LOC730101 promotes darolutamide resistance by competitively inhibiting microRNA miR-1-3p. Moreover, by Hi-C sequencing, we found that LOC730101 is located in a topologically associating domain (TAD) that undergoes specific gene induction in darolutamide-resistant cells. Collectively, our study demonstrates the crucial role of the lncRNA LOC730101 in darolutamide resistance and its potential as a target for overcoming antiandrogen resistance in CRPC.

An investigation of the molecular characterization of the tripartite motif (TRIM) family and primary validation of TRIM31 in gastric cancer

Most TRIM family members characterized by the E3-ubiquitin ligases, participate in ubiquitination and tumorigenesis. While there is a dearth of a comprehensive investigation for the entire family in gastric cancer (GC). By combining the TCGA and GEO databases, common TRIM family members (TRIMs) were obtained to investigate gene expression, gene mutations, and clinical prognosis. On the basis of TRIMs, a consensus clustering analysis was conducted, and a risk assessment system and prognostic model were developed. Particularly, TRIM31 with clinical prognostic and diagnostic value was chosen for single-gene bioinformatics analysis, in vitro experimental validation, and immunohistochemical analysis of clinical tissue microarrays. The combined dataset consisted of 66 TRIMs, of which 52 were differentially expressed and 43 were differentially prognostic. Significant survival differences existed between the gene clusters obtained by consensus clustering analysis. Using 4 differentially expressed genes identified by multivariate Cox regression and LASSO regression, a risk scoring system was developed. Higher risk scores were associated with a poorer prognosis, suppressive immune cell infiltration, and drug resistance. Transcriptomic data and clinical sample tissue microarrays confirmed that TRIM31 was highly expressed in GC and associated with a poor prognosis. Pathway enrichment analysis, cell migration and colony formation assay, EdU assay, reactive oxygen species (ROS) assay, and mitochondrial membrane potential assay revealed that TRIM31 may be implicated in cell cycle regulation and oxidative stress-related pathways, contribute to gastric carcinogenesis. This study investigated the whole functional and expression profile and a risk score system based on the TRIM family in GC. Further investigation centered around TRIM31 offers insight into the underlying mechanisms of action exhibited by other members of its family in the context of GC.

Integrating network pharmacology and computational biology to propose Yiqi Sanjie formula's mechanisms in treating NSCLC: molecular docking, ADMET, and molecular dynamics simulation.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related deaths globally. Current treatments often do not fully meet efficacy and quality of life expectations. Traditional Chinese medicine (TCM), particularly the Yiqi Sanjie formula, shows promise but lacks clear mechanistic understanding. This study addresses this gap by investigating the therapeutic effects and underlying mechanisms of Yiqi Sanjie formula in NSCLC. We utilized network pharmacology to identify potential NSCLC drug targets of the Yiqi Sanjie formula via the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Compounds with favorable oral bioavailability and drug-likeness scores were selected. Molecular docking was conducted using AutoDock Vina with structural data from the Protein Data Bank and PubChem. Molecular dynamics (MD) simulations were performed with Desmond Molecular Dynamics System, analyzing interactions up to 500 nanoseconds using the OPLS4 force field. ADMET predictions were executed using SwissADME and ADMETlab 2.0, assessing pharmacokinetic properties. Using network pharmacology tools, we performed Search Tool for the Retrieval of Interaction Genes/Proteins (STRING) analysis for protein-protein interaction, Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway enrichment, and gene ontology (GO) for functional enrichment, identifying crucial signaling pathways and biological processes influenced by the hit compounds bifendate, xambioona, and hederagenin. STRING analysis indicated substantial connectivity among the targets, suggesting significant interactions within the cell cycle regulation and growth factor signaling pathways as outlined in our KEGG results. The GO analysis highlighted their involvement in critical biological processes such as cell cycle control, apoptosis, and drug response. Molecular docking simulations quantified the binding efficiencies of the identified compounds with their targets-CCND1, CDK4, and EGFR-selected based on high docking scores that suggest strong potential interactions crucial for NSCLC inhibition. Subsequent MD simulations validated the stability of these complexes, supporting their potential as therapeutic interventions. Additionally, the novel identification of ADH1B as a target underscores its prospective significance in NSCLC therapy, further expanded by our comprehensive bioinformatics approach. Our research demonstrates the potential of integrating network pharmacology and computational biology to elucidate the mechanisms of the Yiqi Sanjie formula in NSCLC treatment. The identified compounds could lead to novel targeted therapies, especially for patients with overexpressed targets. The discovery of ADH1B as a therapeutic target adds a new dimension to NSCLC treatment strategies. Further studies, both in vitro and in vivo, are needed to confirm these computational findings and advance these compounds towards clinical trials.

Auranofin and reactive oxygen species inhibit protein synthesis and regulate the level of the PLK1 protein in Ewing sarcoma cells.

Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways.

A phase 1, first-in-human, open-label study evaluating the safety, tolerability, pharmacokinetics, and efficacy of TT125-802 in patients with advanced solid tumors.

TPS3171 Background: TT125-802 is a selective, potent, orally bioavailable small molecule inhibitor of the bromodomain of CBP and p300, two highly homologues lysine acetyltransferases. CBP/p300 act as transcriptional co-activators with multiple domains and functions, including well-documented roles in cell cycle regulation and several critical tumorigenic pathways (1-3). TT125-802 has shown mono-therapy efficacy in preclinical models of castration-resistant prostate cancer, KRAS-mutated colorectal and non-small cell lung cancer (4,5). Furthermore, TT125-802 is capable to interfere with transcriptional resistance mechanisms limiting targeted therapies (6) which will be explored clinically upon determination of a safe, well-tolerated, and biologically active single-agent dose. Methods: This study aims to establish the safety, tolerability, and pharmacokinetic profile of TT125-802. The maximum tolerated dose/recommended dose for expansion (MTD/RDE) of repeat daily dosing for TT125-802 monotherapy will be determined in subjects with advanced solid tumors in a sequential escalating cohorts design, (1 cycle = 21 days) using the Bayesian Logistics Regression Model (BLRM) with sentinel patients (2-day window for first patient enrolled in each cohort). Cohorts will consist of 3 to 6 subjects who will be followed over a dose-limiting toxicity (DLT) period of at least 21 days. TT125-802 will be administered orally once daily; additional schedules may be evaluated. Dose escalation is planned to follow two-fold increments. It may be adapted upon dose level revision and guided by pharmacokinetic analysis. Additional subjects (up to 6) may be enrolled in at least two monotherapy cohorts deemed safe and tolerable to optimize the recommended phase 2 dose (RP2D). Up to a total of 20 additional subjects may be enrolled. Translational/pharmacodynamic endpoints: Exploratory endpoints will provide proof-of-principle and early biomarker data for CBP/p300 inhibition. These analyses will include: 1. Single-cell gene expression evaluation of peripheral blood mononuclear cells (PBMCs) at multiple time points, 2. anagen hair bulb bulk RNAseq analysis, and 3. paired tumor biopsies will be analyzed with single-cell RNA sequencing. Together, these investigations are expected to support optimal dose determination. Additionally, circulating tumor DNA might be evaluated to define molecular responses. Outlook: The recommended phase 2 dose will be used further to explore the efficacy of TT125-802 as monotherapy. TT125-802 will be evaluated with targeted therapies (for example, KRASG12C/D, BRAF, EGFR, etc., inhibitors) to exploit the blocking of transcriptional escape mechanisms driven by CBP/p300. 1. Iyer, 2007. 2. Dutta, 2016. 3. Attar, 2017. 4. AACR2023 posters #6268. 5. AACR poster #3907. 6. AACR2023 poster #3907. Clinical trial information: 2022-500849-24-00 .

Proteo-genomic characterization of cirrhosis-associated liver cancers reveals potential subtypes and therapeutic targets.

This study aimed to identify potential subtypes of hepatocellular carcinoma (HCC) associated with cirrhosis and to investigate key markers using bioinformatic analysis of gene expression datasets-0. Three data sets (GSE17548, GSE56140, and GSE87630) were extracted from the Gene Expression Omnibus (GEO) database and normalized using the Limma package in R. Principal component analysis (PCA) and cluster analysis was performed to examine data distribution and identify subtypes. Differential gene expression analysis was performed using the Limma software package. Protein-protein interaction analysis and functional annotation were performed using the STRING database and Cytoscape software. Important signaling pathways and processes were identified using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Analysis. The analysis revealed different subtypes of HCC associated with cirrhosis and identified several key genes, including CCNB2, MCM4, and CDC20, with strong binding power and prognostic value. Functional annotation indicated involvement in cell cycle regulation and metabolic pathways. ROC analysis showed high sensitivity and specificity of these genes in predicting HCC prognosis. These results suggest that CCNB2, MCM4, and CDC20 may serve as potential biomarkers for predicting HCC prognosis in patients with cirrhosis and provide insights into the molecular mechanisms of HCC progression.

Regulation of eukaryotic transcription initiation in response to cellular stress

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Abstract PS17-06: A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic breast cancer

Abstract Background: Activation of the phosphoinositide 3-kinase (PI3K) pathway is a prominent resistance mechanism to endocrine therapy and CDK4/6 inhibition (CDK4/6i) in ER+, HER2- breast cancer. There is reciprocal crosstalk between PI3K and cell cycle regulatory pathways. Triplet therapy that targets ER, CDK4/6, and PI3K prevents or delays disease progression in preclinical models of ER+, HER2- breast cancer. However, the toxicities of triplet therapy hinder its clinical development. Copanlisib (COP) is an intravenous (IV) PI3Ki with potent activity against α and δ isoforms. Intermittent high-dose PI3Ki is expected to be less toxic and more effective than continuous daily dosing. We set out to determine the recommended phase 2 dose (RP2D) for the COP/Fulvestrant (FUL)/Abemaciclib (ABE) combination. Methods: Eligible patients (pts) included men or women with ER+, HER2- metastatic breast cancer (MBC), with no more than 1 prior chemotherapy in the metastatic setting, and no limits on prior endocrine or targeted therapy. Prior FUL, CDK4/6i and PI3K/mTORi were allowed. Pts with HbA1c >8.5% were excluded. Pts received FUL 500 mg IM standard dosing and COP/ABE at assigned doses. Premenopausal women also received a GnRH agonist. Dose-limiting toxicity (DLT) was assessed during the first 28-day cycle. Adverse events (AEs) were graded according to NCI-CTCAE v5.0. Dose escalation/de-escalation was based on continual reassessment (CRM) and the RP2D was defined as the dose with the highest probability of the DLT rate within the acceptable 25~35%. Results: Between June 2020 and June 2023, 24 pts with ER+, HER2- MBC were enrolled. Table 1 shows the dose levels (DLs) and DLTs. The first 10 pts were enrolled to Part A on two consecutive DLs of COP (DL1: 45 mg IV D1 and D15, n=7; DL2: 45 mg IV D1, D8, and D15, n=3), with ABE fixed at 100 mg PO BID continuously. DL2a exceeded the maximum tolerated dose (MTD). DL1a was tolerable based on Cycle 1 AEs. However, during subsequent cycles, only 1 of the 7 pts in DL1a maintained the intended dose of ABE due to neutropenia, others reduced ABE to 50mg bid (n=5) or discontinued (n=1). This triggered a protocol amendment, adding Part B with ABE administered 5 days on and 2 days off (5-on/2-off) intermittent schedule. 14 pts were enrolled to Part B in 2 consecutive DLs of COP (DL1b: n=7; DL2b: n=7), the same as in Part A. ABE was fixed at 100 mg PO Bid 5-on/2-off. DL2b exceeded the MTD. DL1b was tolerable based on Cycle 1 AEs. In addition, in subsequent cycles, only 1 of 7 pts on DL1b required dose reduction of ABE to 50mg bid. RP2D is therefore defined as DL1b. Grade (G) 4 AEs were rare. The most common all cycle G3 AEs included transient hypertension (45%), rash (30%), anemia (25%), neutropenia (25%), and AST elevation (20%). As of 6/14/2023, 22 pts, with a median of 1 prior metastatic regimen (range 0-5), prior CDK4/6i in 18 (82%), liver mets in 8 (36%), and PIK3CA mutation in 8 (50%) of 16 pts with known mutation status, were evaluable for response. There were 5 partial responses and 3 stable diseases lasting for ≥24 weeks. Clinical benefit rate was 36.4% (8/22, 95%CI: 17.19% ~ 59.34%). Responses were observed in pts regardless of prior CDK4/6i, FUL, or PIK3CA mutation. Conclusion: COP/FUL/intermittent ABE can be safely administered with fair overall tolerability. Preliminary anti-tumor activity was observed, which will be further examined in the randomized phase II trial (NCT 03939897). Table 1 Dose-limiting Toxicities by Dose Level Citation Format: Cynthia Ma, Jingqin Luo, Jessica Moss, Maryann Kwa, Ritesh Parajuli, Katia Khoury, Emily Douglas, Ashley Frith, Caron Rigden, Foluso Ademuyiwa, Shana Thomas, Brittney Haas, Gerburg Wulf, Claire Dees, Rabih Said. A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic breast cancer [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 PS17-06.

Abstract PO5-06-11: RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis

Abstract Background: Ribosome is a complex structure in cells for protein synthesis. It has been considered always have a constant composition. Recent studies have revealed that the Ribosome composition of tumor cells is heterogeneous. The Ribosome protein composition, post-translational modification, rRNA modification, and rRNA variants can lead to the existence of specialized "onco-ribosomes" in tumor cells, which regulates the rate of translation and protein synthesis, and can affect the occurrence and development of tumors significantly. The abnormal expression of multiple Ribosome proteins in triple-negative breast cancer may be the target of its diagnosis and treatment, also, an independent factor in prognosis evaluation. Methods: To learn the expression levels of Ribosome biogenesis related genes in normal breast cells and abnormal breast cells with different pathological subtypes of breast cancer, the following studies have been done: 1. Bioinformatics analysis on TCGA database and single-cell sequencing data; 2. IHC detection on TNBC tissue and adjacent tissues; 3. Determine the expression level of RPL27A in TNBC cells, and analyze the correlation between the expression of RPL27A and clinical information of patients. To explore the effect of RPL27A on Ribosome biogenesis, protein synthesis and other signaling pathways through Proteomics and molecular biology experiments have been designed and tested. The effects of RPL27A on malignant biological behaviors such as proliferation, migration, infiltration, and clonal formation of TNBC cells were studied through cell biology experiments and mouse experiments. Results: The analysis of TCGA database and single-cell sequencing data showed that the RPL27A was significantly increased in TNBC group. Immunohistochemical experiments showed that RPL27A was highly expressed in TNBC tissue and was impressively positively correlated with tumor size, lymph node metastasis, and bone metastasis. There was significantly negatively correlated with overall survival (OS) and disease free survival (DFS). It can be an independent factor for evaluating the prognosis of TNBC. By knocking out RPL27A in TNBC cell line, label free proteomics analysis showed that Ribosome biogenesis, protein synthesis, cell cycle regulation and other signaling pathways were obviously down regulated. Cell biology experiments have implied that the high expression of RPL27A in TNBC promotes cell proliferation, enhances cell migration, infiltration, and cloning ability, leads to more malignant biological behavior of tumor cells. Further research demonstrates that the high expression of RPL27A in TNBC significantly activated the EIF3C signal, cause changes in the composition of Ribosome in TNBC cells, promoted the formation of "onco-ribosomes", and distinctly increased the rate of protein synthesis. The study of PDX mice and Organoid showed that the knock out of RPL27A significantly reduced the growth rate and transfer ability of TNBC. Conclusion: The obvious over expressing of RPL27A in TNBC, which can significantly promote the growth and metastasis of tumor cells by influencing the Ribosome biogenesis process. Citation Format: Weipeng Zhao, Jingyi Zhang, Ye Zhu, Xin Yang, Xichuan Li. RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis [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 PO5-06-11.

Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

Integrative analysis of the bladder cancer from a cell cycle NCAM1 perspective at both single cell and bulk resolution.

Bladder cancer (BLCA) is a prevalent and deadly form of urinary cancer, and there is a need for effective therapies, particularly for muscle-invasive bladder cancer (MIBC). Cell cycle inhibitors show promise in restoring control of the cell cycle in BLCA cells, but their clinical prognosis evaluation is limited. Transcriptome and scRNA-seq data were collected from the Cancer Genome Atlas Program (TCGA)-BLCA and GSE190888 cohort, respectively. R software and the Seurat package were used for data analysis, including cell quality control, dimensionality reduction, and identification of differentially expressed genes. Genes related to the cell cycle were obtained from the genecards website, and a protein-protein interaction network analysis was performed using cytoscape software. Functional enrichment analysis, immune infiltration analysis, drug sensitivity analysis, and molecular docking were conducted using various tools and packages. BLCA cell lines were cultured and transfected for in vitro experimental assays, including RT-qPCR analysis, and CCK-8 cell viability assays. We identified 32 genes as independent risk or protective factors for BLCA prediction. Functional enrichment analysis revealed their involvement in cell cycle regulation, apoptosis, and various signaling pathways. Using these genes, we developed a nomogram for predicting BLCA survival, which displayed high prognosis stratification efficacy in BLCA patients. Four cell cycle associated key genes identified, including NCAM1, HBB, CKD6, and CTLA4. We also identified the main cell types in BLCA patients and investigated the functional differences between epithelial cells based on their expression levels of key genes. Furthermore, we observed a high positive correlative relationship between the infiltration of cancer-associated fibroblasts and the risk score value. Finally, we conducted in vitro experiments to demonstrate the suppressive role of NCAM1 in BLCA cell proliferation. These findings suggest that cell cycle associated genes could serve as potential biomarkers for predicting BLCA prognosis and may represent therapeutic targets for the development of more effective therapies. Hopefully, these findings provide valuable insights into the molecular mechanisms and potential therapeutic targets in BLCA from the perspective of cell cycle. Moreover, NCAM1 was a novel cell proliferation suppressor in the BLCA carcinogenesis.

Canonical pathways for validating steroid-associated osteonecrosis in mice

The present study aimed to establish and evaluate a preclinical model of steroid-associated osteonecrosis (SAON) in mice. Sixteen 24-week-old male C57BL/6 mice were used to establish SAON by two intraperitoneal injections of lipopolysaccharide (LPS), followed by three subcutaneous injections of methylprednisolone (MPS). Each injection was conducted on working day, with an interval of 24 h. Six cycles of injections were conducted. Additional twelve mice (age- and gender-matched) were used as normal controls. At 2 and 6 weeks after completing induction, bilateral femora and bilateral tibiae were collected for histological examination, micro-CT scanning, and bulk RNA sequencing. All mice were alive until sacrificed at the indicated time points. The typical SAON lesion was identified by histological evaluation at week 2 and week 6 with increased lacunae and TUNEL+ osteocytes. Micro-CT showed significant bone degeneration at week 6 in SAON model. Histology and histomorphometry showed significantly lower Runx2+ area, mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR/BS), type H vessels, Ki67+ (proliferating) cells, and higher marrow fat fraction, osteoclast number and TNFα+ areas in SAON group. Bulk RNA-seq revealed changed canonical signaling pathways regulating cell cycle, angiogenesis, osteogenesis, and osteoclastogenesis in the SAON group. The present study successfully established SAON in mice with a combination treatment of LPS and MPS, which could be considered a reliable and reproducible animal model to study the pathophysiology and molecular mechanism of early-stage SAON and to develop potential therapeutic approaches for the prevention and treatment of SAON.

Abstract 1662: Evaluation of TEAD inhibitor, VT03989 treatment on growth of aggressive meningioma preclinical models

Abstract Background: Aggressive meningiomas (WHO grade II/III) are incurable brain tumors and despite surgery and radiation therapy, tumors recur frequently without approved systemic treatment options. Hence, there is an unmet need for targeted inhibition therapy and biomarkers that predict vulnerability to targeted therapies in aggressive meningioma patients. Hippo signaling pathway has been shown to be dysregulated in meningioma, resulting in activation of its target TEAD as a transcriptional factor. We hypothesize that targeted inhibition of TEAD in recurrent high-grade meningioma with NF2 loss will lead to increased inhibition of tumor growth. Here, we investigated the effect of VT3989, a potent TEAD inhibitor, in meningioma cell lines with NF2 wild-type or NF2 loss genotype. Methods: Two meningioma cell-lines: IOMM-Lee (NF2 wild-type) and MG309, a grade III meningioma patient-derived cell line with NF2 loss were treated with various doses of VT3989 and cell viability was assessed. Relative expression of direct targets of TEAD including CTGF and CYR61 was determined with quantitative PCR (qPCR) to ensure target engagement. Global transcriptomic changes were examined via RNAseq analysis on RNA isolated from MG309 cells treated with VT03989 compared to vehicle treated cells. Results: VT3989 treatment significantly decreased viability of both IOMM-Lee and MG309 cells. Canonical downstream targets of TEAD such as CTGF, CYR61, BIRC5, BCL-2 were downregulated in VT3989 treated cells compared to control. Furthermore, VT03989 treatment resulted in downregulation in cell cycle regulation and DNA damage response (DDR) pathway genes. Ongoing work is focused on combining VT3989 with agents targeting cell cycle and DDR pathways. Conclusion: VT3989 treatment led to downregulation of TEAD direct targets CTGF and CYR61 and inhibited cell viability of both IOMM-Lee cell and MG309 meningioma lines. RNA-seq analysis identified cell cycle and DDR pathway genes to be downregulated upon TEAD inhibition. Citation Format: Siddhi Desai, Anil Thaghalli Shivanna, Mariya Stavnichuk, Elizabeth Hayes, Sonam Patel, James McNamara, Anita DeSantis, Nader Sanai, An-Chi Tien, Shwetal Mehta. Evaluation of TEAD inhibitor, VT03989 treatment on growth of aggressive meningioma preclinical models [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 1662.

Effect of Herniarin on Cell Viability, Cell Cycle, and Erk Protein Levels in Different Stages of Bladder Cancer Cells.

This study examines the potential of herniarin from tarragon, as an agent with multifaceted effects on bladder cancer cells and investigates herniarin's impact on cell viability, migration, cell cycle regulation, apoptosis induction, and Erk signaling pathways in bladder cancer cell lines, including RT-112 (grade 1, non-invasive), HTB9 (grade 2, invasive), and HT1376 (grade 3, invasive), through comprehensive in vitro experiments. The compound causes cell cycle arrest at distinct phases in different cell lines: G1/S arrest in RT112 cells, G2/M arrest in HTB9 cells, and S phase arrest in HT1376 cells. Furthermore, herniarin induces caspase-mediated apoptosis in various cell lines and simultaneously modulates protein levels of apoptotic and anti-apoptotic proteins, indicating its potential as a therapeutic agent. Herniarin's influence also extends to Erk signaling, a crucial pathway that regulates cell growth and differentiation. In conclusion, this study reveals herniarin's potential as a versatile agent in the treatment of bladder cancer.

Transcriptomic Profiling for Prognostic Biomarkers in Early-Stage Squamous Cell Lung Cancer (SqCLC).

Squamous cell lung carcinoma (SqCLC) is associated with high mortality and limited treatment options. Identification of therapeutic targets and prognostic biomarkers is still lacking. This research aims to analyze the transcriptomic profile of SqCLC samples and identify the key genes associated with tumorigenesis, overall survival (OS), and a profile of the tumor-infiltrating immune cells. Differential gene expression analysis, pathway enrichment analysis, and Gene Ontology analysis on RNA-seq data obtained from FFPE tumor samples (N = 23) and healthy tissues (N = 3) were performed (experimental cohort). Validation of the results was conducted on publicly available gene expression data using TCGA LUSC (N = 225) and GTEx healthy donors' cohorts (N = 288). We identified 1133 upregulated and 644 downregulated genes, common for both cohorts. The most prominent upregulated genes were involved in cell cycle and proliferation regulation pathways (MAGEA9B, MAGED4, KRT, MMT11/13), while downregulated genes predominately belonged to immune-related pathways (DEFA1B, DEFA1, DEFA3). Results of the survival analysis, conducted on the validation cohort and commonly deregulated genes, indicated that overexpression of HOXC4 (p < 0.001), LLGL1 (p = 0.0015), and SLC4A3 (p = 0.0034) is associated with worse OS in early-stage SqCLC patients. In contrast, overexpression of GSTZ1 (p = 0.0029) and LILRA5 (p = 0.0086) was protective, i.e., associated with better OS. By applying a single-sample gene-set enrichment analysis (ssGSEA), we identified four distinct immune subtypes. Immune cell distribution suggests that the memory T cells (central and effector) and follicular helper T cells could serve as important stratification parameters.

E2F1 mediates competition, proliferation and response to cisplatin in cohabitating resistant and sensitive ovarian cancer cells.

Tumor heterogeneity is one of the key factors leading to chemo-resistance relapse. It remains unknown how resistant cancer cells influence sensitive cells during cohabitation and growth within a heterogenous tumors. The goal of our study was to identify driving factors that mediate the interactions between resistant and sensitive cancer cells and to determine the effects of cohabitation on both phenotypes. We used isogenic ovarian cancer (OC) cell lines pairs, sensitive and resistant to platinum: OVCAR5 vs. OVCAR5 CisR and PE01 vs. PE04, respectively, to perform long term direct culture and to study the phenotypical changes of the interaction of these cells. Long term direct co-culture of sensitive and resistant OC cells promoted proliferation (p < 0.001) of sensitive cells and increased the proportion of cells in the G1 and S cell cycle phase in both PE01 and OVCAR5 cells. Direct co-culture led to a decrease in the IC50 to platinum in the cisplatin-sensitive cells (5.92 µM to 2.79 µM for PE01, and from 2.05 µM to 1.51 µM for OVCAR5). RNAseq analysis of co-cultured cells showed enrichment of Cell Cycle Control, Cyclins and Cell Cycle Regulation pathways. The transcription factor E2F1 was predicted as the main effector responsible for the transcriptomic changes in sensitive cells. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured vs monoculture. Furthermore, an E2F1 inhibitor reverted the increase in proliferation rate induced by co-culture to baseline levels. Our data suggest that long term cohabitation of chemo-sensitive and -resistant cancer cells drive sensitive cells to a higher proliferative state, more responsive to platinum. Our results reveal an unexpected effect caused by direct interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors.