Expression Pathway Analysis Research Articles

<b>Computational Analysis to Identify Novel Drug Targets for Esophageal Cancer</b>

Cancer is a multigene and widespread disease. Increasing drug resistance leads to the development of new therapeutic targets. Recent research indicates that various cellular components called Stress Granules (SGs) are engaged in the cancer-related signaling pathway. The phosphoinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling, considered a master regulator in cancer, has been shown through genomic profiling studies to play a key role in Esophageal Cancer (EC). In this study, we performed the in-silico analysis of an RNA sequencing dataset to investigate the effects of omipalisib, a PI3K/mTOR inhibitor, on EC cell lines. Our objective was to identify novel molecular targets, particularly stress granule-related proteins, that contribute to drug resistance in EC. Using computational approaches including differential gene expression analysis, pathway analysis, and functional enrichment, we examined the transcriptomic changes in response to omipalisib treatment. Our analysis revealed downregulation of the PI3K/mTOR signaling pathway and upregulation of compensatory pathways such as FOXO and JAK-STAT signaling in response to omipalisib. Notably, we identified 16 stress granule-related proteins that were significantly upregulated, suggesting their potential role in drug resistance mechanisms. These findings provide new insights into the molecular mechanisms underlying drug resistance in EC and highlight potential novel targets for therapeutic intervention. Currently, EC is limited by the number of potential drugs for treatment, poor prognosis, and is prone to chemotherapeutic resistance to existing clinically proven drugs. Our computational analysis offers valuable insights into targeting stress granules for cancer drug discovery, potentially enhancing the development of new therapeutic strategies for EC. These results provide a strong foundation for future experimental validation and drug development efforts aimed at overcoming resistance to EC treatment. Received: 1 July 2024 | Revised: 20 September 2024 | Accepted: 20 November 2024 Conflicts of Interest The authors declare that they have no conflicts of interest to this work. Data Availability Statement The data that support the findings of this study are openly available in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE143462 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE143462. Author Contribution Statement Vinod Jangid: Conceptualization, Methodology, Software, Validation, Formal analysis, Data curation, Writing - original draft, Visualization. Chandrasekar Narayanan Rahul: Conceptualization, Methodology, Validation, Investigation,Writing - original draft. Aarthi Rashmi B: Validation, Writing - review & editing. Kanagaraj Sekar:Validation, Writing - review & editing, Supervision, Project administration.

First-in-class mitogen-activated protein kinase (MAPK) p38a: MAPK-activated protein kinase 2 dual signal modulator with anti-inflammatory and endothelial-stabilizing properties

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38a mitogen-activated protein kinase (MAPK) near the binding site of its proinflammatory substrate, mitogen-activated protein kinase–activated protein kinase (MK)2. UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a-binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by surface plasmon resonance, 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells in vitro, and greater lung protection in vivo. GEn-1124 improved survival from 10%–40% in murine acute lung injury induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide instillation and febrile-range hyperthermia and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in tumor necrosis factor α–treated human pulmonary artery endothelial cells showed that the gene-modifying effects of GEn-1124 were much more restricted to tumor necrosis factor α–inducible genes than those of the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38a and MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex and dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance StatementWe describe a novel analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream proinflammatory and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in acute respiratory distress syndrome.

Abstract PR010: High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer

Abstract Background: Metastatic prostate cancer is an androgen receptor (AR) driven disease for which multiple AR targeted therapies are FDA approved, leading to improvements in patient outcomes. However, development of treatment resistance remains universal, driven by AR alterations as well as lineage state transitions that bypass AR signaling and culminate in neuroendocrine prostate cancer (NEPC) with poor prognosis. Liquid biopsies could enable longitudinal molecular analysis to identify such lineage transitions, but bulk RNA sequencing of circulating tumor cells (CTCs) has been limited by the challenge of isolating sufficiently pure samples for global signaling pathway analysis. We developed a novel approach to CTC purification that results in purity comparable to tissue biopsies and performed the first large-scale CTC RNA-seq study in a multi-institutional cohort of patients with metastatic prostate cancer. Using this approach, we identified multiple prostate cancer lineage states associated with prognosis. Methods: 273 blood samples were collected from 117 patients with histologically confirmed metastatic prostate cancer treated at the University of Wisconsin Carbone Cancer Center, William S. Middleton Memorial Veterans Hospital, UC San Diego Moores Cancer Center and Dana Farber Cancer Institute. CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA sequencing. A modification of the ESTIMATE algorithm was used to infer sample tumor purity, and CIBERSORTx was used to infer immune content. Overall survival (OS) was defined as date of death or last contact relative to first CTC sample collection. Results: 146 samples from 70 patients met our >50% purity threshold for gene expression pathway analysis. Single sample pathway analysis identified four CTC transcriptional phenotypes: luminal A (high AR signaling, intermediate proliferation), luminal B (high AR signaling, high proliferation), low proliferation, and neuroendocrine. Compared to patients with low CTC burden/low tumor purity (median OS NR, n=63), patients with luminal A (n=21) and low proliferation (n=12) phenotypes had similar survival, while patients with luminal B (n=18) and NE (n=3) had markedly shorter survival (LumB: median OS 6 months, HR 9.1, log rank p<0.0001, NE: median OS 3.7 months, HR 11.8, log rank p=0.0019). Conclusion: We report the largest CTC RNA sequencing cohort of patients with metastatic prostate cancer and demonstrate that CTC RNA-seq identifies prostate cancer lineage states mirroring those described in tissue profiling. In addition to an NE phenotype concordant with tissue histology and associated with markedly inferior overall survival, we identified a more common luminal B CTC subtype defined by persistent AR signaling and high proliferation and associated with poor prognosis comparable to NEPC. Citation Format: Marina N Sharifi, Jamie M Sperger, Amy K Taylor, Katharine E Tippins, Shannon R Reese, Viridiana Carreno, Katherine R Kaufmann, Alex H Chang, Luke A Nunamaker, Charlotte Linebarger, Kyle T Helzer, Matthew Bootsma, Grace C Blitzer, John Floberg, David Kosoff, Rana R McKay, Xiao X Wei, Shuang G Zhao, Joshua M Lang. High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR010.

Exploring the impact of digestive physicochemical parameters of adults and infants on the pathophysiology of Cryptosporidium parvum using the dynamic TIM-1 gastrointestinal model

BackgroundHuman cryptosporidiosis is distributed worldwide, and it is recognised as a leading cause of acute diarrhoea and death in infants in low- and middle-income countries. Besides immune status, the higher incidence and severity of this gastrointestinal disease in young children could also be attributed to the digestive environment. For instance, human gastrointestinal physiology undergoes significant changes with age, however the role this variability plays in Cryptosporidium parvum pathogenesis is not known. In this study, we analysed for the first time the impact of digestive physicochemical parameters on C. parvum infection in a human and age-dependent context using a dynamic in vitro gastrointestinal model.ResultsOur results showed that the parasite excystation, releasing sporozoites from oocysts, occurs in the duodenum compartment after one hour of digestion in both child (from 6 months to 2 years) and adult experimental conditions. In the child small intestine, slightly less sporozoites were released from excystation compared to adult, however they exhibited a higher luciferase activity, suggesting a better physiological state. Sporozoites collected from the child jejunum compartment also showed a higher ability to invade human intestinal epithelial cells compared to the adult condition. Global analysis of the parasite transcriptome through RNA-sequencing demonstrated a more pronounced modulation in ileal effluents compared to gastric ones, albeit showing less susceptibility to age-related digestive condition. Further analysis of gene expression and enriched pathways showed that oocysts are highly active in protein synthesis in the stomach compartment, whereas sporozoites released in the ileum showed downregulation of glycolysis as well as strong modulation of genes potentially related to gliding motility and secreted effectors.ConclusionsDigestion in a sophisticated in vitro gastrointestinal model revealed that invasive sporozoite stages are released in the small intestine, and are highly abundant and active in the ileum compartment, supporting reported C. parvum tissue tropism. Our comparative analysis suggests that physicochemical parameters encountered in the child digestive environment can influence the amount, physiological state and possibly invasiveness of sporozoites released in the small intestine, thus potentially contributing to the higher susceptibility of young individuals to cryptosporidiosis.

Abstract C106: Enhanced characterization of molecular attributes through multi-omics analysis and comprehensive evaluation of global and local genomic ancestry in colorectal cancer among Hispanic-Latinos

Abstract Introduction: Colorectal cancer (CRC) remains a significant public health challenge, representing the second leading cause of cancer-related mortality in the United States. Although mortality rates have generally decreased, the Hispanic/Latino (H/L) population in the Los Angeles area still experiences mortality rates up to 20% higher than those of their Caucasian American counterparts. Additionally, they are often diagnosed at a younger age and with more advanced disease stages, highlighting significant disparities in CRC outcomes. Currently, there are limited studies integrating clinical and multi-omics data from H/L populations, which are crucial for understanding the implications of colorectal tumorigenesis and addressing these disparities effectively. Methods: Clinical and genomic sequencing data were obtained from 60 primary CRC Tumor/Normal (T/N) samples within the Hispanic/Latino (H/L) population in the Los Angeles area through the PE-CGS network. For comparison, we analyzed 3,578 samples from non-Hispanic Whites (NHW) obtained from public databases. Additionally, we retrieved three CRC samples with spatial transcriptomic (ST) data from the 10xGenomics database. Using Whole Exome and RNA sequencing data, we performed somatic mutations, somatic copy number alterations (SCNAs), differential gene expression, cellular pathways, gene fusions, and global & local genomic ancestry analyses. The ST data underwent analysis utilizing newly released bioinformatics tools. Clinical and genomic data were integrated. Results: In these studies, distinct Amerindian (AMR) genomic ancestry proportion, age at diagnosis, and tumor localization patterns were observed, alongside significant mutations in key genes such as APC, TP53, and KRAS. The studies revealed evident cancer heterogeneity, influenced by factors such as mutation type, microsatellite instability, tumor subsite, and ethnicity. Additionally, several previously well-defined SCNAs were detected in the majority of tumors. Comparative analysis of gene expression and cellular pathways between H/L and NHW samples revealed distinct patterns. Furthermore, we identified gene fusions that potentially contribute to oncogenic activity in CRC. Ancestry analysis pointed to a predominant AMR ancestry. Notably, distinct associations of genetic ancestry with CRC tumor clinicopathological characteristics were identified. ST analysis delineated separate cancer, immune, and stroma components within the tumors. Conclusion: Our research has explored the molecular profiling of CRC tumors in H/L patients, providing crucial insights into the characterization of CRC tumors at the DNA and RNA levels, as well as the complex clinical and genomic diversity within our target population. Additionally, it offers valuable insights into the heterogeneity of CRC tumors and the tumor microenvironment. These findings establish the groundwork for subsequent projects, potentially leading to precision medicine approaches. Citation Format: Enrique I. Velazquez-Villarreal, Brigette Waldrup, Yonatan Amzaleg, Yuxin Jin, Mackenzie Postel, Donna Loza, Serina Ovalle, Elizabeth Quino, Carmen Chavez, Julie Culver, Mariana C. Stern, Heinz-Josef Lenz, David W. Craig, John D. Carpten. Enhanced characterization of molecular attributes through multi-omics analysis and comprehensive evaluation of global and local genomic ancestry in colorectal cancer among Hispanic-Latinos [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C106.

MEST promotes immune escape in gastric cancer by downregulating MHCI expression via SHP2

BackgroundImmune escape is a major obstacle to T-cell-based immunotherapy for cancers such as gastric cancer (GC). Mesoderm-specific transcript (MEST) is a tumor-promoting factor that regulates multiple oncogenic signaling pathways. However, the role of MEST-mediated immune escape is unclear. MethodsBioinformatics analysis of MEST expression and enrichment pathways were performed Quantitative reverse transcription PCR (qPCR) or western blot was used to detect the expression of MEST, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2), Major histocompatibility class I (MHCI)-related genes. Cell function was assessed by Cell Counting Kit (CCK)-8, Transwell, Lactate dehydrogenase (LDH) kit, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). Xenograft nude mice and immune-reconstructed mice were used to test the effects of different treatments on tumor growth and immune escape in vivo. ResultsMEST was upregulated in GC and promoted tumor proliferation, migration, and invasion. Rescue experiments revealed that TNO155 treatment or knockdown of SHP2 promoted the killing ability of CD8+ T cells and the expression of granzyme B (GZMB) and interferon-gamma (IFN-γ), and MEST overexpression reversed the effect. In vivo experiments confirmed that MEST promoted tumor growth, knockdown of MEST inhibited immune escape in GC, and that combination treatment with anti-PD-1 improved anti-tumor activity. ConclusionIn this study, we demonstrated that MEST inhibited IFN-γ secretion from CD8+ T cells by up-regulating SHP2, thereby downregulating MHCI expression in GC cells to promote immune escape and providing a new T cell-based therapeutic potential for GC.

Adipocytes impact on gastric cancer progression: Prognostic insights and molecular features.

Adipocytes, especially adipocytes within tumor tissue known as cancer-associated adipocytes, have been increasingly recognized for their pivotal role in the tumor microenvironment of gastric cancer (GC). Their influence on tumor progression and patient prognosis has sparked significant interest in recent research. The main objectives of this study were to investigate adipocyte infiltration, assess its correlation with clinical pathological features, develop a prognostic prediction model based on independent prognostic factors, evaluate the impact of adipocytes on immune cell infiltration and tumor invasiveness in GC, and identify and validate genes associated with high adipocyte expression, exploring their potential diagnostic and prognostic value. To explore the relationship between increased adipocytes within tumor tissue and prognosis in GC patients as well as the associated mechanisms and potential biomarkers, using public databases and clinical data. Using mRNA microarray datasets from the Gene Expression Omnibus database and clinical samples from Jiangsu Provincial Hospital, survival and regression analyses were conducted to determine the relevant prognostic factors in GC. Feature gene selection was performed using least absolute shrinkage and selection operator and support vector machine recursive feature elimination algorithms, followed by differential gene expression analysis, gene ontology, pathway analysis, and Gene Set Enrichment Analysis. Immune cell infiltration was analyzed using the CIBERSORT algorithm. Tumor adipocyte infiltration correlated with poor prognosis in GC, leading to the development of a highly accurate and discriminative prognostic prediction model. Key genes, ADH1B, SFRP1, PLAC9, and FABP4, were identified as associated with high adipocyte expression in GC. The diagnostic and prognostic potential of these identified genes was validated using independent datasets. Downregulation of immune cells was observed in GC with high adipocyte expression. GC with high intratumoral adipocyte expression demonstrated aggressive tumor biology and a poorer prognosis. The genes ADH1B, SFRP1, PLAC9, and FABP4 have been identified as holding diagnostic and prognostic significance in GC. These findings strongly support the use of adipocyte expression as a valuable indicator of tumor invasiveness and anticipated patient outcomes in GC.

Lipid-encapsulated mRNA encoding an extended serum half-life interleukin-22 ameliorates metabolic disease in mice

ObjectiveInterleukin (IL)-22 is a potential therapeutic protein for the treatment of metabolic diseases such as obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease due to its involvement in multiple cellular pathways and observed hepatoprotective effects. The short serum half-life of IL-22 has previously limited its use in clinical applications; however, the development of mRNA-lipid nanoparticle (LNP) technology offers a novel therapeutic approach that uses a host-generated IL-22 fusion protein. In the present study, the effects of administration of an mRNA-LNP encoding IL-22 on metabolic disease parameters was investigated in various mouse models. MethodsC57BL/6NCrl mice were used to confirm mouse serum albumin (MSA)-IL-22 protein expression prior to assessments in C57BL/6NTac and CETP/ApoB transgenic mouse models of metabolic disease. Mice were fed either regular chow or a modified amylin liver nonalcoholic steatohepatitis–inducing diet prior to receiving either LNP-encapsulated MSA-IL-22 or MSA mRNA via intravenous or intramuscular injection. Metabolic markers were monitored for the duration of the experiments, and postmortem histology assessment and analysis of metabolic gene expression pathways were performed. ResultsMSA-IL-22 was detectable for ≥8 days following administration. Improvements in body weight, lipid metabolism, glucose metabolism, and lipogenic and fibrotic marker gene expression in the liver were observed in the MSA-IL-22–treated mice, and these effects were shown to be durable. ConclusionsThese results support the application of mRNA-encoded IL-22 as a promising treatment strategy for metabolic syndrome and associated comorbidities in human populations.

Abstract 2523: Predictive biomarkers for neoadjuvant FLOT4 chemotherapy response in gastroesophageal cancer: Results of multi-omics approach

Abstract Background: The FLOT4 chemotherapy regimen is a standard preoperative treatment for gastroesophageal junction (GEJ) and gastric cancer (GC), yet the response to this treatment is variable. Identification of predictive biomarkers is crucial for optimizing therapeutic strategies. This study aims to identify predictive biomarkers for FLOT4 treatment efficacy using a multi-omics approach. Methods: We conducted a preliminary differential gene expression analysis in diagnostic tissue pre-treatment and surgical resection post-treatment in six patients (two GEJ and four GC) who all exhibited a partial pathological response (G2) to neoadjuvant FLOT4 chemotherapy. Utilizing DESeq2 for differential gene expression and David pathway analysis for functional insights, we have begun to unravel the complex molecular response to treatment. Upcoming assays include blood gDNA and tumor tissue whole exome sequencing (WES), comprehensive methylation profiling, miRNA characterization, and proteomics via LC-MSMS. Results: Our preliminary data suggest alterations in gene expression associated with cell adhesion, extracellular matrix interaction, and immune response pathways. Three common genes (CXCL2, CXCL3, CXCL1) were identified among upregulated pathways in GEJ patients, while one common gene (IL1A) was observed in GC patients. These findings suggest a complex network of biological processes influenced by FLOT4. In-depth results, including whole-exome sequencing, methylation patterns, and miRNA profiles, will be completed and presented at the conference. Conclusion: These initial findings propose that changes in gene expression related to cellular architecture and immune response are indicative of a partial response to FLOT4 in both GEJ and GC patients. The identified genes and pathways may serve as potential biomarkers for predicting treatment outcomes. Future Directions: By the time of the conference, we anticipate including a more extensive patient cohort with complete response or no response to FLOT4, and comparative analyses with a FOLFOX treatment group. The integration of forthcoming WES, methylation, miRNA, and proteomics data will provide a more comprehensive molecular predictive model. Keywords: FLOT4 chemotherapy, gastroesophageal junction cancer, gastric cancer, predictive biomarkers, differential gene expression. Citation Format: Amira Al-Fahdi, Shoaib Al Zadjali, Ibrahim Al Haddabi, Anoopa Pullanhi, Muna Al Jabri, Aida Al Yayahee, Nada Al Shuaili, Nahad Al Mahrouqi, Mansour Al Moundhri. Predictive biomarkers for neoadjuvant FLOT4 chemotherapy response in gastroesophageal cancer: Results of multi-omics approach [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 2523.

Abstract 3304: Modulation of enhancer accessibility by SMARCA2 PROTACs synergize with TEAD inhibitors to suppress growth of SMARCA4 mutant lung cancers

Abstract The SWI/SNF chromatin remodeling complex is frequently mutated in non-small cell lung cancer with a frequency of 33% in advanced stage disease, making it the most commonly altered complex in lung cancer. Among the various subunits of the SWI/SNF complex, SMARCA4 and ARID1A are by far the most frequently mutated. Importantly, multiple clinical reports have shown that SMARCA4 mutant lung cancers have one of the worst prognosis among genetically defined lung cancer subtypes and lack response to both immunotherapy and KRAS G12C inhibitors. Recent reports, and our own data, have identified the paralogue SMARCA2 to be synthetic lethal to SMARCA4 suggesting SMARCA2 could be a high value therapeutic target. Unfortunately, the discovery of selective inhibitors of SMARCA2 has so far been challenging. To overcome this hurdle, we have recently developed novel, potent and selective SMARCA2 degrading small molecules based on proteolysis targeting chimera (PROTAC) technology. We demonstrated that YD23, our lead SMARCA2 PROTAC, potently and selectively induced degradation of SMARCA2. Importantly, we showed that YD23 selectively inhibits growth of SMARCA4 mutant lung cancer cells. Mechanistically, we demonstrated that YD23 decreased chromatin accessibility only in SMARCA4 deficient cells. In particular, SMARCA2 degradation profoundly decreased chromatin accessibility at enhancers of a number of genes with cell cycle and cell growth regulatory roles. Gene expression profiling and pathway analysis indicated that cell cycle genes were downregulated by YD23 consistent with the reduced chromatin accessibility at their cis-regulatory regions. We further showed that YD23 robustly inhibited growth of SMARCA4-mutant xenograft tumors. While this is promising, SMARCA2 degradation by itself does not induce regression of tumors highlighting the need for identification of efficacious combination strategies. Using integrative gene expression, ATAC-Seq and CUT&RUN assays, we uncover a critical role for YAP/TEAD transcription factor signaling axis in mediating pro-growth phenotypes in SMARCA4 mutant tumors. Finally, we show that the combination of TEAD inhibitors synergizes with YD23 to induce profound tumor growth inhibition. In conclusion, our study provides a mechanistic basis for the anti-tumor action of SMARCA2 degraders and introduces a novel combination strategy for future clinical development against a genetically defined subtype of lung cancer with dismal prognosis. Citation Format: Sasi Kotagiri, Nicholas Blazanin, Mohammad Qudrattulah, Yuanxin Xi, Jing Wang, Yonathan Lissanu. Modulation of enhancer accessibility by SMARCA2 PROTACs synergize with TEAD inhibitors to suppress growth of SMARCA4 mutant lung cancers [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 3304.

Abstract 7632: Final survival outcomes and post-hoc tumor gene expression pathway analyses of complete responders from a phase Ib clinical trial of HSP90 inhibitor onalespib and paclitaxel in patients with advanced triple-negative breast cancer

Abstract Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for stabilization of client proteins over-activated in triple-negative breast cancer (TNBC). Over-expression of HSP90 client proteins has been implicated in paclitaxel resistance, therefore, it was hypothesized that the potent HSP90 inhibitor onalespib could improve paclitaxel efficacy. As TNBCs are a heterogenous population of tumors, it is important to establish biomarkers of response to this treatment combination. Methods: Patients with TNBC were treated with intravenous (IV) paclitaxel and IV onalespib at doses ranging from 120 mg/m2 to 260 mg/m2 on days 1, 8, and 15 of a 28-day cycle utilizing a standard 3+3 design. The primary objectives were to determine dose-limiting toxicities and maximum tolerated dose. Secondary objectives included determination of overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Retrospectively, archival tumor tissue was obtained from study patients and processed for RNA expression analysis by NanoString nCounter platform with the Breast Cancer 360 panel. Post-hoc gene expression analysis was performed through Qiagen Ingenuity Pathway Analysis (IPA). Linear models were employed to detect differential expression between conditions while considering cartridge effect. Results: 31 patients were enrolled in this study, of which 90% received prior taxane therapy. The established recommended phase II dose was 260 mg/m2 onalespib when given with 80 mg/m2 paclitaxel. ORR was 20%, median DOR was 5.6 months. Three patients achieved complete responses (CR), all of whom had received prior taxane therapy. Nanostring profiling analysis revealed that patients who had a CR to treatment expressed higher levels of several breast cancer-associated genes also known to be dependent upon HSP90 activity compared to patients with progressive disease (PD) as their best treatment response. These genes include FGFR4, JAK2, PIK3R1, TGFB3, and TGFBR2 (all p<0.05). Tumor genes overexpressed in patients with PD versus CR included IRF6, MMP7, and KRT17 (all p<0.01). Additionally, IPA showed patients with CR to have substantial upregulation of PD-1, PD-L1, CTLA-4, p53, HER2, and cyclin/cell cycle regulatory signaling pathways compared to patients with PD (all p<0.01). Conclusions: Combination treatment with onalespib and paclitaxel had an acceptable toxicity profile and showed anti-tumor activity in patients with advanced TNBC. Gene expression analysis of patient tumor samples suggest that TNBCs with greater activation of immune checkpoint pathways and those with proteins dependent on HSP90 activity, including p53 and HER2, may be more susceptible to HSP90 inhibition. ClinicalTrials.gov study identification: NCT02474173 Citation Format: Dionisia Marie Quiroga, Himanshu Savardekar, Emily Schwarz, Nicole O. Williams, Courtney Johnson, Adam Brufsky, Mara Chambers, Saveri Bhattacharya, Maria Patterson, Sagar D. Sardesai, Daniel Stover, Maryam Lustberg, Anne Noonan, Mathew Cherian, Darlene M. Bystry, Kasey Hill, Min Chen, Mitch A. Phelps, Julie Stephens, Lianbo Yu, Bhuvaneswari Ramaswamy, Robert Wesolowski, William E. Carson. Final survival outcomes and post-hoc tumor gene expression pathway analyses of complete responders from a phase Ib clinical trial of HSP90 inhibitor onalespib and paclitaxel in patients with advanced triple-negative breast 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 7632.

RhoB expression associated with chemotherapy response and prognosis in colorectal cancer

PurposeTo examine the role of RhoB expression in relation to chemotherapy response, clinical outcomes and associated signaling pathways in colorectal cancer patients.Materials and methodsThe study included 5 colon cancer cell lines, zebrafish embryos and 260 colorectal cancer patients treated with 5-fluorouracil (5-FU) and oxaliplatin (OXL). The methods consisted of CRISPR/Cas9, reactive oxygen species (ROS), caspase-3 activity, autophagy flux, in-silico RNA sequencing and immunohistochemistry. Gene expression analysis and pathway analysis were conducted using RNA-seq data.ResultsAll cancer lines tested, including SW480, SW480-KO13 (RhoB knockout), SW480-KO55 (RhoB knockout), HCT116 and HCT116-OE (RhoB overexpressed), exhibited cytotoxicity to 5-FU and OXL. RhoB knockout cell lines demonstrated significantly reduced migration compared to the control cell lines. Furthermore, RhoB played a role in caspase-3-dependent apoptosis, regulation of ROS production and autophagic flux. The mRNA sequencing data indicated lower expression levels of oncogenes in RhoB knockout cell lines. The zebrafish model bearing SW480-KO showed a light trend toward tumor regression. RhoB expression by immunohistochemistry in patients was increased from normal mucosa to tumor samples. In patients who received chemotherapy, high RhoB expression was related to worse survival compared to low RhoB expression. Furthermore, the molecular docking analysis revealed that OXL had a higher binding affinity for RhoB than 5-FU, with a binding affinity of -7.8 kcal/mol and HADDOCK predicted molecular interactions between RhoB and caspase 3 protein. Gene-set enrichment analysis supported these findings, showing that enrichment of DNA damage response pathway and p53 signaling in RhoB overexpression treatment group, while the RhoB knockout treatment group exhibited enrichment in the negative regulation pathway of cell migration.ConclusionRhoB was negatively associated with chemotherapy response and survival in colorectal cancers. Therefore, RhoB inhibition may enhance chemotherapeutic responses and patient survival.

Vaginal candida infection is associated with host molecular signatures of neutrophil activation in the adjacent ectocervical mucosa in Kenyan sex workers

AbstractProblemOvergrowth of candida species in the human vaginal mucosa causes inflammation, which could render the mucosal barrier more susceptible to HIV infection. Here, we investigated whether this condition also affects the ectocervical mucosa, a potential site of HIV entry, in women at high risk of HIV infection.Method of studyRetrospective medical data and ectocervical tissue samples were obtained from a cohort of Kenyan sex workers. Among 108 women, seven had signs of vaginal candida infection by wet smear microscopy and/or the presence of characteristic discharge. Women lacking these two criteria served as controls. Host transcriptomic profiling and quantitative in situ image analysis of epithelial barrier markers and CD4+ cell distribution were performed.ResultsThe candida group had 162 differentially expressed genes out of 15 435 genes as compared with the control group. Among these 162 genes, 147 were upregulated and 15 were downregulated. Gene expression pathway analysis indicated associations with an upregulated inflammatory response, defined primarily by markers of neutrophil activation. Transcription factor analysis revealed upregulation of pathways related to RELA/REL/NFKB1, JUN and STAT1 in the candida group. In situ image analysis of ectocervical tissue samples showed no differences between groups in terms of epithelial height, expression of epithelial junction proteins (E‐cadherin, claudin‐1, zonula occludens 1, and desmoglein‐1), or epithelial CD4+ cell distribution.ConclusionsVaginal candida infection was associated with inflammation and neutrophil infiltration, but not with severe epithelial disruption or CD4+ cell infiltration, in the ectocervical mucosa.

Analysis of Artistic Instruction and Emotional Expression Pathways in College Piano Performance in the Internet Era

Abstract As computer science advances, it intersects intriguingly with the realm of music acoustics, particularly in enhancing piano performance through technological means. This paper delves into an innovative approach to piano learning and creation, focusing on emotional expression’s nuances. We have devised a system capable of precise musical tone recognition and sound quality evaluation by adopting Mel Frequency Cepstral Coefficients (MFCC) for the nuanced extraction of piano sounds and integrating dynamic fuzzy neural networks. Our findings show an impressive accuracy rate, with musical tone misidentification below 2.58% and sound quality assessment errors within a 5% margin. This work not only sets a new benchmark in piano performance analysis but also paves the way for revolutionary teaching methods in music education, with profound implications for artistic instruction and emotional expression.

Compound 4f, a novel brain-penetrant reversible monoacylglycerol inhibitor, ameliorates neuroinflammation, neuronal cell loss, and cognitive impairment in mice with kainic acid-induced neurodegeneration.

Neuroinflammation, a hallmark of neurodegenerative diseases, is associated with neuronal cell loss and cognitive dysfunction. Monoacylglycerol lipase (MAGL) is involved in neuroinflammation in the brain via the degradation of endocannabinoid 2-arachidonoylglycerol to arachidonic acid, a precursor of some eicosanoids; therefore, MAGL inhibitors are expected to have anti-inflammatory effects. We recently developed a reversible, selective, central nervous system penetrant, and orally available MAGL inhibitor, compound 4f. Compound 4f (1 mg/kg) robustly increased 2-arachidonoylglycerol levels and decreased arachidonic acid levels in the mouse brain. To examine whether compound 4f can suppress neuroinflammation and neuronal cell loss, kainic acid (KA)-injected mice were used as a neuroinflammation model in this study. Compound 4f (1 mg/kg) significantly decreased the cytokine and chemokine expression levels and suppressed neuronal cell loss in the hippocampi of mice. Compound 4f also ameliorated cognitive impairment in KA-injected mice. The cannabinoid receptor 1 antagonist, AM251, and cannabinoid receptor 2 antagonist, AM630, partly blocked the neuroprotective effects of compound 4f in the hippocampi of KA-injected mice. Gene expression profiles and pathway analyses revealed that compound 4f reversed the KA-induced changes in the expression of genes related to inflammation and neurotransmission. These results indicate that the selective and reversible MAGL inhibitor, compound 4f, can be used as a potential therapeutic agent for the treatment of neurodegenerative diseases.

Identification of key biomarkers and therapeutic targets in sepsis through coagulation-related gene expression and immune pathway analysis.

Sepsis, characterized by a widespread and dysregulated immune response to infection leading to organ dysfunction, presents significant challenges in diagnosis and treatment. In this study, we investigated 203 coagulation-related genes in sepsis patients to explore their roles in the disease. Through differential gene expression analysis, we identified 20 genes with altered expression patterns. Subsequent correlation analysis, visualized through circos plots and heatmaps, revealed significant relationships among these genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that these genes are involved in immune response activation, coagulation, and immune receptor activity. Disease Ontology (DO) enrichment analysis further linked these genes to autoimmune hemolytic anemia and tumor-related signaling pathways. Additionally, the CIBERSORT analysis highlighted differences in immune cell composition in sepsis patients, revealing an increase in neutrophils and monocytes and a decrease in inactive NK cells, CD8 T cells, and B cells. We employed machine learning techniques, including random forest and SVM, to construct a diagnostic model, identifying FCER1G and FYN as key biomarkers. These biomarkers were validated through their expression levels and ROC curve analysis in an independent validation cohort, demonstrating strong diagnostic potential. Single-cell analysis from the GSE167363 dataset further confirmed the distinct expression profiles of these genes across various cell types, with FCER1G predominantly expressed in monocytes, NK cells, and platelets, and FYN in CD4+ T cells and NK cells. Enrichment analysis via GSEA and ssGSEA revealed that these genes are involved in critical pathways, including intestinal immune networks, fatty acid synthesis, and antigen processing. In conclusion, our comprehensive analysis identifies FCER1G and FYN as promising biomarkers for sepsis, providing valuable insights into the molecular mechanisms of this complex condition. These findings offer new avenues for the development of targeted diagnostic and therapeutic strategies in sepsis management.

Utilizing bioinformatics tools for analyzing high-throughput data in biomedical research

Bioinformatics has become crucial in biomedical research, enabling the processing of massive volumes of high- throughput data generated by various omics technologies. This work investigates the use of bioinformatics tools to process, analyze, and interpret omics data, including genomics, transcriptomics, proteomics, and metabolomics. It provides an overview of widely used bioinformatics methodologies and algorithms for data preparation, quality control, differential expression analysis, pathway analysis, and functional annotation. The study also highlights current trends and challenges in bioinformatics, such as integrating multi-omics data and developing machine learning algorithms for predictive modeling. This work aims to encourage academics to utilize bioinformatics methods to gain insights into complex biological systems and enhance our understanding of human health and disease.

Psychosocial stressors and breast cancer gene expression in the Black Women's Health Study.

Prior studies indicate that the physiologic response to stress can affect gene expression. We evaluated differential gene expression in breast cancers collected from Black women with high versus low exposure to psychosocial stressors. We analyzed tumor RNA sequencing data from 417 Black Women's Health Study breast cancer cases with data on early life trauma and neighborhood disadvantage. We conducted age-adjusted differential gene expression analyses and pathway analyses. We also evaluated Conserved Transcriptional Response to Adversity (CTRA) contrast scores, relative fractions of immune cell types, T cell exhaustion, and adrenergic signaling. Analyses were run separately for estrogen receptor positive (ER+; n = 299) and ER- (n = 118) cases. Among ER+ cases, the top differentially expressed pathways by stress exposure were related to RNA and protein metabolism. Among ER- cases, they were related to developmental biology, signal transduction, metabolism, and the immune system. Targeted analyses indicated greater immune pathway enrichment with stress exposure for ER- cases, and possible relevance of adrenergic signaling for ER+ cases. CTRA contrast scores did not differ by stress exposure, but in analyses of the CTRA components, ER- breast cancer cases with high neighborhood disadvantage had higher pro-inflammatory gene expression (p = 0.039) and higher antibody gene expression (p = 0.006) compared to those with low neighborhood disadvantage. There are multiple pathways through which psychosocial stress exposure may influence breast tumor biology. Given the present findings on inflammation and immune response in ER- tumors, further research to identify stress-induced changes in the etiology and progression of ER- breast cancer is warranted.

Single-cell resolution of human airway epithelial cells exposed to bronchiolitis obliterans-associated chemicals.

Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.

Effects of Dapagliflozin on Myocardial Gene Expression in BTBR Mice with Type 2 Diabetes.

Dapagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is approved for the treatment of type 2 diabetes, heart failure, and chronic kidney disease. DAPA-HF and DELIVER trial results demonstrate that the cardiovascular protective effect of dapagliflozin extends to non-diabetic patients. Hence, the mechanism-of-action may extend beyond glucose-lowering and is not completely elucidated. We have previously shown that dapagliflozin reduces cardiac hypertrophy, inflammation, fibrosis, and apoptosis and increases ejection fraction in BTBR mice with type 2 diabetes. We conducted a follow-up RNA-sequencing study on the heart tissue of these animals and performed differential expression and Ingenuity Pathway analysis. Selected markers were confirmed by RT-PCR and Western blot. SGLT2 had negligible expression in heart tissue. Dapagliflozin improved cardiac metabolism by decreasing glycolysis and pyruvate utilization enzymes, induced antioxidant enzymes, and decreased expression of hypoxia markers. Expression of inflammation, apoptosis, and hypertrophy pathways was decreased. These observations corresponded to the effects of dapagliflozin in the clinical trials.