Whole-transcriptome Sequencing Research Articles

The ncRNA-mediated regulatory networks of defensins and lysozymes in Riptortus pedestris: involvement in response to gut bacterial disturbances

Insects depend on humoral immunity against intruders through the secretion of antimicrobial peptides (AMPs) and immune effectors via NF-κB transcription factors, and their fitness is improved by gut bacterial microbiota. Although there are growing numbers of reports on noncoding RNAs (ncRNAs) involving in immune responses against pathogens, comprehensive studies of ncRNA-AMP regulatory networks in Riptortus pedestris, which is one of the widely distributed pests in East Asia, are still not well understood under feeding environmental changes. The objective of this study employed the whole-transcriptome sequencing (WTS) to systematically identify the lncRNAs (long noncoding RNA) and circRNAs (circular RNA) and to obtain their differential expression from the R. pedestris gut under different feeding conditions. Functional annotation indicated that they were mainly enriched in various biological processes with the GO and KEGG databases, especially in immune signaling pathways. Five defensin (four novel members) and eleven lysozyme (nine novel members) family genes were identified and characterized from WTS data, and meanwhile, phylogenetic analysis confirmed their classification. Subsequently, the miRNA–mRNA interaction network of above two AMPs and lncRNA-involved ceRNA (competing endogenous RNA) regulatory network of one lysozyme were predicted and built based on bioinformatic prediction and calculation, and the expression patterns of differentially expressed (DE) defensins, and DE lysozymes and related DE ncRNAs were estimated and selected among all the comparison groups. Finally, to integrate the analyses of WTS and previous 16S rRNA amplicon sequencing, we conducted the Pearson correlation analysis to reveal the significantly positive or negative correlation between above DE AMPs and ncRNAs, as well as most changes in the gut bacterial microbiota at the genus level of R. pedestris. Taken together, the present observations provide great insights into the ncRNA regulatory networks of AMPs in response to rearing environmental changes in insects and uncover new potential strategies for pest control in the future.

Transcriptomic Profiling of Peripheral B Cells in Antibody Positive Sjogren's Patients Reveals Interferon Signature.

Sjögren's disease (SjD) is a common systemic autoimmune disease that affects mainly women. Key pathologic features include the infiltration of exocrine glands by lymphocytes and the activation of B lymphocytes with the production of autoantibodies. We aimed to analyze the transcriptome of circulating B cells from patients with SJD and healthy controls to decipher the B-cell-specific contribution to SJD. RNA from peripheral blood B cells of five untreated female patients with SjD and positive ANA, positive anti-SSA (both Ro-52 and Ro-60), positive anti-SSB and positive rheumatoid-factor, and five healthy controls was subjected to whole-transcriptome sequencing. A false discovery rate of < 0.1 was applied to define differentially expressed genes (DEG). RNA-sequencing identified 56 up and 23 down DEG. Hierarchal clustering showed a clear separation between the two groups. Ingenuity pathway analysis revealed that these genes may play a role in interferon signaling, chronic mycobacterial infection, and transformation to myeloproliferative disorders. We found upregulated expression of type-I and type-II interferon (IFN)-induced genes, as well as genes that may contribute to other concomitant conditions, including infections and a higher risk of myeloproliferative disorders. This adds insight into the autoimmune process and suggests potential targets for future functional and prognostic studies.

Comprehensive molecular profiling identifies actionable biomarkers for patients from Thailand and the United Arab Emirates with advanced malignancies.

Comprehensive molecular profiling of tissue samples that can help guide therapy management is not widely available across the globe. Comprehensive molecular profiling through Caris Molecular Intelligence involves the analysis of DNA through next-generation sequencing, chromogenic or fluorescent in situ hybridization, pyrosequencing, and copy number alterations; RNA through whole-transcriptome sequencing and multiplex PCR of RNA; and protein through immunohistochemistry. Here we describe the experience of molecular profiling of tumor tissue samples from patients diagnosed with advanced solid tumors and treated in two countries, the United Arab Emirates and Thailand. Tumor cancer cases submitted to Caris Life Sciences (Phoenix, Arizona, USA) for molecular profiling from the UAE and Thailand were retrospectively analyzed (data accessed between 2019 and 2020) for their molecular alterations and clinical biomarkers, without regard to ethnicity. A total of 451 samples from 35 distinct types of advanced cancers were examined for mutations, amplifications, overexpression, exon copy number alterations, microsatellite instability, deficient mismatch repair, tumor mutational burden, and fusions. Interrogating each step of the biological pathway, from DNA to RNA to distinct protein, identified an alteration with an associated therapy for 75% of these tumor samples. The most common alterations identified included elevated PDL-1 that can be targeted with an immune checkpoint inhibitors and amplification of HER2 for which a variety of anti HER2 therapies are available. Comprehensive molecular profiling in patients with advanced malignancies can help optimize therapeutic management allowing for improved prognostic outcome.

CircHIF1A induces cetuximab resistance in colorectal cancer by promoting HIF1α-mediated glycometabolism alteration

Epidermal growth factor receptor (EGFR)-targeted therapy is an important treatment for RAS wild-type metastatic colorectal cancer (mCRC), but the resistance mechanism remains unclear. Here, the differential expression of circRNAs between Cetuximab sensitive and resistant cell lines was analyzed using whole-transcriptome sequencing. We identified that the expression of circHIF1A was significantly higher in LIM1215-R than in LIM1215. When treated with Cetuximab, downregulation of circHIF1A level weakened the proliferation and clonal formation ability of LIM1215-R, caused more cells to enter G0-G1 phase, and significantly reduced the basal respiration, ATP production, and maximal respiration, as well as the glycolytic capacity and glycolytic reserve. The response rate and prognosis of circHIF1A-positive patients were inferior to those of negative patients. Mechanistically, circHIF1A can upregulate the level of hypoxia-inducible factor 1 A (HIF1A) by competitively binding to miR-361-5p, inducing the overexpression of enzymes such as glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). In a xenograft model, inhibition of circHIF1A expression increased the sensitivity to Cetuximab treatment. In conclusion, circHIF1A can promote HIF1α-mediated glycometabolism alteration to induce Cetuximab resistance in CRC. It has the potential to become a screening indicator for the Cetuximab beneficial population in mCRC and a new therapeutic target for enhancing treatment efficacy.

Pan-Cancer Interrogation of B7-H3 (CD276) as an Actionable Therapeutic Target across Human Malignancies.

B7-H3 (CD276) is a transmembrane glycoprotein of the B7 immune checkpoint superfamily that has emerged as a promising therapeutic target. To better understand the applicability of B7-H3-directed therapies, we analyzed 156,791 samples comprising 50 cancer types to interrogate the clinical, genomic, transcriptomic, and immunological correlates of B7-H3 mRNA expression. DNA (592-gene/whole-exome) and RNA (whole-transcriptome) sequencing was performed from samples submitted to Caris Life Sciences (Phoenix, AZ). B7-H3 high versus low expression was based on top and bottom quartiles for each cancer type. Patients' overall survival was determined from insurance claims data. Pathway analysis was performed using Gene Set Enrichment Analyses (GSEA). Immune cell fractions were inferred using quanTIseq. B7-H3 is expressed across several human malignancies including prostate, pancreatic, ovarian, and lung cancers. High B7-H3 expression is associated with differences in overall survival, possibly indicating a prognostic role of B7-H3 for some cancers. When examining molecular features across all cancer types, we did not identify recurrent associations between B7-H3 expression and genetic alterations in TP53, RB1, and KRAS. However, we find consistent enrichment of EMT, Wnt, TGF-beta, and Notch signaling pathways. Additionally, tumors with high B7-H3 expression are associated with greater proportions of M1 macrophages, but lower fractions of CD8+ T cells. We have begun to define the genomic, transcriptomic, clinical, and immunological features associated with B7-H3 expression in 50 cancer types. We report novel clinical and molecular features of B7-H3-high tumors which may inform how current B7-H3 therapeutics should be deployed and prioritized.

Abstract PO5-13-06: Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes

Abstract Background It has been recently demonstrated that some HER2-low, defined as immunohistochemistry (IHC) 1+ or 2+ with no gene amplification by FISH, breast cancer (BC) patients respond to trastuzumab deruxtecan (T-DXd). Results of the DAISY trial also suggested clinical activity with T-DXd in some of those classified as HER2-0 cancers by IHC, indicating an unmet need to better identify cancers that may truly respond to T-DXd. We compared ERBB2 (HER2) mRNA expression obtained from whole-transcriptome sequencing with IHC/FISH assignment of HER2 status. Furthermore, we constructed a classifier using ERBB2 expression and alterations in other genes as a proof of concept to illustrate classification of BC samples into HER2-0 versus HER2-low status. Methods We chose consecutive HR positive/HER2-negative BC samples that were submitted for OncoExTraTM testing between April 2020 and October 2022. The OncoExTra test uses tumor-normal whole exome and whole transcriptome sequencing to detect somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. In addition, we investigated the expression profile of select genes for this analysis. We utilized OncoExTra data to examine the relationship between HER2 IHC status and gene expression, mutation and copy number profiles of selected genes, and built a classifier to predict HER2 status. Results A total of 279 BC samples were analyzed, including 106 HER2-IHC 0, 120 HER2-IHC 1+ and 53 HER2-IHC 2+. Of these, 273 were primary and 6 were metastatic samples. The distributions of patient age and tumor fraction were similar among these categories (p=0.54 and p=0.41, respectively; one-way ANOVA). ERBB2 expression differed among all three HER2-IHC categories and was lowest in HER2-0 and highest in HER2-2+ (HER2-0 vs HER2-1+, p&amp;lt; 0.001; HER2-0 vs HER2-2+, p&amp;lt; 0.0001; HER2-1+ vs HER2-2+, p=0.011; HER2-0 vs HER2-low, p&amp;lt; 0.0001; Mann-Whitney U test in all comparisons). Five genes were mutated in more than 10% of samples: PIK3CA (45.5%), GATA3 (15.4%), CDH1 (12.5%), MAP3K1 (11.1%) and TP53 (10.8%). Mutations in two genes, GATA3 and PTEN, appeared to differ in frequency between the HER2-0 and HER2-low categories (Table 1). We selected 4 genes with mutations, 2 genes with copy number changes, and expression from ERBB2, ESR1, CD274, HLA-A, HLA-B and HLA-C to train a logistic regression classifier to predict HER2 IHC status. The classifier was trained on 163 samples, hyperparameter tuning was done using 55 samples, and testing was performed on 61 samples. Preliminary results indicated that of the 28 HER2-IHC 0 samples in the test set, 17 (65%) were classified as HER2-low. Further development and evaluation of the classifier using a blinded testing set is currently in progress. Conclusion In this cohort of BC samples, ERBB2 mRNA expression was positively associated with HER2 protein IHC status. Additional studies are required to determine whether a classifier including ERBB2 mRNA expression could be used to identify patients for T-DXd therapy. The relative enrichment of PTEN alterations in HER2-IHC 0 samples suggests that mTOR/AKT inhibitors may sometimes be appropriate in patients who are not eligible for T-DXd therapy, and further investigation should be considered. Table 1. Number (frequency) of altered genes by HER2 IHC status. Citation Format: Gargi Basu, Niru Chennagiri, Turgut Dogruluk, David Hall, Jess Hoag, Cynthia Flannery, Snehal Thakkar, Melanie Palomares, Frederick Baehner, Lajos Pusztai. Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes [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 PO5-13-06.

Abstract GS03-07: Protocol-defined biomarker analysis in the PALLAS (AFT-05) adjuvant trial: Genomic subtype derived from RNA sequencing of HR+/HER2- early breast cancer

Abstract Background. The phase 3 PALLAS trial (NCT02513394) compared two years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. Genomic subtype (PAM50 intrinsic subtype) measured from whole-transcriptome RNA sequencing data was defined in the protocol of the PALLAS trial as the primary biomarker for analysis of prediction and prognosis. Clinical data have been previously presented (Gnant et al, JCO 2022), and the trial now has 5-year median follow-up. Methods. As part of trial eligibility, all participants in PALLAS provided a tumor tissue block prior to randomization (surgical if primary resection, core biopsy if neoadjuvant treatment) for translational analyses (TRANS-PALLAS). The biorepository and laboratory were blinded to identity and processed samples in random order, to minimize bias. Nucleic acids were extracted from samples with sufficient tumor tissue and cellularity (&amp;gt;25 mm2 with ≥20% cancer nuclei). The Genome Sequencing Center at Washington University St. Louis performed whole-transcriptome RNA sequencing. Libraries were prepared from 1 µg DNase-1 treated total RNA, if total RNA DV200 &amp;gt;28 (Agilent Bioanalyzer), using an unbiased library protocol of RNA HyperPrep kit with RiboErase (HMR) (Kapa Biosystems, Wilmington, MA). 100 bp paired-end sequencing was performed on NovaSeq 6000 using S4 Reagent Kit (Illumina, San Diego, CA), with 48 libraries pooled per lane. Intrinsic subtype was determined using Bioclassifier package (Research PAM50 script, Parker et al.) only for the analysis population of primary breast cancer samples that had not been exposed to prior neoadjuvant therapy. Invasive disease-free survival (IDFS) will be visualized using Kaplan-Meier plots, with log-rank test between groups. Cox models of proportional hazards will be developed to evaluate prognosis adjusted for known clinical covariates, or for predictive interactions. The pre-defined level of significance is a two-sided 0.05. Results. From the total study population of 5796 enrolled patients, 4655 tissue blocks had sufficient tumor content to process for RNA, with 3931 yielding sufficient RNA for sequencing, and 2669/4655 (57.3%) submitted tissue blocks had DV200 ≥28 and were successfully sequenced. Clinical unblinding revealed 2370 unique patients (1182 in the palbociclib treatment arm and 1188 in the control arm) with intrinsic subtype defined from their untreated primary tumor: 1555 (65.6%) luminal A, 287 (12.1%) luminal B, 167 (7.0%) HER2-enriched, 310 (13.1%) basal-like, 51 (2.2%) normal-like. We will report the results for association of molecular subtype, proliferation score, and Risk of Recurrence (ROR) scores with invasive disease-free survival (IDFS) by treatment arm at the meeting. Conclusions. The TRANS-PALLAS cohort represents one of the largest biorepositories of HR+/HER2- early breast cancer reflecting contemporary systemic management in the framework of a prospectively randomized global trial. Required tumor block submission in this phase 3 trial yielded data from unbiased whole-transcriptome RNA sequencing of the primary tumor prior to treatment from 41% of the PALLAS participants. The proportion of luminal A cancers was unexpectedly high (66%), indicating a lower-risk distribution of cancers in this population. The planned analyses of prediction and prognosis are ongoing and those results will be presented at the time of the meeting. Support: AFT, ABCSG, Pfizer, ECOG-ACRIN, NSABP Foundation, GBG, BIG; Clinicaltrials.gov Identifier: NCT02513394; https://acknowledgments.alliancefound.org Citation Format: Daniel Stover, Dominik Hlauschek, Erica Mayer, W. Fraser Symmans, Mark Watson, Iros Barozzi, Martin Filipits, Karla Ballman, Meritxell Bellet- Ezquerra, Justin Balko, Gabor Rubovszky, Nicholas Zdenkowski, Adam Brufsky, Guenther Steger, Claudine Isaacs, Sibylle Loibl, Fernando Henao, Meredith Regan, Yuan Liu, Christian Fesl, Patrick O'Brien, Angela DeMichele, Michael Gnant, Otto Metzger. Protocol-defined biomarker analysis in the PALLAS (AFT-05) adjuvant trial: Genomic subtype derived from RNA sequencing of HR+/HER2- early 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 GS03-07.

High-throughput molecular assays for inclusion in personalised oncology trials - State-of-the-art and beyond.

In the last decades, the development of high-throughput molecular assays has revolutionised cancer diagnostics, paving the way for the concept of personalised cancer medicine. This progress has been driven by the introduction of such technologies through biomarker-driven oncology trials. In this review, strengths and limitations of various state-of-the-art sequencing technologies, including gene panel sequencing (DNA and RNA), whole-exome/whole-genome sequencing and whole-transcriptome sequencing, are explored, focusing on their ability to identify clinically relevant biomarkers with diagnostic, prognostic and/or predictive impact. This includes the need to assess complex biomarkers, for example microsatellite instability, tumour mutation burden and homologous recombination deficiency, to identify patients suitable for specific therapies, including immunotherapy. Furthermore, the crucial role of biomarker analysis and multidisciplinary molecular tumour boards in selecting patients for trial inclusion is discussed in relation to various trial concepts, including drug repurposing. Recognising that today's exploratory techniques will evolve into tomorrow's routine diagnostics and clinical study inclusion assays, the importance of emerging technologies for multimodal diagnostics, such as proteomics and in vivo drug sensitivity testing, is also discussed. In addition, key regulatory aspects and the importance of patient engagement in all phases of a clinical trial are described. Finally, we propose a set of recommendations for consideration when planning a new precision cancer medicine trial.

Abstract PO2-06-10: Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative breast cancer patients

Abstract Introduction: Immune checkpoint inhibitors (ICI) are an important therapeutic option for patients with triple negative breast cancer (TNBC). However, identification of patients most likely to respond is challenging. PD-L1 positivity by immunohistochemistry is the standard biomarker used for ICI therapy selection in TNBC. However, other biomarkers, such as analysis of the tumor microenvironment (TME) may be more accurate in predicting response. The XernaTM TME Panel uses RNA sequencing data and machine learning to analyze the TME, utilizing the angiogenic and immunogenic biology of the TME to classify tumors into four TME subtypes. In this study, the distribution of Xerna TME subtypes and associated genomic alterations in TNBC were investigated for their potential use in therapy selection. Methods: A total of 203 TNBC patient samples underwent tumor-normal whole-exome, whole-transcriptome sequencing testing with the OncoExTraTM assay. The whole-transcriptome expression data were analyzed using the Xerna TME Panel to assign each sample to one of four subtypes: Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A). The IA and IS subtypes both have high immune scores that may be particularly sensitive to ICI therapy. Actionable alterations, defined as those with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies, were also identified and associations across Xerna subtypes were explored. Results: Approximately half (100 of 203; 49.3%) of the patient samples had high (IA+IS) immune subtypes (Table 1). Targetable alterations associated with an FDA-approved therapy were present in 114 (56.2%) patients. No biomarkers were significantly associated (p &amp;lt; 0.05) with high (IA+IS) versus low (ID+A) immune scores. Biomarkers associated with ICI response, namely mismatch repair gene alterations (MSH2/3/6, MLH1/3, PMS1/2), high tumor mutational burden (TMB-high) and microsatellite instability were detected in only 6 (3.0%), 3 (1.5%) and 1 (0.5%) patient samples respectively, and all but 1, an MSH6 alteration, were in high immune subtype samples. Conclusions: The Xerna TME Panel classified 49.3% of TNBC patient tumors to IA or IS, suggesting they may respond to ICI therapy. Many (56.2%) patient tumors harbored alterations associated with FDA-approved therapies, providing the potential for novel combination therapies. These findings warrant further study and clinical validation in TNBC patients treated with ICI therapy. Table 1. Frequency of actionable biomarkers that were present in at least 10 (5%) TNBC patient samples. Citation Format: Gargi Basu, Janine Lobello, Snehal Thakkar, Jessica Aldrich, Matthew Halbert, Patrick Eimerman, Cynthia Flannery, Nishitha Therala, David Hall, Daniel Pointing, Lea Vohar, Roman Luštrik, Luka Ausec, Mark Uhlik, Seema Iyer, Laura Benjamin, Frederick Baehner. Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative breast cancer patients [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 PO2-06-10.

Exploring the Mechanisms of Yishen Tongluo Decoction on Repairing DNA Damage in Mouse Spermatogonia Cells Based on Whole Transcriptome Sequencing.

The aim of this study was to investigate the potential mechanism through which Yishen Tongluo decoction (YSTL) repairs DNA damage caused by benzo(a)pyrene diol epoxide (BPDE) in mouse spermatocytes (GC-2). The GC-2 cells were divided randomly into the control group, BPDE group, and low-, medium-, and high-dose YSTL groups of YSTL decoction. A comet assay was used to detect the DNA fragment index (DFI) of cells in each group. Based on the DFI results, whole transcriptome sequencing was conducted, followed by trend analysis, gene ontology (GO) enrichment analysis, kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and ceRNA network analysis. Compared with the control group, the BPDE group reported a significant increase in the DNA fragmentation index (DFI) (p < .05). Compared with the BPDE group, the low-, high- and medium-dose YSTL groups had a significantly reduced DFI (p < .05). Whole-transcriptome sequencing revealed seven differentially expressed circRNAs, 203 differentially expressed miRNAs, and 3,662 differentially expressed mRNAs between the control group and the BPDE group. There was a total of 12 differentially expressed circRNAs, 204 miRNAs, and 2150 mRNAs between the BPDE group and the traditional Chinese medicine group. The pathways involved include DNA repair pathway, nucleotide excision repair pathway, base excision repair pathway, etc. The ceRNA network reported that Hmga2 was the core protein involved, novel_cir_000117 and mmu-miR-466c-3p were located upstream of Hmga2, and they were regulatory factors associated with Hmga2. Finally, we conclude that YSTL decoction may repair sperm DNA damage caused by BPDE through the novel_cir_000117-mmu-miR-466c-3p-Hmga2 pathway.

RET Alterations Differentiate Molecular Profile of Medullary Thyroid Cancer.

Medullary thyroid cancer (MTC) is a rare cancer originating from parafollicular C cells of the thyroid gland. Therapeutically relevant alterations in MTC are predominantly reported in RET oncogene, and lower-frequency alterations are reported in KRAS and BRAF. Nevertheless, there is an unmet need existing to analyze the MTC in the Indian cohort by using in-depth sequencing techniques that go beyond the identification of known therapeutic biomarkers. Here, we characterize MTC using integrative whole-exome and whole-transcriptome sequencing of 32 MTC tissue samples. We performed clinically relevant variant analysis, molecular pathway analysis, tumor immune-microenvironment analysis, and structural characterization of RET novel mutation. Mutational landscape analysis shows expected RET mutations in 50% of the cases. Furthermore, we observed mutations in known cancer genes like KRAS, HRAS, SF3B1, and BRAF to be altered only in the RET-negative cohort. Pathway analysis showed differential enrichment of mutations in transcriptional deregulation genes in the RET-negative cohort. Furthermore, we observed novel RET kinase domain mutation Y900S showing affinity to RET inhibitors accessed via molecular docking and molecular dynamics simulation. Altogether, this study provides a detailed genomic characterization of patients with MTC of Indian origin, highlighting the possible utility of targeted therapies in this disease.

Dynamic Whole-Transcriptome landscape of Acute Bilirubin Encephalopathy in Newborns

Hyperbilirubinemia in newborns may progress to acute bilirubin encephalopathy (ABE), posing short- and long-term health risks. Despite extensive research identifying numerous mRNAs, lncRNAs, circRNAs, and miRNAs associated with brain injury, their roles in neonatal bilirubin-induced brain injury remain elusive. This study employed whole-transcriptome sequencing to ascertain the differentially expressed (DE) RNA profiles in a newborn ABE rat model, followed by bioinformatic analysis. A time-series competing endogenous RNA (ceRNA) regulatory network was established, and the expression trends of 9 arbitrarily chosen RNAs were verified through quantitative real-time polymerase chain reaction(qRT-PCR). In comparison with the control group, we identified 595, 888, and 1448 DE mRNAs; 22, 37, and 37 DE miRNAs; 1945, 1869, and 1997 DE lncRNAs; and 31, 28, and 36 DE circRNAs at 6hours, 12hours, and 24hours, respectively. Predominantly, these DERNAs contribute to biological functions and pathways associated with inflammation, immunity, metabolism, cell death, and neurodevelopmental regulation. Moreover, we constructed ceRNA networks of DE lncRNA/circRNA-DE miRNA-DE mRNA based on time series. The qRT-PCR expression trends for the selected 9 RNAs were generally similar to the RNA-seq outcomes. This investigation uniquely delineated the temporal expression patterns of mRNA and non-coding RNA in ABE, establishing ceRNA networks and identifying potential molecular mechanisms underlying bilirubin-induced hippocampal damage. Nonetheless, further studies are warranted to corroborate these findings in humans.

A novel protein encoded by circLARP1B promotes the proliferation and migration of vascular smooth muscle cells by suppressing cAMP signaling

Background and aimsCircular RNA (circRNA) is closely related to atherosclerosis (AS) incidence and progression, but its regulatory mechanism in AS needs further elucidation. AS development is significantly influenced by abnormal vascular smooth muscle cells (VSMCs) growth and migration. This study explored the potential protein role encoded by circLARP1B on VSMC proliferation and migration. MethodsWe performed whole-transcriptome sequencing in human normal arterial intima and advanced atherosclerotic plaques to screen for differentially expressed circRNAs. The sequencing results were combined with database analysis to screen for circRNAs with coding ability. Real-time quantitative polymerase chain reaction was utilized to identify the circLARP1B expression level in atherosclerotic plaque tissues and cells. CircLARP1B-243aa function and pathway in VSMCs growth and migration were studied by Scratch, Transwell, 5-ethynyl-2'-deoxyuridine, Cell Counting Kit-8, and Western Blot experiments. ResultsWe found that circLARP1B was downregulated in atherosclerotic plaque tissue and promoted the proliferation and migration of VSMCs. circLARP1B encodes a novel protein with a length of 243 amino acids. Through functional experiments, we confirmed the role of circLARP1B-243aa in enhancing VSMCs migration and proliferation. Mechanistically, circLARP1B-243aa promotes VSMCs migration and growth by upregulating Phosphodiesterase 4C to inhibit the cyclic adenosine monophosphate signaling pathway. ConclusionsOur outcomes suggested that circLARP1B could promote VSMCs growth and migration through the encoded protein circLARP1B-243aa. Therefore, it could be a treatment target and biomarker for AS.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

Identification and bioinformatics analysis of lncRNAs in serum of patients with ankylosing spondylitis

ObjectivesThe aim of this study was to explore the long non-coding RNA (lncRNA) expression profiles in serum of patients with ankylosing spondylitis (AS). The role of these lncRNAs in this complex autoimmune situation needs to be evaluated.MethodsWe used high-throughput whole-transcriptome sequencing to generate sequencing data from three patients with AS and three normal controls (NC). Then, we performed bioinformatics analyses to identify the functional and biological processes associated with differentially expressed lncRNAs (DElncRNAs). We confirmed the validity of our RNA-seq data by assessing the expression of eight lncRNAs via quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 20 AS and 20 NC samples. We measured the correlation between the expression levels of lncRNAs and patient clinical index values using the Spearman correlation test.ResultsWe identified 72 significantly upregulated and 73 significantly downregulated lncRNAs in AS patients compared to NC. qRT-PCR was performed to validate the expression of selected DElncRNAs; the results demonstrated that the expression levels of MALAT1:24, NBR2:9, lnc-DLK1-35:13, lnc-LARP1-1:1, lnc-AIPL1-1:7, and lnc-SLC12A7-1:16 were consistent with the sequencing analysis results. Enrichment analysis showed that DElncRNAs mainly participated in the immune and inflammatory responses pathways, such as regulation of protein ubiquitination, major histocompatibility complex class I-mediated antigen processing and presentation, MAPkinase activation, and interleukin-17 signaling pathways. In addition, a competing endogenous RNA network was constructed to determine the interaction among the lncRNAs, microRNAs, and mRNAs based on the confirmed lncRNAs (MALAT1:24 and NBR2:9). We further found the expression of MALAT1:24 and NBR2:9 to be positively correlated with disease severity.ConclusionTaken together, our study presents a comprehensive overview of lncRNAs in the serum of AS patients, thereby contributing novel perspectives on the underlying pathogenic mechanisms of this condition. In addition, our study predicted MALAT1 has the potential to be deeply involved in the pathogenesis of AS.

Abstract LB432: Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal brain tumor types with a five-year survival rate of 6.9%. In 2023, more than 14,490 Americans were expected to receive a diagnosis. Although great progress has been achieved in protein target identification and treatment over the last few years, current treatment options are limited, and tumor recurrence occurs at a high rate due to treatment resistance. Aptamers, short single-stranded oligonucleotide ligands with high-affinity binding to molecular targets, are well suited tools for exploring novel targets in highly heterogeneous tumors with complex etiologies like GBM, where clinically relevant biomarkers are limited. In this study, aptamer-based cancer-specific drug target identification was performed by Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) Biotargeting System. In short, a GBM-specific library was enriched through a Systematic Evolution of Ligands by EXponential enrichment (SELEX) process using micro dissected Formalin-Fixed Paraffin-Embedded (FFPE) tissues from GBM patients, along with whole body normal tissue for counterselection. Microdissection of tissue allows the partitioning of different types of cells to focus on specific cells of interest. The enriched libraries have been evaluated on clinical GBM and normal brain control samples through aptamer mediated tissue staining, resulting in preferential binding of enriched libraries compared to controls in the vast majority of GBM patients with little binding to normal brain samples. The final library was subsequently applied in an in-house developed library-based affinity pull-down assay using lysates from FFPE tissue of the same group of GBM patients and normal brain samples, followed by unbiased Mass Spectrometry for protein target identification. Combining the results from this proteomics analysis and data from the internal Whole Transcriptome Sequencing (WTS) database, we were able to identify several targets that are highly expressed in GBM and other cancer lineages. Antibody staining provided further validation of the expression levels and the cellular localization. The targets identified in this study include not only potential novel therapeutic targets, but also well-studied biomarkers and targets with available drugs already in clinical trials, demonstrating the platform has great potential for identifying high affinity binding and selectivity compared to traditional proteomics and other methods in drug target discovery. Citation Format: Xixi Wei, Myunggi An, Radhika Santhanam, Barbara Richardson, Lucas Vu, Justin Saul, Anna Walton, Michael Gee, Daniel Martin, Amanda Hubbard, Alexis Rodin, Matthew Rosenow, Tassilo Hornung. Identification of novel drug targets using the ADAPT biotargeting system on FFPE tissue from patients with glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB432.

Abstract LB435: Large panel organoid drug testing combined with biomarker analysis: Unveiling prospects for mechanism of action identification and preclinical patient stratification

Abstract Introduction The development of oncology drugs has a persistently high attrition rate of greater than 95%, requiring the development and implementation of novel models and technologies aimed at better predictive abilities during the preclinical phase. Among these, organoid technologies, which involve propagating tumor stem cells in a near-native state, have proven to markedly advance drug testing with enhanced precision. Our previously developed assay-ready organoid technology has paved the way for large panel organoid screening, allowing organoids to augment the efficiency of drug development pipelines, boost patient-derived model usage in preclinical studies, and facilitate accurate predictions of drug action mechanisms. Here, we present a proof-of-concept (POC) large scale organoid experiment, studying the responses of 50 models to the cytotoxic agent paclitaxel, using next generation sequencing data to analyze the drug mechanism of action (MOA) and identify biomarkers that predict drug responses. Methods A panel of 50 organoid models was characterized for paclitaxel response in 9-dose-response curves by measuring ATP levels after a 5-day exposure. Models were genomically profiled by whole-exome sequencing (n = 47) and whole-transcriptome sequencing (n = 44) to analyze the association between organoid pharmacology data and genomic information and elucidate genes and pathways that may be related with, or predictive of, the treatment responses. We performed biomarker analysis on gene expression, mutation, and copy number alteration levels. Results By assessing organoid sensitivity to paclitaxel, we observed no significant indication-specific difference in organoid responses across 7 cancer types. The 50 models were then categorized into responders (AUC &amp;lt; 1.5; n=15) and non-responders (AUC &amp;gt; 2.6; n=14) for biomarker discovery. Differential expression analysis and pathway activation analysis revealed genes and pathways involved in the cell cycle and DNA replication. DNA recombination differentiates responders from non-responders, while genes involved in epithelial cell proliferation, differentiation, and wound healing are associated with drug resistance. Mutation of proto-oncogene RB Transcriptional Corepressor 1, RB1 (a negative regulator of the cell cycle), was found to be a biomarker enriched in non-responders to paclitaxel, corroborating earlier clinical findings. Using the identified profile, we predicted responses for non-tested models in our biobank and validated our approach. Conclusion Large panel organoid drug screening in combination with biomarker analysis brings great opportunities for increasing drug pipeline efficiency. The POC here presented highlights how such a screen can identify MOAs and identify relevant biomarker profiles to be used in clinical trial patient stratification. Citation Format: Linda Xue, Liza Wijler, Marten Hornsveld, Sheng Guo, Leo Price, Marrit Putker, Ludovic Bourre. Large panel organoid drug testing combined with biomarker analysis: Unveiling prospects for mechanism of action identification and preclinical patient stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB435.

Integrative analyses of whole-transcriptome sequencing reveals CeRNA regulatory network in pulmonary hypertension treated with FGF21

Noncoding RNAs have been shown to play essential roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs regulated by hypoxia and FGF21 were selected through intersection analysis. Based on prediction databases and sequencing data, differentially co-expressed mRNAs, miRNAs, lncRNAs, and circRNAs were further screened, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. The ceRNA regulatory network of lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. In addition, quantitative real-time PCR experiments were employed to verify the whole-transcriptome sequencing data. The results of luciferase reporter assays highlighted the relationship between miR-449c-5p and XR_878320.1, miR-449c-5p and Stab2, miR-449c-5p and circ_mtcp1, which suggesting that miR-449c-5p may be a key regulator of FGF21 in the treatment of PH. Taken together, this study provides potential biomarkers, pathways, and ceRNA regulatory networks in HPH treated with FGF21 and will provide an experimental basis for the clinical application of FGF21 in PH.

Molecular Profiling and the Impact of Treatment on Outcomes in Adenoid Cystic Carcinoma Type I and II.

Adenoid cystic carcinoma (ACC) is an uncommon salivary gland cancer with no approved therapies available to treat advanced, incurable disease. Recent molecular profiling efforts have identified two important subtypes: the more aggressive ACC-I is characterized by Notch pathway alterations and MYC amplification whereas ACC-II demonstrates a more indolent phenotype and TP63 overexpression. This retrospective observational cohort study involved de-identified samples from 438 patients with ACC with tumor samples sent for commercially-available molecular profiling (Caris Life Sciences). Next-generation whole-exome and whole-transcriptomic sequencing was performed on primary and metastatic samples. Immunostaining for PD-L1 and RNA deconvolution (quanTIseq) was used to explore the tumor immune microenvironment (TME). Real-world clinical and survival outcome metrics were extracted from insurance claims data. MYC expression was 1.61-fold higher (39.8 vs. 24.7; P < 0.0001) among NOTCH1-mutant ACC-I tumors, whereas MYB/L1 fusion rates were similar among ACC-I/II. The median B-cell fraction in the TME was higher among ACC-II (7.1% vs. 5.8%; P < 0.01), although infiltrating T cells subsets were low among either ACC subgroup (both <1%). When pooling systemic treatment categories, ACC-I patients had worse outcomes with available therapies (HR, 3.06; 95% confidence interval, 1.65-5.68; P < 0.01), with no significant difference in overall survival between ACC-I/II based on chemotherapy or VEGFR tyrosine kinase inhibitor exposure in smaller subsets. We confirmed the previously reported associations with MYC and TP63 in the prognostically relevant subgroups of ACC-I and -II, respectively, and report immunologic differences among these subtypes. Survival outcomes are comparatively worse in ACC-I regardless of treatment type.

Deciphering the role of non-coding RNAs involved in sorafenib resistance

Sorafenib is an important treatment strategy for advanced hepatocellular carcinoma (HCC). Unfortunately, drug resistance has become a major obstacle in sorafenib application. In this study, whole transcriptome sequencing (WTS) was conducted to compare the paired differences between non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and mRNAs, in sorafenib-resistant and parental cells. The overlap of differentially expressed ncRNAs (DENs) between the SMMC7721/S and Huh7/S cells and their parental cells was determined. 2 upregulated and 3 downregulated lncRNAs, 2 upregulated and 1 downregulated circRNAs, as well as 10 upregulated and 2 downregulated miRNAs, in both SMMC7721/S and Huh7/S cells, attracted more attention. The target genes of these DENs were then identified as the overlaps between the differentially expressed mRNAs achieved using the WTS analysis and the predicted genes of DENs obtained using the “co-localization” or “co-expression,” miRanda, and RNAhybrid analysis. Consequently, the potential regulatory network between overlapping DENs and their target genes in both SMMC7721/S and Huh7/S cells was explored. The “lncRNA-miRNA-mRNA” and “circRNA-miRNA-mRNA” networks were constructed based on the competitive endogenous RNA (ceRNA) theory using the Cytoscape software. In particular, lncRNA MED17-203-miRNA (miR-193a-5p, miR-197-3p, miR-27a-5p, miR-320b, miR-767-3p, miR-767-5p, miR-92a-3p, let-7c-5p)-mRNA,” “circ_0002874-miR-27a-5p-mRNA” and “circ_0078607-miR-320b-mRNA” networks were first introduced in sorafenib-resistant HCC. Furthermore, these networks were most probably connected to the process of metabolic reprogramming, where the activation of the PPAR, HIF-1, Hippo, and TGF-β signaling pathways is governed. Alternatively, the network “circ_0002874-miR-27a-5p-mRNA” was also involved in the regulation of the activation of TGF-β signaling pathways, thus advancing Epithelial-mesenchymal transition (EMT). These findings provide a theoretical basis for exploring the mechanisms underlying sorafenib resistance mediated by metabolic reprogramming and EMT in HCC.