Expression Of Protein-coding Genes Research Articles

Transposable elements alter gene expression and may impact response to cisplatin therapy in ovarian cancer.

Cisplatin is widely employed for cancer treatment; therefore, understanding resistance to this drug is critical for therapeutic practice. While studies have delved into differential gene expression in the context of cisplatin resistance, findings remain somewhat scant. We performed a comprehensive investigation of Transposable Elements (TEs) expression and their impact in host genes in two cisplatin-treated ovarian cancer cell lines. RNA-seq, ATAC-seq, and in-depth bioinformatics analysis were used to compare cisplatin-sensitive and -resistant ovarian cancer cell lines. Our results reveal that cisplatin therapy alters the expression of protein-coding genes, but also key TEs, including LINE1, Alu, and endogenous retroviruses, in both cisplatin-sensitive and -resistant cell lines. By co-expressing with downstream genes or by creating chimeric transcripts with host genes at their insertion sites, these TEs seem to control the expression of protein-coding genes, including tumor-related genes. Our model uncovers TEs influencing the expression of cancer genes and cancer pathways. Collectively, our findings indicate that TEs alterations associated with cisplatin treatment occur in critical cancer genes and cellular pathways synergically. This research highlights the importance of considering the entire spectrum of transcribed elements in the genome, especially TE expression, for a complete understanding of complex models like cancer response to treatment.

The Long Non-Coding RNA Gene AC027288.3 Plays a Role in Human Endometrial Stromal Fibroblast Decidualization.

During the secretory phase of the menstrual cycle, endometrial fibroblast cells begin to change into large epithelial-like cells called decidual cells in a process called decidualization. This differentiation continues more broadly in the endometrium and forms the decidual tissue during early pregnancy. The cells undergoing decidualization as well as the resulting decidual cells, support successful implantation and placentation during early pregnancy. This study was carried out to identify new potentially important long non-coding RNA (lncRNA) genes that may play a role in human endometrial stromal fibroblast cells (hESF) undergoing decidualization in vitro, and several were found. The expression of nine was further characterized. One of these, AC027288.3, showed a dramatic increase in the expression of hESF cells undergoing decidualization. When AC027288.3 expression was targeted, the ability of the cells to undergo decidualization as determined by the expression of decidualization marker protein-coding genes was significantly altered. The most affected markers of decidualization whose expression was significantly reduced were FOXO1, FZD4, and INHBA. Therefore, AC027288.3 may be a major upstream regulator of the WNT-FOXO1 pathway and activin-SMAD3 pathways previously shown as critical for hESF decidualization. Finally, we explored possible regulators of AC027288.3 expression during human ESF decidualization. Expression was regulated by cAMP and progesterone. Our results suggest that AC027288.3 plays a role in hESF decidualization and identifies several other lncRNA genes that may also play a role.

An unexpected path for Malat1 in neurons: trafficking out of the nucleus for translation.

The Malat1 (metastasis-associated lung adenocarcinoma transcript 1) long noncoding RNA is highly and broadly expressed in mammalian tissues, accumulating in the nucleus where it modulates expression and pre-mRNA processing of many protein-coding genes. In this issue of Genes & Development, Xiao and colleagues (doi:10.1101/gad.351557.124) report that a significant fraction of Malat1 transcripts in cultured mouse neurons are surprisingly exported from the nucleus. These transcripts are packaged with Staufen proteins in RNA granules and traffic down the lengths of neurites. They then can be released in a stimulus-dependent manner to be locally translated into a microprotein that alters neuronal gene expression patterns.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Unboxing the network among long non-coding RNAs and TGF-β signaling in cancer.

Deeper analysis of molecular mechanisms arising in tumor cells is an unmet need to provide new diagnostic and therapeutic strategies to prevent and treat tumors. The transforming growth factor β (TGF-β) signaling has been steadily featured in tumor biology and linked to poor prognosis of cancer patients. One pro-tumorigenic mechanism induced by TGF-β is the epithelial-to-mesenchymal transition (EMT), which can initiate cancer dissemination, enrich the tumor stem cell population, and increase chemoresistance. TGF-β signals via SMAD proteins, ubiquitin ligases, and protein kinases and modulates the expression of protein-coding and non-coding RNA genes, including those encoding larger than 500 nt transcripts, defined as long non-coding RNAs (lncRNAs). Several reports have shown lncRNAs regulating malignant phenotypes by directly affecting epigenetic processes, transcription, and post-transcriptional regulation. Thus, this review aims to update and summarize the impact of TGF-β signaling on the expression of lncRNAs and the function of such lncRNAs as regulators of TGF-β signaling, and how these networks might impact specific hallmarks of cancer.

Abstract 7355: Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome

Abstract Existing evidence shows that some long non-coding RNAs (lncRNAs) can recruit chromatin regulators and form R-loops to mediate CCTCC-binding factor (CTCF) binding at topologically associated domain (TAD) boundaries to activate transcription of target genes. However, it remains unclear to what extent such lncRNA-dependent chromatin organization events occur genome-wide in normal and cancer cells. By curated public data of lncRNA, CTCF, RAD21 profiles and integrative analysis of multi-omics data using innovative computational approaches, we investigated global associations of lncRNAs and R-loops with activities of transcription factors and chromatin regulators in gene expression regulations across multiple human cell types. We developed a comprehensive computational pipeline for cross-cell-type analysis of lncRNA and protein-coding gene expression profiles and transcription factor binding profiles. We identified a total of 542 candidate genomic regions where lncRNAs play a pivotal role in regulating target genes by facilitating CTCF/cohesin binding. R-loops are enriched at CTCF/cohesin binding sites, suggesting their potential contribution to CTCF binding. Additionally, our findings underscore the significant cell-type specificity of CTCF binding patterns across the genome. This work adds knowledge to cancer genome organization and gene regulation, and builds foundations for further functional and mechanistic studies of lncRNAs and R-loops in the human genome. Citation Format: Shengyuan Wang, Zhenjia Wang, Yuh-Hwa Wang, Hui Li, Peter Todd Stukenburg, Chongzhi Zang. Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome [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 7355.

Abstract 2270: UALCAN Mobile, An app for cancer gene expression data analysis

Abstract Cancer is a complex disease effecting different organs and a major cause of death and a burden on the society. Multiple molecular alteration occur during cancer initiation and disease progression and metastasis. Recent advances in technology led to generation of large amount of molecular data including transcriptome. These large datasets can be used to analyze and identify sub-class specific cancer biomarkers and targets. However, there is a need for the development user friendly tools for its analysis and dissemination of the large scale cancer molecular data. Earlier, we had developed and upgraded web based comprehensive proteogenomic platform UALCAN (ualcan.path.uab.edu), which allows users to analyze and integrate the disparate data to better understand the gene, proteins, and pathways perturbed in cancer and make discoveries. UALCAN web platform enables intuitive analysis and discovery by cancer research community. In the current study, we describe the development of UALCAN Mobile application (App) that will provide cancer transcriptomic data obtained from The Cancer Genome Atlas (TCGA) project, to evaluate protein-coding gene expression based on various stratification including stage, grade, race, gender and molecular-subtypes across over 30 types of cancers. The UALCAN Mobile app aims at providing large cancer dataset on the go. In order to find causative gene expression changes, identify biomarkers and therapeutic targets, UALCAN Mobile will assist as the data will be easily mobile and transportable with this App. Here, we describe the development of UALCAN Mobile and its utility to the cancer research community. UALCAN Mobile App can be found at both iOS/Apple and Android play store and is free to download and use. Citation Format: Sooryanarayana Varambally, Darshan Shimoga Chandrashekar, Gopi Chand Puli, Santhosh Kumar Karthikeyan, Upender Manne, Chad J. Creighton, Sidarth Kumar. UALCAN Mobile, An app for cancer gene expression data analysis [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 2270.

Unraveling the Significance of DGCR8 and miRNAs in Thyroid Carcinoma.

MicroRNAs (miRNAs) act as negative regulators for protein-coding gene expression impacting cell proliferation, differentiation, and survival. These miRNAs are frequently dysregulated in cancer and constitute classes of blood-based biomarkers useful for cancer detection and prognosis definition. In thyroid cancer (TC), the miRNA biogenesis pathway plays a pivotal role in thyroid gland formation, ensuring proper follicle development and hormone production. Several alterations in the miRNA biogenesis genes are reported as a causality for miRNA dysregulation. Mutations in microprocessor component genes are linked to an increased risk of developing TC; in particular, a recurrent mutation affecting DGCR8, the E518K. In this review, we explore these novel findings and resume the current state-of-the-art in miRNAs in thyroid carcinomas.

Genetic imputation of kidney transcriptome, proteome and multi-omics illuminates new blood pressure and hypertension targets

Genetic mechanisms of blood pressure (BP) regulation remain poorly defined. Using kidney-specific epigenomic annotations and 3D genome information we generated and validated gene expression prediction models for the purpose of transcriptome-wide association studies in 700 human kidneys. We identified 889 kidney genes associated with BP of which 399 were prioritised as contributors to BP regulation. Imputation of kidney proteome and microRNAome uncovered 97 renal proteins and 11 miRNAs associated with BP. Integration with plasma proteomics and metabolomics illuminated circulating levels of myo-inositol, 4-guanidinobutanoate and angiotensinogen as downstream effectors of several kidney BP genes (SLC5A11, AGMAT, AGT, respectively). We showed that genetically determined reduction in renal expression may mimic the effects of rare loss-of-function variants on kidney mRNA/protein and lead to an increase in BP (e.g., ENPEP). We demonstrated a strong correlation (r = 0.81) in expression of protein-coding genes between cells harvested from urine and the kidney highlighting a diagnostic potential of urinary cell transcriptomics. We uncovered adenylyl cyclase activators as a repurposing opportunity for hypertension and illustrated examples of BP-elevating effects of anticancer drugs (e.g. tubulin polymerisation inhibitors). Collectively, our studies provide new biological insights into genetic regulation of BP with potential to drive clinical translation in hypertension.

Genome-wide identification and in silico characterization of major RNAi gene families in date palm (Phoenix dactylifera)

BackgroundDates contain various minerals that are essential for good health. The major RNA interference (RNAi) gene families play a vital role in plant growth and development by controlling the expression of protein-coding genes against different biotic and abiotic stresses. However, these gene families for date palm are not yet studied. Therefore, this study has explored major RNAi genes and their characteristics in date palm.ResultsWe have identified 4 PdDCLs, 7 PdAGOs, and 3 PdRDRs as RNAi proteins from the date palm genome by using AtRNAi genes as query sequences in BLASTp search. Domain analysis of predicted RNAi genes has revealed the Helicase_C, Dicer_dimer, PAZ, RNase III, and Piwi domains that are associated with the gene silencing mechanisms. Most PdRNAi proteins have been found in the nucleus and cytosol associated with the gene silencing actions. The gene ontology (GO) enrichment analysis has revealed some important GO terms including RNA interference, dsRNA fragmentation, and ribonuclease_III activity that are related to the protein-coding gene silencing mechanisms. Gene regulatory network (GRN) analysis has identified PAZ and SNF2 as the transcriptional regulators of PdRNAi genes. Top-ranked 10 microRNAs including Pda-miR156b, Pda-miR396a, Pda-miR166a, Pda-miR167d, and Pda-miR529a have been identified as the key post-transcriptional regulators of PdRNAi genes that are associated with different biotic/abiotic stresses. The cis-acting regulatory element analysis of PdRNAi genes has detected some vital cis-acting elements including ABRE, MBS, MYB, MYC, Box-4, G-box, I-box, and STRE that are linked with different abiotic stresses.ConclusionThe results of this study might be valuable resources for the improvement of different characteristics in date palm by further studies in wet-lab.

Regulation of BTB (POZ) Structural Domain 6b by MicroRNA-222b in Zebrafish Embryos after Exposure to Di(2-ethylhexyl)phthalate at Low Concentrations.

Di-(2-ethylhexyl) phthalate (DEHP) is a sort of endocrine disruptor that induces abnormal physiological and biochemical activities such as epigenetic alterations, apoptosis, and oxidative stress. MicroRNAs (miRNAs) are a class of short noncoding RNAs that may regulate the expression of many protein-coding genes when organisms are exposed to environmental chemicals. miR-222b is a differentially expressed miRNA after DEHP exposure. miRNA-mRNA prediction suggested that BTB (POZ) structural domain 6b (BTBD6B) might be a target mRNA of miR-222b, and DEHP exposure altered its expression. However, the correlation between miR-222b and BTBD6B has not been experimentally confirmed. The aim of this study was to investigate the regulation of BTBD6B by miR-222b in zebrafish embryos under the effect of low concentration of DEHP. Dual fluorescent protein assays and dual luciferase reporter gene assays confirmed the interaction between miR-222b and the 3'-untranslated region (3'-UTR) of BTBD6B. Ectopic expression assays showed that miR-222b could negatively regulate BTBD6B in ZF4 cells. However, the relative expression of miR-222b and BTBD6B was significantly higher at both transcriptional and post-transcriptional levels in zebrafish embryos exposed to low concentrations of DEHP. The results of this study improved our understanding of the molecular mechanism of DEHP exposure toxicity. It identified that the aberrant expression of miR-222b/BTBD6B may be one of the mechanisms of DEHP toxicity, which can provide a theoretical reference and scientific basis for environmental management and biological health risk assessment.

Relationship of MicroRNAs with Transposable Elements in the Type 1 Diabetes Development

The review article describes the involvement of epigenetic factors in type 1 diabetes mellitus (T1DM) etiopathogenesis. The disease is characterized by changes in expression of microRNAs that affect the transcription of genes involved in autoimmune reactions, destruction of beta cells and insulin production. However, the cause of the observed epigenetic changes is still unclear. In evolution, the sources of microRNA genes are transposable elements, which occupy up to 45 % of the entire human DNA sequence and are drivers of epigenetic regulation in ontogenesis. They are sources of transcription factor sequences and binding sites for them. Features of the genome distribution of transposable elements can cause changes in the number of 5’VNTR (variable number of tandem repeats) — repeats of insulin promoter region and HERV insertions into HLA genes, which affects their expression. Therefore, I assume that the cause of the development of type 1 diabetes mellitus may be an imbalance in transcription activation of transposons, which contributes to changes in the expression of specific microRNAs and protein-coding genes, and also contributes to autoimmune response development. Triggers for this may be individual features of genome distribution of transposons, viral infections and stress. An analysis of the scientific literature confirms my proposed mechanisms for T1DM development, since the global role of retroelements in hormonal regulation, the sensitivity of transposable elements to exogenous viral infections and stress, and HERV-W expression of the majority of patients with T1DM with activation of the autoimmune response have been proven. Analysis of the MDTE DB (miRNAs derived from transposable elements database) database showed the transposon origin of 12 T1DM-associated microRNAs (miR-192, miR-224, miR-31, miR-320c, miR-326, miR-340, miR-342, miR-44661, miR-548c, miR-652, miR-95), the use of which can become the basis for targeted therapy for T1DM.

Interpretable AI models for predicting distant metastasis development based on genetic data: Kidney cancer example

Kidney cancer has a high metastatic potential with up to 30% of patients developing distant metastasis after surgery. We assessed the value of AI models in predicting the metastatic potential of clear cell renal cell carcinoma (ccRCC), based on the genetic data. Tissue samples from patients with both metastatic and non-metastatic squamous cell carcinoma were analyzed, focusing on the expression and methylation levels of specific protein-coding (PC) and microRNA (miRNA) genes. Using quantitative PCR and data classification techniques, we found a correlation between metastasis and reduced expression of PC-genes CA9, NDUFA4L2, EGLN3, and BHLHE41, as well as increased methylation in miRNA genes MIR125B-1, MIR137, MIR375, MIR193A, and MIR34B. AI models were built for predicting distant metastases based on the expression values and methylation status of selected genes. One model is based on solving a regression problem and is non-interpretable, while another one is based on proposed decision rules and is interpretable. The quality of the models was assessed using sensitivity and specificity metrics, and cross-validation technology was used to ensure the reliability of the results.

Large-scale functional inference for skin-expressing lncRNAs using expression and sequence information.

Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes and have been shown to play important roles in inflammatory skin diseases. However, we still have limited understanding of the functional impact of lncRNAs in skin, partly due to their tissue specificity and lower expression levels compared with protein-coding genes. We compiled a comprehensive list of 18,517 lncRNAs from different sources and studied their expression profiles in 834 RNA-Seq samples from multiple inflammatory skin conditions and cytokine-stimulated keratinocytes. Applying a balanced random forest to predict involvement in biological functions, we achieved a median AUROC of 0.79 in 10-fold cross-validation, identifying significant DNA binding domains (DBDs) for 39 lncRNAs. G18244, a skin-expressing lncRNA predicted for IL-4/IL-13 signaling in keratinocytes, was highly correlated in expression with F13A1, a protein-coding gene involved in macrophage regulation, and we further identified a significant DBD in F13A1 for G18244. Reflecting clinical implications, AC090198.1 (predicted for IL-17 pathway) and AC005332.6 (predicted for IFN-γ pathway) had significant negative correlation with the SCORAD metric for atopic dermatitis. We also utilized single-cell RNA and spatial sequencing data to validate cell type specificity. Our research demonstrates lncRNAs have important immunological roles and can help prioritize their impact on inflammatory skin diseases.

Relationship of transposable elements with long non-coding RNAs and peptides in carcinogenesis

It has been proven that 98 % of the human genome is transcribed. The main part of resulting molecules after their processing function as various RNA molecules, among which the best known are long noncoding RNA (lncRNA) and microRNA. There are 126,000 lncRNA genes in humans that regulate transcription, translation, histone modifications, heterochromatin formation, splicing, microRNA expression and formation, and matrix RNA (mRNA) post-transcriptional modifications. An important property of lncRNAs is their mutual and self-regulation by peptides formed during their translation, which also affect the expression of protein-coding genes. This property may be due to origin of lncRNAs from transposable elements and is a conservative evolutionary characteristic of lncRNA, as one of properties in formation of new genes for variability and adaptation. The role of lncRNAs originating from retroelements and microRNAs formed during their processing in the specific regulation of genes involved in carcinogenesis has been proven. The peptides formed during lncRNA translation can be used as universal tools for targeted therapy of malignant neoplasms. Analysis of the scientific literature made it possible to describe 21 lncRNAs that are translated to form peptides involved in specific tumors pathogenesis. Since the ability of lncRNA to self-regulate by products of its own translation, which is characteristic of all lncRNAs, is also a property of transposable elements, it is promising to study transposons and their relationship with lncRNAs for designing new therapeutic models.

Circulatory proteins relate cardiovascular disease to cognitive performance: a Mendelian randomisation study

AbstractBackgroundMechanistic research suggests synergistic effects of cardiovascular disease (CVD) and dementia pathologies on cognitive decline. The objective of this study was to investigate the causal relationship between cardiovascular disease (CVD) related proteins and cognitive decline.MethodWe applied Mendelian randomisation (MR) and colocalization analysis to investigate the causal relationships of 90 CVD‐related proteins measured by the Olink CVD I panel with cognitive traits. Genetic instruments for circulatory protein concentrations were obtained from a genome‐wide association study (GWAS) meta‐analysis from the SCALLOP consortium (N = 17,747) based on three sets of criteria: 1) protein quantitative trait loci (pQTL); 2) cis‐pQTL (pQTL within ±500kb from the coding gene); and 3) brain‐specific cis‐expression QTL (cis‐eQTL) which accounts for coding gene expression based on GTEx8. Genetic associations of cognitive performance were obtained from GWAS for either: 1) general cognitive function constructed using Principal Component Analysis (N = 300,486); or, 2) g‐Factor constructed using genomic structural equation modelling (N = 11,263‐331,679). Findings for candidate causal proteins were replicated using a separate protein GWAS in Icelanders (N = 35,559).ResultA higher concentration of genetically predicted circulatory myeloperoxidase (MPO) was nominally associated with better cognitive performance (P<0.05) using different selection criteria for genetic instruments. Particularly, brain‐specific cis‐eQTL predicted MPO, which accounts for protein coding gene expression in brain tissues, was associated with general cognitive function (βWald = 0.22, PWald = 2.4×10−4). The posterior probability for colocalization (PP.H4) of MPO pQTL with the g Factor was 0.58. Findings for MPO were replicated using a GWAS on cognitive score in an Icelandic population. Although we did not find evidence for colocalization, we found that higher genetically predicted concentrations of cathepsin D and CD40 were associated with better cognitive performance and higher genetically predicted concentration of CSF‐1 were associated with poorer cognitive performance.ConclusionThe study suggests that proteins primarily identified for their role in cardiovascular diseases may be involved in shared pathways with cognitive reserve or cognitive decline, and may confer therapeutic targets for neurodegenerative diseases such as Alzheimer’s disease.

Molecular Classification of Relapsed DLBCL Reveals Novel Biologic Subgroups

Background Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults. Though standard immunochemotherapy regimens can result in clinical remission and cure in a majority of patients, approximately 30% of patients are primary treatment resistant or eventually relapse (rrDLBCL), and their prognosis is very poor. Recent molecular classification by large scale genomic and transcriptomic profiling of newly diagnosed DLBCL tumors has resulted in a deeper understanding of disease drivers, however, the molecular heterogeneity of rrDLBCL is yet to be fully explored. Therefore, in this study, we used an unsupervised approach to better define transcriptional and genetic programs in rrDLBCL samples hypothesizing that molecular stratification within this high-risk group may lead to a better understanding of this disease, which remains an unmet need, and a more personalized therapeutic approach. Methods Data used in this study included RNA (n=143) and whole exome (n=126) sequencing data from available FFPE tumor samples at the time of a relapse (any line of treatment, r1-r10 relapse timepoints included in analysis, one per patient), consented to the Molecular Epidemiology Resource (n=61), banked in the Mayo Lymphoma Biobank (n=50), or consented to the CC-122-ST-001 clinical trial (n=32, NCT01421524). Unsupervised clustering was performed on protein coding gene expression values using the non-negative matrix factorization (NMF) approach. NMF was carried out by performing 200 runs on cluster sizes from 2 to 6. Cell of origin (COO) was determined using the method described by Reddy et al. The tumor microenvironment was analyzed using CIBERSORTx and Lymphoma Microenvironment Classification (LME). Genetic classification was done using LymphGen. Results In order to identify unique transcriptional programs in rrDLBCL, we implemented NMF and consensus clustering to define an ideal quantity of stable patient clusters. We identified 4 rrDLBCL patient clusters (rrC1-rrC4) as the most stable solution, based on consensus clustering selection criteria of iterative NMF runs. While relapse timepoint was not associated with individual clusters, we did observe significant differences in tumor characteristics between the clusters. COO classification found that rrC1 and rrC4 were enriched for GCB-DLBCL while rrC3 was enriched for ABC-DLBCL classification (P < .001). To explore the biologic programs in each cluster, we used pathFindR, which leverages protein-protein interaction networks to identify disease related pathways, rrC1 and rrC4 were defined by pathways involved in cell cycle and mismatch repair; oxidative phosphorylation in rrC2; and MAPK and NF-kB in rrC3. Next, LME classification was done to define patterns in the tumor microenvironment and we found a significant difference between groups (P < .001), with rrC2 and rrC4 tumors enriched for a depleted TME, while rrC1 and rrC3 had a more immune rich or inflammatory TME. This was supported by CIBERSORTx analysis identifying significant differences (P > .001) in abundance of CD8 T cells, memory CD4 T cells, and M1 macrophages between clusters, with rrC4 having the lowest abundance. We then explored genetic patterns in each cluster. Gene level mutation enrichment analysis showed BCL7A mutation exclusively in C4 (6/6, P = .03). The clusters were not associated with LymphGen classification. Lastly, our recently published high risk signature classification that is associated with early clinical failure and is characterized by metabolic dysregulation, a depleted TME, T53 alterations, and poor outcome, was significantly different between the groups (P = .03) with enrichment for high-risk cases in rrC4. In summary, we show for the first time that rrDLBCL patients can be classified into four gene expression clusters that are associated with distinct pathway, TME, and genetic programs. These clusters should now be tested to learn if they can help select patients for newer therapies for rrDLBCL such as CAR-T and bispecific antibodies.

Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer

Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 were not previously reported. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-β/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine.

Abstract 18175: LncRNA in Monocytes of Patients With Type 2 Diabetes and Established Cardiovascular Disease Modulate the Expression of Protein-Coding Genes

Introduction: Monocytes contribute to cardiovascular complications of type 2 diabetes (T2D) through various mechanisms. Objectives: We investigated diabetes-induced changes in long non-coding RNAs (lncRNA) from monocytes of patients with T2D and established cardiovascular disease. Methods: We performed polyA capture and paired-end RNA sequencing of monocytes from 120 patients with T2D and 37 non-diabetes controls. Monocytes were sorted using FACSAria2 flow cytometer. Alignments and gene counts were done with STAR to reference GRCh38 using Gencode (v41) gene annotations. The batch correction was done with CombatSeq and differential expression analysis with EdgeR. The false discovery was controlled with the Benjamini-Hochberg procedure. Pathway analysis was done with the IPA software using differentially expressed genes (DEGs) with a p-value < 0.05. Co-expression analysis was done with cdsR and Cytoscape. Results: We found two significantly upregulated lncRNAs in monocytes from diabetes patients (ENSG00000287255 and ENSG00000289424, FDR = 0.035). Pathway analysis on DEGs revealed a network affecting cellular movement, growth, and development that includes directly interacting plasma membrane genes ITGB4 and CDH1, nuclear genes HIF1a, ZEB1, ETS1, TWIST1, SNAI2, NFKB complex, and lncRNA genes SNHG29 and FBXL19-AS1 (p < 1*10^ -25 ). Analysis within T2D patients revealed one lncRNA upregulated only in monocytes from patients with microvascular disease (ENSG00000261654 on chr1, FDR =0.022). Interestingly, co-expression analysis uncovered correlated expression clusters between lncRNAs and protein-coding genes, with one interesting interaction identified between the highly connected MCR1 and SNHG7, a lncRNA shown to respond to IGF-1 (insulin-like growth factor 1) . Conclusion: Monocytes from patients with diabetes have differentially expressed lncRNA genes that potentially impact diabetes-related pathways.

Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor

Pathogenic mutations in mitochondrial DNA cause severe and often lethal multi-system symptoms in primary mitochondrial defects. However, effective therapies for these defects are still lacking. Strategies such as employing mitochondrially targeted restriction enzymes or programmable nucleases to shift the ratio of heteroplasmic mutations and allotopic expression of mitochondrial protein-coding genes have limitations in treating mitochondrial homoplasmic mutations, especially in non-coding genes. Here, we conduct a proof of concept study applying a screened DdCBE pair to correct the homoplasmic m.A4300G mutation in induced pluripotent stem cells derived from a patient with hypertrophic cardiomyopathy. We achieve efficient G4300A correction with limited off-target editing, and successfully restore mitochondrial function in corrected induced pluripotent stem cell clones. Our study demonstrates the feasibility of using DdCBE to treat primary mitochondrial defects caused by homoplasmic pathogenic mitochondrial DNA mutations.