Revealing the History and Mystery of RNA-Seq

Advances in RNA-sequencing technologies have led to the development of intriguing experimental setups, a massive accumulation of data, and high demand for tools to analyze it. To answer this demand, computational scientists have developed a myriad of data analysis pipelines, but it is less often considered what the most appropriate one is. The RNA-sequencing data analysis pipeline can be divided into three major parts: data pre-processing, followed by the main and downstream analyses. Here, we present an overview of the tools used in both the bulk RNA-seq and at the single-cell level, with a particular focus on alternative splicing and active RNA synthesis analysis. A crucial part of data pre-processing is quality control, which defines the necessity of the next steps; adapter removal, trimming, and filtering. After pre-processing, the data are finally analyzed using a variety of tools: differential gene expression, alternative splicing, and assessment of active synthesis, the latter requiring dedicated sample preparation. In brief, we describe the commonly used tools in the sample preparation and analysis of RNA-seq data

O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition

Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase cyclin-dependent kinase 9 (CDK9) is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics, and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity/flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.Peer reviewe

O-GlcNAc transferase couples MRE11 to transcriptionally active chromatin to suppress DNA damage

Background: Transcription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects. Methods: We used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples. Results: Inhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors. Conclusions: Both OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments.Peer reviewe

CDK9 inhibition activates innate immune response through viral mimicry

Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition‐induced cancer cell‐selective anti‐proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over‐expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis‐spliced RNA. Double‐stranded RNA (dsRNA)‐activated kinase can recognize these mis‐spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor‐induced anti‐proliferative effects. Using time‐resolved transcriptional profiling (SLAM‐seq), targeted proteomics, and ChIP‐seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB‐driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late‐stage prostate cancer, a currently lethal disease

Inhibition of O-GlcNAc transferase activates tumor-suppressor gene expression in tamoxifen-resistant breast cancer cells

In this study, we probed the importance of O-GlcNAc transferase (OGT) activity for the survival of tamoxifen-sensitive (TamS) and tamoxifen-resistant (TamR) breast cancer cells. Tamoxifen is an antagonist of estrogen receptor (ERa), a transcription factor expressed in over 50% of breast cancers. ERa-positive breast cancers are successfully treated with tamoxifen; however, a significant number of patients develop tamoxifen-resistant disease. We show that in vitro development of tamoxifenresistance is associated with increased sensitivity to the OGT small molecule inhibitor OSMI-1. Global transcriptome profiling revealed that TamS cells adapt to OSMI-1 treatment by increasing the expression of histone genes. This is known to mediate chromatin compaction. In contrast, TamR cells respond to OGT inhibition by activating the unfolded protein response and by significantly increasing ERRFI1 expression. ERRFI1 is an endogenous inhibitor of ERBB-signaling, which is a known driver of tamoxifen-resistance. We show that ERRFI1 is selectively downregulated in ERa-positive breast cancers and breast cancers driven by ERBB2. This likely occurs via promoter methylation. Finally, we show that increased ERRFI1 expression is associated with extended survival in patients with ERa-positive tumors (p = 9.2e-8). In summary, we show that tamoxifen-resistance is associated with sensitivity to OSMI-1, and propose that this is explained in part through an epigenetic activation of the tumor-suppressor ERRFI1 in response to OSMI-1 treatment.Peer reviewe

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism

Emerging frontiers in androgen receptor research for prostate Cancer: insights from the 2nd international androgen receptor Symposium

Continued exploration of the androgen receptor (AR) is crucial, as it plays pivotal roles in diverse diseases such as prostate cancer (PCa), serving as a significant therapeutic focus. Therefore, the Department of Urology Dresden hosted an international meeting for scientists and clinical oncologists to discuss the newest advances in AR research. The 2nd International Androgen Receptor Symposium was held in Dresden, Saxony, Germany, from 26–27.04.2024, organised by Dr. Holger H.H. Erb. Following the format of the first meeting, more than 35 scientists from 8 countries attended the event to discuss recent developments, research challenges, and identification of venues in AR research. An important new feature was the involvement of PhD students and young investigators, acknowledging the high scientific quality of their work. The symposium included three covers: new advances from clinical research, basic and translational research, and novel strategies to target AR. Moreover, based on its increasing clinical relevance, a PSMA theranostic mini-symposium was added at the end of the AR symposium to allow the audience to discuss the newest advances in PSMA theranostic. This report focuses on the highlights and discussions of the meeting

Lipid degradation promotes prostate cancer cell survival

Prostate cancer is the most common male cancer and androgen receptor (AR) is the major driver of the disease. Here we show that Enoyl-CoA delta isomerase 2 (ECI2) is a novel AR-target that promotes prostate cancer cell survival. Increased ECI2 expression predicts mortality in prostate cancer patients (p = 0.0086). ECI2 encodes for an enzyme involved in lipid metabolism, and we use multiple metabolite profiling platforms and RNA-seq to show that inhibition of ECI2 expression leads to decreased glucose utilization, accumulation of fatty acids and down-regulation of cell cycle related genes. In normal cells, decrease in fatty acid degradation is compensated by increased consumption of glucose, and here we demonstrate that prostate cancer cells are not able to respond to decreased fatty acid degradation. Instead, prostate cancer cells activate incomplete autophagy, which is followed by activation of the cell death response. Finally, we identified a clinically approved compound, perhexiline, which inhibits fatty acid degradation, and replicates the major findings for ECI2 knockdown. This work shows that prostate cancer cells require lipid degradation for survival and identifies a small molecule inhibitor with therapeutic potential.</p

Low free 25-hydroxyvitamin D and high vitamin D binding protein and parathyroid hormone in obese Caucasians. A complex association with bone?

Background Studies have shown altered vitamin D metabolism in obesity. We assessed differences between obese and normal-weight subjects in total, free, and bioavailable 25-hydroxyvitamin D (25(OH) D, 25(OH) D-Free, and 25(OH) D-Bio, respectively), vitamin D binding protein (DBP), parathyroid hormone (PTH) and bone traits. Methods 595 37-47-year-old healthy Finnish men and women stratified by BMI were examined in this cross-sectional study. Background characteristic and intakes of vitamin D and calcium were collected. The concentrations of 25(OH) D, PTH, DBP, albumin and bone turnover markers were determined from blood. 25(OH) D-Free and 25(OH) D-Bio were calculated. pQCT was performed at radius and tibia. Results Mean +/- SE (ANCOVA) 25(OH) D-Free (10.8 +/- 0.6 vs 12.9 +/- 0.4 nmol/L; P = 0.008) and 25(OH) DBio (4.1 +/- 0.3 vs 5.1 +/- 0.1 nmol/L; P = 0.003) were lower in obese than in normal-weight women. In men, 25(OH) D (48.0 +/- 2.4 vs 56.4 +/- 2.0 nmol/L, P = 0.003), 25(OH) D-Free (10.3 +/- 0.7 vs 12.5 +/- 0.6 pmol/L; P = 0.044) and 25(OH) D-Bio (4.2 +/- 0.3 vs 5.1 +/- 0.2 nmol/L; P = 0.032) were lower in obese. Similarly in all subjects, 25(OH) D, 25(OH) D-Free and 25(OH) D-Bio were lower in obese (P Conclusions The associations between BMI and 25(OH) D, 25(OH) D-Free, and 25(OH) D-Bio, DBP, and PTH suggest that obese subjects may differ from normal-weight subjects in vitamin D metabolism. BMI associated positively with trabecular bone traits and CSI in our study, and slightly negatively with cortical bone traits. Surprisingly, there was a negative association of free and bioavailable 25(OH) D and some of the bone traits in obese women.Peer reviewe