title: Circadian Rhythms of Metabolite Abundance in Drosophila are Largely Driven by Time of Feeding

first: Daniel

last: Cavanaugh

address: 1050 W Sheridan Rd., Life Sciences Building Room 215

department: Biology

summary: Organisms exhibit daily oscillations in metabolite abundance. These oscillations could arise from circadian clock control of metabolic pathways in peripheral tissues, or may be secondary to rhythmic food intake, which is primarily controlled by central circadian clocks in the brain. To determine the relative contribution of central and peripheral clocks and behavioral cycles to metabolic rhythms in the fruit fly, Drosophila melanogaster, we conducted large-scale metabolite profiling with fine temporal resolution across multiple days in control flies with intact molecular clocks and in flies where CRISPR/Cas9 gene editing was used to specifically eliminate molecular circadian clock function in the fat body, a peripheral metabolic tissue, or the brain. As these latter flies lack feeding rhythms due to central circadian clock dysfunction, we also included an experimental cohort of flies which lacked central brain clocks but were subjected to time-restricted feeding (TRF) protocols to impose feeding rhythms. Single-nuclei RNA sequencing confirmed selective molecular clock elimination following fat body manipulations, which was associated with predicted alterations in clock gene expression and an attenuation of time-of-day differences in the abundance of fat body transcripts involved in key metabolic pathways. Interestingly, we identified few rhythmically expressed metabolites in flies that were allowed ad libitum food access. In contrast, flies which lacked brain clocks but were raised on TRF exhibited robust circadian abundance rhythms across a large range of metabolites. These findings suggest that metabolic rhythms in Drosophila are more strongly regulated by feeding cycles than by direct circadian clock control of metabolic pathways despite the presence of metabolic genes that exhibit local-clock dependent modulation of expression across the day.

overall_design: Single-nuclei RNA-seq (Parse Biosciences) on abodomen tissue of adult male Drosophila with disrupted molecular clocks in the fat body (to>perCRISPR) and control flies (to>Cas9) at Circadiant Time (CT) 0 and CT12.

first: Madelyn

middle: R

last: Cusick

first: Amanda

middle: J

last: Samaras


title: RNY1 partitions into extracellular vesicles and ribonucleoprotein particles and regulates macrophage programming by airway fluids during inflammation.

status: Public on Jun 22 2026

city: Nashville

first: Cherie

middle: Evelyn

last: Saffold

state: Tennessee

address: 1211 21st Ave S

department: Pathology, Microbiology, and Immunology

country: USA

institute: Vanderbilt University Medical Center

type: Expression profiling by high throughput sequencing

summary: YRNAs are small noncoding RNAs that are abundant in the extracellular space. Prior research has shown that YRNAs are present in most biofluids and that their levels fluctuate during disease. Yet our understanding of YRNA dynamics in the context of biofluid extracellular particles during disease processes is limited. In this study, we found that RNY1, one of two murine YRNAs, increases in airway fluid during allergen-induced lung inflammation and correlates with neutrophil infiltration. Using RNase sensitivity assays and size exclusion chromatography, we determined that airway fluid RNY1 is protected by both EVs and protein-containing structures in distinct extracellular compartments. In contrast the other murine YRNA, RNY3, is only enriched in BALF EVs, suggesting that RNY1 is uniquely partitioned in heterogenous extracellular particles. Both EV-enriched and protein-enriched RNY1-containing extracellular compartments were able program robust pro-inflammatory transcriptional responses in target macrophages. RNY1 contributed to this programming, as macrophages treated with a protein-enriched compartment isolated from RNY1-/- mice demonstrated lower induction of interferon-associated genes. Together, this study demonstrates that YRNAs are heterogeneously packaged in complex biofluids and contribute to intercellular signaling axes during inflammation.

overall_design: RNA-seq profiling of BMDMs treated with EV- or Protein-enriched BALF fractions derived from RNY1 +/+ or RNY1 -/- mice with OVA-induced airway inflammation

middle: E

first: Antiana

middle: C

last: Richardson

first: Heather

middle: H

last: Pua


title: Causal splicing variants revealed by deep-learning integration of single-cell sQTL mapping under influenza infection

city: Durham

first: Liuyang

last: Wang

state: NC

address: 0049 CARL Building

institute: Duke University

summary: Fulfilling the promise of human genetics in elucidating disease requires identifying causal variants and genes underlying genetic association signals. Molecular quantitative trait locus (molQTL) analyses, e.g. expression QTL (eQTL) and splicing QTL (sQTL), link genetic variants to intermediate molecular phenotypes, but pinpointing causal variants and their regulatory effects remains challenging. Here, we integrate sQTL analysis with deep-learning-based splicing effect annotation to identify causal genetic variants and elucidate their functional mechanisms affecting human phenotypes. Using a single-cell GWAS method (scHi-HOST) on 96 lymphoblastoid cell lines (LCLs) with and without influenza A virus (IAV) infection, we discovered ~43,000 sQTL-SNP-junction pairs associated with 217 genes during IAV infection. Integrating sQTLs with AI splice prediction and statistical fine-mapping, we uncovered 57 likely causal variants that affect cis-acting molecular splicing components (5’ donor, 3’ acceptor). Among these, we experimentally validated a causal sQTL signal affecting poly (ADP-ribose) polymerase 2 (PARP2). The causal variant, rs2297616, alters the 5’ splice donor site in the second intron of PARP2, resulting in two protein isoforms differing by 13 amino acids. The derived A allele was associated with the longer protein isoform and increased IAV levels in LCLs. CRISPR editing validated the causal effect of this variant on both protein length and IAV infection. Lastly, these 57 putative causal sQTLs were further linked to over a hundred GWAS traits, including many variants associated with autoimmune diseases. Our work provides a catalog of causal sQTL with direct splicing impacts, providing causal mechanistic insights from genotype to disease susceptibility.

overall_design: 96 human lymphoblastoid cell lines (LCLs) from the 1000 Genomes project were pooled and analyzed using droplet-based single-cell RNA-seq in 2 samples.

first: Benjamin

last: Schott

first: Guinevere

last: Connelly

first: Dennis

last: Ko


title: A 24-hour light-dark time-series bulk RNA-seq dataset of the mouse lacrimal gland

city: weifang

first: Hongyu

last: Li

state: shandong Province

address: 7166 Baotong West Street, Weicheng District, Weifang City, Shandong Province

country: China

institute: Shandong Second Medical University

summary: The lacrimal gland is a major exocrine organ responsible for tear secretion and is essential for maintaining ocular surface homeostasis. However, time-resolved transcriptomic resources for this tissue remain limited. Here, we generated a bulk RNA-seq time-series dataset from extraorbital lacrimal glands of male C57BL/6J mice maintained under a controlled 12:12 light-dark schedule. Tissue samples were collected at 3-h intervals across a 24-h cycle, yielding eight time points with three biological replicates per time point. Libraries were prepared from total RNA and sequenced as 150-bp paired-end reads. This dataset provides a reusable temporal transcriptomic resource for downstream studies of lacrimal gland biology, ocular surface physiology, and gene-expression dynamics under defined light-dark conditions.

overall_design: Bulk RNA-seq profiling of mouse extraorbital lacrimal glands collected at eight Zeitgeber time points across a 24-h light-dark cycle: ZT0, ZT3, ZT6, ZT9, ZT12, ZT15, ZT18, and ZT21. Three biological replicates were collected at each time point, resulting in 24 libraries.

first: Ting

first: Chuyao

first: Aijun

last: Deng

first: Xinwei

last: Jiao


title: Multi-omic analysis reveals distinct endothelial gene expression profiles induced by pathological flow [miRNA-Seq]

status: Public on Jun 23 2026

city: Newcastle upon Tyne

first: Graham

last: Smith

address: Framlington Place

department: Bioinformatics Support Unit

country: United Kingdom

institute: Newcastle University

type: Non-coding RNA profiling by high throughput sequencing

summary: Aims -Coronary artery disease (CAD) remains a leading cause of mortality globally. Atherosclerosis develops at arterial sites exposed to disturbed, low, or oscillatory shear stress, while acute coronary syndromes-triggered by plaque rupture or erosion predominantly occurs in regions subjected to elevated shear stress. While the response of endothelial cells to disturbed flow has received significant attention, the response to elevated flow is less well studied.  Methods and Results - To address this shortfall, we conducted a comprehensive multi-omic analysis of primary human coronary artery endothelial cells (HCAECs) cultured for 72 hours under oscillatory shear stress (OSS), physiological laminar shear stress (LSS), and elevated shear stress (ESS). This identified 2,175 shear-regulated genes, with 1,218 uniquely responsive to OSS and 665 to ESS. Both OSS and ESS induced significant changes in RNA isoform selection, affecting 580 and 848 genes respectively. Signalling pathways regulating CAD pathogenesis including HIPPO, TGFβ/BMP, and IRF, showed altered RNA isoform selection which may influence plaque development and plaque erosion. 65% of LD-filtered CAD-associated genetic variants from two recent multi-population meta-analyses contained at least one OSS or ESS-regulated gene within 250Kb. Proteomic analysis identified 289 proteins differentially expressed under OSS and 171 under ESS, with notable discordance between mRNA and protein changes observed in 28.7% (OSS vs LSS) and 16.6% (ESS vs LSS) genes. Additionally, 40 shear-responsive microRNAs were identified, potentially contributing to 71% of this discordance. Conclusion- Both oscillatory and elevated flow trigger distinct HCAEC gene expression programmes, affecting mRNA, microRNA, splicing, and protein expression, and modulating pathways central to CAD pathogenesis.

overall_design: Confluent monolayers of three independent batches of male HCAECs were exposed to oscillatory ±0.5Pa 1Hz (OSS), normal physiological laminar shear stress (1.5Pa LSS) and elevated laminar shear stress (7.5Pa ESS) for 72 hours using our established parallel plate flow chambers. In brief, low passage HCAECs were seeded at high density onto gelatin-coated slides and cultured for 72 hours to ensure confluence and allow stable cell-cell junctions to form and extracellular matrix deposition to occur. Subsequently these HCAECs were cultured under flow for 72 hours before lysis for either RNA or protein purification. The ability to run >25  flow chambers at a time allowed the samples for all analyses for each individual donor to be prepared in a single experiment, allowing direct comparison across the different omic analyses for each HCAEC donor.

first: Stephen

last: White

first: Giulio

last: Vidotto

first: Robert

last: Beal

first: Sara

last: Luzzi

first: Jon

last: Humphries

first: Martin


title: DNA Methylatransferase Inhibition Prevents Platinum-Induced Ovarian Cancer Stem Cell Enrichment

city: Bloomington

first: Heather

last: O'Hagan

state: Indiana

institute: Indiana University

summary: Platinum-based therapy is the standard first-line treatment for high-grade serous ovarian cancer (HGSC). However, most patients develop resistance and recurrence despite an initial response to therapy. Ovarian cancer stem cells (OCSCs) are enriched in recurrent tumors and contribute to platinum resistance. These tumors also show promoter DNA hypermethylation, and DNA methyltransferase inhibitors (DNMTis) have been shown to restore sensitivity in platinum-resistant ovarian cancer cells. Here, we demonstrated that combining DNMTi with platinum prevented the platinum-induced enrichment of OCSCs and identified NF-κB and STAT3 signaling pathways as potential regulators of platinum-induced OCSC enrichment. STAT3 was active at baseline in OC cells and platinum treatment alone activated NF-κB while maintaining STAT3 activity. Platinum combined with DNMTi decreased STAT3 activation, while still inducing NF-κB activation. Knockdown experiments demonstrated that the presence of both NF-κB and STAT3 was necessary for platinum-induced OCSC enrichment. Analysis of STAT3 and NF-κB subunit p65 CUT&RUN data showed increased binding in introns and intergenic regions in response to platinum. Additionally, DNMTi enriched NF-κB binding at endogenous retroviruses (ERVs), which correlated with changes in expression of nearby genes when DNMTi was combined with platinum. We conclude that combining DNMTi with platinum modulates STAT3 and NF-κB activation and genomic binding, potentially influencing target gene expression and preventing platinum-induced enrichment of OCSCs.

overall_design: RNA-seq profiling of PEO1 cells mock-treated or treated with platinum-based agent cisplatin (Cis) and/or DNA methyltransferase inhibitor decitabine (DAC)

middle: M

first: Truc

middle: T

last: Vuong


title: DICER1-related tumor predisposition mutations lead to a gain of 3p-miRNA function, HERVH activity and increased metastatic potential

city: Castellón de la Plana

first: Guillermo

last: Peris

state: CAstellón

address: Av. Vicent Sos Baynat, s/n

department: Llenguatges i Sistemes Informàtics

country: Spain

institute: Universitat Jaume I

summary: The DICER1 gene is mutated in cancer, including in DICER1-related tumour predisposition. Cancer-associated hotspot mutations have been reported in both catalytic domains of DICER and are predicted to disrupt miRNA biogenesis. To understand how these hotspot mutations contribute to cancer development, we generate cell lines harbouring single amino acid substitutions within the catalytic RNase IIIa (S1344L) or RNase IIIb (D1709N) domains of the endogenous DICER1 gene. Here we show that both mutations result in a widespread loss of 5p miRNAs, and an increase in 3p passenger strands loading into AGO2. The shared similarities between both mutants can be attributed to the structural proximity of the S1344 residue to the RNase IIIb catalytic centre. Functionally, we find that changes in the repertoire of miRNAs loaded into AGO2 result in altered gene expression, impacting critical pathways for cancer development, including metastatic potential. Additionally, our results indicate that inactivating the processing activity of DICER does not result in genomic instability. Instead, mutations cause upregulation of transposable elements, including the human endogenous retrovirus H through miRNA-independent mechanisms. This suggests that both canonical and non-canonical DICER functions are important to understand DICER1-related tumour predisposition.

overall_design: RNA sequencing of tumour and normal tissue obtained with FFPE (formalin-fixed paraffin-embedded).

first: Katrina

last: Gordon

first: Nicolás

last: Bellora

first: Jeroen

last: Witteveldt

first: Joanna

last: Wojtus

first: Felix

last: Mueller

first: Katharina

last: Kases

first: Pilar

middle: G

last: Marchante

first: Shelagh

last: Boyle

last: Heras

first: Atlanta

last: Cook

first: Cei

last: Abreu-Goodger

last: Macías


title: Organoid modeling of lung branching morphogenesis and epithelial lineage specification [scRNA-Seq]

city: Guangzhou

first: Ruifang

last: Zhang

state: Guangdong

address: Haizhu District

institute: Guangzhou Laboratory

summary: In this study, multi-level branching lung organoids (BLOs) were generated from primary tip epithelial cells. Single-cell RNA sequencing demonstrated that BLOs comprise proximal airway and distal alveolar epithelial cell populations, faithfully modeling the developmental trajectory of distal lung epithelial lineages in vitro.

overall_design: We generated multi-level branching lung organoids (BLOs) from primary tip epithelial cells and analyzed their transcriptional profiles using single-cell RNA sequencing (scRNA-seq).


title: Lineage Plasticity Driven by GATA6 Loss Fuels Colorectal Cancer Metastasis [H3K27ac_CUT&RUN]

city: New York

first: Norihiro

last: Goto

state: NY

address: 413 East 69th Street

department: Department of Medicine

institute: Weill Cornell Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Colorectal cancer (CRC) liver metastases are the leading cause of CRC-related mortality, yet the genetic and epigenetic drivers underlying this process remain poorly understood. Here, we established a pro-metastatic CRC organoid library through serial orthotopic transplantation of liver metastasis–derived organoids. Integrative RNA-seq and ATAC-seq analyses identified a pro-metastatic signature characterized by multilineage plasticity, including fetal-like and basal-like/squamous transcriptional programs. Motif and transcription factor activity analyses identified GATA6 as a key regulator of these epigenetic alterations. GATA6 expression is downregulated in liver metastases, and its genetic ablation enhances liver metastasis with minimal effects on primary tumor growth. Mechanistically, GATA6 loss triggers pro-metastatic transcriptional programs, including fetal-like and basal-like/squamous states, accompanied by LGR5⁻ cell generation. This is mediated by direct repression of HNF4A and increased H3K27ac, and occurs independently of SOX17. Together, these findings identify GATA6 loss as a central regulator of multilineage plasticity that drives liver metastasis in CRC

overall_design: We performed H3K27ac CUT&RUN for control and GATA6-KO AKP orgnaoids.

first: Saori

first: Omer

last: Yilmaz


title: Dynamic m6A Epitranscriptomic Remodeling Coordinates Host Transcriptional Reprogramming during MPXV Infection

city: Kunming

first: Ruixia

last: Tang

address: Jiaoling Road 935

institute: Chinese  Academy  of Medical Sciences & Peking Union Medical College

summary:
Background: The global emergence of monkeypox virus (MPXV) highlights the urgent need for a deeper understanding of host–pathogen interactions. Although transcriptional responses to MPXV infection have been characterized, the role of epitranscriptomic regulation particularly N6‑methyladenosine (m6A) modification remains largely unexplored.
Methods: We performed an integrated analysis of time‑series transcriptomic and m6A methylome profiles using whole blood samples collected from MPXV‑infected rhesus macaques at 7, 14, and 21 days post‑infection, with distinct animals used at each time point. Host gene expression and m6A modification dynamics were examined over the course of infection. Differential expression and differential m6A modification analyses were conducted, followed by integrative pathway and immune cell signature profiling.
Results: MPXV infection induced sustained host reprogramming, characterized by suppression of immune pathways and activation of metabolic processes. A global increase in m6A modifications was observed, accompanied by upregulation of the methyltransferase METTL3 and downregulation of demethylases (FTO, ALKBH5) and readers (YTHDF1-3). Knockdown of METTL3 or YTHDF2 reduced viral replication, suggesting a proviral role for this regulatory circuit. Integrative analysis identified 38 genes with coordinated changes in both transcription and m6A modification across all three time points. Focusing on literature-curated pathogenic pathways, we further identified 11 dual-regulated host factors. Notably, DNAJB1 was the only gene shared between these two independent selection strategies. m6A peaks near transcription start sites and within 5′UTR positively correlated with gene expression, whereas coding region modifications showed weak negative correlations. Immune lineage signatures showed gradual declines in T cell, NK, and monocyte/macrophage signatures with a progressive increase in B cell signatures. Cross‑dataset comparison confirmed core m6A regulatory trends despite heterogeneity across tissues and viral strains.
Conclusions: This study reveals m6A epitranscriptomic remodeling as a key correlate of the host response to MPXV infection and nominates DNAJB1 alongside the other 10 dual‑regulated genes as candidate host factors for further mechanistic investigation.

overall_design: This study analyzed whole blood samples from 12 adult rhesus macaques (Macaca mulatta), including 9 MPXV-infected animals and 3 mock-infected controls. For each infected animal, longitudinal samples were collected at three time points post-infection: day 7, day 14, and day 21. Total RNA was extracted from each sample and subjected to two complementary high-throughput sequencing assays: (1) RNA-seq for transcriptome profiling, and (2) m6A-MeRIP-seq for epitranscriptomic mapping of N6-methyladenosine modifications. Thus, each biological sample generated two types of sequencing libraries. All experimental groups included three biological replicates per condition (3 control animals and 3 infected animals per time point). The control group received sterile PBS injection. No technical replicates or dye-swaps were performed.

first: Jindi

last: Huang

first: Wenhai

last: Yu

first: Shuaiyao

last: Lu

first: Jienan

last: Zhou

first: Kaiyun

last: Ding

first: Junbin

last: Wang

first: Cong

first: Xiaoqing

last: Fu

first: Tingfu

last: Du

first: Qiangming

last: Sun


title: Lineage Plasticity Driven by GATA6 Loss Fuels Colorectal Cancer Metastasis [GATA6_CUT&RUN]

overall_design: We performed GATA6 CUT&RUN in control and GATA6-KO AKP organoids.


title: Spatially-Resolved Multiomic Atlas of Leiomyosarcoma Identifies Two Clinically Relevant Epigenetically-Driven Cell States [CUT&RUN]

city: Houston

first: Zhao

last: Zheng

state: TX

address: 1901 East Rd

department: Genomic Medicine

institute: md anderson cancer center

summary: Leiomyosarcoma is a smooth muscle–derived malignancy marked by significant clinical heterogeneity. The extent and nature of cellular heterogeneity and molecular underpinnings remain poorly understood. To address this at transcriptomic and epigenomic levels, we performed single-nucleus multiome sequencing on untreated primary leiomyosarcoma tissues. Malignant cells segregated almost exclusively into two previously unrecognized and epigenetically distinct states: a dedifferentiated, mesenchymal-like subtype (MES) and a differentiated smooth muscle–enriched subtype (SMC). Chromatin accessibility profiling revealed strong enrichment of nuclear factor I (NFI) transcription factor motifs in MES cells, whereas AP-1 family motifs—most prominently FOSL2—were selectively accessible in SMC cells. Established leiomyosarcoma cell lines faithfully recapitulated these subtypes, and targeted depletion of NFI or AP-1 factors suppressed proliferation, invasion, and in vivo tumor growth, demonstrating functional dependency on these transcriptional programs. Spatial transcriptomics across 328 tissue cores from 128 leiomyosarcomas showed that immunosuppressive macrophages preferentially cluster around MES regions, revealing a subtype-specific tumor–immune niche. Clinically, MES-dominant tumors were associated with significantly worse patient outcomes. Through an epigenetic inhibitor screen, we identify and validate SMARCA4/2 inhibition as a promising therapeutic vulnerability for MES leiomyosarcomas. Together, this work defines two epigenetically driven, transcription factor–regulated, and clinically relevant states of leiomyosarcoma, revealing mechanistic underpinnings of tumor heterogeneity and uncovering actionable therapeutic strategies.

overall_design: CUT&RUN of H3K4me3, NFIA, NFIB, NFIC, NFIX, BACH2, FOSL2

first: Denu

last: Ryan

first: Zheng

last: Zhao


title: Spatially-Resolved Multiomic Atlas of Leiomyosarcoma Identifies Two Clinically Relevant Epigenetically-Driven Cell States

overall_design: ChIP-seq of H3K4me3, NFIA, NFIB, NFIC, NFIX, BACH2, FOSL2


title: Divergent 3D genome architecture of male germ cells across vertebrates [Hi-C]

city: Cerdanyola del Vallès

first: Laia

last: Marín-Gual

address: Campus UAB

institute: Universitat Autònoma de Barcelona

summary: The evolutionary plasticity of higher-order genome organization in vertebrates—and its transmission through the germ line—is central to understanding genome function and evolution. Yet, the mechanisms regulating these processes remain poorly characterized across lineages. Here, we integrate fluorescence-activated cell sorting, in situ Hi-C, and single-cell RNA sequencing to investigate germ line genome architecture in eutherians, marsupials, and reptiles, —lineages that last shared a common ancestor ~320 million years ago. We uncover lineage-specific chromatin folding patterns in germ cells, shaped by chromosome morphology and genome size, which constrain DNA loop formation and inter-chromosomal interactions during meiosis. We also explore the relationship between 3D genome remodeling and gene regulation in the context of meiotic sex chromosome inactivation. In the tammar wallaby, we identify regions of the X that escape MSCI, suggesting incomplete silencing in marsupials. These findings provide high-resolution insights into the evolution of germ line chromatin architecture and the co-evolution of genome structure and function across vertebrates.

sample_organism:
Notamacropus eugenii
Pogona vitticeps

platform_organism:
Notamacropus eugenii
Pogona vitticeps

overall_design: Germ cell populations from tammar wallaby and bearded dragon individuals were isolated by Fluorescence-Activated Cell Sorting. Hi-C from isolated populations (spermatogonia, primary spermatocytes and round spermatids) from tammar wallaby and bearded dragon. Single-cell RNA-seq of whole testes from tammar wallaby and bearded dragon

first: Lucía

last: Álvarez-González

first: Laura

last: González-Rodelas

first: Fleur

last: Coppen

first: Ashley

last: Milton

first: Rachel

last: O'Neill

first: Andrew

last: Pask

first: Marilyn

middle: B

last: Renfree

first: Arthur

last: Georges

first: Paul

middle: D

last: Waters

first: Aurora

last: Ruiz-Herrera


title: Divergent 3D genome architecture of male germ cells across vertebrates [scRNA-seq]


title: Molecular modulators of CDK4/6 inhibitor response in experimental glioma identified through genome-wide CRISPR-Cas9 screening

city: Tübingen

first: Daniel

middle: J.

last: Merk

address: Otfried-Müller-Str. 27

country: Germany

institute: Hertie-Institute for Clinical Brain Research

type: Methylation profiling by genome tiling array

summary: Glioblastoma harbors frequent alterations in the retinoblastoma (RB1) pathway, providing a genetic rationale for therapeutic targeting with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. The NOA-20 trial did not reveal a progression-free survival benefit of CDK4/6 inhibition plus radiation therapy in newly diagnosed, MGMT-unmethylated glioblastoma. In fact, a monotherapy with CDK4/6 inhibitors has not demonstrated efficacy in solid tumors. We aimed at discovering response modulators to CDK4/6 inhibition, paving the way for rational combination therapies. We conducted genome-wide CRISPR-Cas9 screens in human glioma cell lines and stem-like cells (LN229, LN18, LNZ308, T98G, and GS-9) under CDK4/6 inhibition, employing knockout (Brunello library) and activation strategies (Calabrese library), followed by genetic and pharmacological validation of selected candidate genes in vitro and ex vivo (primary cultures) as well as the investigation of one functionally-instructed combination therapy in vivo. Loss of AMBRA1 and gain of function of CCNE1 reduced sensitivity to CDK4/6 inhibition in glioma cells, whereas disruption of CHEK1 or FAM122A resulted in synthetic lethality in combination with CDK4/6 inhibition. AMBRA1-deficient glioma cells exhibited increased sensitivity to CHK1 inhibition, revealing a context-specific vulnerability. Combined inhibition of CHK1 and CDK4/6 led to synergistic anti-glioma activity in vitro, ex vivo, and in vivo.

overall_design: DNA methylation profiling was peformed on 6 human glioma cell lines.

first: Ghazaleh

last: Tabatabai


title: DNA Methylatransferase Inhibition Prevents Platinum-Induced Ovarian Cancer Stem Cell Enrichment [RNA-Seq]

first: Heather

last: O'Hagan

overall_design: RNA-seq profiling of OVCAR3 cells mock-treated or treated with platinum-based agent cisplatin (Cis) and/or DNA methyltransferase inhibitor decitabine (DAC)

first: Truc

last: Vuong


title: High Throughput Characterization of Eukaryotic 2A-Like Peptides Identifies Novel Leucine-Associated Reduction in Protein Abundance

city: Cleveland

first: Kenneth

last: Matreyek

state: Ohio

address: 2103 Cornell Rd, Room 5104

department: Pathology

institute: Case Western Reserve University

summary: Virally-derived ribosomal skipping 2A peptides are a popular tool for protein co-production.  Despite their use in over 9,000 publications, the biochemical and biophysical properties underlying the skipping mechanism remain largely unexplored.  We identified 4,218 2A-like peptides originating from non-viral organisms.  We developed and utilized the Trifluorescent Reporter fluorescent tool for high-throughput multiplexable analysis of ribosomal skipping, and tested 3,271 2A-like peptide sequences.  We identified peptides that skipped, failed to skip, and skipped but failed to restart translation, in addition to peptides that induced a protein reduction phenotype.  A poly-leucine stretch in a conserved alpha-helix N-terminal to the conserved GDxExNPGP motif drove both the skipping and reduction phenotypes, with the number and location of leucine residues being the determinant of which phenotype was observed.  Analysis of the native eukaryotic protein contexts revealed that reduction may be harnessed as an expression regulator.  The high-throughput approach used in this work greatly expands the functional knowledge of what biophysical and biochemical characteristics lead to ribosomal skipping, including a latent eukaryotic ‘leucine stall-helix’ motif.

overall_design: Sort-Seq data for HEK293T cells genomically containing a trifluorescent construct comprising mCherry N-terminal to mKG N-terminal to miRFP670.  DNA sequences corresponding to 2A-like peptides are positioned in a flexible loop of mKG and alter fluorescent outputs based on ribosomal skipping activity.  Samples were quartile FACS sorted and sequenced to identify the contained 2A-like peptide.

first: John

last: Snell

middle: A


title: DNA Methylatransferase Inhibition Prevents Platinum-Induced Ovarian Cancer Stem Cell Enrichment [Cut & Run]

overall_design: CUT&RUN analysis of STAT3 and p65 in OVCAR3 cells mock-treated or treated with platinum-based agent cisplatin (Cis) and/or DNA methyltransferase inhibitor decitabine (DAC)


title: RNA-Seq analysis of p27+/+ and p27-/- mouse embryonic fibroblasts grown in full medium for 24 h

city: Toulouse

first: Arnaud

last: Besson

address: 118 route de Narbonne

department: MCD UMR5077

country: France

institute: Centre de Biologie Integrative - CNRS

summary: The goal was to determine the gene expression changes occuring upon knockout of the CDKN1B gene (p27Kip1) by RNA-Seq.

overall_design: Two lines of p27+/+ (n = 8) and one line of p27-/- (n = 4) grown in full medium for 24 h were used for transcriptomics analyses. Cells were lysed in TriReagent (T9424, Sigma Aldrich) and RNA was extracted using Direct-zol RNA Miniprep Plus Kits (Zymo Research) according to the manufacturer’s protocol. RNA integrity was verified on 0.8% agarose gel and quantified using a Nanodrop spectrophotometer. cDNA was synthetized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA-Seq samples were sequenced using Illumina Novaseq (paired-end, 2 x 150 bp long reads, 10M reads/sample). The quality of each raw sequencing file (fastq) was verified with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Fastq were aligned the reference mouse genome (GRCm39) and counted on the GENCODE annotation from database (gtf GRCm39) using STAR aligner (STAR_2.5.4a).

first: Leo

last: Rolland

first: Marion

last: Aguirrebengoa


title: A Distinct CAR-T Cell Phenotype Mediates Therapeutic Response at Limited Doses

city: Berlin

first: Schayan

last: Yousefian

address: Hannoversche Str. 28

institute: Charité Universitätsmedizin Berlin

summary: Chimeric antigen receptor (CAR) T cells are typically administered at high doses to ensure enduring therapeutic responses. However, achieving high cell numbers to meet end-product specifications is often challenging, posing substantial medical and socioeconomic barriers. Lower doses may be administered, but response rates are rather low, and the molecular and cellular mechanisms underlying therapeutic efficacy at limited doses remain unknown. To address this gap, we conducted deep phenotyping of anti-CD19 CAR-T cell products and their starting leukapheresis materials from a phase I/II clinical dose-escalation trial targeting B-cell malignancies. Patients responding to CAR-T cells despite low doses exhibited a dose-dependent enrichment of CAR-T cells with functional effector or effector memory-like states. The absolute number of these cells emerged as a robust biomarker of therapeutic response across varying dose levels. Notably, the manufacturing of CAR-T cells capable of mediating therapeutic responses at low doses was determined by T cell levels and T cell-supportive myeloid cells present in the starting material. Conversely, regulatory myeloid cell states contributed to the production of dysfunctional CAR-T cells and subsequent therapy failure. Our study provides critical insights into the phenotype, mechanisms, and biomarkers of CAR-T cells that drive therapeutic responses at low doses, with significant medical and socioeconomic implications.

overall_design:
Pre-manufacturing PBMCs from 28 patients enrolled in the HD-CAR-1 trial were subjected to single-cell proteo-genomics analysis using the BD Rhapsody platform on two consecutive days. In brief, samples were thawed at 37 °C, washed and cell suspensions were resuspended in 2% FSC 0.5 mM EDTA PBS (FACS buffer). Cells were stained with a live-dead mix including DAPI and Caspase-3. After a short incubation with the live-dead mix at 4 °C, 25k T cells and 25k non-T cells were sorted for each sample using a FACSAria III sorter (BD Biosciences). The sorted cells were then pooled for each sample respectively prior to labeling with hashing (sample-tag) antibodies according to the manufacturer's instructions. Incubation with hashing antibodies was performed on ice for 30 minutes. After three rounds of washing, samples were stained with the Abseq master mix (83 oligonucleotide-labeled antibodies; from BD Biosciences) for 30 minutes on ice. Subsequently, samples were washed three times before continuing with the single-cell capture protocol using the BD Rhapsody scRNA-seq platform (BD Biosciences). For each cartridge sorted cells from 4-5 sorted were pooled and loaded. In total 6 cartridges were prepared. Single-cell capture and preparation of hashing (sample-tag), mRNA and Abseq libraries were performed according to the manufacturer's instructions. Quality control of libraries was conducted using the Qubit dsDNA HS (High Sensitivity) kit and the 4200 TapeStation system (Agilent). Libraries of RNA, Abseq and sample tags(SMK) were pooled and sequenced using the Novaseq 6000 system (Illumina). Sample and assay demultiplexing is performed by the computational pre-preprocessing pipeline of BD (v.2.0). Cartridge 1 contains patient samples: P2-01, P1-05, P2-02, P1-07, P2-04. Cartridge 2 contains patient samples: P2-08, P1-12, P1-11, P2-09, P2-07. Cartridge 3 contains patient samples: P1-01, P2-13, P2-16, P2-14. Cartridge 4 contains patient samples: P2-18, P2-20, P1-02, P2-21, P1-02. Cartridge 5 contains patient samples: P1-03, P2-05, P1-04, P1-03, P2-17, P2-06. Cartridge 6 contains patient samples: P1-10, P1-08, P2-11, P2-10.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to legal restrictions on sharing personally identifiable sequence data.
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first: Simon

last: Haas

first: Michael

last: Schmitt


title: Characterizing circulating pharmacodynamic biomarkers of IFNβ-1a biologics [low]

city: Silver Spring

first: Lakshmi Manasa

middle: Sakuntala

last: Chekka

state: Maryland

address: 10903 New Hampshire Avenue

department: DARS, CDER

institute: U.S. FDA

summary: The current project is designed to evaluate the utility of proteomics in identifying pharmacodynamic (PD) biomarkers for biosimilarity assessment. This is an extension of the data submitted in GSE207945 which profiled longitudinal plasma samples from healthy subjects treated with single therapeutic doses/high doses of IFNβ-1a (30 μg) or Pegylated IFNβ-1a (125 μg) or placebo, to identify candidate PD biomarkers for the two biologics (n=248 for IFNβ-1a and N=528 for pegIFNβ-1a). The current project is a continuation study profiling longitudinal plasma samples from healthy subjects treated with a low dose of IFNβ-1a (7.5 μg) and pegIFNβ-1a (31.25 μg).  In the current study, we reproduced the previous PD biomarker candidate responses at an intermediate/lower dose IFNβ-1a (15 μg) and pegIFNβ-1a (62.5 μg), and further characterized their PD response using characteristics such as significant area under the effect curve, dose-response, temporal profile and return to baseline, variability and sensitivity of the biomarker with sensitive doses on the steep portion of the dose-response curve and mechanism of action using the three tested dose-groups (high, intermediate and low) in the study.

overall_design: The study includes data on >580 proteins from 12 subjects treated with IFNβ-1a (7.5 µg), 12 with pegIFNβ-1a (31.25 µg). The 12 subjects on IFNβ-1a were tested at 10 timepoints over 6 days, and the rest were tested at 12 time points over 13 days. Replicates for 3 random samples were included on the array along with two covid covalacent plasma samples.

first: Lakshmi

last: Manasa Sakuntala Chekka

first: Paula

last: Hyland


title: Sequential inflammatory and matrisome programs drive remodeling of the mouse carotid-jugular arteriovenous fistula

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

summary: We studied the transformation of the murine jugular vein after creation of carotid-jugular arteriovenous fistulas using bulk and single-cell RNA sequencing.

overall_design: The outflow veins of carotid-jugular AVFs were collected at early (~3 days), mid (~10 days), and late (~21 days) time points after AVF creation. Contralateral jugular veins were also harvested as controls. Total RNA was isolated for bulk RNA sequencing. For single-cell sequencing, vessels were collected in cold cell culture media and enzymatically digested to generate single-cell suspensions. Samples were run using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics) with a target capture of 10,000 cells.

first: Laisel

last: Martinez

first: Roberto

middle: I

last: Vazquez-Padron


title: DICER1 syndrome-associated mutations lead to 3p-miRNA gain of function and increased metastatic potential [miRNA-Seq]

city: Edinburgh

first: Jeroen

last: Witteveldt

state: midlothian

address: The King's Buildings, Ashworth Laboratories

department: IIIR

institute: The University of Edinburgh

summary: The DICER1 gene is mutated in cancer, including Dicer1 syndrome, a rare tumour predisposition syndrome. Cancer-associated hotspots mutations have been reported in both catalytic domains of Dicer, and are predicted to disrupt miRNA processing activity. To understand these hotspot mutations in cancer development, we have generated cell lines harbouring single amino acid substitutions within either the RNAse IIIa (S1344L) and the RNAse IIIb (D1709N) domains of the endogenous Dicer1 gene. We show that both mutations result in a widespread loss of 5p miRNAs, but, unexpectedly, an increase in 3p passenger strands loading into Ago2. Similarities between both mutants can be explained as the S1344 residue is structurally also part of the RNase IIIb catalytical site. Functionally, we found that changes in the repertoire of miRNAs loaded into Ago2 result in altered gene expression, impacting critical pathways for cancer development, including metastatic potential. Our results indicate that inactivating the processing activity of Dicer does not result in genomic instability. Instead, mutations cause a specific upregulation of human endogenous retrovirus H (HERVH) and 3p miRNAs, which have the potential to be used as markers for Dicer1 syndrome tumours.

overall_design: miRNA profiling of AGO2 immunoprecipitation from wild type PA-1 cells and PA-1 cell lines with two different  mutants in the protein Dicer. Homozygous mutations of S1344L and D1709N.

first: Katrina

last: Gordon

first: Nicolas

last: Bellora

first: Joanna

last: Wojtus

first: Felix

last: Mueller

first: Katharina

last: Kases

first: Pilar

last: Marchante

first: Guillermo

last: Peris

first: Shelagh

last: Boyle

first: Sara

last: Heras

first: Atlanta

last: Cook

first: Cei

last: Abreu-Goodger

last: Macias


title: DICER1 syndrome-associated mutations lead to 3p-miRNA gain of function and increased metastatic potential [small RNA]

overall_design: smallRNA profiling of wild type PA-1 cells and PA-1 cell lines with three different  mutants in the protein Dicer. Homozygous mutations of S1344L and D1709N and a compound heterozygous mutation of D1709N and a Dicer heterozygous.


title: Maternal trans-vaccenic acid reshapes neonatal T cell development and immune imprinting (LIME-seq)

city: Pasadena

first: Cheng-Wei

last: Ju

state: CA

address: 1200 E California Blvd

institute: California Institute of Technology

summary: How maternal nutrition influences neonatal immune development and imprinting through breastfeeding remains largely unclear. Here, we report that maternal supplementation with 25 trans-vaccenic acid (TVA) – the predominant naturally occurring trans-fatty acid in human breast milk (HBM) – promotes neonatal T cell development in mice, particularly expanding the naïve CD4+ T cell population with enhanced readiness. This implements a shift from T helper (Th)2- biased neonatal immune responses to Th1-skewing, resulting in improved adaptive immunity against viral infection and lung inflammation. Mechanistically, TVA via breastfeeding reprograms neonatal naïve CD4+ 30 T cells through a G protein coupled receptor (GPR43)-CCCTC-binding factor axis for enhanced cooperation with transcription factor TBX21, which also exhibits a longlasting immune imprinting effect. Furthermore, TVA levels in HBM from mothers of preterm infants inversely correlate with the incidence of inflammation-associated bronchopulmonary dysplasia in their preterm infants. Our findings establish the multifaceted benefits of maternal 35 nutrition and breastfeeding via TVA in promoting infant immune homeostasis and protective immunity.

overall_design: For each mouse line, eight-week-old C57BL/6J male and female mice were fed either a TVA-enriched diet or a control diet for two weeks prior to mating. Breeding pairs were maintained on their respective diets throughout gestation and lactation. Offspring were continuously maintained on the same diet and inbred to establish and propagate each dietary lineage. Fecal samples were collected from two-week-old pups for LIME-seq analysis (n = 5 per group)

first: Yiqian

last: Pan

first: Hao

last: Fan

first: Chuan

last: He


title: DICER1 syndrome-associated mutations lead to 3p-miRNA gain of function and increased metastatic potential

last: Witteveldt

overall_design: RNA-seq profiling of wild type PA-1 cells and PA-1 cell lines with three different  mutants in the protein Dicer. Homozygous mutations of S1344L and D1709N and a compound heterozygous mutation of D1709N.


title: A human-specific microRNA controls the timing of excitatory synaptogenesis (miRNA mimics)

city: Zurich

first: Gerhard

last: Schratt

address: Winterthurerstrasse 190

department: HEST

country: Switzerland

institute: ETH Zürich

summary: Neural circuit development in the human cortex is considerably prolonged in comparison to non-human primates, a trait that contributes to the remarkable cognitive capacity of modern humans. Here, we explore the regulatory role of non-coding RNAs, which dramatically expanded during brain evolution, in synapse development of human-induced pluripotent stem-cell derived neurons. We found that inhibition of a human-specific microRNA, miR-1229-3p, alters the trajectory of human neuronal maturation and enhances excitatory synaptic transmission. Transcriptome analysis following miR-1229 knockdown revealed a downregulation of mitochondrial DNA (mtDNA) encoded genes. We further show that miR-1229 regulates mitochondrial morphology, mtDNA abundance as well as mitophagy, and that stimulation of mitochondrial metabolism rescues decreased calcium buffering in miR-1229-3p depleted neurons. Accordingly, miR-1229 directly targets an entire network of genes involved in mitochondrial function and ER-associated protein homeostasis. Our findings reveal an important function of human-specific miR-1229-3p in developmental timing of human synaptogenesis and generally implicate non-coding RNAs in the control of human connectivity and cognition.

overall_design: To find direct targets of miR-1229-3p, we overexpressed miRNA mimics into SH-Sy5y cells. Specifically, we transfected a control condition (40pmol of Neg. Ctrl.), a miR-1229 10nM condition (20pmol of Neg. Ctrl. & 20pmol of miR-1229-3p precursor) and a miR-1229 20nM condition (40pmol miR-1229-3p precursor).

first: Michael

last: Soutschek


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer [BETi_DNMTi_human_RNAseq]

city: Ann Arbor

first: Karan

last: Bedi

state: MI

address: 1600 Huron Pkwy

department: Biostatistics

institute: University of Michigan

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Neuroendocrine prostate cancer (NEPC) is the most lethal form of lineage plasticity in prostate cancer. BET bromodomain inhibitors (BETi) previously were shown to block genes associated with lineage plasticity. Extensive rewiring of DNA methylation is also associated with NEPC tumors. We sought to determine whether combined BET protein and DNA methyltransferase inhibition (DNMTi) would result in greater anti-tumor activity than single agent treatments alone. To test this, we treated NEPC cell line models with 500nM ZEN (BETi), 100nM dAZA (DNMTi), or the combination daily for 72hrs. We also tested the BETi + DNMTi combination in vivo using an NEPC PDX (LTL331R) with 50mg/kg ZEN and 0.8mg/kg dAZA. Combined BETi and DNMTi was more effective than single agent treatment in suppressing growth in NEPC models compared to single agent treatments. To identify the gene expression changes underlying the superior viability effect of the combo treatment, we peformed RNAseq on treated cells. We also performed RNAseq on a PDX model that reliably transitions from adenocarcinoma (LTL331) to NEPC (LTL331R) in order to uncover the gene expression changes associated with NEPC lineage plasticity and to determine whether BETi and/or DNMTi reverses these gene expression changes.

overall_design: RNAseq was performed on untreated adenocarcinoma PDXs (LTL331) and untreated NEPC PDXs (LTL331R);  NEPC cell line models, LTL331R (derived from PDX) and NCI-H660 were treated with BET inhibitor (500nM ZEN), DNA methyltransferase inhibitor (100nMdAZA), or the combination (500nM ZEN + 100nM dAZA) daily for 72 hrs with corresponding vehicle (DMSO) controls. Combination treatment was also tested in vivo using the NEPC PDX LTL331R where mice were treated with 50mg/kg ZEN daily by oral gavage, 0.8mg/kg dAZA for five consecutive days followed by a 9-day holiday by IP injection, or the combination with corresponding vehicle controls.

first: William

middle: K

last: Storck

first: Raymond

last: Cavalcante

first: Diana

last: Flores

first: Anbarasu

last: Kumaraswamy

first: Zhi

last: Duan

first: Joel

last: Yates

first: Joshi

last: Alumkal


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer [ChIP-seq]

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Loss of the tumor suppressors TP53 and RB1 is common in tumors exhibiting lineage plasticity; however, mechanisms by which TP53/RB1 loss promotes this phenotype remain poorly understood, and effective treatments are limited. To identify the histone acetylation alterations that occur with TP53/RB1 loss chromatin reprogramming, we performed H3K27Ac ChIP-seq on TP53/RB1-deficient vs. TP53/RB1-intact cell lines.

overall_design: H3K27Ac ChIP-seq was performed on TP53/RB1-deficient (TKO; DKOcr298) and TP53/RB1-intact (SKO) cells in order to uncover the histone acetylation changes induced by TP53/RB1 loss.

first: Eva

last: Rodansky


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer [ATAC-seq]

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Loss of the tumor suppressors TP53 and RB1 is common in tumors exhibiting lineage plasticity; however, mechanisms by which TP53/RB1 loss promote this phenotype remain poorly understood, and effective treatments are limited. Thus we used multi-omic profiling of TP53/RB1 loss prostate cancer models to identify alterations in chromatin accessibility, DNA methylation, and gene expression associated with lineage plasticity.

overall_design: ATAC-seq was performed on DMSO-treated RB1-deficient (DKO; DKOcr105), TP53/RB1-deficient (TKO; DKOcr298), and TP53/RB1-intact (SKO) cells in order to uncover the chromatin accessibility changes induced by TP53/RB1 loss


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer [ERRBS]

type: Methylation profiling by high throughput sequencing

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Loss of the tumor suppressors TP53 and RB1 is common in tumors exhibiting lineage plasticity; however, mechanisms by which TP53/RB1 loss promotes this phenotype remain poorly understood, and effective treatments are limited. Thus we used multi-omic profiling of TP53/RB1 loss prostate cancer models to identify alterations in chromatin accessibility, DNA methylation, and gene expression associated with lineage plasticity. BET bromodomain inhibitors previously were shown to block genes associated with lineage plasticity. We sought to determine whether they could block pathways activated upon TP53/RB1 loss as well. TP53/RB1-deficient cells also harbored widespread DNA methylation changes that silenced pathways linked with lineage plasticity, which we found could be reversed by DNA methyltransferase (DNMT) inhibitors. Combined BET bromodomain and DNMT inhibition was more effective than single agent treatment in suppressing growth in TP53/RB1 loss models compared to single agent treatments. To identify the DNA methylation changes underlying the superior viability effect of the combo treatment, we peformed enhanced reduced representation bisulfite sequencing (ERRBS) on treated cells.

overall_design: ERRBS was performed on DMSO-treated RB1-deficient (DKO; DKOcr105), TP53/RB1-deficient (TKO; DKOcr298), and TP53/RB1-intact (SKO) cells in order to uncover the DNA methylation induced by TP53/RB1 loss; TKO and DKOcr298 were treated with BET inhibitor (500nM ZEN), DNA methyltransferase inhibitor (100nMdAZA), or the combination (500nM ZEN + 100nM dAZA) daily for 72 hrs with corresponding vehicle (DMSO) controls.


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Neuroendocrine prostate cancer (NEPC) is the most lethal form of lineage plasticity in prostate cancer. BET bromodomain inhibitors (BETi) previously were shown to block genes associated with lineage plasticity. Extensive rewiring of DNA methylation is also associated with NEPC tumors. We sought to determine whether combined BET protein and DNA methyltransferase inhibition (DNMTi) would result in greater anti-tumor activity than single agent treatments alone. To test this, we treated NEPC cell line models with 500nM ZEN (BETi), 100nM dAZA (DNMTi), or the combination daily for 72hrs. We also tested the BETi + DNMTi combination in vivo using an NEPC PDX (LTL331R) with 50mg/kg ZEN and 0.8mg/kg dAZA. Combined BETi and DNMTi was more effective than single agent treatment in suppressing growth in NEPC models compared to single agent treatments. To identify the DNA methylation changes underlying the superior viability effect of the combo treatment, we peformed enhanced reduced representation bisulfite sequencing (ERRBS) on treated cells.

overall_design: ERRBS was performed on NEPC cell line models, LTL331R (derived from PDX) and NCI-H660,  treated with BET inhibitor (500nM ZEN), DNA methyltransferase inhibitor (100nMdAZA), or the combination (500nM ZEN + 100nM dAZA) daily for 72 hrs with corresponding vehicle (DMSO) controls. Combination treatment was also tested in vivo using the NEPC PDX LTL331R where mice were treated with 50mg/kg ZEN daily by oral gavage, 0.8mg/kg dAZA for five consecutive days followed by a 9-day holiday by IP injection, or the combination with corresponding vehicle controls.


title: The retrotransposable element-silencing factor Daxx controls microglia identity and function in the adult brain

city: Bonn

first: Giuliano

last: Crispatzu

address: Venusberg-Campus 1, Gebäude 99, Venusberg Campus 1/Gebäude 99

department: Nuclear Function in CNS pathophysiology

institute: DZNE

summary: Death domain-associated protein (Daxx) is one of the two main H3.3 chaperones in mammalian cells and acts as key repressor of RTEs of viral origin. In the present study, we found that Daxx is an IFN-stimulated gene mediating repression of RTEs and IFN-stimulated genes (ISGs) upon inflammatory stimuli in microglia. Using both bulk and single-cell genomics, we demonstrated that shortly after Daxx loss microglia displayed co-expression of DNA damage/p53, IFN/developmental and DAM-like signatures, suggesting lowering of the epigenetic barrier and increased plasticity. With time, Daxx-deficient microglia become restricted to a DAM-like/ApoeHIGH profile, associated with decreased synaptic puncta and altered behavior. Finally, brains from older microglia-specific Daxx KO mice accumulate ApoE protein along with lysosomal markers. Together, our data revealed a role of Daxx in maintenance of the homeostatic state of microglia in the adult brain, with implications for our understanding of the relationship between loss of epigenetic barriers and neuroinflammation and its potential implications for brain function and aging.

overall_design: To investigate the functional role of Daxx in the microglia, we generate a tamoxifen-inducible Daxx conditional knockout mouse by crossing Cx3cr1-CreERT2 mouse line with Daxx conditional allele (Cx3cr1-CreERT2, Daxxflox/flox). The microglia were isolated from mice brain at 3 days after tamoxifen administration for CUT&Tag sequencing.

first: Paolo

last: Salomoni

first: Xin

last: Yan


title: Quantitative characterization of the aging intestine secretome reveals novel secreted proteins that act in the extracellular space to regulate lifespan

city: Stanford

first: Daniel

last: Richard

address: 290 Jane Stanford Way

department: Genetics

institute: Stanford University

summary: Secreted proteins are essential to modulate the extracellular space and communicate to distal cells or tissues. But the importance of extracellular proteins in aging has been understudied. Here we use proximity labeling followed by quantitative proteomics to systematically characterize the intestine and neuron secretome in C. elegans. We identify many previously unknown intestine secreted proteins that change with age, and we validate the secretion of these proteins by in vivo cell biology. One of these secreted proteins, ACP7, is well conserved in humans. Overexpressing ACP7 extends lifespan in a secretion-dependent manner, and we find that ACP7 acts as a phosphatase in the extracellular space. Finally, we identify additional proteins along the secretion pathway that regulate lifespan, including VAPA – a protein localized to small secretory vesicles in the intestine. Our systematic characterization of tissue-specific secretome during aging uncovers novel proteins that impact lifespan and highlights the role of extracellular enzymes in aging.

overall_design: Bulk RNA-Seq of C. elegans to determine how the overexpression of a secreted phosphatase, ACP7, from the intestine impacts the animal's transcriptome as compared to wild-type N2 animals and the overexpression of a phosphatase inactive ACP7-7A.

first: Jason Wayne

last: Miklas

first: Daniel Joseph

first: Brandon

last: Ameglio

first: Anne

last: Brunet


title: Dendritic cell dysfunction independently predicts poor prognosis in acute lymphoblastic leukemia

city: Monrovia

first: Srividya

last: Swaminathan

address: 1218 S Fifth Ave, BRC2064

institute: Beckman Research Institute of City of Hope

summary: We have used scRNA-seq to evaluate the production and maturation of DC1-6 subsets to be disrupted in children and adults with ALL. While conventional DC1 and DC2 are reduced, cells within all DC subsets in patients have impaired antigen presentation and produce less cytokines needed for initiating T-cell surveillance than their normal counterparts in healthy people. ALL DCs are unable to induce T-cell proliferation. Overexpression of MYC in leukemia cells, in part by abrogating NK surveillance, disrupts DC homeostasis and reduces the ability of DCs to induce T-cell immunity. Importantly, immature, dysfunctional DCs with impaired antigen presentation independently predict poor prognosis in patients with high-risk ALL. Thus, phenotyping DC subsets in bone marrow or peripheral blood at ALL diagnosis or relapse could inform the course of treatment.

overall_design: Following the manual removal of pathogenic B cells from B-ALL samples and pathogenic T cells from T-ALL samples through magnetic microbead separation, we employed scRNA-seq for the remainnig immune compartments.

first: Swaminathan

last: Srividya

first: Kumar

last: Anil

first: Hamane

last: Kory


title: Combined BET Bromodomain and DNMT Inhibition Targets Lineage Plasticity in Prostate Cancer [BETi_DNMTi_mouse_RNAseq]

first: Karan

last: Bedi

summary: Lineage plasticity is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Loss of the tumor suppressors TP53 and RB1 is common in tumors exhibiting lineage plasticity; however, mechanisms by which TP53/RB1 loss promotes this phenotype remain poorly understood, and effective treatments are limited. Thus we used multi-omic profiling of TP53/RB1 loss prostate cancer models to identify alterations in chromatin accessibility, DNA methylation, and gene expression associated with lineage plasticity. BET bromodomain inhibitors previously were shown to block genes associated with lineage plasticity. We sought to determine whether they could block pathways activated upon TP53/RB1 loss as well. TP53/RB1-deficient cells also harbored widespread DNA methylation changes that silenced pathways linked with lineage plasticity, which we found could be reversed by DNA methyltransferase (DNMT) inhibitors. Combined BET bromodomain and DNMT inhibition was more effective than single agent treatment in suppressing growth in TP53/RB1 loss models compared to single agent treatments. To identify the gene expression changes underlying the superior viability effect of the combo treatment, we peformed RNAseq on treated cells.

overall_design: RNAseq was performed on DMSO-treated RB1-deficient (DKO; DKOcr105), TP53/RB1-deficient (TKO; DKOcr298), and TP53/RB1-intact (SKO) cells in order to uncover the transcriptomic changes induced by TP53/RB1 loss; TKO and DKOcr298 cells were treated with BET inhibitors (500nM ZEN or 1µM JQ1) for 24hrs with corresponding vehicle (DMSO) controls; TKO and DKOcr298 were treated with BET inhibitor (500nM ZEN), DNA methyltransferase inhibitor (100nMdAZA), or the combination (500nM ZEN + 100nM dAZA) daily for 72 hrs with corresponding vehicle (DMSO) controls.

first: William

last: Storck

first: Raymond

last: Cavalcante


title: Analysis of gene expression profiles in matched cancer-associated and non-tumoral fibroblasts from patients with head and neck squamous cell carcinoma

city: Woluwé

first: Jérôme

last: Ambroise

address: Avenue Hippocrate 54

department: Institut de Recherche Expérimentale et Clinique

country: Belgium

institute: Université catholique de Louvain

summary: Among stromal components, cancer-associated fibroblasts (CAFs) constitute the predominant non-malignant population in the HNSCC microenvironment, where their abundance correlates with advanced disease and poor prognosis. CAFs support tumor progression via extracellular matrix remodeling and paracrine signaling through cytokines, growth factors, exosomes and miRNA. Here, we carried out a transcriptomic analysis of 6 couples of CAFs and matching non-tumoral fibroblasts (NFs) derived from HNSCC patients in order to reveal gene expression changes between CAF and NF cell populations and identify potential CAF-specific therapeutic targets.

overall_design: CAFs were derived from biopsy or surgical resection pieces of treatment-naive HNSCC patients operated in the Cliniques universitaires Saint-Luc (Brussels, Belgium), and patient-matched non-tumoral fibroblasts (NFs) were collected from a concomitant biopsy of the inner side of the cheek. CAFs were cultured in DMEM-GlutaMAX supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.5 nM human recombinant basic fibroblast growth factor (b-FGF, Cat#78003, STEMCELL Technologies) and then maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37°C. NFs were grown in human fibroblast expansion basal medium (Cat#M106500, Gibco) supplemented with 2% low serum growth supplement (LSGS, Cat#S00310, Thermo Fisher Scientific) and 1% penicillin/streptomycin. The study was approved by the ethics committee from UCLouvain (approval ID UCL-ONCO 2015-02, Informed consent amendment 4, Version 5 dated on 04/03/2021) in accordance with the principles of the Declaration of Helsinki, and all patients gave their written informed consent.

first: Cyril

last: Corbet

first: Sandra

last: Schmitz

first: Jean-Pascal

last: Machiels


title: Analysis of cetuximab-induced gene expression changes in head and neck squamous cell carcinoma cell lines

summary: Despite recent advances in immunotherapy and anti-epidermal growth factor receptor (EGFR) treatments, the therapeutic landscape for locally advanced HNSCC has changed little over recent decades, and clinical outcome remains poor. Cetuximab, monoclonal antibody targeting EGFR, offers only modest and often transient clinical responses, owing to the persistence of a therapy-tolerant cell population that seeds residual disease and eventual therapeutic failure. Clinical evidence across cancer types indicates that depth of response to targeted therapy correlates with prolonged progression-free survival, highlighting the importance of residual disease as a driver of resistance. However, the molecular features enabling HNSCC cells to tolerate EGFR blockade before the acquisition of stable resistance remain incompletely understood. To uncover early transcriptional adaptations to EGFR inhibition, we performed bulk RNA-sequencing on FaDu and HN5 HNSCC cells treated with cetuximab for 72 hours.

overall_design: FaDu and HN5 cell lines were treated with 1 µg/mL cetuximab for 72h before RNA extraction and bulk RNA-sequencing.


title: Nodal/Smad2 signaling sustains developmental pausing by repressing Pparg -mediated lipid metabolism [RNA-Seq]

city: Toronto

first: Giacomo

last: Furlan

state: ONTARIO

address: 25 Orde St

country: Canada

institute: Lunenfeld-Tanenbaum Research Institute

summary: Cells and organisms can enter transient dormant states to survive unfavorable conditions during development, physiological adult states and disease. One paradigmatic case of dormancy is diapause, whereby embryos transiently pause development and enter a state of suspended animation. In mammals, diapause occurs pre-implantation at the blastocyst state and involves global growth suppression and metabolic rewiring towards lipid usage as energy source. The molecular regulation of diapause remains poorly understood, including whether it occurs by default or requires active signaling. Here, we identify the canonical TGF-β signaling pathway as an essential driver of transcriptional and metabolic reprogramming in diapause. TGF-β signaling was thought to only be required post-implantation, but we show that the ligand Nodal and its downstream effector Smad2 are essential for the survival of paused embryonic stem cells (ESCs) and blastocysts. Mechanistically, we found that Smad2 represses a Pparg, a transcription factor that is a master regulator of lipid storage, a process incompatible with pausing.  Ablation of Pparg in Smad2-deficient ESCs rescues their survival and prevents excess lipid buildup in pausing conditions. These findings establish Nodal/Smad2 signaling as pivotal for sustaining embryonic diapause and suggest that the crosstalk between TGF-β signaling and the Pparg pathway may be broadly relevant for metabolic rewiring in dormancy contexts.

overall_design: WT, Smad2KO_1 and Smad2KO_2 ESCs were grown on gelatin for 12 hours in FBS/LIF media, under control and pausing conditions (200 nM INK128). For each conditions, 4 replicates were collected

first: Evelyne

last: Collignon

first: Miguel

last: Ramalho Santos


title: Evaluation of cetuximab-induced gene expression changes in residual PDX tumors of head and neck squamous cell carcinoma

summary: Cetuximab, a monoclonal antibody targeting EGFR, offers only modest and often transient clinical responses in recurrent or metastatic HNSCC, owing to the persistence of a therapy-tolerant cell population that seeds residual disease and eventual therapeutic failure. Clinical evidence across cancer types indicates that depth of response to targeted therapy correlates with prolonged progression-free survival, highlighting the importance of residual disease as a driver of resistance. However, the molecular features enabling HNSCC cells to tolerate EGFR blockade before the acquisition of stable resistance remain incompletely understood. We analyzed residual tumors from cetuximab-treated patient-derived xenograft (PDX) models of HNSCC (#UCLHN04 and #HNC004) to identify gene expression changes associated with anti-EGFR tolerance in HNSCC cells.

overall_design: PDX-bearing nude mice were weekly treated with 30 mg/kg cetuximab (or vehicle). Residual tumors were defined as those demonstrating tumor size stabilization for at least four weeks following cetuximab treatment, representing a state of maximal therapeutic response. RNA-seq analysis was performed on samples coming from vehicle- and cetuximab-treated PDX, revealing major changes of expression at the mRNA level. This research complies with all relevant ethical regulations. These in vivo experiments received the approval of the ethics committee from UCLouvain (approval ID 2022/UCL/MD/024) and were carried out according to national care regulations.The study for PDX model generation was initially approved by the ethics committee from UCLouvain (approval ID UCL/MD/2012/09July/314) in accordance with the principles of the Declaration of Helsinki, and all patients gave their written informed consent.


title: Nodal/Smad2 signaling sustains developmental pausing by repressing Pparg -mediated lipid metabolism [ATAC-seq]


title: Defining the relationships among four tests with antibody-mediated rejection in heart transplants

city: Edmonton

first: Jessica

last: Chang

address: 250 Heritage Medical Research Centre

department: Medicine

institute: University of Alberta

summary: Four tests assess antibody-mediated rejection (ABMR) in heart transplants: standard of care local endomyocardial biopsy (EMB) histology, EMB molecular analysis (Molecular Microscope® Diagnostic System - MMDx), donor-derived cell-free DNA (dd-cfDNA), and donor-specific antibody (DSA). These tests have become critical for the development and use of novel therapies for ABMR. The Trifecta-Heart study (NCT04707872) aimed to define the relationships among these tests. MMDx diagnosed more ABMR, TCMR, and No rejection (NR) than histology, a pattern confirmed in a validation set from the larger INTERHEART study (NCT02670408). When correlations and partial correlation modeling were used to defined the hierarchy of relationships among all tests, MMDx ABMR correlated more strongly with dd-cfDNA and DSA than did histologic ABMR. In MMDx-histology discrepancies, biopsies with no histologic rejection but with MMDx ABMR had elevated DSA and dd-cfDNA, and biopsies with MMDx NR but histology ABMR had low DSA and dd-cfDNA. DSA-negative ABMR by MMDx or histology had elevated dd-cfDNA, similar to DSA-positive ABMR. Most cases with molecular injury and high dd-cfDNA also had concurrent molecular rejection. These inter-test relationships will be useful in guiding ABMR management, including monitoring of treatment responses and relapses, especially when discrepancies occur.

overall_design: The study compared standard of care local histology diagnosis to the results of three central tests assessing ABMR: central DSA (One Lambda Inc.), dd-cfDNA (Natera, Inc.), and MMDx rejection assessments in 214 EMBs. Results were primarily expressed as positive-negative to facilitate analysis and to simulate the way all tests are used for clinical decision-making.


title: Dissecting Resistance to CDK4/6 Inhibition in Experimental Glioma via Genome-wide CRISPR-Cas9 Screening

first: Daniel

last: Merk

summary: CDK4/6 inhibitors exhibit therapeutic potential in several tumor entities. Their clinical efficacy in glioblastoma might be limited by intrinsic and acquired resistance mechanisms. Understanding underlying genetic and molecular factors of this resistance might inform the design of effective therapeutic strategies. Genome-wide CRISPR-Cas9 screens offer a powerful approach to systematically identify resistance drivers and synthetic lethal interactions, paving the way for rational combination therapies.We conducted genome-wide CRISPR-Cas9 knockout and activation screens in human glioma cell lines LN229 and GS9 under CDK4/6 inhibition using Brunello and Calabrese libraries. Top hits were validated using functional assays. Furthermore, we conducted bulk RNA sequencing of treated and untreated cells for transcriptomic insights into drug response modifiers and molecular pathways involved.Functional genomics identified Ambra1 and CCNE1 as key drivers of resistance. Both Ambra1 knockout and CCNE1 overexpression, conferred resistance across both long-term and stem-like glioma models. Additionally, knockout of CHEK1 and FAM122A uncovered synthetic lethal interactions with CDK4/6 inhibition. Functional validation confirmed that glioma cells rely on CHEK1 or FAM122A for cell cycle progression, with cell line specific sensitivity. Transcriptomic analyses consistently highlighted alterations in pathways associated with cell cycle regulation, DNA replication, and the Fanconi anemia pathway. Of note, several differentially expressed genes overlapped with screen hits, supporting the robustness of our findings. Notably, pharmacological inhibition of CHEK1, both in vitro and ex vivo, enhanced sensitivity to CDK4/6 inhibition in a sequence-dependent manner, indicating a critical role of the sequence of drug administration.

overall_design: Human glioma cell lines were treated with the CDK4/6 inhibitor abemaciclib or corresponding DMSO concentrations for 48 hours.

first: Ghazaleh

last: Tabatabai


title: In vivo chemical reprogramming is associated with a toxic accumulation of lipid droplets hindering rejuvenation

city: Boston

first: Wayne

last: Mitchell

state: MA

address: 77 Avenue Louis Pasteur

institute: Brigham and Women's Hospital

summary: Partial reprogramming has emerged as a promising strategy to reset the epigenetic landscape of aged cells towards more youthful profiles. Recent advancements have included the development of chemical reprogramming cocktails that can lower the epigenetic and transcriptomic age of cells and upregulate mitochondrial biogenesis and oxidative phosphorylation. However, the ability of these cocktails to affect biological age in a mammalian aging model has yet to be tested. Here, we have characterized the effects of partial chemical reprogramming on mitochondrial structure and function in aged mouse fibroblasts and tested its in vivo efficacy in genetically diverse male UM-HET3 mice. This approach increases the size of mitochondria, alters cristae morphology, causes an increased fusing of mitochondrial networks, and speeds up movement velocity. At lower doses, the chemical reprogramming cocktail can be safely administered to middle-aged mice using implantable osmotic pumps, albeit with no effect on the transcriptomic age of kidney or liver tissues and only a modest effect on the expression of OXPHOS complexes. However, at higher doses, the cocktail causes a drastic reduction in body weight necessitating euthanasia. In the livers and kidneys of these animals, we observe significant increases in lipid droplet accumulation, as well as changes in mitochondrial morphology in the livers that are associated with mitochondrial stress. Thus, partial chemical reprogramming may induce mitochondrial stress and lead to significant lipid accumulation, which may cause toxicity and hinder the rejuvenation of cells and tissues in aged mammals.

overall_design: 12-month-old male UM-HET3 mice were treated with 7c (repsox, valproate, tranylcypromine, DZNep, CHIR99021, forskolin, TTNPB) or vehicle (50% DMSO / 50% PEG300) for 28 days using subcutaneously-implanted osmotic minipumps. After 28 days, the animals were euthanized, total RNA was isolated from flash-frozen liver and kidney tissues, and mRNA-seq was performed to evaluate the impact of 7c treatment on transcriptomic biomarkers of biological age.

first: Cecilia

last: de Magalhaes

first: Alexander

last: Tyshkovskiy

first: Yushi

last: Uchida

first: Ludger

last: Goeminne

first: Takaharu

last: Ichimura

first: Emery

middle: L

last: Ng

first: Alibek

last: Moldakozhayev

first: Joseph

middle: V

last: Bonventre

first: Vadim

middle: N

last: Gladyshev


title: Gene transcriptomics reveals that HOXC9 regulates the progression of Esophageal Squamous Cell Carcinoma

city: Zhengzhou

first: Yangyang

last: Ji

state: Henan

address: No. 1, Jianshe East Road, Erqi District, Zhengzhou City, Henan Province

institute: The First Affiliated Hospital of Zhengzhou University

summary: Homeobox C9 (HOXC9) has been found to play a critical role in tumor progression. However, its regulatory function in esophageal squamous cell carcinoma (ESCC) remains unclear. We analyzed the gene expression profiles of KYSE30 cells stably overexpressing HOXC9 using RNA-seq.

overall_design: To investigate the regulatory effect of HOXC9 on whole genome gene expression, we analyzed the gene expression profiles of control or HOXC9 overexpressing KYSE30 cells using RNA-seq. The aim of this study is to reveal how HOXC9 affects ESCC malignant progression.

first: Cai

last: Zhang


title: Transcriptomic profiling of bone marrow cells from CML mouse models and newly diagnosed patients treated with SOS1 inhibitor BI-3406 and/or imatinib

city: Salamanca

first: Rósula

last: Garcia Navas

state: Salamanca

address: Campus Miguel de Unamuno, P. Universidad de Coimbra s/n. Lab.1

institute: Centro de Investigación del Cáncer

summary: Despite the success of BCR-ABL1 tyrosine kinase inhibitors (TKIs) like imatinib in treating chronic myeloid leukemia (CML), therapeutic resistance and disease persistence remain significant challenges. This study investigated the potential of BI-3406, a selective SOS1 inhibitor (a key activator of RAS signaling), either alone or in combination with imatinib. Using preclinical CML models, including transgenic mice and patient samples, it was observed that BI-3406 and imatinib administration were well-tolerated. Although both drugs partially reduced splenomegaly and leukocytosis individually, combination therapy showed a significantly greater therapeutic effect. This combination extended survival, normalized spleen architecture, and sharply reduced leukemic stem and progenitor cells, suggesting a synergistic impact on leukemic burden and stem cell compartments. In vitro assays confirmed that BI-3406 potentiated the cytotoxic effects of imatinib in CML cell lines, increasing apoptosis. Notably, in primary bone marrow cells from patients with the imatinib-resistant T315I mutation, BI-3406 not only synergized with ponatinib but also restored sensitivity to imatinib, offering a potential strategy to overcome drug resistance. RNA sequencing revealed that BI-3406 alone triggered a compensatory anabolic transcriptional program, while imatinib suppressed key proliferative and metabolic pathways. However, combination therapy produced more profound reprogramming, simultaneously reinforcing anti-proliferative effects and activating immune and inflammatory pathways. These conserved effects across murine and human samples support the translational potential of this dual-targeting strategy. In conclusion, SOS1 inhibition with BI-3406, in combination with TKIs, dismantles oncogenic signaling, suppresses leukemic stemness, and reprograms the immune microenvironment. This dual-targeting approach holds promise as a novel therapeutic strategy to enhance TKI efficacy and overcome resistance in CML.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-seq was performed on bone marrow cells from two experimental systems:  (1) A transgenic murine model of chronic myeloid leukemia (CML), based on the hematopoietic expression of the human p210BCR/ABL fusion oncogene. Mice were generated by crossbreeding a transgenic line (Tec-p210BCR/ABL) harbors a floxed Sos1 allele (Sos1^fl/fl) and a knockout of Sos2, with CreERT2-mediated deletion of SOS1 upon tamoxifen administration, as previously described (PMCID: PMC9406065). All mice were on a C57BL/6J background and housed under identical conditions. Treatment with vehicle, imatinib (60 mg/kg), BI-3406 (50 mg/kg), or the combination was administered orally for five consecutive weeks (5 days on / 2 days off per week), starting when peripheral blood neutrophil levels exceeded 40%, typically at 5–6 months of age. Bone marrow was collected post-treatment, and total RNA was extracted for sequencing.  (2) Primary bone marrow mononuclear cells from newly diagnosed, untreated CML patients were isolated by Lymphoprep density-gradient centrifugation. Cells were treated ex vivo with vehicle (DMSO), imatinib (500nM), BI-3406 (1 µM), or their combination for 48 hours. Total RNA was then isolated for sequencing.  This dataset includes untreated and treated samples from both murine and human bone marrow, allowing for cross-species comparative transcriptomic analysis of SOS1 and BCR-ABL1 inhibition in CML

first: Rosula

last: García-Navas

first: Carmela

last: Gómez Rodríguez

first: Belén

last: Zamora-Valdivieso

first: Sonsoles

last: Calvo-Jiménez

first: Nuria

last: Calzada

first: Magdalena

last: Sierra

first: Fermin

last: Sánchez-Guijo

first: Kaja

last: Kostyrko

first: Robyn

last: Schenk

first: Marco

last: Hofmann

first: Alberto

last: Fernández Medarde

first: Eugenio

last: Santos

first: Anabel

last: Clemente-Serrano


title: The mRNA profile of HT29 cells after treatment with YM155

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: To investigate the molecular mechanisms by which YM155, a survivin inhibitor, suppresses tumor progression, HT29 human colon cancer cells were treated with YM155 for 24 hours. The aim was to elucidate how YM155 affects the expression of various cancer-related genes, including survivin.

overall_design: HT29 cells were treated with YM155 for 24 hours. Each sample was run in triplicate.

first: Chitose

last: Oneyama

last: Okuzaki


title: Single-cell transcriptomic profiling of mouse colon in early-stage amebic colitis

city: Shanghai

first: Shaokun

last: Pan

state: Shanghai

address: 131, Dong'an Rd

institute: Fudan University

summary: We have employed a single cell sequencing approach using scRNAseq to study amoeba infection of colon.

overall_design: Following amoeba infection, all cells from colon of mice were isolate and analyzed  usig scRNA-seq.

first: Xunjia

last: Cheng

first: Hongze

last: Zhang


title: CASZ1 regulates the rate at which outer hair cells mature and is required for hearing

city: Bethesda

first: Robert

last: Morell

state: MD

address: 35A Convent Drive, Room 1F-103

institute: National Institute on Deafness and Other Communication Disorders

summary: The transcriptional activator ATOH1 is a master regulator of the development of mechanosensory hair-cells (HCs) in the ear. We report that the ATOH1 target gene Casz1 encodes a transcription factor that regulates the rate of outer HC (OHC) maturation by gene repression. Genetic deletion of Casz1 during (but not after) development of the mouse cochlea caused: hearing loss; abnormal organization of mechanosensory stereocilium bundles and abnormally low F-actin density in cuticular plates in OHCs; progressive loss of OHCs; and mild morphological alterations in inner HCs. RNA sequencing revealed that Casz1 deletion delayed downregulation of genes expressed in immature OHCs, including the actin regulator-encoding gene Coro2a, and accelerated upregulation of genes expressed in mature OHCs. Coro2a knockdown restored the density of cuticular plate F-actin in Casz1 mutant OHCs. Our data indicate that CASZ1 regulates transcriptional and morphological maturation of OHCs, and that CASZ1 in maturing HCs is necessary for hearing.

overall_design: A floxed allele of the Casz1 gene was conditionally deleted (Casz1-cKO) in the inner ears of mice carrying a Sox10-Cre transgene. Total RNA was extracted from the organs of Corti of postnatal day 4 Casz1-cKO mice (n=42) and wild-type (Casz1-WT) mice (n=39). The extracted RNA was pooled into 5 samples per genotype (7-9 mice per sample) to obtain sufficiently high RNA concentration for sequencing. The RNA samples were reverse-transcribed, and the resulting cDNA was sequenced. Gene expression differences between the two genotypes were evaluated using EdgeR.

first: Yoko

last: Nakano

first: Elizabeth

last: Driver

first: Susan

last: Wiechert

first: Braulio

last: Peguero

first: Erich

last: Boger

first: Chantal

last: Allamargot

first: Anelika

last: Doetzlhofer

middle: J

first: Matthew

middle: W

last: Kelley

first: Botond

last: Banfi


title: Lineage Plasticity Driven by GATA6 Loss Fuels Colorectal Cancer Metastasis [RNA-seq_GATA6KO]

city: New York

first: Norihiro

last: Goto

address: 413 East 69th Street

institute: Weill Cornell Medicine

summary: Colorectal cancer (CRC) liver metastases are the leading cause of CRC-related mortality, yet the specific genetic and epigenetic drivers that promote this process remain poorly understood. Here, we established a pro-metastatic CRC organoid library through serial orthotopic transplantation of organoids derived from mouse liver metastases. Integrative RNA-seq and ATAC-seq analyses of these organoids uncovered distinct transcriptomic and chromatin accessibility profiles correlating with metastatic potential. These pro-metastatic epigenetic changes were characterized by reactivation of fetal-like programs and downregulation of LGR5+ intestinal stem cell signatures. Notably, they converged on the silencing of the transcription factor GATA6, which occurs in liver metastases in both mice and humans. Genetic ablation of GATA6 in mouse models significantly enhanced liver metastasis while having minimal impact on primary tumor growth. Mechanistically, the loss of GATA6 induced the pro-metastatic epigenetic state and promoted the generation of LGR5- cancer cells. Together, these findings demonstrate that GATA6 loss drives liver metastatic progression by reprogramming cancer cells into a pro-metastatic epigenetic state through the hijacking of developmental programs.

overall_design: We established organoids from liver metastases and transplanted them into the colons of immunocompetent mice again. As controls, we also established organoids from the primary tumours and retransplanted them as well. We repeated these transplants for three rounds, thereby establishing multiple lines of organoids from both primary tumours and liver metastases in each round.

first: Saori

first: Omer

last: Yilmaz


title: Lineage Plasticity Driven by GATA6 Loss Fuels Colorectal Cancer Metastasis [ATAC-seq_met]


title: Transposable Element-Driven Expansion of Enhancer RNA Repertoires Underlies Regulatory Innovation and Polyploid Adaptation in Cereal Crops [PolyA RNA-seq]

city: shanghai

first: yijing

last: zhang

address: 2005 Songhu Road

department: Biochemistry

summary: Cereal genomes have undergone repeated polyploidization and transposable element (TE) proliferation, collectively generating complex regulatory landscapes. The evolutionary trajectories and functional implications of these landscapes, however, remain largely unexplored. By employing chromatin-bound RNA sequencing across seven cereal species, we systematically mapped 45,952 regulatory element transcripts (RETs), including 32,867 distal RETs corresponding to enhancer RNAs (eRNAs). Our analysis reveals that 56% of lineage-specific eRNAs originate from TE expansions, suggesting TEs as significant reservoirs of species-specific regulatory innovation in cereals. Notably, we uncovered a remarkable similarity in defense-related function, root-specific expression, and TE-derived origin of eRNAs across ancient and recent evolutionary layers of Triticeae, suggesting recurrent recruitment of TE-derived root-associated regulatory elements during Triticeae evolution. Furthermore, we found that young eRNA pairs in hexaploid wheat with high sequence similarity, many originating from RLG_famc8.3 and DTC_famc4.3, exhibit pronounced root specificity and coordinated expression, suggesting a targeted amplification and refinement of the successful ancestral regulatory strategy established after Triticeae divergence. To facilitate community access, we developed Cereal-eRNAdb (http://bioinfo.cemps.ac.cn/Cereal-eRNAdb/), a comprehensive database integrating 69,426 eRNAs with functional annotations across 296 samples. Our work indicates that TE-mediated innovation of root-specific eRNAs as a candidate mechanism that may contribute to Triticeae adaptation and provides a foundational resource for exploiting regulatory variation in cereal crop breeding.

sample_organism:
Hordeum vulgare
Oryza sativa
Secale cereale
Triticum aestivum
Triticum urartu
Zea mays
Aegilops tauschii

overall_design: Seedlings (above-ground) of common wheat (Triticum aestivum cultivars ‘Chinesespring’‘AK58’ and ‘Fielder’), barley (Hordeum vulgare cultivar ‘Morex’), rye (Secale cereale cultivar ‘Weining’), maize (Zea mays cultivar ‘B73’) and rice (Oryza sativa cultivars ‘ZH11’ and ‘Jap’) were harvested after 9-day growth. The roots, leaf and embryos of three wheat species (Triticum aestivum cultivar ‘Chinese Spring’, Triticum urartu cultivar ‘G1812’ and Aegilops tauschii cultivar ‘AL8-78’) were collected as follows: roots in the Hoagland solution were harvested after 9-day growth; the fresh immature embryos were isolated 14 days post anthesis; leaf were were harvested after 9-day growth. All the above materials were flash frozen in liquid nitrogen followed by nuclei isolation. Nuclei were isolated from 1 mL tissue powder solubilized in 20 mL ice-cold Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextron T40, 20 mM HEPES, pH7.4, 10 mM MgCl2, 0.75% Triton X-100, 0.5 mM EDTA, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF). The mixture was incubated on ice for 5 min, then filtered through 110 µm nylon mesh and a 40 µm cell strainer. The crude nuclei were spun down at 3,500 g for 5 min at 4°C, followed by washes with 150 µL of resuspension buffer (50% Glycerol, 0.5 mM EDTA, 25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF, 0.4 U/µL RNase inhibitor) and 300 µL of wash buffer (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 M urea, 0.5 mM EDTA, 1% Tween 20, 1 mM DTT, 8 mM β-mercaptoethanol, 2 µg/mL pepstatin A, 2 µg/mL aprotinin, 2 mM PMSF, 0.4 U/µL RNase inhibitor). Again, the obtained nuclei were washed with another 150 µL of resuspension buffer and 150 µL of wash buffer. CB RNA was extracted from washed nuclei using TRIzol reagent and subjected to genomic DNA removal by TURBOTM DNase (Life Technologies). After purification with RNA Clean & ConcentratorTM-5 kit (Zymo Research, R1013), ~1 µg CB RNA was subjected to rRNA depletion by a riboPOOL kit (siTOOLs Biotech, PanPlant-10 nmol) and polyA RNA removal by oligo (dT) beads (NEB, S1419). PolyA RNA was enriched from total RNA by oligo(dT) beads. Purified CB RNA and PolyA RNA were transformed into cDNA libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765) and sequenced on an Illumina NovaSeq platform.

first: Yijing


title: Trans-vaccenic acid in breastmilk primes neonatal T cell to against virus infection

city: Chicago

first: Hao

last: Fan

state: IL

address: 900 E 57TH ST

institute: University of Chicago

summary: Nutrients in breastmilk can play a critical role in neonatal immune development, we employing a combination of single cell transcriptomics and epigenomic analysis to test how the milk nutrient Trans-vaccenic acid affect neonatal spleen immune cell development and increase the ability to against virus.

overall_design: Challenge 10-day-old C57 mice of the CON and TVA groups with influenza A virus, 7 days after infection, lungs were collected from the mice, five mice samples of each group were pooled,  single lung infiltrating cells were used for sequencing


title: The retrotransposable element-silencing factor Daxx controls microglia identity and function in the adult brain

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: Death domain-associated protein (Daxx) is one of the two main H3.3 chaperones in mammalian cells and acts as key repressor of RTEs of viral origin. In present study, we found that Daxx is an IFN-stimulated gene mediating repression of RTEs and IFN-stimulated genes (ISGs) upon inflammatory stimuli in microglia. Using both bulk and single-cell genomics, we demonstrated that shortly after Daxx loss microglia displayed co-expression of DNA damage/p53, IFN/developmental and DAM-like signatures, suggesting lowering of the epigenetic barrier and increased plasticity. With time, Daxx-deficient microglia become restricted to a DAM-like/ApoeHIGH profile, associated with decreased synaptic puncta and altered behavior. Finally, brains from older microglia-specific Daxx KO mice accumulate ApoE protein along with lysosomal markers. Together, our data revealed a role of Daxx in maintenance of the homeostatic state of microglia in the adult brain, with implications for our understanding of the relationship between loss of epigenetic barriers and neuroinflammation and its potential implications for brain function and aging.

overall_design:
To investigate the functional role of Daxx in the microglia, we generate a tamoxifen-inducible Daxx conditional knockout mouse by crossing Cx3cr1-CreERT2 mouse line with Daxx conditional allele (Cx3cr1-CreERT2, Daxxflox/flox). The microglia will be isolated from mice brain at different time points after tamoxifen administration for RnA-seq analysis.
Please note that each sample records along with Sample Tags and demultiplexing information is included in the 'sample_metadata.txt' and 'Readme.md.txt'.

first: Paolo

last: Salomoni


title: Transposable Element-Driven Expansion of Enhancer RNA Repertoires Underlies Regulatory Innovation and Polyploid Adaptation in Cereal Crops [CB RNA-seq]

platform_organism:
Hordeum vulgare
Oryza sativa
Secale cereale
Triticum aestivum
Triticum urartu
Zea mays
Aegilops tauschii

last: Zhang


title: Cohesin and NuRD Antagonistically Drive Alternative Neuronal Fates via PLZF

city: Cambridge

first: Dongyeop

last: Lee

address: 31 Ames St

institute: HHMI/MIT

summary: Diverse genetic and epigenetic factors cooperate to specify cellular fates during development. Establishing these fates is especially critical in the nervous system, which comprises diverse neuronal cell types. How genomic architecture interfaces with epigenetic regulators to drive transcriptional programs underlying neuronal fates remains poorly understood. Here we show that cohesin, a protein complex that shapes genomic architecture, promotes GABAergic fate specification in a subset of neurons in the nematode Caenorhabditis elegans. This process is facilitated by EOR-1, a homolog of the human promyelocytic leukemia zinc finger (PLZF) transcription factor. The nucleosome remodeling and deacetylase (NuRD) complex and TRA-4, another PLZF homolog, promote tyraminergic fate in the normally GABAergic neurons when cohesin or EOR-1 function is lost, revealing an antagonistic mechanism determining alternative neuronal fates. These findings highlight a critical interplay among genome architecture, epigenetic remodeling and transcriptional regulation in neuronal fate specification and, given the evolutionary conservation of these factors, suggest a mechanism underlying neural development across species.

overall_design: Wild-type, coh-1(n6618), eor-1(cs28), tra-4(bc250) coh-1(n6618) and eor-1(cs28); tra-4(bc250) strains expressing nIs883 (tyraminergic GFP/octopaminergic mCherry reporter) were used for RNA-Seq. Three biological replicates were prepared independently on the same days. Embryos were released by bleaching adult worms in 2-3 HG/NA22 plates per condition. The released embryos were incubated in M9 buffer at 20°C by shaking until the majority of embryos developed to the comma to 2-fold stages. Embryos were collected by centrifugation at 180xg for 1 min, and the supernatant was discarded. Embryos in 100 µl M9 buffer were stored at -80°C. Total RNA was isolated using the QIAGEN RNeasy kit (QIAGEN). Embryos in M9 buffer were resuspended in 800 µl of RLT buffer (QIAGEN RNeasy kit) containing 1% β-mercaptoethanol (Mallinckrodt), and dissociated using a BeadBug microtube homogenizer (Benchmark Scientific) with zirconium beads (0.5 mm, Benchmark Scientific) for 30 sec. Dissociated embryos and beads were centrifuged at 15,800xg for 10 min at room temperature to separate debris. The supernatant was used to isolate total RNA according to the manufacturer's instructions. Sequencing libraries were prepared by the MIT BioMicro Center, and sequencing was performed using the NEXTSeq500 sequencing platform with a 75 nt set up. Differentially expressed genes were analyzed using WBcel235 as a reference genome, STAR (v2.5.3), RSEM (v1.3.1) and DESeq2 (v1.24.0).

first: Takashi

last: Hirose

first: H

middle: R

last: Horvitz


title: Xenium spatial transcriptomics reveals C10 tumor transcriptomic changes induced by Smarta cell

first: Jia

last: Nie

address: Building 37, Room 3016

department: Center for Cancer Research

institute: National Cancer Institute

summary: To elucidate how antigen-specific CD4 T cells indirectly repress tumor growth independently of other lymphocytes, we sought to gain insight into the changes they induced in the tumor micro-environment (TME) using the 10x Genomics Xenium technology

overall_design: C10 tumors from Rag1–/– mice or another genetic mutant mice, treated with either PBS(Ctrl) or Smarta on day 4 post-tumor transplant, were dissected at day 10 and subjected to formalin-fixed and paraffin-embedded (FFPE) processing. Two 5 μm FFPE tissue sections were used for Xenium spatial transcriptomics analysis, each including one tumor from each condition from one of two independent experiments.


title: Trans-vaccenic acid in breastmilk primes neonatal T cell immunity

summary: Nutrients in breastmilk can play a critical role in neonatal immune development, we employing a combination of single cell transcriptomics and epigenomic analysis to test how the milk nutrient Trans-vaccenic acid affect neonatal spleen immune cell development.

overall_design: Spleens were collected from 2-week-old C57 mice of the CON and TVA groups, five mice samples of each group were pooled,  single splenic cells were used for sequencing


title: The retrotransposable element-silencing factor Daxx controls microglia identity and function in the adult brain [RNA-seq]

address: Venusberg-Campus 1, Building 99

institute: DZNE

overall_design: To investigate the functional role of Daxx in the microglia, we generate a tamoxifen-inducible Daxx conditional knockout mouse by crossing Cx3cr1-CreERT2 mouse line with Daxx conditional allele (Cx3cr1-CreERT2, Daxxflox/flox). The microglia will be isolated from mice brain at different time points after tamoxifen administration for RNA-seq analysis.

first: Hang-mao