title: The Alb-R26Met liver cancer mouse model recapitulates molecular features and heterogeneity of hepatocellular carcinoma patients

city: Marseille

first: Abdessamad

last: EL KAOUTARI

state: France

address: 27 boulevard Lei Roure

country: France

institute: INSERM

summary: We have generated an inside-out genetic mouse model of liver cancer, the Alb-R26Met mice, in which slightly increased levels of the wild-type form of the MET Receptor Tyrosine Kinase (RTK) occurs specifically in the liver following the deletion of a stop cassette by the Cre recombinase. This genetic combination is obtained by crossing the R26stopMet with Alb-Cre mice. The Alb-R26Met mice spontaneously develop liver tumours and model the whole tumorigenic program. These tumours correspond to HCC patients belonging to the proliferative-progenitor subset. Importantly, the Alb-R26Met model recapitulates several features of HCC patients, including the molecular heterogeneity, the temporal heterogeneity of tumour onset, and the resistance to RTK inhibitors used in the clinic for HCC treatment. This unique genetic setting offered us the possibility to compare the transcriptomic profile of  Alb-R26Met non-tumoral livers with Alb-R26Met advanced liver tumours. Outcomes offer an overall vision of how gene expression switches during a tumorigenic process modelled by the Alb-R26Met mice. Importantly, as tumorigenesis in the Alb-R26Met genetic setting occurs in an immune-competent context, outcomes offers as well the possibility to explore the profound changes in the immune microenvironment in the liver tumours, by performing deconvolution of RNAseq data.

overall_design: mRNA of a total of 6  non-tumoral livers from  Alb-R26Met mice, and 12 advanced stage tumours from Alb-R26Met, were submitted for bulk RNA-sequencing.

first: Floriane

last: Cannet

first: Celia

last: Sequera

first: Paula

last: Michea Veloso

last: El Kaoutari

first: Jean-Paul

last: Borg

first: Flavio

last: Maina


title: Development of a potent epigenetic editor targeting human PCSK9 with durable reduction of cholesterol in mice and non-human primates [RNA-seq]

status: Public on Feb 05 2025

city: Boston

first: Frederic

last: Tremblay

state: MA

address: 201 Brookline Ave, Suite 1101

country: USA

institute: Chroma Medicine

type: Expression profiling by high throughput sequencing

summary: DNA methylation in gene promoter regions is a conserved, innate epigenetic mechanism to control gene expression. Transient application of epigenetic editors designed to induce DNA methylation has been shown to silence genes in cultured cells and in mice. Here we report the development of a potent, efficacious, and specific human PCSK9-targeting epigenetic editor. A single administration of lipid nanoparticles encapsulating mRNA encoding the epigenetic editor which precisely targets the PCSK9 locus was sufficient to drive near-complete silencing in transgenic mice expressing human PCSK9. Silencing was durable for at least one year and was fully maintained following partial hepatectomy-induced liver regeneration. In addition, we showed reversibility of epigenetic editing in previously silenced mice upon treatment with a targeted epigenetic activator designed to demethylate the PCSK9 locus. Importantly, a single administration of the epigenetic editor robustly, potently, and durably decreased circulating PCSK9 (~90%) in cynomolgus monkeys with concomitant reduction in low-density lipoprotein cholesterol (~70%). These findings support the development of an epigenetic editor targeting PCSK9 for the treatment of hypercholesterolemia and demonstrate the broad therapeutic potential of precise, efficacious, durable, and reversible epigenetic editing in vivo

overall_design: RNA-Seq in PXB primary human hepatocytes of No Treatment, epigenetic editor effector without gRNA (Effector Only), and PCSK9-EE at day 15.

first: Qiang

last: Xiong

first: Shrijal

middle: S

last: Shah

first: Chih-Wei

last: Ko

first: Kenneth

last: Kelly

first: Mary

last: Morrison

first: Cristiana

last: Giancarlo

first: Ricardo

middle: N

last: Ramirez

first: Erica

middle: M

last: Hildebrand

first: Sarah

middle: B

last: Voytek

first: Gabriel

middle: K

last: El Sebae

first: Shane

middle: H

last: Wright

first: Liam

last: Lofgren

first: Scott

last: Clarkson

first: Samantha

middle: J

last: Linder

first: Songlei

last: Liu

first: Taesun

last: Eom

first: Shefal

last: Parikh

first: Yuki

last: Weber

first: Salette

last: Martinez

first: Padma

last: Malyala

first: Sahar

last: Abubucker

first: Ari

middle: E

last: Friedland

first: Morgan

middle: L

last: Maeder

first: Angelo

last: Lombardo

first: Vic

last: Myer

first: Aron

last: Jaffe


title: Gut Dysbiosis Impacts Brain Metastasis Outgrowth and Immune Response Dynamics

city: Dallas

first: Siyuan

last: Zhang

address: 6001 Forest Park Rd

department: Department of Pathology

institute: UT Southwestern Medical Center

summary: We used 16S V3/V4 region amplification to evaluate the composition of bacteria species in mouse fecal pellets after vehicle or ABX treatment and before and after fecal matter transplant.

overall_design: To evaluate gut microbiome changes after ABX treatment, fecel pellets were collected from young-adult (12 weeks old) wild type C57Bl/6 mice after 21 days of vehicle or antibiotics treatment (to induce gut microbiota depletion). In one sequencing round, we sequenced a total of 6 different fecal samples (3 control, 3 depleted gut microbiota (ABX)). To evaluate fecal matter transplant (FMT) efficacy, ~12 weeks old C57BL/6 mice were first pre-treated for 14 days with vehicle or ABX. Following the 14-day pre-treatment, all mice were switched to regular water and received PBS via oral gavage for a 2-day washout period. Next, mice from vehicle and ABX pre-treatment groups were randomized and cohoused. For FMT, fresh fecal pellets were collected from mice in the vehicle pre-treatment group. 2-3 pellets were placed in 1 mL of sterile PBS in a 1.5 mL tube and vortexed into a homogenate. The homogenates were then centrifuged at 500 g for 5 minutes and supernatant was collected. All mice received 200 uL of fecal matter supernatant by oral gavage. This procedure was repeated every other day for a seven-day period. Mouse stool was collected post-vehicle/ABX treatment and post FMT/cohouse for 16s rRNA analysis. Mouse fecal DNA extraction was performed using the ZymoBIOMICS DNA Miniprep Kit (Cat# D4300). 16S V3/V4 region amplification, sample indexing and library preparation were performed following the Illumina 16S Metagenomic Sequencing Library Preparation (PN 15044223 Rev. B). Amplicons were indexed using the Nextera XT Index Kit and pooled into a library for Illumina sequencing.

last: Golomb


title: Combinatorial Extracellular Matrix Tissue Chips for Optimizing Mesenchymal Stromal Cell Microenvironment and Manufacturing

status: Public on Feb 04 2025

city: Palo Alto

first: Ishita

last: Jain

address: 3801 Miranda Avenue

institute: Stanford University

summary: Despite the therapeutic potential of mesenchymal stromal cells (MSC), there is limited understanding of optimal extracellular matrix (ECM) environments to manufacture these cells. We developed tissue chips to study the effects of multi-factorial ECM environments under manufacturable stiffness ranges and multi-component ECM compositions. Manufacturing qualities of cell expansion potential, immunomodulation, and differentiation capacity were examined. The results show stiffness effects, with 900 kPa substrates supporting higher proliferation and osteogenic differentiation, along with anti-inflammatory IL10 expression, whereas 150 kPa substrates promoted adipogenic differentiation at 150 kPa, suggesting that optimal ECM environments may differ based on manufacturing goals. ECM biochemistries containing fibronectin and laminin further modulated MSC manufacturing qualities across various stiffnesses. Proteomic and transcriptomic analyses revealed unique ECM combinations that induced higher levels of angiogenic and immunomodulatory cytokines, compared to single factor ECMs. These findings demonstrate that optimized ECM environments enhance MSC manufacturing quality.

overall_design: Short term gene changes were assessed in MSCs seeded on different substrate stiffnesses on various ECM combinations. After 48 hours in growth media, cells were lysed and RNA was extracted using GeneJET RNA isolation kit (Fisher Scientific) followed by RNA sequencing by Novogene Corporation (n=4).

first: Jain

last: Ishita

first: Chan

last: Alex

first: Yang

last: Guang

first: He

last: Hao

first: Lam

last: Johnny

first: Sung

last: Kyung

first: Huang

last: Ngan


title: In-vitro gene signature of NHEK's treated with caspase inhibitor Q-VD-Oph or DMSO

status: Public on Feb 08 2025

city: Balitmore

first: Charles

middle: Scott

last: Kirby

state: Maryland

address: 1551 Orleans Street, Koch CRBII Suite 216

institute: Johns Hopkins

summary: To investigate the effect of caspase inibition in human keratinocytes

overall_design: Total RNA from NHEK’s treated with either Q-VD-OPh(n=3) or DMSO (n=3) were submitted to Novogene for Sequencing

first: Nasif

last: Islam

first: Eric

last: Wier

first: Luis

middle: A

last: Garza


title: Gut Dysbiosis Impacts Brain Metastasis Outgrowth and Immune Response Dynamics [CITE-seq]

summary: We used CITE-seq (10x Gemoics-based) to profile and compare the transcriptomes and cell surface expression of a set of immune markers of brain immune cells from young-adult C57BL/6 mice with and without antibiotics induced gut microbiota depletion (ABX) during brain metastasis. Brain immune cells were collected from naive brains (D00) and at different time points throughout brain metastasis outgrowth (4 (D04), 8 (D08) and 14 (D14) days post injection). We sequenced a total of 24 different mouse brains (3 biological replicates per group: (1) D00 control, (2) D00 ABX, (3) D04 control, (4) D04 ABX, (5) D08 control, (6) D08 ABX, (7) D14 control, (8) D14 ABX). We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Then, we stained each brain with it's own unique hashing antibody so that we could subsequently pool samples. Two groups were collapsed into one to generate four samples, each loaded into one well on the 10x Chromium (D00 control + D00 ABX, D04 control + D04 ABX, D08 control + D08 ABX, D14 control + D14 ABX).  Sequencing libraries were prepared for each of the four collapsed groups (6 samples per group). Following sequencing, each sample is separated in silico by it's unique hashing antibody.

overall_design: Naive brains were obtained from ~12 week old female C57BL/6 mice with vehicle or ABX treatment. Brains with metastases were collected from ~12 week old female C57BL/6 mice with vehicle or ABX treatment at 4, 8 and 14 days post intra-carotid injection. Cell suspensions were prepared by percoll gradient centrifugation to obtain suspensions enriched for immune cells. We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Then, we stained each brain with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium. Two groups were collapsed into one to generate four samples, each loaded into one well on the 10x Chromium (D00 control + D00 ABX, D04 control + D04 ABX, D08 control + D08 ABX, D14 control + D14 ABX).  Sequencing libraries were prepared for each of the four collapsed groups (6 samples per group).

first: Samantha


title: CAV2-expressing Nerves induce Metabolic Switch toward Mitochondrial Oxidative Phosphorylation to Promote Cancer Stemness

city: tianjin

first: Zhang

last: ze

address: Huanhu Xi Road, Tiyuan Bei, Hexi District, Tianjin

country: China

institute: Tianjin Medical University Cancer Institute and Hospital

summary: Cancer cells and the nervous system engage in a dynamic interplay, significantly influencing initiation and progression in head and neck squamous cell carcinoma (HNSCC). Our findings highlight that cancer cells drive an increase in caveolin-2 (Cav2) expression within trigeminal ganglia and associated neural fibers in the tumor milieu, fostering a reciprocal attractant relationship between tumor cells and nerves. Notably, the knockout of Cav2, either globally or specifically in sensory neurons or glial cells, markedly attenuates the growth of orthotopically implanted tongue tumors. Moreover, Cav2-expressing nerves are implicated in shifting cancer cell metabolism towards mitochondrial oxidative phosphorylation, a process involved in maintenance of cancer stem cells (CSCs). Our results also demonstrate that Cav2-expressing nerves confer stemness properties to cancer cells. Disruption of Cav2 expression, both globally and in specific neural cell types, impedes tumorigenesis and progression in a 4-NQO- induced HNSCC mouse model.This interplay observed between cancer cells, neurons, and glial cells suggests a potential mechanism through which tumor-associated nerves might influence cancer stemness via metabolic reprogramming. This highlights a possible new direction for anticancer therapy that warrants further investigation.

overall_design:
After 72 hours of co-culture with wild TG or Cav2 -/- TG, total RNA of SCC15 cells was extracted using TRIzol reagent, and RNA quality was assayed by Nanodrop and Bioanalyzer. Three biological replicates with good quality RNA were used to make cDNA libraries.

Please note that the records have been updated with raw data files on Apr 21, 2025.

first: Ruoyan

last: Liu

first: Zhonghao

last: Sun

first: Chunli

last: Wang


title: CHR11 promotes the production of siRNAs from endogenous protein-coding genes

status: Public on Feb 06 2025

city: Gif-sur-Yvette

first: Thomas

last: Blein

address: Avenue des Sciences

institute: CNRS

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNA sequencing was performed to determine the accumulation of endogenous siRNAs in wildtype and chr11 chr17 double mutant plants in response to TCV.

overall_design: small RNA-seq of Arabidopsis thaliana leaves leaves non-infected and infected with Turnip crinkle virus (TCV) in WT (Col-0) or chr11chr17 double mutant

first: Taline

last: Elmayan

first: Aurélie

last: Christ

first: Hervé

last: Vaucheret


title: Development of a potent epigenetic editor targeting human PCSK9 with durable reduction of cholesterol in mice and non-human primates [hybrid capture methylseq]

first: Frederic

last: Tremblay

type: Genome binding/occupancy profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Hybrid capture methylation sequencing using custom hybrid capture panels from a human cell line (HeLa) and human PCSK9 transgenic mouse liver tissue to measure on-target methylation at the PCSK9 TSS region

first: Qiang

last: Xiong

first: Shrijal

last: Shah

first: Chih-Wei

last: Ko

first: Kenneth

last: Kelly

first: Mary

last: Morrison

first: Cristiana

last: Giancarlo

first: Ricardo

last: Ramirez

first: Erica

last: Hildebrand

first: Sarah

last: Voytek

first: Gabriel

last: El Sebae

first: Shane

last: Wright

first: Liam

last: Lofgren

first: Scott

last: Clarkson

last: Linder

first: Songlei

first: Taesun

last: Eom

first: Shefal

last: Parikh

first: Yuki

last: Weber

first: Salette

last: Martinez

first: Padma

last: Malyala

first: Sahar

last: Abubucker

first: Ari

last: Friedland

first: Morgan

last: Maeder

first: Angelo

last: Lombardo

first: Vic

last: Myer

first: Aron

last: Jaffe


title: The genomes and epigenomes of aquatic plants (Lemnaceae) promote triploid hybridization and clonal reproduction [ChIP-Seq]

city: Cold Spring Harbor

first: Robert

last: Martienssen

state: NY

address: 1 Bungtown Rd

department: Delbruck Bldg.

institute: Cold Spring Harbor Laboratory

summary: The Lemnaceae (duckweeds) are the world’s smallest but fastest growing flowering plants. Prolific clonal propagation facilitates continuous micro-cropping for plant-based protein and starch production, and holds tremendous promise for sequestration of atmospheric CO2. Here, we present chromosomal assemblies, annotations, and phylogenomic analysis of Lemna genomes that uncover candidate genes responsible for the unique metabolic and developmental traits of the family, such as anatomical reduction, adaxial stomata, lack of stomatal closure, and carbon sequestration via crystalline calcium oxalate. Lemnaceae have selectively lost genes required for RNA interference, including Argonaute genes required for reproductive isolation (the triploid block) and haploid gamete formation. Triploid hybrids arise commonly among Lemna, and we have found mutations in highly-conserved meiotic crossover genes that could support polyploid meiosis. Further, mapping centromeres by chromatin immunoprecipitation suggests their epigenetic origin despite divergence of underlying tandem repeats and centromeric retrotransposons. Syntenic comparisons with Wolffia and Spirodela reveal that diversification of these genera coincided with the “Azolla event” in the mid-Eocene, during which aquatic macrophytes reduced high atmospheric CO2 levels to those of the current ice age. Facile regeneration of transgenic fronds from tissue culture, aided by reduced epigenetic silencing, makes Lemna a powerful biotechnological platform, as exemplified by recent engineering of high-oil Lemna that outperforms oil seed crops.

sample_organism:
Lemna minor
Lemna japonica
Lemna turionifera

overall_design: Chromatin immuno-precipitation sequencing of Lemna minor, Lemna turionifera, and Lemna japonica centromeric histone 3

first: Evan

last: Ernst

first: Bradley

last: Abramson

first: Kenneth

last: Acosta

first: Phuong

middle: T

last: Hoang

first: Cristian

last: Mateo-Elizalde

first: Veit

last: Schubert

first: Buntora

last: Pasaribu

first: Patrice

last: Albert

first: Nolan

last: Hartwick

first: Kelly

last: Colt

first: Anthony

last: Aylward

first: Umamaheswari

last: Ramu

first: James

last: Birchler

first: Ingo

first: Eric

last: Lam

first: Todd

middle: P

last: Michael


title: A functional single cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumors. [in vitro]

city: Leuven

first: Samantha

last: Pretto

address: ON4 Herestraat 49

department: Center for Cancer Biology

country: Belgium

institute: VIB-KU Leuven

summary: Rewiring T cell metabolism can improve intratumoral infiltration and function. By performing a CRISPR/Cas9 metabolic survey in CD8+ T cells, we identified 83 targets enriched at the primary and/or metastatic niche in the context of pancreatic cancer. We applied single-cell RNA sequencing to disclose transcriptome changes associated with each metabolic perturbation. This revealed Elongation of Very-Long-chain fatty acids protein 1 (Elovl1) as a metabolic target to sustain proliferation and both effector and memory functions in CD8+ T cells. Accordingly, Elovl1 inactivation in adoptively transferred T cells combined with αPD-1 showed therapeutic efficacy in resistant pancreatic and melanoma tumors. Loss of Elovl1 in T cells rewired lipid metabolism and changed the plasma membrane composition mainly through SREBP2 activation, leading to higher cholesterol content and stronger TCR signaling. Finally, ELOVL1 in CD8+ T cells correlated with αPD-1 response in melanoma patients. Altogether, Elovl1 targeting synergizes with αPD-1 to promote effective anti-tumor T cell responses

overall_design: OT-I T cells transduced with a lentiviral vector containing a non-targeting guide or a sgRNA targeting Elovl1, were used to perferm adoptive cell transfer in mice bearing orthotopic KPC_OVA tumors. The day after aPD-1 therapy was initiated. 7 days from ACT, control and Elovl1 KO OT-I T cells were sorted from the primary tumor. Sorting via Flow cytometry (FACS), was based on CD90.1 expression (CD90.1 being the reporter gene of the lentiviral vector). Sorted cells underwent bulk RNA sequencing. Each replicate represent cells sorted from a different mouse. Sample 1 (pool of T46 and T47)  and sample 2 (pool of T49 and T51) are pool of cells sorted from 2 tumors each. We sorted and isolated the cells separately and pooled the extracted RNA to increase the RNA quantity.

first: Lotte

last: Vanheer

first: Pierre

last: Bourdely

first: Mario

last: Di Matteo

first: Massimiliano

last: Mazzone


title: Development of a potent epigenetic editor targeting human PCSK9 with durable reduction of cholesterol in mice and non-human primates [whole genome methyl-seq]

first: Frederic

last: Tremblay

overall_design: WGMS in PXB primary human hepatocytes of epigenetic editor effector without gRNA (Effector Only), and PCSK9-EE at day 15.

first: Qiang

last: Xiong

first: Shrijal

last: Shah

first: Chih-Wei

last: Ko

first: Kenneth

last: Kelly

first: Mary

last: Morrison

first: Cristiana

last: Giancarlo

first: Ricardo

last: Ramirez

first: Erica

last: Hildebrand

first: Sarah

last: Voytek

first: Gabriel

last: El Sebae

first: Shane

last: Wright

first: Liam

last: Lofgren

first: Scott

last: Clarkson

last: Linder

first: Songlei

last: Liu

first: Taesun

last: Eom

first: Shefal

last: Parikh

first: Yuki

last: Weber

first: Salette

last: Martinez

first: Padma

last: Malyala

first: Sahar

last: Abubucker

first: Ari

last: Friedland

first: Morgan

last: Maeder

first: Angelo

last: Lombardo

first: Vic

last: Myer

first: Aron

last: Jaffe


title: A functional single cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumors. [in vivo]

first: Samantha

last: Pretto

first: Qian

last: Yu

first: Pierre

last: Bourdely

first: Bernard

last: Thienpont

first: Mario

last: Di Matteo

first: Massimiliano

last: Mazzone


title: In situ targeted gene expression analysis of healing skin on SD0 post-wounding in Wildtype and Rnasel knock out wounds

first: Charles

last: Kirby

summary: We generated a spatial transcriptomics dataset of wildtype (C57BL/6, n = 3)and Rnasel knockout (Rnaseltm1Slvm, n =3) mice at scab detachment day 0, to investigate specific type of cell populations and their gene expressions.

overall_design: The dorsal side of male and female 3-week-old (21 days) mice were shaved at approximately 8-11g of weight. Using surgical scissors  approximately 1.26 cm2 X 1.25cm2 of skin was excised creating wounds deep into the fascia. Using the 10X Genomics Xenium platform  we generated a spatial transcriptomics dataset of 100 custom genes and 379 genes from the 2023 "mMulti_v1" list   from wildtype (C57BL/6 n = 3)and Rnasel knockout (Rnaseltm1Slvm n =3) mice at scab detachment day 0.

first: Kirby

last: Charles S.

first: Lee

last: Sam S.

first: Garza

last: Luis A.


title: Identification of genetic modifiers of vEDS mice using whole-genome SNP data

status: Public on Feb 09 2025

city: Baltimore

first: Caitlin

last: Bowen

address: 1800 Orleans

summary:
Aortic dissection or rupture is a major cause of mortality in vascular Ehlers Danlos Syndrome (vEDS), a connective tissue disorder caused by heterozygous mutations in the COL3A1 gene. C57BL6/J (BL6) mice carrying the Col3a1G938D/+ mutation recapitulate the vEDS vascular phenotype and die suddenly of aortic rupture/dissection. However, 129S6/SvEvTac (129) mice expressing the same Col3a1G938D/+ mutation show near-complete life-long protection from vascular rupture.
To identify genetic modifiers of vascular risk in vEDS, we performed genome-wide genotyping of intercrossed BL6/129 vEDS mice stratified by survival and identified a significant protective locus encompassing a variant in Map2k6, encoding Mitogen-Activated Protein Kinase Kinase 6 (M2K6), a p38-activating kinase.

overall_design: We genotyped 91 mixed background vEDS mice with a mild phenotype (coded as controls, 62 females and 29 males) , and 96 mixed background vEDS mice with a severe phenotype (coded as cases, 29 females, 67 males). We used a linkage disequilibrium (LD) block-pruned set of 615 SNPs to perform logistic regression GWAS, using sex as a covariate.

first: Harry

middle: C

last: Dietz


title: Ancient synteny links metabolism with erythroid development

status: Public on Feb 10 2025

city: Oxford

first: Robert

last: Beagrie

address: Headley Way

department: Molecular Haematology Unit

country: United Kingdom

institute: MRC Weatherall Institute of Molecular Medicine

summary: In order to examine the link between Nprl3 regulation of cellular metabolism and erythropoiesis, we performed RNA-seq and identified gene expression changes in Nprl3 +/- and Nprl3 -/- mouse fetal liver erythroblasts compared with wild type Nprl3 +/+ erythroblasts.

overall_design: Poly(A)+ mRNA was analysed from four Nprl3 +/+ fetal livers (2 male, 2 female), two Nprl3 +/- fetal livers (2 male) and two Nprl3 -/- fetal livers (1 male, 1 female).

first: Alexandra

last: Preston

first: Hal

last: Drakesmith


title: A functional single cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumors [CRISPR]

overall_design: OT-I T cells transduced with a metabolic CRISPR library were sorted from the primary tumor, Lung metastasis, liver metastasis, peritoneal metastasis, draining and non-draining lymph nodes and spleen of ova expressing Pancreatic cancer (KPC_OVA) bearing mice. Sorting via Flow cytometry (FACS), was based on CD90.1 expression (CD90.1 being the reporter gene of the library vector). Sorted cells underwent genomic extraction, sgRNA amplification and NGS sequencing.

last: di Matteo


title: A functional single cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumors [CROPseq]

overall_design: OT-I T cells transduced with a metabolic CRISPR library were sorted from the primary tumor of ova expressing Pancreatic cancer (KPC_OVA) bearing mice. Sorting via Flow cytometry (FACS), was based on CD90.1 expression (CD90.1 being the reporter gene of the library vector). Sorted cells underwent scRNA sequencing.


title: Comprehensive Interrogation of Synthetic Lethality in the DNA Damage Response

status: Public on Feb 03 2025

city: Zürich

first: Markus

last: Schröder

state: Zürich

address: Otto-Stern-Weg 7

department: Biology, Molecular Health Sciences

country: Switzerland

institute: ETH Zürich

summary: The DNA damage response (DDR) is a multi-faceted network of pathways that preserves genome stability. Unraveling the complementary interplay between these pathways remains a challenge. Here, we comprehensively mapped genetic interactions for all core DDR genes using combinatorial CRISPRi screening. We discovered myriad new connections, including interactions between cancer genes and small molecule targets. We focused on two of the strongest interactions: FEN1/LIG1:WDR48 and FANCM:SMARCAL1. First, we found that WDR48 works with USP1 to restrain overactive translesion synthesis in FEN1/LIG1-deficient cells, and that a preclinical inhibitor of USP1 specifically kills FEN1-deficient cells. Second, we found that SMARCAL1 and FANCM suppress DNA double-strand break (DSB) formation at TA-rich repeats in late-replicating regions that otherwise escape into mitosis and cause nuclear fragmentation. Our dataset provides a springboard for further mechanistic investigations into connections between DDR factors and suggests multiple interactions that could be exploited in cancer therapy.

overall_design: MRE11 ChIP-seq

first: John

last: Fielden

first: Sebastian

last: Siegner

first: Danielle

last: Gallagher

first: Maria

middle: R

last: Dello Stritto

first: Lena

last: Kobel

first: Moritz

middle: F

last: Schlapansky

first: Petr

last: Cejka

first: Marco

last: Jost

first: Jacob

last: Corn


title: Long-read sequencing disentangles isoform complexity at allele-specific loci

city: Munich

first: Daniel

last: Andergassen

address: Biedersteiner Straße 29

department: Institute of Pharmacology and Toxicology

country: Germany

institute: Technical University of Munich

summary: In recent years, long-read sequencing technologies have detected transcript isoforms with unprecedented accuracy and resolution. However, it remains unclear whether long-read sequencing can effectively disentangle the isoform landscape of complex allele-specific loci that arise from genetic or epigenetic differences between alleles. Here, we combine the PacBio Iso-Seq workflow with the established phasing approach WhatsHap to assign long reads to the corresponding allele in polymorphic F1 mouse hybrids. Upon comparing the long-read sequencing results with matched short reads, we observed general consistency in the allele-specific information and were able to confirm the imprinting status of known imprinted genes. We then explored the complex imprinted Gnas locus known for allele-specific non-coding and coding isoforms and were able to benchmark historical observations. This approach also allowed us to detect isoforms  from both the active and inactive X chromosomes of genes that escape X chromosome inactivation. The described workflow offers a promising framework and demonstrates the power of long-read transcriptomic data to provide mechanistic insight into complex allele-specific loci.

overall_design:
To capture the allele-specific expression landscape through long-read sequencing, we bred C57BL/6J (BL6) females carrying a heterozygous deletion of the proximal A-repeat (Xist −/+) with a CAST/EiJ (CAST) male. We then collected RNA from a whole brain sample from a resultant F1 female (BL6ΔXist × CAST) and performed long-read sequencing.
The allele-specific analysis was conducted by counting the reads from each allele and calculating a ratio. This analysis was compared to short-read sequencing allele-specific analysis and does not include replicates.

first: Lison

last: Lemoine

first: Sarah

last: Hoelzl

first: Tim

last: Hasenbein

first: Elisabeth

last: Graf


title: Serum miRNA-186-3P and miRNA-382-3P constitute a novel Diagnostic miRNA signature for palindromic rheumatism

status: Public on Feb 02 2025

city: Ningbo

first: Junhong

last: He

state: Zhejiang

address: 1059 Zhongshan East Road

summary:
Background: Palindromic rheumatism (PR) is a unique disease characterized by the intermittent inflammation of different joints that may progress to a variety of immune-related diseases. Unclear diagnostic criteria have limited the research on its pathogenesis and treatment options. Recently, microRNAs (miRNAs) have been used in the diagnosis of various diseases; however, the role of miRNAs in PR diagnosis remains unexplored. Using next-generation high-throughput sequencing (NGS), this study aimed to screen miRNAs specifically expressed in the serum of patients with PR to construct a diagnostic signature and verify its diagnostic efficacy.
Methods: Patients with PR (N=4), patients with rheumatoid arthritis (RA; N=3), and healthy controls (N=3) were included in an exploration cohort. Differentially expressed miRNAs were screened using NGS to construct diagnostic signatures and bioinformatics tools were used to perform target gene enrichment analysis of the top 25 differentially expressed miRNAs, both upregulated and downregulated. RT-qPCR was used to verify the differential expression of the diagnostic signatures in the three validation cohorts of patients with PR (N=27) and RA (N=30), and healthy controls (N=31), and the diagnostic efficiency of the diagnostic signatures was evaluated using receiver operator characteristic (ROC) curves.
Results: A total of 130 differentially expressed miRNAs–35 upregulated and 95 downregulated miRNAs–were found in the PR exploration cohort, which differed between both RA and healthy controls. miRNA-186-3p showed the largest upregulated difference, and miRNA-382-3p showed the largest downregulated difference, and consequently, were selected to construct the diagnostic signature. In the ROC analysis of the validation cohort, the diagnostic signature produced an area under the ROC curve (AUC) of 1 (95% CI 1.000-1.000) compared with healthy controls. For patients with RA, the diagnostic signature produced an AUC of 0.915 (95% CI 0.842-0.988). However, miRNA-186-3p and miRNA-382-3p levels were not associated with disease activity in patients with PR.
Conclusion: A diagnostic signature comprising miRNA-186-3p and miRNA-382-3p can effectively diagnose and differentiate PR from RA. This study provides a basis for the creation of a clinical miRNA signature for the diagnosis of PR.

overall_design: This study aimed to screen differentially expressed miRNAs in PR samples and compare them to those in RA and healthy control samples using next-generation high-throughput RNA sequencing to construct miRNA signatures for PR diagnosis and verify their diagnostic efficacy. Additionally, bioinformatics tools were used to perform target gene enrichment analysis on differentially expressed miRNAs, offering new insights into defining PR diagnostic criteria and studying its pathogenesis.

first: Fangfang

last: Yuan

first: Zefu

last: Weng

first: Qiong

last: Yang

first: Jing

last: Luo

first: Lina

last: Ying

first: Haiyan

last: Huang

first: Xin

last: Zhang

first: Yahui

last: Chen

first: Jixia

last: Lin


title: Combined loss of obscurin and obscurin-like 1 in murine hearts results in diastolic dysfunction, altered metabolism and deregulated mitophagy.

status: Public on Feb 01 2025

city: La Jolla

first: Stephan

last: Lange

state: CA

address: 9500 Gilman Dr

department: Medicine

institute: UC San Diego

summary: RNAseq raw data generated from total cardiac RNA of cardiac specific Obscurin x Obsl1 knockout mice

overall_design: Three biological replicates were analyzed for four genotypes (control [CTL], obscurin knockout [Obsc], Obsl1 knockout [OL1], obscurin x Obsl1 double knockout [dKO])

first: Matúš

last: Soták

first: Emma

last: Börgeson


title: ABCG8-mediated sterol efflux increases cancer cell progression through LRP6/Wnt/beta-catenin signaling pathway in radiotherapy-resistant triple negative breast cancer cells (RT-R-TNBC), RT-R-MDA-MB-231

city: Jinju

first: Hye Jung

last: Kim

address: 816 Bungil 15

department: College of Medicine, Department of Pharmacology

country: South Korea

institute: Gyeongsang National University

summary: ATP-binding cassette (ABC) transporters are known to be increased in various tumor cells, including breast cancer. They are responsible for mediating drug resistance, leading to treatment failure. In the present study, gene expression array analysis revealed that among ABC transporter subtypes, ABCG8 was most increased in radiotherapy-resistant triple negative breast cancer (RT-R-TNBC) compared to TNBC. ABCG8 is known to be involved in sterol efflux, but its role in cancer is not well known. Therefore, we investigated the effect of ABCG8 on tumor progression in RT-R-TNBC. RT-R-MDA-MB-231 exhibited increased cholesterol levels in both cells and surrounding media by inducing SREBP1 and FASN. ABCG8 siRNA increased intracellular cholesterol but decreased media cholesterol, indicating an accumulation of cholesterol inside cells. Additionally, RT-R-MDA-MB-231 showed increased levels of β-catenin, which was significantly reduced by ABCG8 knockdown. Moreover, ABCG8 knockdown led to cell cycle arrest in the G2/M phase by reducing PLK1 and Cyclin B1. RT-R-MDA-MB-231 also exhibited increased phosphor-LRP6 and LRP6 levels, which were decreased by ABCG8 siRNA. Interestingly, LRP6 siRNA decreased β-catenin, PLK1, and Cyclin B1. In addition, feedback mechanisms such as LXR and IDOL were decreased in RT-R-MDA-MB-231 cells. This study suggests for the first time the role of ABCG8 and the cholesterol exported by ABCG8, not inside the cells, affects cancer progression through the LRP6/Wnt/beta-catenin signaling pathway in RT-R-TNBC. The regulation of this pathway may offer a potential therapeutic strategy for treating RT-R-TNBC.

overall_design: RNA-seq profiling of MDA-MB-231 and radiotherapy resistant MDA-MB-231 (RT-R-MDA-MB-231)

first: Hana

last: Jin

first: Young Shin

last: Ko

first: Seung Pil

last: Yun

first: Sang Won

last: Park

first: Ju-Yeong

last: Won

first: Nam Binh

last: Nguyen

first: Yaeram

first: Vedaste

last: Nsanzimana


title: Immune responses of lung mesenchymal cells during pneumonia

first: Single Cell

last: Sequencing Core

address: 72 E. Concord St

institute: BU Single Cell Sequencing Core

summary: Mesenchymal cell roles during respiratory infection are not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single cell suspensions of mice that were naïve (after receiving only saline vehicle), infected (after intratracheal instillation of pneumococcus 24 hours previously), or recovered from infection (after non-lethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into five well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naïve and recovered groups overlapped in dimension reduction plots, suggesting mesenchymal cells return to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response conserved across cell-types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similar gene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrate diverse and specialized immune activities of lung mesenchymal cells during pneumonia.

overall_design: We used flow cytometry to isolate all cells from the lung that were not leukocytes, epithelial cells, or endothelial cells (i.e., CD45-CD326-CD31-), to compare cell-types and transcriptomes between young adult mice that were naïve, experiencing acute pneumonia, or fully recovered with a remodeled lung immune system (10, 12). All mice across these three groups received the same types of surgeries and intratracheal instillations, but the instillates differed. The control group, labeled as “Naïve,” received only the sterile saline vehicle. The active infection group, labeled as “Pneumonic,” received pneumococcus 24 hours prior to euthanasia. The recovered group, labeled as “Resolved”, had pneumococcus infections a month previously, yielding lungs with no ongoing infection or inflammation but a remodeled resident immune system that provides improved protection against further infections. Cells were barcoded using 10x Chromium Genomics, and all three groups were sequenced together using Next Generation Sequencing, before cell-specific transcriptomes were analyzed using CeLDA, Seurat, and SPRING (27-30).

first: Alicia

last: Soucy

first: Joseph

last: Mizgerd


title: Single-cell transcriptomics revealed e-cigarettes-induced vascular remodeling by enhancing Tcf21 expression

status: Public on Jan 31 2025

city: Beijing

first: 唐

last: 伟评

address: 2 Anzhen Rd, Chaoyang District,

institute: Beijing Institute of Heart Lung and Blood Vessel Diseases

summary: Electronic cigarettes (e-cigs) are battery-powered new tobacco products attracting wide attention due to their potential in promoting smoking cessation and uncertainty in long-term health effects. According to the scientific statement from the American Heart Association (AHA), e-cig use may be a potential threat to the cardiopulmonary system and populational studies suggested that e-cig aerosol could enhance arterial stiffness Laboratory evidence suggested that e-cigs could accelerate atherosclerosis and atherosclerosis or aortic aneurysm in mice, while the exact mechanisms of e-cig-related vascular effects remained largely unknown. Here, we investigate the sub-chronic effects of e-cig aerosol on vascular structure, function, and changes in single-cell transcriptome in mice.

overall_design:
C57BL/6J male mice aged 8 weeks inhaled filtered air (as control) or e-cig (RELX, containing glycerin, propylene glycol, spices, and 3% nicotine) 1 h per day in the exposure chamber for consecutive 90 days. The whole aorta samples collected from mice were used for single-cell transcriptome.

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first: Ruiyang

last: Ding

first: Xiaoke

last: Ren

first: Qinglin

last: Sun

first: Shiqian

last: Liu

first: Linyuan

last: Huang

first: Zhiwei

first: Junchao

last: Duan


title: TFE3 fusion proteins drive oxidative metabolism, ferroptosis resistance and general RNA synthesis in translocation renal cell carcinoma. [Cut and Tag/ChIP-seq]

city: Illkirch-Graffenstaden

first: Guillaume

last: Davidson

address: 1 Rue Laurent Fries

institute: I.G.B.M.C. - Institut de génétique et de biologie moléculaire et cellulaire

summary: The oncogenic mechanisms by which TFE3 fusion proteins drive translocation renal cell carcinoma (tRCC) are poorly characterised. Here, we integrated loss and gain of function experiments with multi-omics analyses in tRCC cell lines and patient tumors. High nuclear accumulation of NONO-TFE3 or PRCC-TFE3 fusion proteins promotes their broad binding across the genome, engaging a core set of M/E-box-containing regulatory elements to activate specific gene expression programs as well as promiscuous binding to active promoters to stimulate mRNA synthesis. Within the core program, TFE3 fusions directly regulate genes involved in ferroptosis resistance and oxidative phosphorylation metabolism (OxPhos) increasing functional OxPhos levels. Consequently, human tRCC tumors display high OxPhos scores that persist during their epithelial to mesenchymal transition (EMT). EMT of tRCC tumours was further associated with presence of mesenchymal tRCC cancer cells and myofibroblast cancer-associated fibroblasts (myCAFs) that are both hallmarks of poor prognostic outcomes. We provide unique insights into how broad genomic binding of TFE3 fusion proteins promotes tRCC tumourigenesis by regulating OxPhos and ferroptosis resistance and more generally stimulating RNA synthesis.

overall_design: Cut and Tag for TFE3 and ChIP-seq for H3K27ac were performed in 4 tRCC cell lines (UOK109, TF1, UOK120 and UOK146) and modified HEK293T cells using 2 different antibodies for TFE3.

first: Alexandra

last: Helleux

first: Anas

last: Fadloun

first: Antonin

last: Lallement

first: Christelle

last: Thibault-Carpentier

first: Gabriel

last: Malouf

first: Irwin


title: Ergosterol-Induced Immune Response in Barley Involves Phosphorylation of Phosphatidylinositol Phosphate Metabolic Enzymes and Activation of Diterpene Biosynthesis

city: Cologne

first: Alga

last: Zuccaro

state: NRW

address: Zuelpicher Str. 47b

department: Institute for Plant Sciences

institute: University of Cologne

summary: Lipids play crucial roles in plant-microbe interactions, functioning as structural components, signaling molecules, and microbe-associated molecular patterns (MAMPs). However, the mechanisms underlying lipid perception and signaling in plants remain largely unknown. Here, we investigate the immune responses activated in barley (Hordeum vulgare L.) by lipid extracts from the beneficial root endophytic fungus Serendipita indica and compare them to responses elicited by chitohexaose and the fungal sterol ergosterol. We demonstrate that S. indica lipid extract induces hallmarks of pattern-triggered immunity (PTI) in barley. Ergosterol emerged as the primary immunogenic component and was detected in the apoplastic fluid of S. indica-colonized barley roots. Notably, S. indica colonization suppresses the ergosterol-induced burst of reactive oxygen species (ROS) in barley. By employing a multi-omics approach, which integrates transcriptomics, phosphoproteomics, and metabolomics, we provide evidence for the phosphorylation of phosphatidylinositol phosphate (PIP) metabolic enzymes and activation of diterpene biosynthesis upon exposure to fungal lipids. Furthermore, we show that phosphatidic acid (PA) enhances lipid-mediated apoplastic ROS production in barley. These findings indicate that plant lipids facilitate immune responses to fungal lipids in barley, providing new insights into lipid-based signaling mechanisms in plant-microbe interactions.

overall_design: To identify candidate genes involved in downstream pathways activated in response to lipid MAMP treatment of barley roots, we investigated transcriptomic changes upon treatment with ergosterol or S. indica lipids compared to solvent control treatment at 2 hpt.

first: Pia

last: Saake


title: YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1 mediated reprogramming of bone mesenchymal stem cells

city: Vienna

first: Florian

last: Halbritter

address: Zimmermannplatz 10

country: Austria

institute: St. Anna Children's Cancer Research Institute (CCRI)

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Development of a potent epigenetic editor targeting human PCSK9 with durable reduction of cholesterol in mice and non-human primates

city: Boston

first: Frederic

last: Tremblay

address: 201 Brookline Ave, Suite 1101

institute: Chroma Medicine

type: Methylation profiling by high throughput sequencing

sample_organism:
Macaca fascicularis
Homo sapiens
Mus musculus


title: YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1 mediated reprogramming of bone mesenchymal stem cells [RNA-seq]

summary: Ewing sarcoma (EwS) is an aggressive cancer of adolescents in need of effective treatment. Insulin-like growth factor (IGF) 1 is an autocrine growth factor for EwS, but only 10% of patients responded to IGF-1 receptor blockade. Although presumed to originate from mesenchymal progenitors during bone development, targeting of the EwS driver oncogene EWS::FLI1  to the mesenchymal lineage in a mouse model does not result in tumor formation but to skeletal malformations and perinatal death. We report that transient exposure to IGF-1 concentrations mimicking serum levels during puberty reprogrammes limb-derived mesenchymal cells of EWS::FLI1-mutant mice to stable transformation and tumorigenicity. We identify a modular mechanism of IGF-1-driven tumor promotion in the early steps of EwS pathogenesis, in which Yap1 plays a central role. Pharmacologic Yap1/Tead inhibition reverses the transformed phenotype of EWS::FLI1 expressing cells. Our data provide a rationale for combined IGF-1R and YAP/TEAD inhibition in the treatment of EwS patients.

overall_design: 15 RNA-seq datasets from two sets of experiments: Experiment #1, 3 WT vs 4 EFPrx1 MSCLCs; Experiment #2, 2 EFPrx1 vs 2 ha-EFPrx1 vs 4 hr-EFPrx1 MSCLCs

first: Rahil

last: Noorizadeh

first: Barbara

last: Sax

first: Branka

last: Radic-Sarikas

first: Valerie

last: Fock

first: Maximilian

last: Kauer

first: Aleksandr

last: Bykov

first: Veveeyan

last: Suresh

first: Michaela

last: Schlederer

first: Lukas

last: Kenner

first: Gerhard

last: Weber

first: Wolfgang

last: Mikulits

first: Richard

last: Moriggl

first: Heinrich

last: Kovar


title: YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1 mediated reprogramming of bone mesenchymal stem cells [ATAC-seq]

overall_design: 12 ATAC-seq datasets from WT, EFPrx1, ha-EFPrx1, and hr-EFPrx1 MSCLCs


title: Antibody-mediated targeting of human microglial LILRB4 attenuates amyloid-dependent pathology

city: Hangzhou

first: Jinchao

last: Hou

address: Binsheng Road

department: Anesthesiology

institute: Zhejiang University

summary: Microglia help limit the progression of Alzheimer’s disease (AD) by constrainingβ-Amyloid (Aβ) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. LILRB4 expression and function in microglia has not been investigated. Here, we find that LILRB4 is highly expressed in microglia of patients with AD. Using mice that accumulate Aβ andcarry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping Aβ plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb)reduced Aβ load,mitigated Aβ-related behavioral abnormalities, enhanced microglia phagocytosis and attenuated expression of interferon-induced genes.In vitrobinding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction.In silicomodeling, biochemical and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

overall_design: To invest the blocking effect of anti-LILRB4 in vivo, we treat the ILT Telo Tg x 5XFAD mice with the Ctrl IgG2a or anti-LILRB4 for eight weeks and determine the amyloid pathology and microglia transcriptome at the end of the treatment.

first: Yun

last: Chen

first: Marco

last: Colonna


title: Interpretable deep learning reveals the sequence rules of Hippo signaling (ATAC-Seq)

city: Overland Park

first: Khyati

middle: Sudhir

last: Dalal

state: KS

address: 75th street, APT 106, 6329 W

institute: Stowers Institute for Medical Research

summary: How specific cells respond to signaling pathways is largely encoded in the DNA sequence. However, the sequence rules result from complex interactions between signaling and cell-type-specific transcription factors and are considered intractable by traditional methods. Here, we leverage interpretable deep learning on high-resolution data and extensive validation experiments to identify the sequence rules for the Hippo pathway in mouse trophoblast stem cells. We show that Tead4 and Yap1 engage in two types of cooperativity. First, their binding is enhanced by cell-type-specific transcription factors, including Tfap2c, in a distance-dependent manner. Second, a strictly-spaced Tead double motif is a canonical Hippo pathway element that mediates strong Tead4 cooperativity through transient protein-protein interactions on DNA. These mechanisms occur genome-wide and allow us to predict how small sequence changes alter the activity of enhancers in vivo. This illustrates the power of interpretable deep learning to decode canonical and cell type-specific sequence rules of signaling pathways.

overall_design: Assay for transposase-accessible chromatin DNA-sequencing (ATAC-seq) in wild-type, native(non-fixed) cells of mouse Trophoblast stem cells (TSCs)(Singh and Gerton 2021).

first: Charles

last: McAnany

first: Melanie

last: Weilert

first: Cathy

last: McKinney

first: Sabrina

last: Krueger

first: Julia

last: Zeitlinger


title: Oncohistone H3.3K27M-driven CREB5/ID1 Axis Mediates the Malignant Cell States of Diffuse Intrinsic Pontine Glioma (DIPG)

first: Qiaoran

last: Xi

address: Haidian District

department: School of Life Sciences

institute: Tsinghua University


title: The significance of PAX5 in Merkel cell carcinoma

city: Ulm

first: Emil

last: Chteinberg

address: Albert-Einstein-Allee 11

institute: Ulm University Medical Center


title: The significance of PAX5 in Merkel cell carcinoma (RNA-Seq II)

summary:
Merkel cell carcinoma (MCC) is a highly malignant skin cancer that expresses epithelial-, neuroendocrine-, and lymphoid-associated genes. Here, we focused on B-cell differentiation, which is characterised by the co-expression of PAX5 and TdT. PAX5 is the master regulator of B-cell commitment and is expressed in 65% of MCC cases. Yet, little is known about the underlying molecular biology of the frequently reported PAX5 expression in MCC.
Multi-omics analyses, including RNA next-generation sequencing, RT-qPCR, immunohistochemistry, and western blotting, were performed to assess PAX5 expression in MCC. Differential DNA methylation analysis at 61,043 PAX5 binding sites in enhancer and promoter elements was performed to detect differences between n=14 MCC tissues and n=91 various normal B-cell populations.
RNA analysis revealed full-length PAX5 expression in MCC at the transcriptional level using both PAX5 transcription start sites. PAX5 protein expression was found in 40 of 41 MCCs and six out of seven MCC cell lines. DNA methylation array analysis revealed 1,084 hypermethylated loci of enhancer and promoter elements located in PAX5 binding sites in MCC. Of these, 702 loci were associated with 257 genes which are not expressed. The PAX5-associated regulatory elements of these 257 genes were enriched for interferon regulatory factor 4 (IRF4) and SPi-proto-oncogene (SPI1) binding motifs. Neither IRF4 or SPI1 could be detected in MCC on a RNA or protein level. Thus, because of the absence of these transcription factors we conclude that full length PAX5 alone cannot induce B-cell differentiation.

overall_design: gene expression profiling analysis of RNA-seq data for Merkel cell carcinoma tissues, Merkel cell carcinoma cell lines and one pre-B-cell acute lymphoblastic leukemia cell line

last: Kolarova

last: Vogt

first: Amanda

last: Macamo

first: Felix

last: Bormann

first: Helene

last: Kretzmer

first: Ernst

last: Speel

first: Joost

last: van den Oord

first: Christof

last: Schneider

first: Stephan

last: Stilgenbauer

first: Jürgen

last: Becker

first: Véronique

last: Winnepenninckx

first: Erik

last: Biessen

first: Martin

last: Zenke

first: Anna

last: Kurz

first: Reiner

last: Siebert

first: Axel

last: zur Hausen


title: Interpreting regulatory mechanisms of Hippo signaling through a deep learning sequence model

first: Khyati

last: Dalal

summary: How specific cells respond to signaling pathways is largely encoded in the DNA sequence. However, the sequence rules result from complex interactions between signaling and cell type-specific transcription factors and are considered intractable by traditional methods. Here, we leverage interpretable deep learning on high-resolution data and extensive validation experiments to identify the sequence rules for the Hippo pathway in mouse trophoblast stem cells. We show that TEAD4 and YAP1 engage in two types of cooperativity. First, their binding is enhanced by cell type-specific transcription factors, including TFAP2C, in a distance-dependent manner. Second, a strictly-spaced Tead double motif is a canonical Hippo pathway element that mediates strong Tead4 cooperativity through transient protein-protein interactions on DNA. These mechanisms occur genome-wide and allow us to predict how small sequence changes alter the activity of enhancers in vivo. This illustrates the power of interpretable deep learning to decode canonical and cell type-specific sequence rules of signaling pathways. These super series include ChIP-nexus data for TF binding (TEAD4, YAP1, CDX2, TFAP2C, GATA3), H3K27ac Chip-seq, ATAC-seq, RNA-seq, and Nascent RNA captured through the TT-seq method in mouse trophoblast stem cells.

overall_design:
For RNA seq: STAR count table, RSEM-TPM table, and edgeR analysis of wild-type and CRISPR mutated Tead4 single motif to TEAD4 double motif at chr17:6,827,739-6,828,394 genomic region (putative enhancer of Ezrin gene)

ChIP-nexus of TEAD4 and Pol II along with H3K27ac ChIP-seq performed in wild-type and CRISPR mutated cells. The mutation was at the putative enhancer region of the Rin3 gene (chr12:102,261,820-102,262,484), where the TEAD4 single motif moved away from the TFA2PC motif from 20bp in wild-type to 60 bp in CRISPR mutated cells. Also, the ChIP-nexus of TEAD4 was performed in wild-type and CRISPR-mutated cells. The mutation was at the putative enhancer region of the Ezrin gene (chr17:6,827,739-6,828,394 ), where the Tead4 single motif was replaced with the TEAD4 double motif.


title: Single-cell genomics of the mouse olfactory cortex reveals contrasts with neocortex and ancestral signatures of cell type evolution

city: Providence

first: Alexander

last: Fleischmann

state: RI

address: 185 meeting street

department: Neuroscience

institute: Brown University

summary: Understanding the molecular logic of cortical cell-type diversity can illuminate cortical circuit function and evolution. Here, we performed single-nucleus transcriptome and chromatin accessibility analyses to compare neurons across three- to six-layered cortical areas of adult mice and across tetrapod species. We found that, in contrast to the six-layered neocortex, glutamatergic neurons of the three-layered mouse olfactory (piriform) cortex displayed continuous rather than discrete variation in transcriptomic profiles. Subsets of piriform and neocortical glutamatergic cells with conserved transcriptomic profiles were distinguished by distinct, area-specific epigenetic states. Furthermore, we identified a prominent population of immature neurons in piriform cortex and observed that, in contrast to the neocortex, piriform cortex exhibited divergence between glutamatergic cells in lab versus wild-derived mice. Finally, we showed that piriform neurons displayed greater transcriptomic similarity to cortical neurons of turtles, lizards, and salamanders than to those of the neocortex. In summary, despite over 200 million years of co-evolution alongside the neocortex, olfactory cortex neurons retain molecular signatures of ancestral cortical identity.

overall_design:
Nuclei from micro-dissected cortical areas were isolated in C57Bl/6 mice and wild-derived mice, and 10x multiome or 10x RNA sequencing 3.1 version (only for some wild-derived mice replicates) were performed. Areas collected were:

A = anterior piriform cortex
P = posterior piriform cortex
T = agranular insular cortex
N = primary somatosensory cortex

W = data from wild-derived mice
1, 2, 3... = biological replicate ID

first: Sara

last: Zeppilli

first: Alonso

last: Ortega Gurrola

first: Pinar

last: Demetci

first: Robin

last: Attey

first: Noga

last: Zilkha

first: Tali

last: Kimchi

first: Ritambhara

last: Singh

first: Maria

last: Tosches

first: Anton

last: Crombach

first: Tuan

last: Pham

first: Robert

middle: S

last: Datta

first: David

last: Brann


title: Interpretable deep learning reveals the sequence rules of Hippo signaling (ChIP-Seq)

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-nexus) for Tead4, Yap1, Tfap2c, Cdx2, Gata3 and Pol II Factors, as well as H3K27ac ChIP-seq experiment, in wild-type mouse trophoblast stem cells (Singh and Gerton 2021). ChIP-nexus for Tead4,Pol II and ChIP-seq H3K27ac in CRISPR mutatated cell lines at genomic region of chr12:102,261,820-102,262,484 (putative enhancer for Rin3 gene consisting Tfap2c and Tead motifs) & chr17:6,827,739-6,828,394 (putative enhancer for Ezrin gene consisting for Tead double motif)

Chromatin immunoprecipitation DNA-sequencing (ChIP-nexus) for Tead4, in wild-type mouse embryonic stem cells (R1-ESCs).

first: Charles

last: McAnany

first: Melanie

last: Weilert

first: Cathy

last: McKinney

first: Sabrina

last: Krueger

first: Julia

last: Zeitlinger


title: Salsalate improves the anti-tumor efficacy of Lenvatinib in MASH-driven hepatocellular carcinoma.

city: Hamilton

first: Russta

last: Fayyazi

state: ON

address: 1280 Main St W

country: Canada

institute: McMaster University

summary: Rates of hepatocellular carcinoma (HCC) are increasing rapidly due to the epidemic of metabolic dysfunction-associated steatohepatitis (MASH). In addition to increased incidence, emerging evidence suggests that MASH driven HCC is associated with poor survival outcomes potentially due to the complex liver microenvironment which is characterized by hypoxia, steatosis, and fibrosis. Lenvatinib, is a multi-tyrosine kinase inhibitor, that is a standard of care therapy for unresected HCC, but 5-year survival rates are less than 20%. Therefore, developing treatments that inhibit cancer growth kinetics and target the tumor microenvironment to improve the therapeutic response in MASH-HCC are needed.  Salsalate is a rheumatoid arthritis therapy that stimulates the AMP-activated protein kinase (AMPK) increasing fatty acid oxidation while reducing de-novo lipogenesis, fibrosis and cell proliferation pathways. Thus, we hypothesized that Salsalate could improve the therapeutic efficacy of Lenvatinib in MASH-HCC. In the current study, we show that treatment of human HCC cells with clinically relevant concentrations of Lenvatinib and Salsalate synergistically suppress proliferation and clonogenic survival, activate AMPK and inhibit the mTOR-HIF1a and Erk1/2 signaling pathways. In orthotopic xenograft and MASH-HCC mouse models Lenvatinib and Salsalate combination therapy suppressed angiogenesis and steatosis and fibrosis. RNA-sequencing revealed combination therapy enhanced mitochondria fatty acid oxidation and suppressed glycolysis, angiogenesis, fibrosis and cell cycle progression with regulatory network analysis suggesting a potential role for Activating transcription factor 3 (ATF3) and ETS-proto-oncogene-1 (ETS1). These data suggest that Lenvatinib and Salsalate combination therapy may have therapeutic potential for MASH-HCC due to effective metabolic rewiring and growth inhibition, leading to improvements in the liver microenvironment and inhibition of HCC proliferation.

overall_design:
To investigate transcriptomic effects of Lenvatinib-mesylate, Salicylic acid, or combination treatment in vitro, bulk RNA-seq data was obtained from Hep3B cells (n = 3 per condition). Hep3B is a human HCC immortalized cell line. Dosage for each group was as follows: Control = DMSO-VEH, Salicylic acid = 1mM , Lenvatinib = 100nM, Salicylic acid + Lenvatinib (combination) = 1mM Salsalate + 100nM Lenvatinib. Cells were treated for 9h. Cells reached no more than 80% confluence.
To investigate transcriptomic effects of Lenvatinib-mesylate, Salsalate, or combination treatment in vivo, bulk RNA-seq data was obtained from  non-tumoral liver tissue from a FAT-NASH murine model (WD-CCl4 model, n = 6 per condition). Mice underwent model development for 24wks before allocation into 4 groups: vehicle, Salsalate treatment (300mg/kg/day), Lenvatinib treatment (2.5mg/kg/day), or Salsalate+Lenvatinib treatment (combination, 300mg/kg/day + 2.5mg/kg/day). Treatment groups had drug administered 6 days per week for 6 weeks (experiment endpoint for all groups = 30 weeks).

first: Evangelia

last: Tsakiridis

first: Gregory

last: Steinberg

first: Jaya

last: Gautam

first: Elham

last: Ahmadi

first: James

last: Lally

first: Hannah

last: She

first: Simon

last: Wang

first: Daniel

last: Del Rosso

first: Celina

last: Valvano

first: Olga-Demetra

last: Biziotis

first: Fiorella

last: Di Pastena

first: Brandon

last: Meyers

first: Paola

last: Muti

first: Theodoros


title: TFE3 fusion proteins drive oxidative metabolism, ferroptosis resistance and general RNA synthesis in translocation renal cell carcinoma. [RNA-seq]

first: Guillaume

last: Davidson

overall_design: RNA-seq was performed in triplicates for 4 tRCC cell lines (UOK109, TF1, UOK120 and UOK146) following siCtrl or siTFE3 and in HEK293T following overexpression of GFP, TFE3, NONO-TFE3 or PRCC-TFE3.

first: Alexandra

last: Helleux

first: Gabriel

last: Malouf

first: Irwin


title: Enzyme-mediated proximity labeling reveals the co-translational targeting of DLGAP5 mRNA to the centrosome during mitosis

first: Gang

address: 5th, Yiheyuan Road, Beijing

institute: Peking University

summary: Subcellular RNA localization is a conserved mechanism in eukaryotic cells and plays critical roles in diverse physiological processes including cell proliferation, differentiation, and embryo development. Nevertheless, the characterization of centrosome-localized mRNAs remains under-explored due to technical difficulties. In this study, we utilize APEX2-mediated proximity labeling to map centrosome-proximal transcriptome, identifying DLGAP5 mRNA as a novel centrosome-localized transcript during mitosis. Using a combination of drug perturbation, truncation, deletion, and mutagenesis, we demonstrate that microtubule binding of nascent MBD1 polypeptide is required for centrosomal transport of DLGAP5 mRNA. Our data also reveals that mRNA targeting efficiency is tightly linked to the coding sequence length. Thus, our study provides a transcriptomic resource for future investigation of centrosome-localized RNAs, and sheds light on mechanisms underlying mRNA centrosomal localization.

overall_design: MERR APEX-seq libraries were generated from the following two cell lines: (1) APEX2-PCNT-EGFP (Centrosome); (2) APEX2-NES (Cytosol). For each cell line, three biological replicates were performed. Total RNAs directly extracted from the cells were set as Input. RNAs labeled by centrosome- or cytosol-localized APEX2 were set as Enrich. RNAs enriched from negative controls omitting probes were set as Control.

first: Mo

last: Li

first: Peng

last: Zou


title: TFE3 fusion proteins drive oxidative metabolism, ferroptosis resistance and general RNA synthesis in translocation renal cell carcinoma


title: Tet proteins regulate differentiation potential across retinal cell types

city: Pittsburgh

first: Shea

middle: Alexandra

last: Heilman

state: PA

address: 3501 Fifth Ave

department: Ophthalmology

institute: University of Pittsburgh

summary: Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet-null(tet2-/-;tet3-/-) zebrafish exhibit extensive retinal defects. Here, we leveraged scRNAseq technologies to better understand cell type-specific deficits and molecular signatures underlying the tet2-/-;tet3-/- phenotype across differentiation stages. Our results revealed phenotypes in developing tet2-/-;tet3-/- retinae including delayed specification of several retinal cell types, reduced maturity across late-stage tet2-/-;tet3-/- cones, expansions of immature subpopulations of both horizontal and bipolar cells, and altered biases of bipolar cell subtype fates at late differentiation stages. Together, these data support Tet proteins as regulators of cell specification, lineage fate commitment, and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue.

overall_design: Embryonic and larval eye cells were collected from 36,48,72, and 120 hpf zebrafish retinae from sibling control (sibCTL) and tet2-/-tet3-/- retinae. Dissociated cells were analyzed via scRNAseq

last: Schriever

first: Dennis

last: Kostka

first: Kristen

last: Koenig

first: Jeffrey

last: Gross


title: CXCR4+ PD-L1+ neutrophils are increased in non-survived septic mice

city: Sao Paulo

first: Guilherme

last: Cebinelli

address: Elias Jafet, 755

country: Brazil

institute: Hospital Israelita Albert Einstein

summary: The dysregulated host response to infections can lead to sepsis, a complex disease characterized by a spectrum of clinical phenotypes associated with the host immune response variability and outcomes. This heterogeneity poses challenges for implementing specific therapeutic approaches. While clinical sepsis phenotypes are well distinguished, the mechanisms driving these heterogeneous responses remain poorly understood. Using an unbiased experimental approach, we analysed immune cell activation profiles in survived and non-survived CLP-septic to gain insights into the immunological mechanisms by which neutrophils contribute to the hyperinflammatory septic phenotype. Our finds reveal that non-survived septic mice exhibit increased frequencies of immature CXCR4+ PD-L1+ neutrophils and monocytes in the bloodstream, accompanied by an accumulation of trafficking-specific CXCR4+ PD-L1+ neutrophils into the lungs. The increased PD-L1 expression on CXCR4+ neutrophils is associated with increase of IFN-gamma signaling pathways. Additionally, the IFN-gamma and LPS promote an activation profile of CXCR4+ PD-L1+ neutrophils, exhibiting a phenotype associated with inflammation and organ damage. Notably, abrogating the IFN-gamma reduced susceptibility to CLP-sepsis and diminished PD-L1 expression on CXCR4+ neutrophils. This study provides molecular and functional insights into the immune cell activation profiles associated with the worsening of the septic hyperinflammatory phenotype experimental model. The CXCR4+ PD-L1+ neutrophils population and elevated plasmatic IFN-gamma levels highlighted here represent promising targets for therapeutic modulation in clinical sepsis hyperinflammatory phenotype.

overall_design: Isolation of leukocytes and single-cell RNA sequencing: Sepsis was induced using the cecal ligation and puncture (CLP) model, as previously described (Rittirsch et al., 2009). The mice were anesthetized by inhalation of 1.5 % of isoflurane, and two punctures were made through the ligated cecum using an 18-gauge needle. After the surgery, 1 mL of saline and tramadol 25 mg/kg was injected subcutaneously. After 6 hours of the surgery, antibiotic treatment with 30 mg/kg ertapenem (Merck Sharp & Dohme) was administered, followed by six additional doses every 12 hours. After 12 hours of the CLP-sepsis induction, 50 uL of blood samples were collected from the retro-orbital plexus of anesthetized mice, and the sepsis survival rate was monitored for up to 7 days. Leukocytes were isolated using positive magnetic isolation of CD45+ cells, following the manufacturer’s instructions (Miltenyi Biotec, cat#130-052-301). Leukocytes from each mouse were the multiplexed with a molecular tag following the 10x Genomics 3’ CellPlex Kit (10x Genomics, cat#1000261), and cells from 10 mice were encapsulated in two lanes of a 10× Chromium instrument, each 5 mice per lane. Libraries were constructed using the Chromium Next GEM Single Cell 3' Kit v3.1 following the manufacturer’s instructions (10x Genomics, cat#1000269), and were subsequently sequenced on the HiSeq 4000 (Illumina). Single-cell RNA sequencing data pre-processing: Base call files were converted in the Cell Ranger to FASTQ files using the mkfastq function. The reads were then aligned to the mm10 Mus musculus transcriptome using the Cell Ranger software pipeline (version 6.1.2) provided by the 10x Genomics to generate raw genes by cell matrices of unique molecular identifier counts for each sample. The samples were then grouped in health controls, survived, and non-survived, and were read into R using the read10x function (Hao et al., 2021), resulting in a count matrix with 32285 genes and 8248 cells. A Seurat object was then created, and a filter was applied to remove the low-quality cells by excluding those based on unique molecular identifier counts (nCount_RNA < 10000), the number of unique genes (nFeature_RNA between 200 and 3000), and the percentage of mitochondrial gene expression (percent.mt < 10). The remaining expression values were then normalized using the SCTransform (Hafemeister & Satija, 2019).

middle: M

first: Maísa

middle: O

last: Leandro

first: Antonio

last: Oliveira

first: Kalil

last: de Lima

first: Paula

last: Donate

first: Cleyson

last: Barros

first: Anderson

last: Ramos

first: Victor

last: Costa

first: Daniele

last: Nascimento

first: Luis Eduardo

last: Damasceno

first: Amanda

last: Tavares

first: André

middle: N

last: Gonçalves

first: Helder

last: Nakaya

first: Thiago

last: Cunha

first: José Carlos

last: Alves-Filho

first: Fernando

middle: Q


title: Competitive behaviours in Serratia marcescens are coordinately regulated by a lifestyle switch frequently inactivated in the clinical environment

status: Public on Jan 29 2025

city: Dundee

first: Sarah

last: Coulthurst

address: Dow Street

institute: University of Dundee, School of Life Sciences

summary: Opportunistic bacterial pathogens require effective mechanisms to compete with other bacteria and to switch between host and environment-adapted states. The Type VI secretion system (T6SS) occurs widely in Gram-negative bacteria and mediates efficient killing of neighbouring competitors. Here we describe the distribution of T6SSs across the genus Serratia and report the unexpected discovery that a highly-conserved antibacterial T6SS is differentially active between closely-related clinical isolates of Serratia marcescens. By combining genomic and experimental approaches, we identified a genus-core two-component system which controls T6SS activity and is subject to very frequent inactivating mutations, exclusively in clinically-derived isolates of S. marcescens. This regulatory system controls a number of lifestyle-related traits at transcriptional and post-translational levels, including T6SS activity, antibiotic production, motility and adhesion. Our data support a model whereby this system represents a conserved, modular switch from sessile to pioneering and aggressive behaviour, which is subject to strong selection pressure in the clinical environment.

overall_design: We observed that near-identical isolates of S. marcescens have different phenotypes relating to anti-bacterial antagonism, motility, and adherence, that correspond with variation in full-length/loss-of-function mutations in betR and reg2. To investigate the role of BetR and Reg2 on S. marcescens, we constructed betR and reg2 mutants from three near-identical isolates in order to compare between full-length and naturally-occuring loss-of-function mutations in these two genes, in otherwise isogenic isolates. Cells were grown for 4.5 hours in liquid LB medium at 37 degrees celcius with shaking, before total RNA was harvested. 4 independent biological replicates for each isolate were performed.

first: David

middle: J

last: Williams


title: Interpretable deep learning reveals the sequence rules of Hippo signaling (RNA-Seq)

first: Khyati

last: Dalal

overall_design: RNA-seq and Nascent RNA captured through TT-seq method (Schwalb et al. 2016) in wild-type mouse trophoblast stem cells (Singh and Gerton 2021). RNA-seq in CRISPR mutatated cell lines at genomic region of chr17:6,827,739-6,828,394 (putative enhancer for Ezrin gene consisting for Tead double motif)

first: Charles

last: McAnany

first: Melanie

last: Weilert

first: Cathy

last: McKinney

first: Sabrina

last: Krueger

first: Julia

last: Zeitlinger


title: Immunotherapy with natural conventional type-1 dendritic cells excels at immune memory induction to eradicate cancer relapse

city: MADRID

first: Ignacio

last: Heras-Murillo

state: MADRID

address: c/Melchor Fernandez Almagro, 3

country: Spain

institute: CNIC

summary: The potential of dendritic cell (DC) vaccination against cancer is not fully achieved. While next generation vaccines using adoptive transfer of natural DCs are explored, little is known about the precise nature of the anti-cancer immune response triggered by different natural DC subsets and their relevance in preventing postsurgical tumor recurrence. Here, we used mouse splenic conventional DC1s (cDC1s) or cDC2s pulsed with tumor cell lysates to generate DC vaccines. cDC1-based vaccination induced a stronger effector and memory CD4+ and CD8+ anti-tumor T cell response, leading to a better control of tumors treated either therapeutically or prophylactically. Using an experimental model of tumor relapse, we showed that adjuvant or neoadjuvant cDC1 vaccination improved anti-tumor immune memory, particularly by increasing the infiltrates of CD4+ tissue resident memory T cells. This translated into complete prevention of tumor relapses. Our findings suggest that cDC1-based vaccination excels at immune memory induction and prevention of cancer recurrence.

overall_design: Gene expression profile at single cell level of tumor infiltrating lymphocytes (TILs, including CD4+ and CD8+ T cells)  from MC38 colorectal carcinoma cell line injected intradermally in C57BL/6 mice at day 5 of growth. CD4+ and CD8+ T cells from 4 groups: 1) primary tumors; 2) tumor rechallenge 30d after primary tumor resection; 3) tumor rechallenge 30d after primary tumor resection: neoadjuvant treatment of primary tumors with cDC1-based anti-cancer vaccination; 4)  tumor rechallenge 30d after primary tumor resection: neoadjuvant treatment of primary tumors with anti-PD1. Two biological replicates (each a pool of 3 mice) for each experimental condition are analyzed. Multiplexing was done using CMO tags.

first: Diego

last: Mañanes

first: Stefanie

last: K Wculek

last: Sancho


title: U2 IP-seq from K562 cells expressing SF3B1-3xFlag WT or SF3B1-3xFlag K700E

status: Public on Jan 28 2025

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

institute: UCLA

summary: The branch site / 3' splice site recognition machinery is a hotspot for disease-associated mutations. A single amino acid substitution K700E in the U2 snRNP-specific protein SF3B1 is frequently associated with cancer and is particularly common among myelodysplastic syndromes. The goal of this study is to employ the U2 IP-seq strategy in examining the differences in branch site selection in cells expressing wild type SF3B1 vs those carrying the K700E mutation.

overall_design: K562 erythroleukemia cell line was engineered in Manley lab to express a 3x-Flag tagged version of SF3B1, either carrying the K700E mutation or retaining the original sequence (Zhang J et al 2019, PMID: 31474574). These cells were grown under standard conditions (in IMDM medium supplemented with 10% FBS), harvested and washed with 1x PBS. High molecular weight nuclear extracts were prepared as previously described (Damianov A et al 2024, PMID: 38537639). Precatalytic complexes carrying branch site sequences were affinity purified either with anti-SF3A2 antibody, each in a triplicate, or with anti-FLAG antibody recognizing the epitope tagged SF3B1. In parallel controls were carried out without antibody, or using an aliquot of the extract without affinity purification. After deproteinization, the U2 snRNA fragments were selectively degraded with antisense DNA oligonucleotide and RNase H. Sequencing libraries carrying inline barcodes were constructed using the iCLIP library reagents as in previous IP-seq experiments (Damianov A et al 2024, PMID: 38537639). and were sequenced in two S4 2x 100 bp lanes. After demultiplexing and removal of PCR duplicates, clusters of mapped reads will be determined. Branch site clusters were annotated separately as in the IP-seq analyses from 293Flp-In cells expressing Flag-tagged proteins. Branch point analysis were also performed as before.

first: Andrey

last: Damianov

first: Chia-Ho

first: Douglas

last: Black


title: A niche driven mechanism determines response and a mutation-independent therapeutic approach for myeloid malignancies

city: New York

first: Ioanna

last: Mosialou

institute: Columbia University

summary: Myeloid cancers such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) remain resistant to standard of care (SOC) and targeted therapies. Here, we identify an oncogenic signal from the niche as a mechanism determining response to all trans-retinoic acid (ATRA), a regimen with disparate results in AML. In samples from clinical trials, responsiveness to ATRA correlates with activation of b-catenin/JAG1 in osteoblastic lineage cells and Notch1 signaling in MDS/AML cells of patients. ATRA inhibits b-catenin activation in patients and leukemic mice. As a result, it specifically suppresses growth and survival and promotes differentiation of MDS/AML cells solely from patients with active b-catenin/JAG1 signaling. This event is independent of cytogenetics and mutational profile. ATRA also improves disease outcome in mice with no evidence of relapse and a superior safety profile to SOC. A circulating skeletal stem cell population expressing activated b-catenin allows patient stratification and monitoring treatment response. A human blocking antibody against JAG1 further improves efficacy, curing mice from leukemia and maintaining the beneficial effects on patient-derived MDS/AML cells. These results demonstrate a niche-directed therapeutic approach, tailored to at least 1/3 of MDS/AML patients that can evade relapse and overcome SOC toxicity, highlighting the therapeutic potential of targeting the niche. b-catenin activation provides an explanation for the differential response to ATRA and a mechanistic biomarker for immediate ATRA repurposing in myeloid malignancies, potentially extending in a broad range of cancers.

overall_design: Rnaseq in primary human osteoblasts from 4 healthy donors treated witth Wnt3a or Wnt3a+ATRA

first: Stavroula

last: Kousteni

first: Raul

last: Rabadan

first: Anqi

last: Wang


title: Lymph node macrophages drive immune tolerance and resistance to cancer therapy by expression of the immune-regulatory cytokine IL-33 [scRNA-seq]

city: Toronto

first: Tracy

last: McGaha

state: Ontario

address: 610 University Ave

department: Immunology

institute: University Health Network

summary: Apoptotic cells are immunosuppressive, creating a potential therapeutic barrier in cancer therapy. In this reportwe investigated responses to apoptotic tumor cell phagocytosis (i.e. efferocytosis) after therapy in the tumor draining lymph node (TDLN). Treatment with cisplatin or the BRAF inhibitor PLX4720 caused a significant increase in accumulation of apoptotic tumor material in the TDLN. We identified the primary phagocyte population responsible for clearing dying tumors in the TDLN was medullary sinus macrophages (MSMs). Tumor cell efferocytosis by MSMs induced an immune suppressive transcriptional program distinct from other macrophage or dendritic cell populations in the TDLN. One of the most differentially induced mRNAs in MSMs wasIl33,coding fora potent immune-regulatory cytokine. Administration of neutralizing anti-IL-33 receptor antibodies or deletion ofIl33in MSMs altered tumor responses to therapy with a significant enhancement of anti-tumor activity compared to controls. Mechanistically, IL-33 activated Treg cells in the TDLN with subsequent suppression of CD8+T cell responses. Importantly, combining IL-33 receptor blockade, BRAF inhibitor, and PD-1 blockade significantly improved tumor regression with enhanced, CD8+T cell dependent immunity. Finally, apoptotic tumor cells induced IL-33 expression in human macrophages, IL-33 expression in sentinel lymph nodes positively corresponded with disease stage and negatively correlated with survival in melanoma and breast cancer. Thus, the data identifies an immune response to therapy that abrogates nascent anti-tumor immunity, providing a previously undescribed functional interaction with broad implications for cancer therapy.

overall_design: Tumor bearing-mice were treated with Cisplatin, one single (Tx1) or seven consecutive (Tx7) PLX4720 treatments. Tumor or TDLN were then harvested, digested and cell sorted.

first: Sara

last: Lamorte

first: Rene

last: Quevedo

first: Robbie

last: Jin

first: Luke

last: Neufeld

first: Kitty

last: Liu

first: M

last: Ciudad

first: Sabelo

last: Lukhele

first: Jessica

last: Bruce

first: Zaid

middle: K

last: Saeed

first: Drew

last: Wakeham

first: Brian

last: Snow

first: Lisa

last: Jones

first: Hal

last: Berman

first: Dana

last: Philpott

first: Stephan

last: Girardin

first: Jennifer

last: Gommerman

first: David

middle: H

last: Munn

first: Tak

middle: W

last: Mak

first: Mikael

last: Karlsson

middle: G

last: Brooks


title: Canonical prolactin signaling and global mRNA expression in the skin of Holstein heifers carrying the SLICK1 allele of the prolactin receptor gene.

city: Davis

first: Anna

middle: Carolina

last: Denicol

address: 450 BIOLETTI WAY, MEYER HALL

department: Animal Science

institute: University of California Davis

summary: The SLICK1 allele of the prolactin receptor gene (SLICK1) is associated with a short hair coat and thermotolerance in cattle. SLICK1 includes a single base pair deletion that creates a premature stop codon and prevents transcription of 120 amino acids involved in JAK/STAT signaling. It is unknown if SLICK1 modifies JAK/STAT signaling or the transcriptional response to prolactin. To investigate PRLR-associated signaling pathways in heterozygous SLICK1+/- Holsteins (slick), we performed immunohistochemistry on skin explants obtained from slick (n=5) and non-slick (n=6) heifers to evaluate phosphorylated (p)STAT1, pSTAT3, and pSTAT5 immunoreactivity (pSTAT1+, pSTAT3+, pSTAT5+) in hair follicles (HF) and sweat glands (SG). In slick skin, more HF had no pSTAT3 immunoreactivity compared to non-slick skin. No difference was found for the proportion of pSTAT1+ or pSTAT5+ HF, nor the proportion of pSTAT1+ and pSTAT3+ SG between genotypes. Within immunoreactive HF and SG, there were no differences between genotypes in the proportion of pSTAT1+, pSTAT3+, or pSTAT5+ cells in HF, or pSTAT1+ and pSTAT3+ cells in SG. Next, we investigated pSTAT3 immunoreactivity and the transcriptome of skin explants after exposure to prolactin in vitro. Skin explants from slick (n=6) and non-slick (n=6) heifers were cultured for 36 h in the presence of 50 ng/mL recombinant ovine prolactin, bisected, and each half underwent immunohistochemistry for pSTAT3 or RNA sequencing. No difference was found between genotypes in the proportion of pSTAT3+ HF or SG, nor the proportion of pSTAT3+ cells within HF or SG. RNA was poly-A enriched and sequenced using Novaseq6000 (Illumina) and 221,342,181 reads were mapped to the bovine genome (bosTau 9). The DESeq package was used to determine the differentially expressed genes (DEG) with P<0.01 and fold-change>1.5. There were 87 upregulated and 79 downregulated DEG in slick compared to non-slick skin. Ingenuity Pathway Analysis identified IL-17, leukocyte extravasation, and wound healing as upregulated signaling pathways, as well as activation of TNF, IL-1β, OSM, IFNγ, IL-17α, and IL-1R and inhibition of SHH and BMP4 upstream of the DEG. Analysis of genomic regions within ± 2 kb of all DEG’s respective transcription start sites revealed enrichment of three binding sites for the OCT1 transcription factor. In conclusion, our results suggest differences in local immune regulation, hair growth inhibition, and tissue remodeling in slick skin.

overall_design: Skin biopsies (6mm^2) were collected from half-sister heifers that were sired by one of two heterozygous slick bulls. Skin explants were cultured in the presence of ovine recombinant prolactin for 36 hours. Gene expression analysis of RNA-seq data from the cultured skin explants was used to determine differentially expressed genes between genotypes.

first: Montana D

last: Altman

first: Alice T

last: Mathews

first: M Belen

last: Rabaglino

first: Russell C

last: Hovey

first: Anna C


title: A niche driven mechanism determines response and a mutation-independent therapeutic approach for myeloid malignancies.

city: shanghai

first: Cui

last: Liang

address: changhai road 168

institute: changhai hospital

summary: Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) remain resistant to standard of care (SOC) and targeted therapies. Here, we identify an oncogenic signal from the niche as a mechanism determining response to all-trans-retinoic acid (ATRA), a regiment with disparate results in AML. In reported clinical trials responsiveness to ATRA correlates with activation of b-catenin/JAG1 in osteoblasts and Notch1 signaling in MDS/AML cells. ATRA inhibits osteoblastic b-catenin activation in patients and leukemic mice and thus suppresses patient MDS/AML cell growth and survival and promotes their differentiation independent of cytogenetics and mutational profile. ATRA also improves disease outcome in mice with no evidence of relapse and a superior safety profile as compared to SOC. A circulating skeletal stem cell population expressing activated b-catenin allows patient stratification and monitoring treatment response. A human blocking antibody against JAG1 further improves efficacy, curing mice from leukemia and maintaining the beneficial effects on patient-derived MDS/AML cells. These findings provide a mechanistic biomarker for immediate ATRA repurposing in MDS/AML and demonstrate the therapeutic potential of targeting the niche to evade relapse and overcome toxicity.

overall_design: We collected bone marrow samples from two AML patients who had received ATRA treatment and compared the gene differences in the bone marrow of AML patients before and after ATRA treatment using RNA-seq.

first: Ioanna

last: Mosialou

first: Abdullah

last: Ali

first: Rossella

last: Labella

first: Brygida

last: Bisikirska

first: lvaro

last: Cuesta-Dominguez

last: Luo

first: Marta

last: Galan-Diez

first: Jiaxin

last: Lin

first: Junfei

last: Zhao

last: Chernak

first: Kazuya

last: Fukasawa

first: Jiayu

last: Su

first: Jason

last: Higa

first: Rachel

middle: A

last: Adams

first: Adam

middle: L

last: Corper

first: Sergey

last: Pampou

first: Catherine

last: Woods

first: Xiaomin

last: Fan

first: Roshan

middle: P

last: Shah

last: Liu

first: Maël

last: Heiblig

first: Steven

last: Kornblau

first: Ellin

first: Joseph

last: Jurcic

first: Azra

last: Raza

first: Stavroula

last: Kousteni


title: Multiple Mechanisms of Termination Modulate the Dynamics of RNAPI Transcription

city: Warsaw

first: Tomasz

middle: Wojciech

last: Turowski

address: Pawinskiego 5a

department: Laboratory of Transcription Mechanisms

country: Poland

institute: Insitute of Biochemistry and Biophysics PAS

summary: Transcription elongation is stochastic, driven by a Brownian ratchet, making it subject to changes in velocity. On the rDNA, multiple polymerases are linked by “torsional entrainment”, generated by DNA rotation. We report that release of entrainment, by co-transcriptional 3’-end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5’-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities are reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are terminated by forward and reverse “torpedoes”; Rat1 and exosome cofactor TRAMP. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Mathematical modeling supported substantial premature termination. These basic insights into transcription in vivo, will be relevant to many systems.

overall_design: samples were analysed in total from a strain carrying HTP tagged RNAPI and untagged strain as a negative control. Duplicate or multiplicate experiments were carried out.

first: Elisabeth

last: Petfalski

first: Marie-Luise

last: Winz

first: Katarzyna

last: Grelewska-Nowotko

first: David

last: Tollervey


title: RNA profiling of lung endothelial cells of mice under normal and high fat diet (HFD).

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

institute: CNIO

summary: In order to analyze the genes and molecular pathways modulated by diet in the lungs of mice under DIO model, we performed RNA-seq on CD45-, CD31+ lung endothelial cells from mice under HFD,  and compared their expression profiles with those under control diet.

overall_design: Lungs from mice under ND and HFD diet (12 weeks) were excised, disaggregated and digested in collagenase for 1 h at 37°C. The cell suspensions were seeded onto plates coated with  fibronectin, collagen I and gelatin. After attachment, cells were negatively selected with anti-CD16/CD32 mAb coupled to magnetic beads, and then positively selected with anti-ICAM-2 coupled to magnetic beads. Cells were seeded onto plates coated with collagen I before RNA extraction. RNA was extracted with trizol and subsequent Qiagen columns. RNA sequencing was performed afterwards.

first: Marta

last: Hergueta

last: Graña-Castro

first: Hector

last: Peinado


title: Lymph node macrophages drive immune tolerance and resistance to cancer therapy by expression of the immune-regulatory cytokine IL-33 [RNA-seq]

first: Tracy

last: McGaha

first: Sara

last: Lamorte

first: Rene

last: Quevedo

first: Robbie

last: Jin

first: Luke

last: Neufeld

first: Kitty

first: M

middle: T

last: Ciudad

first: Sabelo

last: Lukhele

first: Jessica

last: Bruce

first: Zaid

last: Saeed

first: Drew

last: Wakeham

first: Brian

last: Snow

first: Lisa

last: Jones

first: Hal

last: Berman

first: Dana

last: Philpott

first: Stephan

last: Girardin

first: Jennifer

last: Gommerman

last: Munn

first: Tak

last: Mak

first: Mikael

middle: C

last: Karlsson

last: Brooks


title: pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida

status: Public on Jan 30 2025

city: Kiel

first: Hinrich

last: Schulenburg

address: Am Botanischen Garten 9

department: Evolutionary Ecology and Genetics

institute: Kiel University

summary: The bacterial second messenger c-di-GMP is known to influence the formation of biofilms and thereby persistence of pathogenic and beneficial bacteria in hosts. A previous evolution experiment with Pseudomonas lurida MYb11, occasional symbiont of the nematode Caenorhabditis elegans, led to the emergence of host-specialized variants with elevated intracellular c-di-GMP. Thus far, the molecular underpinnings of c-di-GMP-mediated host specialization were unknown in this symbiosis. Therefore, the current study aimed at identifying candidate molecular processes by combining transcriptomic and functional genetic analyses. We found that MYb11 host specialists differentially expressed genes related to attachment, motility and biofilm production, including pelD from the pel gene cluster. pelD deletion resulted in reduced intra-host competitive fitness, lower bacterial numbers in C. elegans and loss of biofilm biomass. Our results identify pelD as a previously unknown key modulator of beneficial symbiont-host associations that acts downstream of c-di-GMP.

overall_design: RNA-seq data of the wild type of P. lurida MYb11 and evolved P. lurida MYb11 with mutations in either wspE, wspF or rph from tryptic soy broth (liquid) and tryptic soy agar (colony). Five replicates each.

first: Anna

last: Czerwinski

first: Julia

last: Löwenstrom

first: Sören

last: Franzenburg

first: Espen

middle: E

last: Groth

first: Nancy

last: Obeng


title: The significance of PAX5 in Merkel cell carcinoma (RNA-Seq I)

first: Emil

last: Chteinberg

summary:
Merkel cell carcinoma (MCC) is a highly malignant skin cancer that expresses epithelial-, neuroendocrine-, and lymphoid-associated genes. Here, we focused on B-cell differentiation, which is characterised by the co-expression of PAX5 and TdT. PAX5 is the master regulator of B-cell commitment and is expressed in 65% of MCC cases. Yet, little is known about the underlying molecular biology of the frequently reported PAX5 expression in MCC.
Multi-omics analyses, including RNA next-generation sequencing, RT-qPCR, immunohistochemistry, and western blotting, were performed to assess PAX5 expression in MCC. Differential DNA methylation analysis at 61,043 PAX5 binding sites in enhancer and promoter elements was performed to detect differences between n=14 MCC tissues and n=91 various normal B-cell populations.
RNA analysis revealed full-length PAX5 expression in MCC at the transcriptional level using both PAX5 transcription start sites. PAX5 protein expression was found in 40 of 41 MCCs and six out of seven MCC cell lines. DNA methylation array analysis revealed 1,084 hypermethylated loci of enhancer and promoter elements located in PAX5 binding sites in MCC. Of these, 702 loci were associated with 257 genes which are not expressed. The PAX5-associated regulatory elements of these 257 genes were enriched for interferon regulatory factor 4 (IRF4) and SPi-proto-oncogene (SPI1) binding motifs. Neither IRF4 or SPI1 could be detected in MCC on a RNA or protein level. Thus, because of the absence of these transcription factors we conclude that full length PAX5 alone cannot induce B-cell differentiation.

overall_design: gene expression profiling analysis of RNA-seq data for Merkel cell carcinoma tissues, Merkel cell carcinoma cell lines and one pre-B-cell acute lymphoblastic leukemia cell line

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