Characterization Of Gene Expression Profiles Research Articles

FLT4 causes developmental disorders of the cardiovascular and lymphovascular systems via pleiotropic molecular mechanisms.

Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD). Distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. Phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development. Here, we show that FLT4 variants identified in TOF patients, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wildtype FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of wildtype human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants. Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms. Proteostatic dysfunction, if confirmed as a mechanism of CHD pathogenesis for other predisposing genes, may identify pathways to therapeutic interventions. Distinguishing mechanistically how variants in FLT4 give rise to CHD may have potential to individualise genetic counselling in affected families.

CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

Abstract 1589: Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is primarily characterized by a desmoplastic reaction, mainly consisting of cancer-associated fibroblasts (CAFs). In order to investigate the heterogeneity of CAFs, we initially conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using pancreas tissue from 12-week-old KPC (K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre) PDAC mouse models. We identified three clusters enriched with myofibroblast CAF (myCAF) gene markers and one cluster enriched with inflammatory CAF (iCAF) gene markers. Next, we performed single-cell RNA sequencing (scRNA-seq) on pancreas tissues from KPC mice. scRNA-seq revealed four distinct fibroblast clusters, each exhibiting characteristic gene expression profiles of normal fibroblasts, iCAF, myCAF and antigen-presenting CAF (apCAF). These findings suggest that CAFs in the myCAF cluster identified in scATAC-seq may have potential to differentiate into either myCAFs or other CAF subtypes based on certain cues. Next, we focus on the expression of myCAF marker genes, particularly two commonly used markers, Tagln and Acta2. Among myCAFs, two subtypes were identified: Tagln-positive and Acta2-positive CAFs, and Tagln-negative and Acta2-positive CAFs. To confirm our findings, we conducted Immunohistochemical analysis (IHC) on KPC pancreas samples. We observed CAFs that were positive for both Talng and αSMA exclusively in close proximity to tumor cells. Fibroblast expressing only αSMA mostly located in normal area. Tagln is known as an actin-binding protein and plays an important role in activated fibroblasts. Orthotopic PDAC mouse models using Tagln homozygous knockout mice showed a significant reduction in tumor weight compared to the wild-type mice (P = 0.0076). Indeed, we found that high TAGLN expression in PDAC samples was associated with poor survival, as demonstrated by the analysis of the TCGA dataset. These data suggest that myCAFs expressing TAGLN contribute to the promotion of PDAC growth. Targeting stromal TAGLN might be a potential therapeutic strategy to counteract PDAC progression. Citation Format: Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Maruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1589.

Construction of an abnormal glycosylation risk model and its application in predicting the prognosis of patients with head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck, and the incidence rate is increasing year by year. Protein post-translational modification, recognized as a pivotal and extensive form of protein modification, has been established to possess a profound association with tumor occurrence and progression. This study employed bioinformatics analysis utilizing transcriptome sequencing data, patient survival data, and clinical data from HNSCC to establish predictive markers of genes associated with glycosylation as prognostic risk markers. The R procedure WGCNA was employed to construct a gene co-expression network using the gene expression profile and clinical characteristics of HNSCC samples. Multiple Cox Proportional Hazards Regression Model (Cox regression) and LASSO analysis were conducted to identify the key genes exhibiting the strongest association with prognosis. A risk score, known as the glycosylation-related genes risk score (GLRS), was subsequently formulated utilizing the aforementioned core genes. This scoring system facilitated the classification of samples into high-risk and low-risk categories, thereby enabling the prediction of patient prognosis. The association between GLRS and clinical variables was examined through both univariate and multivariate Cox regression analysis. The validation of six core genes was accomplished using quantitative real-time polymerase chain reaction (qRT-PCR). The findings demonstrated noteworthy variations in risk scores among subgroups, thereby affirming the efficacy of GLRS in prognosticating patient outcomes. Furthermore, a correlation has been observed between the risk-scoring model and immune infiltration. Moreover, significant disparities exist in the expression levels of diverse immune checkpoints, epithelial-mesenchymal transition genes, and angiogenic factors between the high and low-risk groups.

Expansion modes of primate nervous system structures in the light of the Prosomeric Model

The expansion of human and non-human primate central nervous system structures has been a paramount question for classic and contemporary studies in comparative vertebrate neuroanatomy. These studies can benefit from framing data analysis within the Prosomeric Model, which defines a common Bauplan for all vertebrate species, including mammals. According to this model, the vertebrate nervous system is composed of several Fundamental Morphological Units (FMUs) that are defined and delineated by characteristic gene expression profiles. Thus, the expansion of neural structures can be traced back to heterochronic neurogenesis, cell lineage specification, and axon growth in their corresponding FMUs. In the present article, we exemplify the use of the Prosomeric Model as the proper theoretical framework for analyzing the expansion of the cerebral and cerebellar cortices, the pontine nuclei, the striatum, the nigrostriatal dopaminergic system, the thalamus, and the amygdala in primates compared to rodents. We describe the quantitative (volume and neuron number) and qualitative (cytoarchitectonic and cell type differences) expansion of these structures in primates versus rodents and define different expansion modes. Then, we relate these modes to the developmental primary events of specification and secondary events of histogenesis, like neurogenesis. We conclude that the systematic analysis of the molecular regulation of primary and secondary developmental events in each FMU in rats, primates, and other mammals could provide the necessary insight to identify the causal mechanisms of the expansion modes described in the present article.

IL-6 Reduces Spheroid Sizes of Osteophytic Cells Derived from Osteoarthritis Knee Joint via Induction of Apoptosis

Osteophytes in osteoarthritis (OA) joints contribute to restriction of joint movement, joint pain, and OA progression, but little is known about osteophyte regulators. Examination of gene expression related to cartilage extracellular matrix, endochondral ossification, and growth factor signaling in articular cartilage and osteophytes obtained from OA knee joints showed that several genes such as COL1A1, VCAN, BGLAP, BMP8B, RUNX2, and SOST were overexpressed in osteophytes compared with articular cartilage. Ratios of mesenchymal stem/progenitor cells, which were characterized by co-expression of CD105 and CD166, were significantly higher in osteophytic cells than articular cells. A three-dimensional culture method for cartilage and osteophyte cells was developed by modification of cultures of self-assembled spheroid cell organoids (spheroids). These spheroids cultured in the media for mesenchymal stem cells containing transforming growth factor-β3 showed characteristic morphologies and gene expression profiles of articular cartilage and osteophytes, respectively. The effects of IL-1β, tumor necrosis factor-α, and IL-6 on the spheroids of articular and osteophytic cells were studied. To the best of our knowledge, they provide the first evidence that IL-6 suppresses the spheroid size of osteophytic cells by inducing apoptosis and reducing extracellular matrix molecules. These data show that IL-6 is the suppressor of osteophyte growth and suggest that IL-6 expression and/or activity are implicated in the regulation of osteophyte formation in pathologic joints.

Global characterization of gene expression in the brain of starved immature Rhodnius prolixus.

Rhodnius prolixus is a vector of Chagas disease and has become a model organism to study physiology, behavior, and pathogen interaction. The publication of its genome allowed initiating a process of comparative characterization of the gene expression profiles of diverse organs exposed to varying conditions. Brain processes control the expression of behavior and, as such, mediate immediate adjustment to a changing environment, allowing organisms to maximize their chances to survive and reproduce. The expression of fundamental behavioral processes like feeding requires fine control in triatomines because they obtain their blood meals from potential predators. Therefore, the characterization of gene expression profiles of key components modulating behavior in brain processes, like those of neuropeptide precursors and their receptors, seems fundamental. Here we study global gene expression profiles in the brain of starved R. prolixus fifth instar nymphs by means of RNA sequencing (RNA-Seq). The expression of neuromodulatory genes such as those of precursors of neuropeptides, neurohormones, and their receptors; as well as the enzymes involved in the biosynthesis and processing of neuropeptides and biogenic amines were fully characterized. Other important gene targets such as neurotransmitter receptors, nuclear receptors, clock genes, sensory receptors, and takeouts genes were identified and their gene expression analyzed. We propose that the set of neuromodulatory-related genes highly expressed in the brain of starved R. prolixus nymphs deserves functional characterization to allow the subsequent development of tools targeting them for bug control. As the brain is a complex structure that presents functionally specialized areas, future studies should focus on characterizing gene expression profiles in target areas, e.g. mushroom bodies, to complement our current knowledge.

Individual immune cell and cytokine profiles determine platelet-rich plasma composition

ObjectivePlatelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes.DesignFor each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed.ResultsAll three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content.ConclusionsIndividuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product.Trial registrationThis study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

AML-060 A Truncated Nucleus Isoform of FGFR1 Drives the Transformation of Leukemia Stem Cells in Leukemogenesis

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated in the cytoplasm as a result of chromosome translocations. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. In this study, we used the mouse bone marrow transduction and transplantation model to demonstrate the leukemic transformation ability of this tnFGFR1 and identified its critical downstream targets, FLT3 and KIT, in mouse and acute myeloid leukemia (AML) patient samples. Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq were used to examine the related signaling pathways and regulated target genes. We now show that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1 induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expresses tnFGFR1, which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but sensitive to the FLT3 inhibitor quizartinib (AC220), suggesting an alternative approach for the treatment of SCLL. This study demonstrates a novel model for the transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by tnFGFR1. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Comprehensive analysis of liquid-liquid phase separation-related genes in prediction of breast cancer prognosis.

Objective: Liquid-liquid phase separation (LLPS) is a functional unit formed by specific molecules. It lacks a membrane and has been reported to play a crucial role in tumor drug resistance and growth by regulating gene expression and drug distribution. However, whether LLPS could be used to predict cancer prognosis was not clear. This study aimed to construct a prognostic model for breast cancer based on LLPS-correlated genes (LCGs). Methods: LCGs were identified using the PhaSepDB, gene expression profile and clinical characteristics of breast cancer were obtained from TCGA and cBioportal. The PanCancer Atlas (TCGA) cohort was used as the training cohort to construct the prognostic model, while the Nature 2012 and Nat Commun 2016 (TCGA) cohort and GEO data were used as test cohort to perform external verification. Data analysis was performed with R4.2.0 and SPSS20.0. Results: We identified 140 prognosis-related LCGs (pLCGs) (p< 0.01) in all cohorts, 240 pLCGs (p< 0.01) in the luminal cohort, and 28 pLCGs (p< 0.05) in the triple-negative breast cancer (TNBC) cohort. Twelve genes in all cohorts (training cohort: 5/10-year ROC values were 0.76 and 0.77; verification cohort: 5/10-year ROC values were 0.61 and 0.58), eight genes in the luminal cohort (training cohort: 5/10-year ROC values were 0.79 and 0.75; verification cohort: 5/10-year ROC values were 0.62 and 0.62), and four genes in the TNBC cohort (training cohort: 5/10-year ROC values were 0.73 and 0.79; verification cohort: 5/10-year ROC values were 0.55 and 0.54) were screened out to construct the prognostic prediction model. The 17-gene risk-score was constructed in all cohorts (1/3/5-year ROC values were 0.88, 0.83, and 0.81), and the 11-gene risk-score was constructed in the luminal cohort (1/3/5-year ROC values were 0.67, 0.85 and 0.84), and the six-gene risk-score was constructed in the TNBC cohort (1/3/5-year ROC value were 0.87, 0.88 and 0.81). Finally, the risk-score and clinical factors were applied to construct nomograms in all cohorts (1/3/5-year ROC values were 0.89, 0.79 and 0.75, C-index = 0.784), in the luminal cohort (1/3/5-year ROC values were 0.84, 0.83 and 0.85, C-index = 0.803), and in the TNBC cohort (1/3/5-year ROC values were 0.95, 0.84 and 0.77, C-index = 0.847). Discussion: This study explored the roles of LCGs in the prediction of breast cancer prognosis.

Different Approaches for the Profiling of Cancer Pathway-Related Genes in Glioblastoma Cells.

Deregulation of signalling pathways that regulate cell growth, survival, metabolism, and migration can frequently lead to the progression of cancer. Brain tumours are a large group of malignancies characterised by inter- and intratumoral heterogeneity, with glioblastoma (GBM) being the most aggressive and fatal. The present study aimed to characterise the expression of cancer pathway-related genes (n = 84) in glial tumour cell lines (A172, SW1088, and T98G). The transcriptomic data obtained by the qRT-PCR method were compared to different control groups, and the most appropriate control for subsequent interpretation of the obtained results was chosen. We analysed three widely used control groups (non-glioma cells) in glioblastoma research: Human Dermal Fibroblasts (HDFa), Normal Human Astrocytes (NHA), and commercially available mRNAs extracted from healthy human brain tissues (hRNA). The gene expression profiles of individual glioblastoma cell lines may vary due to the selection of a different control group to correlate with. Moreover, we present the original multicriterial decision making (MCDM) for the possible characterization of gene expression profiles. We observed deregulation of 75 genes out of 78 tested in the A172 cell line, while T98G and SW1088 cells exhibited changes in 72 genes. By comparing the delta cycle threshold value of the tumour groups to the mean value of the three controls, only changes in the expression of 26 genes belonging to the following pathways were identified: angiogenesis FGF2; apoptosis APAF1, CFLAR, XIAP; cellular senescence BM1, ETS2, IGFBP5, IGFBP7, SOD1, TBX2; DNA damage and repair ERCC5, PPP1R15A; epithelial to mesenchymal transition SNAI3, SOX10; hypoxia ADM, ARNT, LDHA; metabolism ATP5A1, COX5A, CPT2, PFKL, UQCRFS1; telomeres and telomerase PINX1, TINF2, TNKS, and TNKS2. We identified a human astrocyte cell line and normal human brain tissue as the appropriate control group for an in vitro model, despite the small sample size. A different method of assessing gene expression levels produced the same disparities, highlighting the need for caution when interpreting the accuracy of tumorigenesis markers.

Correlation between BAP1 Localization, Driver Mutations, and Patient Survival in Uveal Melanoma.

Simple Summary100 uveal melanomas from the UK were analyzed for molecular biomarkers, including alterations of chromosomes 3 and 8, cellular localization of BAP1, and genes known to be mutated in uveal melanoma. Consistent with earlier studies, loss of nuclear BAP1 (nBAP1) predicted shorter overall survival. Tumors with BAP1 loss of function mutations frequently exhibited heterogeneous BAP1 staining, and tumors with ≥25% loss of nBAP1 were a more reliable prognosistic indicator than chromosome 3 loss (LOH3) or chromosome 8q gain. Regardless of mutation class, most BAP1 mutations led to loss of nBAP1 and aberrant expression of cBAP1.Uveal melanoma (UM) is an uncommon but highly aggressive ocular malignancy. Poor overall survival is associated with deleterious BAP1 alterations, which frequently occur with monosomy 3 (LOH3) and a characteristic gene expression profile. Tumor DNA from a cohort of 100 UM patients from Moorfields Biobank (UK) that had undergone enucleation were sequenced for known UM driver genes (BAP1, SF3B1, EIF1AX, GNAQ, and GNA11). Immunohistochemical staining of BAP1 and interphase FISH for chromosomes 3 and 8 was performed, and cellular localization of BAP1 was correlated with BAP1 mutations. Wildtype (WT) BAP1 staining was characterized by nBAP1 expression with <10% cytoplasmic BAP1 (cBAP1). Tumors exhibited heterogeneity with respect to BAP1 staining with different percentages of nBAP1 loss: ≥25% loss of nuclear BAP1 (nBAP1) was superior to chr8q and LOH3 as a prognostic indicator. Of the successfully sequenced UMs, 38% harbored oncogenic mutations in GNA11 and 48% harbored mutations in GNAQ at residues 209 or 183. Of the secondary drivers, 39% of mutations were in BAP1, 11% were in EIF1AX, and 20% were in the SF3B1 R625 hotspot. Most tumors with SF3B1 or EIF1AX mutations retained nuclear BAP1 (nBAP1). The majority of tumor samples with likely pathogenic BAP1 mutations, regardless of mutation class, displayed ≥25% loss of nBAP1. This included all tumors with truncating mutations and 80% of tumors with missense mutations. In addition, 60% of tumors with truncating mutations and 82% of tumors with missense mutations expressed >10% cBAP1.

Exploration of molecular features of PCOS with different androgen levels and immune-related prognostic biomarkers associated with implantation failure.

BackgroundPolycystic ovary syndrome (PCOS), the most common heterogeneous reproductive disease afflicting women of childbearing age, has been recognized as a chronic inflammatory disease recently. Most PCOS patients have hyperandrogenism, indicating a poor prognosis and poor pregnancy outcomes. The molecular mechanism underlying PCOS development is still unknown. In the present study, we investigated the gene expression profiling characteristics of PCOS with hyperandrogenism (HA) or without hyperandrogenism (NHA) and identified immune-related factors that correlated with embryo implantation failure.MethodsPCOS and recurrent implantation failure (RIF) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. ClueGO software was used to perform enrichment analysis of differentially expressed genes (DEGs) in PCOS with varying androgen levels. The Weighted Co-Expression Network Analysis (WGCNA) was used to identify co-expressed modules and shared gene signatures between HA PCOS and RIF. Moreover, the upregulated DEGs of HA PCOS and RIF were intersected with shared gene signatures screening by WGCNA to excavate further key prognostic biomarkers related to implantation failure of HA PCOS. The selected biomarker was verified by qRT-PCR.ResultsA total of 271 DEGs were found in HA PCOS granulosa cell samples, and 720 DEGs were found in NHA PCOS. According to CuleGO enrichment analysis, DEGs in HA PCOS are enriched in immune activation and inflammatory response. In contrast, DEGs in NHA PCOS are enriched in mesenchymal cell development and extracellular space. Using WGCNA analysis, we discovered 26 shared gene signatures between HA PCOS and RIF, which were involved in corticosteroid metabolism, bone maturation and immune regulation. DAPK2 was furtherly screened out and verified to be closely related with the development of HA PCOS, acting as an independent predictor biomarker of the embryo implantation failure. DAPK2 expression was negatively correlated to the embryo implantation rate (r=-0.474, P=0.003). The immune infiltration results suggested that upregulated DAPK2 expression was closely related with NK cell infiltration and macrophage M2, playing an essential role in the pathogenesis of implantation failure in HA PCOS.ConclusionOur research revealed the expression profiling of PCOS with different androgen levels and identified DAPK2 as a critical prognostic biomarker for implantation failure in PCOS.

Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission

It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were collected for analysis. A novel digital color-coded barcode counting technology (NanoStringTM) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p < 0.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function.

Neuroinflammatory Gene Expression Analysis Reveals Pathways of Interest as Potential Targets to Improve the Recording Performance of Intracortical Microelectrodes.

Intracortical microelectrodes are a critical component of brain-machine interface (BMI) systems. The recording performance of intracortical microelectrodes used for both basic neuroscience research and clinical applications of BMIs decreases over time, limiting the utility of the devices. The neuroinflammatory response to the microelectrode has been identified as a significant contributing factor to its performance. Traditionally, pathological assessment has been limited to a dozen or so known neuroinflammatory proteins, and only a few groups have begun to explore changes in gene expression following microelectrode implantation. Our initial characterization of gene expression profiles of the neuroinflammatory response to mice implanted with non-functional intracortical probes revealed many upregulated genes that could inform future therapeutic targets. Emphasis was placed on the most significant gene expression changes and genes involved in multiple innate immune sets, including Cd14, C3, Itgam, and Irak4. In previous studies, inhibition of Cluster of Differentiation 14 (Cd14) improved microelectrode performance for up to two weeks after electrode implantation, suggesting CD14 can be explored as a potential therapeutic target. However, all measures of improvements in signal quality and electrode performance lost statistical significance after two weeks. Therefore, the current study investigated the expression of genes in the neuroinflammatory pathway at the tissue-microelectrode interface in Cd14−/− mice to understand better how Cd14 inhibition was connected to temporary improvements in recording quality over the initial 2-weeks post-surgery, allowing for the identification of potential co-therapeutic targets that may work synergistically with or after CD14 inhibition to improve microelectrode performance.

A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML

BackgroundMyeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated.MethodsStem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes.ResultsFor the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. These leukemia cells show a mixed immunophenotype with a B-cell B220 + Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. We further show that de novo human AML also express tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone.ConclusionsThis study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with an inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Differential Expression and Prognostic Correlation of Immune Related Factors Between Right and Left Side Colorectal Cancer.

BackgroundGrowing evidence suggests that colorectal cancer (CRC) should be considered a heterogeneous disease. The right side (RCC) and left side (LCC) colorectal cancer have different clinical characteristics and immune landscapes. The aim of this study was to analyze differential expression and prognostic correlation of immune-related factors between RCC and LCC.MethodsThe gene expression profile and clinical characteristics of CRC patients were retrieved from The Cancer Genome Atlas data portal (n=525). Using a deconvolution algorithm, immune cell infiltration in RCC and LCC based on the RNA-seq data was analyzed. Differentially expressed genes (DEGs) were obtained by performing differential gene expression analysis. Immune-related DEGs were derived by the intersection with immune-related factors downloaded from the IMMPORT database. To further validate the findings, we applied immunohistochemical (IHC) staining of a CRC tissue microarray (TMA). The distribution of immune cells in RCC and LCC and changes in the expression of immune molecules on their membranes were verified. The expression levels of circulating cytokines were measured by flow cytometry to detect the cytokines secreted by immune cells in RCC and LCC. Furthermore, to reveal the prognostic value of differential immune factors on RCC and LCC patients, survival analysis based on mRNA levels using TCGA cohort and survival analysis using protein levels was performed using our CRC patients.ResultsThe infiltration of immune cells differed between RCC and LCC, the infiltration degree of macrophages M0 was significantly higher in LCC, while the infiltration degree of differentiated macrophages M1 and M2, CD4+ T and CD8+ T cells was significantly higher in RCC. The expression of related molecules by immune cells also differed between RCC and LCC. The expression of 7 genes in RCC was higher than that in LCC, which were CCR5, CD209, CD8A, HCK, HLA-DPB1, HLA-DQA1, HLA-DRA, respectively. Meanwhile, the expression of 2 genes in LCC was higher than in RCC, which were IL-34 and PROCR. Patients with RCC having high expression of HLA-DQA1 mRNA or proteins had better survival and LCC patients with high expression of IL 34 mRNA or protein had better survival.ConclusionsIn this study, we comprehensively compared differences in immune cells and regulating factors between left and right colorectal cancer. Different expression patterns and their effects on survival were identified. The analysis of immune-related factors may provide a theoretical basis for precise immunotherapy of RCC and LCC.

P329: UNRAVELING THE ROLE OF GATA3 IN EARLY HUMAN T CELL DEVELOPMENT AND EARLY T CELL PRECURSOR ALL (ETP-ALL)

Background: ETP-ALL is a subtype of T cell acute lymphoblastic leukemia (T-ALL) and can be identified by a characteristic immunophenotype and gene expression profile. Treatment of relapsed ETP-ALL remains challenging using conventional chemotherapy and little targeted therapies are available. A better understanding in the molecular mechanisms involved in the leukemogenesis and maintenance of ETP-ALL could lead to improved and more targeted treatments. An estimated 9% of ETP-ALL patients carry mutations in GATA3, a transcription factor known to regulate T cell commitment by inducing T lineage genes and inhibiting genes important for other lineages such as NK and B cells. Although these mutations have been described to be loss-of-function mutations, the exact mechanisms through which they contribute to ETP-ALL remain to be elucidated. Aims: We aim to gain insights into the molecular mechanisms through which GATA3 regulates normal human early T cell development and how disruption of GATA3 function, as a result of mutation, can contribute to the emergence of ETP-ALL. Methods: We have studied the effect of the most prevalent GATA3 mutation (c.827G>A, p.R276Q) in ETP-ALL on normal human T cell development using in vitro differentiation cultures. We examined the impact of GATA3 p.R276Q on gene expression, DNA binding and chromatin accessibility through RNA-, CUT&Tag- and ATAC-sequencing respectively, starting from primary CD34+CD1a- early human T cell progenitors isolated from thymus or the ETP-like cell line PER117. Results: While overexpression of wild-type GATA3 in thymic progenitor T cells induces an acceleration of development towards the CD4+CD8ab+ double positive stage, overexpression of the mutant form results in a delay and an accumulation of CD4 immature single positive cells in OP9-DL1 co-cultures. Remarkably, the inhibition of NK cell development, which is an important function of wild-type GATA3, is maintained by GATA3 R276Q. This indicates that GATA3 R276Q is not merely a loss-of-function mutation. RNA-, CUT&Tag- and ATAC sequencing after overexpression in human CD34+CD1a- immature thymocytes or the ETP-ALL-like cell line PER117 revealed that most key regulatory effects are maintained by GATA3 R276Q compared to its wild-type counterpart, including induction of T cell genes and inhibition of stem cell and NK cell genes, binding to its target regions by recognizing GATA motifs and exerting a pioneering function by opening and closing chromatin. Summary/Conclusion: Continuous GATA3-R276Q has a detrimental effect on T cell development in vitro. Since the inhibition on NK cell development is maintained, these findings suggest that this decision is uncoupled from the induction of T cell lineage commitment. Many important effects on RNA expression, DNA binding and chromatin accessibility are maintained by the R276Q mutant compared to wild-type GATA3, pointing out that this mutation not purely acts as a loss-of-function. The exact mechanisms through which GATA3 R276Q contributes to ETP-ALL still remain to be elucidated, but our results show that inhibition of T cell development most likely is one piece of the puzzle that leads to malignant transformation.

Abstract LB059: Subtype classification of pediatric high-grade glioma tumors by comparative transcriptomics

Abstract Pediatric high-grade glioma (pHGG) is a highly malignant and poorly understood cancer driven by diverse genetic and epigenetic mechanisms. Here, we use comparative RNA sequencing, outlier analysis, and spectral clustering approaches to analyze transcriptomic data of 1,543 pediatric brain tumor specimens from the UCSC Treehouse Childhood Cancer Initiative (Treehouse) and Open Pediatric Brain Tumor Atlas (OpenPBTA) to identify subpopulations of pHGG patients with characteristic gene expression profiles. We find that approximately half (45%) of pHGG tumors from OpenPBTA exist in three subgroups defined by high outlier-level expression either of: mitochondrially-encoded 12S and 16S rRNAs; genes enriched in the HSF1-mediated heat shock response and activation pathways; or six C/D box snoRNA (SNORD) genes originating from the paternally-expressed SNORD116 locus involved in Prader-Willi syndrome, a complex neurodevelopmental disorder. Interestingly, the same set of HSF1-dependent pathway genes is also significantly upregulated in a subset (~11%) of pHGG tumors from Treehouse, validating this finding in two independent compendia with different transcript isolation strategies (Treehouse, polyA selection; OpenPBTA, ribodepletion). Our work identifies distinct classes of tumors with outlier-level expression of genes with previously unknown roles in pHGG and provides a framework for subtyping tumors by comparative transcriptomics that is adaptable to any cancer type. We are currently investigating the molecular roles of HSF1-response genes and the imprinted SNORD116 gene cluster in pHGG. Our ongoing research into the biomolecular signatures and mechanisms of the three major tumor classes of pHGG as defined in our study will contribute to a greater understanding of pHGG disease manifestation and progression, and will inform strategies of tailored therapeutic interventions for children with this devastating disease. Citation Format: Gina D. Mawla, A. Geoffrey Lyle, Ellen T. Kephart, Katrina Learned, Holly C. Beale, Joshua E. Goldford, Olena M. Vaske. Subtype classification of pediatric high-grade glioma tumors by comparative transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB059.

Abstract 5837: A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases

Abstract Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. Stem cell transduction and transplantation in syngeneic mice were used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and the results showed that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. RPPA and RNA-Seq examination of the related signaling pathways and target genes revealed that tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expressed tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo suggesting an alternative approach for treatment of SCLL. This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases results with the combined effects of direct protein activation by the full-length kinases and gene regulation by the truncated nucleus derivative tnFGFR1, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile. Citation Format: Baohuan Cai, Yun Liu, Yating Chong, Hualei Zhang, Stephanie Mori, Atsuko Matsunaga, Xuexiu Fang, John Cowell, Tianxiang Hu. A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5837.