Pathway Signatures Research Articles

P74 Guselkumab clinical super-response is associated with faster normalization of blood and skin pathology: data from the Phase 3b GUIDE trial

Abstract The ongoing GUIDE trial (NCT03818035) examines the clinical and immunological impact of an extended dosing interval of guselkumab [an interleukin (IL)-23p19 subunit inhibitor] in patients with moderate-to-severe plaque-type psoriasis. A mechanistic substudy was conducted to explore the molecular differences in the skin and blood of ‘super-responder’ (SRe) and non-SRe patients. SRes were defined as patients achieving a psoriasis area and severity index (PASI) = 0 at both Week [W]20 and W28 following guselkumab treatment. RNA-sequencing was performed on non-lesional (at W0) and lesional skin samples (at W0, W4, W28, and W68) along with serum analyses of matching timepoints. Published gene sets related to psoriasis skin and cytokine-stimulated keratinocytes were used for gene set variation analysis. At W0, ∼1100 protein-coding transcripts were upregulated in lesional vs. non-lesional skin, and ∼3000 protein-coding transcripts were downregulated in lesional vs. non-lesional skin. Following treatment with guselkumab, robust pharmacodynamic changes were observed for immune- and psoriasis inflammation-related gene signatures. Faster changes were observed in SRes vs. non-SRes. Signatures related to psoriasis skin inflammation, IL-17-stimulated keratinocyte response, T-helper (Th)17 cells, and many keratinocyte subtypes (differentiated, proliferating and inflamed keratinocytes) were more significantly reduced in SRes vs. non-SRes as early as W4. By W28, all signatures that were upregulated at baseline in lesional vs. non-lesional skin were normalized in SRes, including molecular scar skin and Th17 pathway signatures. In contrast, among non-SRes, molecular scar and immune-cell-regulated signatures (including macrophage, Th22, mucosal-associated invariant T cells, and CD4+ T cells) were <75% normalized at W28. In both SRes and non-SRes, signatures initially downregulated in lesional vs. non-lesional skin at W0, including those for adipocytes, smooth muscles, meta-analysis-derived psoriasis downregulated genes, and IL-17-stimulated KC downregulated genes, represent the few signatures that did not return to non-lesional skin levels after 68 weeks of guselkumab treatment. Consistent with the effect of guselkumab on inflammatory processes in the skin as observed via RNA sequencing, guselkumab treatment decreased the serum levels of several inflammatory mediators as early as W4. The levels of IL-17A, IL-17F and IL-22 were comparable in SRes and non-SRes, while the reduction in beta defensin-2 level was more pronounced in SRes at W4 and W28. These reductions were maintained to W68. In summary, molecular analyses of SRes and non-SRes in the GUIDE clinical trial show kinetic and quantitative differences in the response to guselkumab treatment.

Transcriptomic insights into the stress signaling and drought tolerance mechanisms in sea-island cotton (Gossypium barbadense)

Drought stress significantly impacts plant growth and agricultural productivity. Elucidating the molecular mechanisms underlying drought stress response and plant tolerance is crucial for developing resilient crops. In Gossypium barbadense (G. barbadense), the specific genetic responses to drought stress remain underexplored. To provide insights into the transcriptomic dynamics and tolerance mechanisms in G. barbadense, we screened a diverse panel of G. barbadense accessions to identify drought-tolerant genotypes and investigate drought-stress responses across root and shoot tissues at two distinct time points. Differentially expressed genes (DEGs) analysis revealed diverse drought-responsive genes across tissue types and treatment time points. Functional enrichment and predictive protein-protein interaction (PPI) network analyses elucidated intricate patterns of drought-stress signaling pathways and transcriptional regulatory mechanisms. These upregulated DEGs were enriched in functional categories such as hormone signal transduction, phosphatidylinositol signaling system, ubiquitin-mediated proteolysis, phenylpropanoid biosynthesis, glutathione metabolism, and carbon metabolism pathways. The PPI network analysis underscores the activation of key signaling genes such as plant U-box E3 ubiquitin ligases (PUBs), protein phosphatase 2 C (PP2Cs), and F-Box genes, as well as transcriptional factors (CBF/NFYA) and various effector genes. These networks revealed the activation of effector genes involved in phenylpropanoid biosynthesis (Thioredoxin like 2–1, 1-Cys), glutathione metabolism (Thioredoxin, GPX6), and carbohydrate/sugar metabolism (GBSSI, AMY1.1). Gene silencing experiments validated the regulatory roles predicted for PUBs and PP2Cs in stress signaling and NFYA transcriptional factor in modifying the plant morphology and physiology to enhance drought tolerance. This research provides critical insights into the genetic signatures of stress signaling and regulatory pathways associated with drought tolerance in G. barbadense. The identified candidate genes are valuable for targeted breeding efforts to enhance drought tolerance and crop yield.

A novel computational approach for the mining of signature pathways using species co-occurrence networks in gut microbiomes

BackgroundAdvances in metagenome sequencing data continue to enable new methods for analyzing biological systems. When handling microbial profile data, metagenome sequencing has proven to be far more comprehensive than traditional methods such as 16s rRNA data, which rely on partial sequences. Microbial community profiling can be used to obtain key biological insights that pave the way for more accurate understanding of complex systems that are critical for advancing biomedical research and healthcare. However, such attempts have mostly used partial or incomplete data to accurately capture those associations.MethodsThis study introduces a novel computational approach for the identification of co-occurring microbial communities using the abundance and functional roles of species-level microbiome data. The proposed approach is then used to identify signature pathways associated with inflammatory bowel disease (IBD). Furthermore, we developed a computational pipeline to identify microbial species co-occurrences from metagenome data at various granularity levels.ResultsWhen comparing the IBD group to a control group, we show that certain co-occurring communities of species are enriched for potential pathways. We also show that the identified co-occurring microbial species operate as a community to facilitate pathway enrichment.ConclusionsThe obtained findings suggest that the proposed network model, along with the computational pipeline, provide a valuable analytical tool to analyze complex biological systems and extract pathway signatures that can be used to diagnose certain health conditions.

Pharmacodynamic effects and exploratory efficacy of afimetoran, a TLR7/8 inhibitor, in patients with cutaneous lupus erythematosus

Background: Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin condition occurring alone or as a manifestation of systemic lupus erythematosus (SLE). There is an unmet need for safe and effective CLE-focused treatments. Afimetoran is an investigational, first-in-class, potent oral inhibitor of toll-like receptors (TLRs) 7/8. Given the involvement of TLRs 7/8 in SLE, afimetoran may have therapeutic potential for CLE. A phase 1b randomized, double-blind, placebo-controlled study (NCT04493541) demonstrated preliminary safety and efficacy of afimetoran in patients with active CLE. We assessed the pharmacodynamics, safety, and efficacy of afimetoran and its impact on CLE pathobiology. Methods: Patients aged 18–65 years with SLE and cutaneous manifestations by EULAR/ACR 2019 classification criteria or biopsy-proven CLE, and modified CLE Disease Area and Severity Index-Activity (CLASI-A) score ≥6, were randomized 2:1 to once-daily afimetoran (30 mg) or placebo for 16 weeks with a 4-week post-treatment follow-up period. The primary endpoints were safety and tolerability; efficacy was exploratory. Transcriptomics and cytokine analyses were performed using peripheral whole blood and serum, respectively, at baseline (pre-dose) and each study visit (weeks 1, 2, 4, 8, 12, 16, and 20 [post-dose]). Statistical analyses compared data from the 13 randomized patients with thirteen age-, ethnicity-, and gender-matched healthy volunteers. Treatment responses were evaluated longitudinally in each treatment arm. The dataset was analyzed using a linear regression model. Gene set variation analysis (GSVA) was performed for pathway enrichment. Results: 13 patients with CLE were randomized (afimetoran, n=8; placebo, n=5); 12 patients completed 16 weeks of treatment and 1 discontinued after 6 weeks due to COVID-19. Compared with placebo, afimetoran demonstrated a favorable safety profile with no serious adverse events, and showed a greater reduction in CLASI-A scores as early as week 4, which persisted to week 20. Improvement in the molecular disease profile was rapid (week 1), sustained through the 16-week treatment period, and the 4-week post-treatment follow-up period. Expression of interferon (IFN) pathway genes was significantly reduced with afimetoran compared with baseline (ΔGSVA enrichment score [ES]=1.57, P<0.0001) and placebo (ΔGSVA ES=0.56, P<0.01); this effect was maintained through week 16 and ≥4 weeks post-treatment. Expression of TLR7 and TLR8 pathway genes and key cytokines (interleukin [IL]-6, tumor necrosis factor α, IL-18, IFNγ, macrophage inflammatory protein-1 alpha [CCL3/MiP1α] or beta [CCL4/MiP1β]) were greatly reduced with afimetoran treatment. GSVA demonstrated robust pharmacodynamic activity on immune cell populations, including immature and activated dendritic cells, macrophages, and inflammatory pathway signatures. Conclusion: In patients with CLE, afimetoran was safe, well tolerated, and demonstrated durable efficacy beyond the treatment period. Afimetoran’s clinical effects and potent pharmacodynamic impact on the molecular disease profile suggest a substantial therapeutic benefit for patients with CLE.

EPCO-06. DIFFUSE MIDLINE GLIOMAS: FROM CELL CYCLE TO THERAPEUTIC OPPORTUNITIES

Abstract The fatal Diffuse Midline Gliomas (DMG) are characterized by an undruggable H3K27M mutation in H3.1 or H3.3. K27M impairs normal development by stalling of differentiation. As replication timing influences gene expression, cell fate, and cellular response to therapeutics, we undertook a multi-omics approach (Repli-seq, cell cycle RNA-seq, single cell RNA-seq) in DMG cells (H3.1K27M and H3.3K27M subgroups) and tumors in comparison to normal brain to gain an appreciation for targetable pathways in DMG. DMG cells presented differential replication timing in each subgroup, which, in turn, correlated with significant differential gene expression including the cholesterol biosynthesis pathway. Consistent with these findings, DMG tumors presented high replication stress and cholesterol biosynthesis pathway signatures. Moreover, DMG cells were specifically vulnerable to an inhibitor of the cholesterol pathway and to a replication stress therapy, singly and in combination. In conclusion, this combinatorial genomics approach revealed insights into therapeutic opportunities for this incurable disease.

Transcriptomic signatures of WNT-driven pathways and granulosa cell-oocyte interactions during primordial follicle activation.

Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis.

Co-Expression Network and Machine Learning Analysis of Transcriptomics Data Identifies Distinct Gene Signatures and Pathways in Lesional and Non-Lesional Atopic Dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin condition with complex origins. Current treatments often yield suboptimal results due to an incomplete understanding of its underlying mechanisms. This study aimed to identify pathway and gene signatures that distinguish between lesional AD, non-lesional AD, and healthy skin. We conducted differential gene expression and co-expression network analyses to identify differentially co-expressed genes (DCEGs) in lesional AD vs. healthy skin, lesional vs. non-lesional AD, and non-lesional AD vs. healthy skin. Modules associated with lesional and non-lesional AD were identified based on the correlation coefficients between module eigengenes and clinical phenotypes (|R| ≥ 0.5, p-value < 0.05). Subsequently, we employed Ingenuity Pathway Analysis (IPA) on the identified DCEGs, followed by machine learning (ML) analysis within the pathway expression framework. The ML analysis of pathway expressions, selected by IPA and derived from gene expression data, identified relevant pathway signatures, which were validated using an independent dataset and correlated with AD severity measures (EASI and SCORAD). We identified 975, 441, and 40 DCEGs in lesional vs. healthy skin, lesional vs. non-lesional, and non-lesional vs. healthy skin, respectively. IPA and ML analyses revealed 25 relevant pathway signatures, including wound healing, glucocorticoid receptor signaling, and S100 gene family signaling pathways. Validation confirmed the significance of 10 pathway signatures, which were correlated with the AD severity measures. DCEGs such as MMP12 and S100A8 demonstrated high diagnostic efficacy (AUC > 0.70) in both the discovery and validation datasets. Differential gene expression, co-expression networks and ML analyses of pathway expression have unveiled relevant pathways and gene signatures that distinguish between lesional, non-lesional, and healthy skin, providing valuable insights into AD pathogenesis.

H3K4me2 distinguishes a distinct class of enhancers during the maternal-to-zygotic transition.

After egg fertilization, an initially silent embryonic genome is transcriptionally activated during the maternal-to-zygotic transition. In zebrafish, maternal vertebrate pluripotency factors Nanog, Pou5f3 (OCT4 homolog), and Sox19b (SOX2 homolog) (NPS) play essential roles in orchestrating embryonic genome activation, acting as "pioneers" that open condensed chromatin and mediate acquisition of activating histone modifications. However, some embryonic gene transcription still occurs in the absence of these factors, suggesting the existence of other mechanisms regulating genome activation. To identify chromatin signatures of these unknown pathways, we profiled the histone modification landscape of zebrafish embryos using CUT&RUN. Our regulatory map revealed two subclasses of enhancers distinguished by presence or absence of H3K4me2. Enhancers lacking H3K4me2 tend to require NPS factors for de novo activation, while enhancers bearing H3K4me2 are epigenetically bookmarked by DNA hypomethylation to recapitulate gamete activity in the embryo, independent of NPS pioneering. Thus, parallel enhancer activation pathways combine to induce transcriptional reprogramming to pluripotency in the early embryo.

Oncogenic pathway signatures predict the risk of progression and recurrence in well-differentiated pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (pNETs) are genomically diverse tumors. The management of newly diagnosed well-differentiated pNETs is limited by a lack of sensitivity of existing biomarkers for prognostication. Our goal was to investigate the potential utility of genetic markers as a predictor of progression-free survival (PFS) and recurrence-free survival (RFS). Whole-exome sequencing of resected well-differentiated, low and intermediate-grade (G1 and G2) pNETs and normal adjacent tissue from patients who underwent resection from 2005 to 2015 was performed. Genetic alterations were classified using pan-genomic and oncogenic pathway classifications. Additional samples with genetic and clinicopathologic data available were obtained from the publicly available International Cancer Genome Consortium (ICGC) database and included in the analysis. The prognostic relevance of these genomic signatures on PFS and RFS was analyzed. Thirty-one patients who underwent resection for pNET were identified. Genomic analysis of mutational, copy number, cytogenetic, and complex phenomena revealed similar patterns to prior studies of pNETs with relatively few somatic gene mutations but numerous instances of copy number changes. Analysis of genomic and clinicopathologic outcomes using the combined data from our study as well as the ICGC pNET cohort (n = 124 patients) revealed that the recurrent pattern of whole chromosome loss (RPCL) and metastatic disease were independently associated with disease progression. When evaluating patients with local disease at the time of resection, RPCL and alterations in the TGFβ oncogenic pathway were independently associated with the risk of recurrence. Well-differentiated pNETs are genomically diverse tumors. Pathway signatures may be prognostic for predicting disease progression and recurrence.

The structure of a continental intraplate volcanic system and controls from shear zones: Insights into the central Hoggar Cenozoic volcanic province, Northwest Africa, from electrical resistivity images

Continental intraplate volcanic systems, with their locations far from plate tectonic boundaries, are not well understood: the crustal and lithospheric mantle structure of these systems remain enigmatic and there is no consensus on the mechanisms that cause melt generation and ascent. The Cenozoic saw the development of numerous volcanic provinces on the African plate, including within the Central Hoggar, located in Northwest Africa, part of the Tuareg shield. The magmatic activity began at approximately 34 Ma and continued throughout the Quaternary. In order to understand the origins and potential mechanisms that generated the intraplate volcanic activity in the Central Hoggar we aim to image the subsurface architecture, in terms of electrical resistivity, from the surface to the lithospheric mantle. To do so we use magnetotelluric measurements from 40 locations to generate a 3-D electrical resistivity model, over an area of about 100 km by 160 km. Low-resistivity features (i.e., conductors) are observed in the crust that are narrow, linear structures congruent with the boundaries of terranes and prominent fault zones (e.g., Azrou N’Fad). They likely reflect the Pan-African mega-shear zones, which were reactivated throughout the tectonic evolution of the region. The model reveals that these faults are lithospheric-scale. The low-resistivity features likely represent the signatures of past fluid pathways and mineralization. A deeper low-resistivity feature is observed in the upper lithospheric mantle directly beneath the Manzaz and Atakor volcanic districts. It may represent local, small-scale metasomatism of the sub-continental lithospheric mantle, and low-percent partial melting, that sits above a regional, large-scale asthenospheric upwelling associated with the Hoggar swell. It is likely the origin point of the fluids responsible for the overlying anomalies. The results highlight the control of the lithospheric-scale, mega-shear zones on the spatial distribution of the recent Cenozoic volcanic activity, which was influenced by the location of pre-existing structural weaknesses.

TGFβ-mediated inhibition of hypodermal adipocyte progenitor differentiation promotes wound-induced skin fibrosis.

Aberrant activation of dermal fibroblasts during wound healing often leads to debilitating fibrotic changes in the skin, such as scleroderma and keloids. However, the underlying cellular and molecular mechanisms remain elusive. Here, we established a wound-induced skin fibrosis (WISF) mouse model in mature adult mice, characterised by excessive deposition of collagen bundles, loss of dermal adipocytes, and enrichment of DPP4+Ly6A+THY1+ hypodermal interstitial adipocyte progenitors (HI-APs) and pericytes, resembling human fibrotic skin diseases. This WISF model exhibited an age-dependent gain of fibrotic characteristics, contrasting with the wound-induced hair neogenesis observed in younger mice. Through comprehensive analyses of the WISF, we delineated a trajectory of fibroblast differentiation that originates from HI-APs. These progenitors highly expressed several extracellular matrix (ECM) genes and exhibited a TGFβ pathway signature. TGFβ was identified as the key signal to inhibit the adipogenic potential and maintain the fibrogenic potential of dermal APs. Additionally, administering a TGFβ receptor inhibitor to wound scar reduced the abundance of ECM-producing APs. Finally, analysis of human scleroderma skin tissues revealed a negative correlation between the expression of AP-, ECM-, and TGFβ pathway-related genes and PPARG. Overall, this study establishes a wound-induced skin fibrosis mouse model and demonstrates that TGFβ-mediated blockage of HI-AP differentiation is crucial for driving fibrotic pathology. Targeting HI-APs and adipogenesis may provide novel avenues for developing disease-modifying therapies for fibrotic skin diseases.

Molecular characterization of the salivary adenoid cystic carcinoma immune landscape by anatomic subsites

Adenoid cystic carcinoma (AdCC) is a slow-growing salivary gland malignancy that relapses frequently. AdCCs of the submandibular gland exhibit unique differences in prognosis and treatment response to adjuvant radiotherapy compared to other sites, yet the role of tumor anatomic subsite on gene expression and tumor immune microenvironment (TIME) composition remains unclear. We used 87 samples, including 48 samples (27 AdCC and 21 normal salivary gland tissue samples) from 4 publicly available AdCC RNA sequencing datasets, a validation set of 33 minor gland AdCCs, and 39 samples from an in-house cohort (30 AdCC and 9 normal salivary gland samples). RNA sequencing data were used for single sample gene set enrichment analysis and TIME deconvolution. Quantitative PCR and multiplex immunofluorescence were performed on the in-house cohort. Wilcoxon rank-sum, nonparametric equality-of-medians tests and linear regression models were used to evaluate tumor subsite differences. AdCCs of different anatomic subsites including parotid, submandibular, sublingual, and minor salivary glands differed with respect to expression of several key tumorigenic pathways. Among the three major salivary glands, the reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway signature was significantly underexpressed in AdCC of submandibular compared to parotid and sublingual glands while this association was not observed among normal glands. Additionally, the NRF2 pathway, whose expression was associated with favorable overall survival, was overexpressed in AdCCs of parotid gland compared to minor and submandibular glands. The TIME deconvolution identified differences in CD4+ T cell populations between AdCC of major and minor glands and natural killer (NK) cells among AdCC of minor, submandibular, and parotid glands while plasma cells were enriched in normal submandibular glands compared to other normal gland controls. Our data reveal key molecular differences in AdCC of different anatomic subsites. The ROS and NRF2 pathways are underexpressed in submandibular and minor AdCCs compared to parotid gland AdCCs, and NRF2 pathway expression is associated with favorable overall survival. The CD4+ T, NK, and plasma cell populations also vary by tumor subsites, suggesting that the observed submandibular AdCC tumor-intrinsic pathway differences may be responsible for influencing the TIME composition and survival differences.

Zeolite Functionalized with Magnesium/Aluminum/Lanthanum Ternary Hydroxide for the Phosphometabolite Profiling of Malignant Neoplastic Serum Samples.

ATP upregulation is a significant driver of aggressive cancer cell phenotypes. Phosphometabolites participate in metabolic pathways and are overexpressed in cancer cell activity. Therefore, developing novel and accurate methods for detecting phosphometabolites in biological fluids is essential. In this research, a novel zeolite composite comprising magnesium, aluminum, and lanthanum hydroxides (MALZ) is developed and used for the first time to enrich phosphorylated metabolites via its inherent interaction with phosphate groups. SEM micrographs show a crystalline cubic structure with a small diameter of 36.62 nm. FTIR analysis confirms the phosphate adsorption and desorption using AMP and ATP as the standards. XRD analysis of MALZ provides structural information about the synthesized composite. Adsorption-desorption parameters, such as pH, shaking time, and MALZ concentration, are optimized to analyze the binding capacity of the fabricated material for phosphorylated metabolites. A kinetic study reveals the rapid and effective AMP and ATP adsorptions on MALZ. The multiple hydroxyl groups of ternary hydroxides and high affinity of lanthanum toward the phosphate group enrich 26 phosphometabolites from serum samples of malignant neoplastic patients. The LC-MS profile shows characteristic phosphometabolites that may act as signatures of cancer-related abnormal metabolic pathways. This study may provide an experimental pathway for detecting metabolites in human body fluids.

Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability

BackgroundAutism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model.MethodsSociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala.ResultsC58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin’s beneficial effects on myelin gene expression.LimitationsOur findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin’s effects need further examination to understand its’ potential as an ASD therapeutic.ConclusionsOur work demonstrates the C58/J mouse model’s utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

1965-LB: Metabolomic and Transcriptomic Signatures of Gestational Diabetes Mellitus (GDM) Suggest Nutritional and Inflammatory Pathways in Intergenerational Transfer of Risk

1965-LB: Metabolomic and Transcriptomic Signatures of Gestational Diabetes Mellitus (GDM) Suggest Nutritional and Inflammatory Pathways in Intergenerational Transfer of Risk

Comprehensive whole genome and transcriptome analysis of patients with advanced solid tumor treated with immune checkpoint inhibitor therapy in the pan-cancer cohorts from the Marathon of Hope Cancer Centres Network Study (MOHCCN).

2645 Background: Immune checkpoint inhibitors (ICI) improve survival in multiple advanced solid tumors but many patients do not benefit. We conducted a comprehensive whole genome transcriptome sequencing (WGTS) analysis to identify predictors of immune sensitivity. Methods: Clinical and molecular data from archival or pre-treatment FFPE tumor tissue were available for analysis from The Marathon of Hope Cancer Centres Network Study (MOHCCN). It includes patients (Pts) with advanced solid tumors and ECOG PS 0 or 1 treated with ICIs targeting PD-1, PD-L1, or CTLA-4 in the INSPIRE (NCT02644369) and OCTANE (NCT02906943) cohorts. Response (R) to ICI was defined as radiological and clinical response without PFS event at 6 months; versus non-response (NR) as radiological or clinical progression within 6 months. Responders without evidence of progression for &gt;12 months were categorized as durable responders (DR). Nucleic acids were sequenced using Illumina NovaSeq 6000 system targeting minimum coverage of 80X and 30X for tumour and normal WGS, respectively and 80M reads for tumour RNA-seq. WGTS data were integrated with clinical data to identify associations with response to ICIs. Also, an analysis of immune cell types was conducted using multiplexed IHC and RNA-Seq deconvolution (CIBERSORT). Results: 59 Pts were included in this analysis: 28 (R) and 31 (NR). The most common tumor types were head and neck (n = 19) and melanoma (n = 7). Median age was 62 years (range 24-81); male 58% and median follow-up was 58 months (range 11-280). The most frequent ICI was pembrolizumab 76%, with 90% of all pts receiving ICI monotherapy and 10% combination. 75% of pts received chemotherapy prior to ICI. Higher tumor mutation burden (TMB) was observed in R vs NR (median 13 vs 5 coding mut/Mb, p=0.001), with the highest TMB in patients with DR (median 14 mut/Mb ). Differential RNA gene expression analysis showed NR had increased expression of several oncogenic pathway signatures, including MYC targets, G2M checkpoint, and E2F targets. Differential abundance analysis of immune cell types did not yield any differences of immune cell populations amongst R versus NR after correction for multiple comparisons. Responders had significant enrichment in mutations in WNT/β-catenin pathway genes in both coding (APC, AMER1, LZTR1, TCF7L2) and promoter regions (CTNNB1). Notably, the 6 Pts with CTNNB1 promoter mutations had significantly increased CTNNB1 gene expression (p = 0.005). Conclusions: ICI responders showed enrichment in coding mutations of several negative regulators of the Wnt/β-catenin pathway and non-coding promoter mutations in CTNNB1 compared with non-responders. Further investigation is ongoing to biologically validate how these mutations in the Wnt/β-catenin pathway may lead to improved response to ICI.

A prognostic model based on selected cell state and cellular community scores in patients with advanced melanoma treated with immune checkpoint inhibitors (Ecotype-ICI score) as a predictor of ICI immunotherapeutic benefits.

9568 Background: We conducted an enhanced immune cell state atlas analysis of the data of patients with melanoma treated with ICI to investigate predictors of therapeutic benefits and mechanisms of resistance. Methods: Leveraging RNA-seq from melanoma patients (n=141) treated with ICI within the ORIEN Avatar project (NCT03977402), we constructed a prognostic model based on selected cell state and cellular community scores, also known as ecotype-ICI score ( Li. AACR 2023). We evaluated the model’s predictive value using transcriptomic data from high-risk melanoma patients treated with adjuvant ipilimumab (n=471) or interferon-α (n=248) as part of the E1609 phase 3 trial (1). RNA-seq data were initially deconvoluted for cellular community signatures using EcoTyper. The six prognostic carcinoma ecotype (CE) signatures identified (CE1, CE2, C6, CE7, CD9, CD10) were utilized for calculating a risk score based on a multivariable Cox model, which was trained using Avatar data. For additional external validation, we analyzed data from patients with metastatic melanoma treated with anti-CTLA4 ( Va. n=40), anti-PD1 ( Liu. n=121), and anti-PD1 alone or combined with anti-CTLA4 ( Gide. n=66). A series of ad hoc survival analyses, including Kaplan-Meier analysis, log-rank tests, and Cox regression were further applied. Results: The ecotype-ICI score showed strong prognostic significance in predicting overall survival (OS) in the entire group of E1609 patients (N=719; Cox P &lt; 0.0001, log-rank P &lt; 0.0001), the ipilimumab cohort (Cox P &lt; 0.000143, log-rank P =0.00011), but less so with interferon (Cox P= 0.06). CE9, characterized by its proinflammatory nature and IFN-gamma signaling, and CE10, noted for its canonical T cell state signatures, both demonstrated a leading predictive value according to multivariable Cox regression analysis. Interestingly, CE2, noted for its lymphocyte deficiency signature, also demonstrated complementary predictive value for outcomes following ipilimumab. Within the validation cohorts of public data sets noted above, the ecotype-ICI scores showed similarly strong prognostic significance in predicting OS (Gide. P = 0.045; Liu. P = 0.029; Va. P = 0.011). Ecotype-ICI also demonstrated a distinct distribution in the responder group (complete or partial response) compared to the progressive disease group, underscoring its potential as a discriminative biomarker for ICI response and survival outcomes. Conclusions: Our analysis has successfully established the utility of the immune cell state atlas in predicting therapeutic benefits with ICIs across diverse ICI treatment regimens in melanoma. We will update our results with data related to gene ontology, KEGG pathways and the most complementary pathway signatures at the meeting. 1. Tarhini, 2020.

The STING agonist IMSA101 enhances chimeric antigen receptor T cell function by inducing IL-18 secretion

As a strategy to improve the therapeutic success of chimeric antigen receptor T cells (CART) directed against solid tumors, we here test the combinatorial use of CART and IMSA101, a newly developed stimulator of interferon genes (STING) agonist. In two syngeneic tumor models, improved overall survival is observed when mice are treated with intratumorally administered IMSA101 in addition to intravenous CART infusion. Transcriptomic analyses of CART isolated from tumors show elevated T cell activation, as well as upregulated cytokine pathway signatures, in particular IL-18, in the combination treatment group. Also, higher levels of IL-18 in serum and tumor are detected with IMSA101 treatment. Consistent with this, the use of IL-18 receptor negative CART impair anti-tumor responses in mice receiving combination treatment. In summary, we find that IMSA101 enhances CART function which is facilitated through STING agonist-induced IL-18 secretion.

Increased H19/miR-675 Expression in Adult T-Cell Leukemia Is Associated with a Unique Notch Signature Pathway.

The Notch pathway is a key cancer driver and is important in tumor progression. Early research suggested that Notch activity was highly dependent on the expression of the intracellular cleaved domain of Notch-1 (NICD). However, recent insights into Notch signaling reveal the presence of Notch pathway signatures, which may vary depending on different cancer types and tumor microenvironments. Herein, we perform a comprehensive investigation of the Notch signaling pathway in adult T-cell leukemia (ATL) primary patient samples. Using gene arrays, we demonstrate that the Notch pathway is constitutively activated in ATL patient samples. Furthermore, the activation of Notch in ATL cells remains elevated irrespective of the presence of activating mutations in Notch itself or its repressor, FBXW7, and that ATL cells are dependent upon Notch-1 expression for proliferation and survival. We demonstrate that ATL cells exhibit the expression of pivotal Notch-related genes, including notch-1, hes1, c-myc, H19, and hes4, thereby defining a critical Notch signature associated with ATL disease. Finally, we demonstrate that lncRNA H19 is highly expressed in ATL patient samples and ATL cells and contributes to Notch signaling activation. Collectively, our results shed further light on the Notch pathway in ATL leukemia and reveal new therapeutic approaches to inhibit Notch activation in ATL cells.