Pathway Signatures Research Articles

T cell expressions of aberrant gene signatures and Co-inhibitory receptors (Co-IRs) as predictors of renal damage and lupus disease activity

BackgroundSystemic lupus erythematosus (SLE) is distinguished by an extensive range of clinical heterogeneity with unpredictable disease flares and organ damage. This research investigates the potential of aberrant signatures on T cell genes, soluble Co-IRs/ligands, and Co-IRs expression on T cells as biomarkers for lupus disease parameters.MethodsComparative transcriptome profiling analysis of non-renal and end-stage renal disease (ESRD) phenotypes of SLE was performed using CD4 + and CD8 + cDNA microarrays of sorted T cells. Comparing the expression of Co-IRs on T cells and serum soluble mediators among healthy and SLE phenotypes.ResultsSLE patients with ESRD were downregulated CD38, PLEK, interferon-γ, CX3CR1, FGFBP2, and SLCO4C1 transcripts on CD4 + and CD8 + T cells simultaneously and NKG7, FCRL6, GZMB/H, FcγRIII, ITGAM, Fas ligand, TBX21, LYN, granulysin, CCL4L1, CMKLR1, HLA-DRβ, KIR2DL3, and KLRD1 in CD8 T cells. Pathway enrichment and PPI network analyses revealed that the overwhelming majority of Differentially Expressed Genes (DEGs) have been affiliated with novel cytotoxic, antigen presentation, and chemokine-cell migration signature pathways. CD8 + GZMK + T cells that are varied in nature, including CD161 + Mucosal-associated invariant T (MAIT) cells and CD161- aged-associated T (Taa) cells and CD161-GZMK + GZMB + T cells might account for a higher level of GZMK in CD8 + T cells associated with ESRD. SLE patients have higher TIGIT + , PD1 + , and lower CD127 + cell percentages on CD4 + T cells, higher TIM3 + , TIGIT + , HLA-DR + cell frequency, and lower MFI expression of CD127, CD160 in CD8 T cells. Co-IRs expression in T cells was correlated with soluble PD-1, PDL-2, and TIM3 levels, as well as SLE disease activity, clinical phenotypes, and immune-therapy responses.ConclusionThe signature of dysfunctional pathways defines a distinct immunity pattern in LN ESRD patients. Expression levels of Co-IRs in peripheral blood T cells and serum levels of soluble PD1/PDL-2/TIM3 can serve as biomarkers for evaluating clinical parameters and therapeutic responses.

Pathway Activation Analysis for Pan-Cancer Personalized Characterization Based on Riemannian Manifold.

The pathogenesis of carcinoma is believed to come from the combined effect of polygenic variation, and the initiation and progression of malignant tumors are closely related to the dysregulation of biological pathways. Quantifying the alteration in pathway activation and identifying coordinated patterns of pathway dysfunction are the imperative part of understanding the malignancy process and distinguishing different tumor stages or clinical outcomes of individual patients. In this study, we have conducted in silico pathway activation analysis using Riemannian manifold (RiePath) toward pan-cancer personalized characterization, which is the first attempt to apply the Riemannian manifold theory to measure the extent of pathway dysregulation in individual patient on the tangent space of the Riemannian manifold. RiePath effectively integrates pathway and gene expression information, not only generating a relatively low-dimensional and biologically relevant representation, but also identifying a robust panel of biologically meaningful pathway signatures as biomarkers. The pan-cancer analysis across 16 cancer types reveals the capability of RiePath to evaluate pathway activation accurately and identify clinical outcome-related pathways. We believe that RiePath has the potential to provide new prospects in understanding the molecular mechanisms of complex diseases and may find broader applications in predicting biomarkers for other intricate diseases.

Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages

In this study, J774A.1 macrophages stimulated by lipopolysaccharide(LPS) and adenosine triphosphate(ATP) were used to establish an in vitro model of pyroptosis, and the intervention mechanism of free total rhubarb anthraquinones(FTRAs) on pyroptosis was investigated. J774A.1 macrophages were cultured in vitro, and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25, 0.5, and 1 μg·mL~(-1)) and ATP(1.25, 2.5, and 5 mmol·L~(-1)). An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8, propidium iodide(PI) apoptotic cell staining, lactate dehydrogenase(LDH), interleukin(IL)-18, and tumor necrosis factor(TNF)-α release. Then, J774A.1 macrophages were randomly divided into six groups: blank control group, LPS+ATP group, high-dose FTRA group, and low, medium, and high-dose FTRA pre-protection group. The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP. Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect. The results showed that the stimulation condition of 0.50 μg·mL~(-1) LPS+5.00 mmol·L~(-1) ATP was the most effective in the in vitro model of macrophage pyroptosis. FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions, alleviate cell damage, lower the positive rate of PI staining, and reduce the release of LDH, IL-18, and TNF-α. FTRAs were able to significantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signature molecules, TLR4, NLRP3, cleaved-caspase-1, and cleaved-caspase-11, but they had no significant effect on ASC proteins. FTRAs were also able to significantly inhibit the mRNA expression of caspase-1, caspase-11, and GSDMD. These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating classical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.

Abstract 3644: Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma

Abstract Background: Tumor progression and therapeutic response are regulated by the tumor microenvironment. Understanding the spatial association and architecture of molecular characteristics and composition of tumor immune microenvironment at single-cell and subcellular resolution encourages improvements in clinical prognosis and immunotherapy benefits. Single-cell Spatial profiling technologies permit the study of transcriptional activity at the spatial, single-cell level and provide abundant, high-resolution information required for the identification of clinical-related features in immuno-oncology. Methods: We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on FFPE human skin squamous cell carcinoma using the CosMx™ Spatial Molecular Imager (SMI). For the selection of regions of interest, four protein markers and DAPI were co-detected on the same tissue slide. Tertiary analysis algorithms were developed for cell typing, co-localization of genes and ligands, cell-cell interaction, and pathway analysis. Results: Thousands of transcripts were simultaneously detected with high sensitivity and specificity on the FFPE skin squamous cell carcinoma tissue section at single-cell subcellular resolution. We investigated the tumor microenvironment including cell types, their spatial distribution and proportion of diversified immune cells in the tumor compartment. The cell typing results revealed many cancerous subpopulations, which resolved spatially from cells that lacked any defining marker genes. We also assessed distances between immune cells and their nearest functional-related neighbors. Moreover, we revealed the ligands co-localization, as well as spatial patterns of direct cell-cell interactions and signaling pathways. We found that some genes have elevated expression in macrophages which are near cancer compared to those are far from cancer cells, empowering the molecular investigation of novel signaling between immune and cancer cells in tumor immune microenvironment. Conclusions: Single-cell spatial measurements of 6,000 RNA and four proteins on the same tissue section, along with a large viewing area on archival FFPE tissue, provide a novel tool to reveal the spatial signature of tumor microenvironment and oncogenic pathways, facilitating the next level of cancer and therapeutic research. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Michael Patrick, Shanshan He, Saskia Ilcisin, David Kroeppler, Patrick Danaher, Jason Reeves, Mark Gregory, Haiyan Zhai, Michael Rhodes, Joseph Beechem. Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma [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 3644.

Abstract 1850: From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase

Abstract Proteomic changes were investigated in a neuroblastoma cell line, SK-N-AS, treated with a small molecule inhibitor, AOH1996, designed to specifically bind proliferating cell nuclear antigen (PCNA) in cancer. PCNA is crucial to DNA replication, DNA repair, transcription, cell cycle control, and chromatin organization through interactions with different proteins. Pathway analysis of differential protein expressions between treated and untreated cells revealed less G2/M DNA damage checkpoint regulation and decreased assembly of RNA polymerase complex. Phosphoproteomic analysis by PTM Signature Enrichment Analysis (PTM-SEA) identified signatures of kinase activities, signaling pathways, and perturbations. Specifically, the histone deacetylase inhibitors belinostat and vorinostat were identified as enriched perturbation signatures. Both inhibitors were tested in combination with AOH1996 in SK-N-AS as well as cell lines derived from breast, lung, ovarian, and pancreatic cancer, revealing drug synergies at specific dose combinations. Western blot analysis showed that belinostat decreased protein expression of PCNA, suggestive of mechanistic synergies with AOH1996+belinostat combination therapies. Our proteomic analysis reveals a therapeutic potential of combining AOH1996 and histone deacetylase inhibitors for treating cancer with possibly lower drug dose and toxicities compared to single drug treatment. Citation Format: Caroline M. Li, Krystine Garcia-Mansfield, Robert G. Lingeman, Long Gu, Robert J. Hickey, Patrick Pirrotte, Linda H. Malkas. From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase [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 1850.

Abstract 577: Deciphering the mechanism of the menin-MLL complex dependency in HCC

Abstract Liver cancer is the third leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most common primary liver cancer (~90%). The 5-year survival rate of patients with advanced HCC is ~18% due to the late prognosis and the moderate clinical benefit of available systemic therapy options. Mutations in chromatin regulators were detected in ~50% of liver cancer. Since many chromatin regulators have been shown a dependency in various cancer types and can modulate therapy response, we focused on identifying new therapeutic targets of HCC among epigenetic modifiers and evaluating them as potential targets for future drug development. We constructed a CRISPR library of 6000 gRNAs targeting 737 genes involved in chromatin-mediated gene regulation. Using the epigenome-focused CRISPR/Cas9 screening in two-dimensional and three-dimensional settings in multiple liver cancer cell lines (HepG2, HLF, PLC/PRF5), we identified a list of epigenetic regulators as potential new targets in HCC. Two of them, MEN1 and ASH2L, are core subunits of the menin-MLL complex mediating H3K4 trimethylation (me3). We validated dependencies of HCC on MEN1 and ASH2L by performing a negative selection CRISPR/Cas9 competitive growth assay in HLF and PLC/PRF5 cell lines. Moreover, treatment of HCC cell lines (HLF, PLC/PRF5, HepG2) with recently developed menin inhibitor SNDX-5613 (revumenib) revealed a dose-dependent reduction in cell proliferation. To determine transcriptional changes associated with disruption of the menin-MLL complex, we performed RNA sequencing of HLF and PLC/PRF5 cells following either inhibition with 5µM SNDX-5613 for 4 days or knockout of the MEN1 or ASH2L gene. We observed the upregulation of KRAS and the oncogenic signature of TGFβ signaling pathways in all menin-MLL complex disrupting conditions. We did not detect global changes in H3K4me3 levels upon the menin inhibition or MEN1 or ASH2L knockouts assessed by immunoblotting. To explore the mechanism of the antiproliferative effect of the menin inhibition on HCC further, we evaluated changes in the binding of menin to chromatin and H3K4me3 levels in HCC cells by performing CUT&RUN following treatment of HLF cells with 5µM SNDX-5613 for 4 days. Menin inhibition led to a drastic removal of menin from the chromatin, while H3K4me3 levels were only mildly decreased. We identified 8765 genes near promoter regions with menin binding and H3K4me3 mark, of which 1952 genes were near regions with simultaneous loss of menin and decreased H3K4me3 levels upon menin inhibition. Integration of our genomics data revealed a list of 30 genes, which are downregulated upon menin inhibition in both HLF and PLC/PRF5 cells (FC≥1.5) and the direct targets of the menin-MLL complex in HLF cells, suggesting their potential role in HCC cell survival. Altogether, we anticipate that menin and ASH2L serve as promising targets and represent an appealing therapeutic strategy for HCC treatment. Citation Format: Margarita M. Dzama, Peyton C. Kuhlers, Jesse R. Raab. Deciphering the mechanism of the menin-MLL complex dependency in HCC [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 577.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [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 6620.

Single nucleotide variants in nuclear pore complex disassembly pathway associated with poor survival in osteosarcoma.

The bone tumor, osteosarcoma, remains challenging to treat in children and young adults, especially when patients present with metastatic disease. Developing new therapies based on genomic data from sequencing projects has proven difficult given the lack of recurrent genetic lesions across tumors. MYC overexpression has been associated with poor outcomes in osteosarcoma. However, other genomic markers of disease severity are lacking. We utilized whole genome sequencing of 106 tumors and matched normal controls in order to define genomic characteristics that correlate with overall survival. Single nucleotide variants were overlaid onto annotated molecular pathways in order to define aberrant pathway signatures specific to aggressive osteosarcoma. Additionally, we calculated differential gene expression in a subsample of 71 tumors. Differentially expressed genes were then queried for known MYC-responsive genes. Molecular pathways specific to nuclear pore complex disassembly (NPCD) show significant correlation with poor overall survival in osteosarcoma when mutations were present. Genes involved in immune response and immune regulation are enriched in the differential expression analysis of samples with and without NPCD pathway aberrations. Furthermore, neither MYC nor MYC-responsive genes show differential expression between NPCD-aberrant and non-aberrant groups. The NPCD pathway mutations are dominated by regulatory region variants rather than protein-altering mutations, suggesting that dysregulation of genetic regulatory networks may be the underlying mechanism for their relation to osteosarcoma phenotype. Overall survival is significantly worse in patients whose tumors show aberrations in the NPCD pathway. Moreover, this difference in survival is not driven by MYC-overexpression, suggesting a novel mechanism for some aggressive osteosarcomas. These findings add light to the evolving understanding of the drivers of osteosarcoma and may aid in the search for new treatments based on patient-specific genetic data.

Abstract IA007: Distinct immune landscapes characterize highly and minimally invasive brain metastases

Abstract IA007: Distinct immune landscapes characterize highly and minimally invasive brain metastases

The prognosis, chemotherapy and immunotherapy efficacy of the SUMOylation pathway signature and the role of UBA2 in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the most common malignant tumors worldwide. Small Ubiquitin-like Modifier (SUMO)-ylation plays a crucial role in tumorigenesis. However, the SUMOylation pathway landscape and its clinical implications in LUAD remain unclear. Here, we analyzed genes involved in the SUMOylation pathway in LUAD and constructed a SUMOylation pathway signature (SUMOPS) using the LASSO-Cox regression model, validated in independent cohorts. Our analysis revealed significant dysregulation of SUMOylation-related genes in LUAD, comprising of favorable or unfavorable prognostic factors. The SUMOPS model was associated with established molecular and histological subtypes of LUAD, highlighting its clinical relevance. The SUMOPS stratified LUAD patients into SUMOPS-high and SUMOPS-low subtypes with distinct survival outcomes and adjuvant chemotherapy responses. The SUMOPS-low subtype showed favorable responses to adjuvant chemotherapy. The correlations between SUMOPS scores and immune cell infiltration suggested that patients with the SUMOPS-high subtype exhibited favorable immune profiles for immune checkpoint inhibitor (ICI) treatment. Additionally, we identified UBA2 as a key SUMOylation-related gene with an increased expression and a poor prognosis in LUAD. Cell function experiment confirmed the role of UBA2 in promoting LUAD cell proliferation, invasion, and migration. These findings provide valuable insights into the SUMOylation pathway and its prognostic implications in LUAD, paving the way for personalized treatment strategies and the development of novel therapeutic targets.

Integrative single-cell expression and functional studies unravels a sensitization to cytarabine-based chemotherapy through HIF pathway inhibition in AML leukemia stem cells.

Relapse remains a major challenge in the clinical management of acute myeloid leukemia (AML) and is driven by rare therapy-resistant leukemia stem cells (LSCs) that reside in specific bone marrow niches. Hypoxia signaling maintains cells in a quiescent and metabolically relaxed state, desensitizing them to chemotherapy. This suggests the hypothesis that hypoxia contributes to the chemoresistance of AML-LSCs and may represent a therapeutic target to sensitize AML-LSCs to chemotherapy. Here, we identify HIFhigh and HIFlow specific AML subgroups (inv(16)/t(8;21) and MLLr, respectively) and provide a comprehensive single-cell expression atlas of 119,000 AML cells and AML-LSCs in paired diagnostic-relapse samples from these molecular subgroups. The HIF/hypoxia pathway signature is attenuated in AML-LSCs compared with more differentiated AML cellsbut is more expressed than in healthy hematopoietic cells. Importantly, chemical inhibition of HIF cooperates with standard-of-care chemotherapy to impair AML growth and to substantially eliminate AML-LSCs in vitro and in vivo. These findings support the HIF pathway in the stem cell-driven drug resistance of AML and unravel avenues for combinatorial targeted and chemotherapy-based approaches to specifically eliminate AML-LSCs.

DOP35 Spatial transcriptomics reveals cellular niches associated with histological inflammation in postoperative Crohn’s Disease

Abstract Background Postoperative Crohn’s disease (CD) recurs in ~70% of patients ≤1 year following ileocolonic resection (ICR), as observed endoscopically. Current understanding of molecular determinants underlying histological intestinal inflammation in patients with postoperative CD recurrence is minimal. To address this issue, we used advanced spatial transcriptomic analyses on intestinal biopsy samples with matched histological and endoscopic assessments from neoterminal ileum, anastomosis, and colon of patients with CD who had undergone colonoscopy following ICR. Methods Formalin-fixed paraffin-embedded (FFPE) tissue biopsies from neoterminal ileum, anastomosis, and colon were prospectively collected from a 15-patient cross-sectional cohort with CD who underwent routine endoscopic assessment following ICR. Spatial transcriptomics was performed on samples using 10X Genomics Visium CytAssist Spatial Gene Expression for FFPE V2.0. Annotation of tissue regions of histologically active inflammation and classification of histologically inactive (Robarts Histopathology Index ≤3 with subscores of 0 for neutrophils in the lamina propria and epithelium) or active disease were performed by an expert gastrointestinal pathologist. R programming language was used for data analysis (Figure). Results Fourteen of the 43 (32.6%) FFPE biopsies had histological evidence of inflammation. Spatial transcriptomics clustering revealed a distinct transcriptional signature by the segregation of 4 clusters ("inflamed clusters") composed mostly (≥69%) of cells from samples defined by histologically active disease. Tissue regions identified as inflamed by expert pathologist annotation contributed to >46% of inflamed clusters’ composition and <15% of remaining clusters. Cell type decomposition and cellular niche analysis revealed immune (e.g., myeloid, T cells, and B cells) and epithelial cells in close proximity sharing a similar microenvironment in 3 inflamed clusters while the 4th cluster was composed of >90% epithelial cells. Differentially expressed genes from the inflamed clusters were similarly expressed across intestinal segments including inflamed anastomotic biopsies and were enriched for signalling pathways involving immune cells recruitment/activation, inflammatory signalling, extracellular matrix organisation, and oxidative stress response. Conclusion This is the first study to explore spatial transcriptomic signatures associated with histological inflammation in patients with postoperative CD recurrence. Our analysis revealed a spatial signature of cellular niches and signalling pathways arising upon histological inflammation, providing insight into therapeutic targets and biomarkers across anatomic locations and the anastomosis.

SPCS, a Novel Classifier System Based on Senescence Axis Regulators Reveals Tumor Microenvironment Heterogeneity and Guides Frontline Therapy for Clear Cell Renal Carcinoma

RationaleThe emerging evidence suggested that senescence regulator genes were involved in multi cancers, which may be utilized as new targets for cancers. However, the dysregulation and clinical impact of senescence regulator genes in clear cell renal cell cancer (ccRCC) were still in foggy. MethodsUsing multiomics data from TCGA-KIRC and other datasets, we comprehensively investigated the function of senescence regulator genes in ccRCC. ccRCC patients could be remodeled into 2 significant different groups basing on senescence regulators expression: senescence-pattern cancer subtype1 (SPCS1) and subtype2 (SPCS2). We further explored clinical characteristics, functional analysis, tumor immune microenvironment, immunotherapy response, genomic mutation and drug sensitivity between the 2 subtypes. Besides, senescence-pattern related risk model was established to determine the patient's prognosis of ccRCC. Finally, the overview of MECP2 function was investigated in multi cancers. ResultsccRCC patients could be divided into SPCS1 (normal aging group) and SPCS2 (Aging disorder group). The 2 subtypes showed significant different clinical characteristics and biological process in ccRCC. SPCS2, an aggressive subtype, comprised higher clinical stage and worse prognosis of ccRCC patients. SPCS2 subtype indicated activated oncogenic signaling pathway and metabolic signatures to prompt cancer expansion. SPCS2 subgroup owned immunocompromised status, which induced immune dysfunction and low ICI therapy response. The genome-copy numbers of SPCS2, including arm-gain and arm-loss was significantly more frequent than SPCS1. In addition, the 2 subtypes argue contrasting drug sensitivity profiles in clinical specimens and matched cell lines. Finally, we constructed a prognostic risk model consisted of each subtype's leading biomarkers, which exerted a satisfied performance for ccRCC patients. ConclusionSenescence regulator-related signature could modify functional pathways and tumor immune microenvironment by genome mutation and pathway interaction. Senescence regulator-related molecular subtype strengthen the understanding of ccRCC’ characterization and guide clinical treatment. Targeting senescence regulators may be regard as a proper way in ccRCC.

TICRR serves as a prognostic biomarker in lung adenocarcinoma with implications in RNA epigenetic modification, DDR pathway, and RNA metabolism.

TOPBP1 interacting checkpoint and replication regulator (TICRR), a hub gene of the Cdk2-mediated initiation step of DNA replication, has been shown an essential role in tumorigenesis by accelerating the DNA replication of tumor cells. RT-qPCR was used to detect the mRNA expression of TICRR in LUAD tumors and adjacent normal tissues. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database of LUAD were acquired to analyze the critical role of TICRR expression in survival prognosis and clinicopathology characters in LUAD. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were performed using the R package. The correlation of TICRR expression with immune cellinfiltration, RNA epigenetic modification, DNA damage repair (DDR) pathway, and cell metabolism of LUAD was further explored to verify significant conclusions. TICRR was significantly upregulated in most cancer types, including LUAD, lung squamous cell carcinoma (LUSC), and others. Cox regression analysis indicated the overexpression of TICRR was associated with poor survival in several cancers. In LUAD, TICRR expression was positively correlated with tumor stage and was increased in smoking, male, and high tumor mutational burden (TMB) patients. Enrichment analysis revealed that TICRR could influence tumor proliferation and prognosis via activating pathways involving cell cycle, DNA repair, DNA replication, cysteine metabolism, oxidative phosphorylation, and ubiquitin-mediated proteolysis pathways. Interestingly, high TICRR expression correlated with DDR pathway signature (34 genes), 37 m6A/m5C regulated genes, and some metabolism-regulated genes. Silencing the TICRR gene affects cysteine metabolism and modifies cancer-related pathways, with decreased cell cycle and increased B/T cell receptor signaling. Our TICRR risk model accurately predicts LUAD patient prognosis, validated across GEO datasets, and is integrated with clinical characteristics via a nomogram, facilitating personalized treatment strategies and enhancing patient management. Taken together, TICRR has emerged as a promising prognostic biomarker in lung adenocarcinoma (LUAD), with implications in immune activation, cell cycle regulation, RNA modification, and tumor energy metabolism. These findings suggest that TICRR could serve as a viable therapeutic target and a reliable prognostic indicator for LUAD.

Integrative transcriptome and metabolome analysis of fluoride exposure induced developmental neurotoxicity in mouse brain

Fluoride could cause developmental neurotoxicity and significantly affect the intelligence quotient (IQ) of children. However, the systematic mechanism of neuronal damage caused by excessive fluoride administration in offspring is largely unknown. Here, we present a comprehensive integrative transcriptome and metabolome analysis to study the mechanism of developmental neurotoxicity caused by chronic fluoride exposure. Comparing the different doses of fluoride treatments in two generations revealed the exclusive signature of metabolism pathways and gene expression profiles. In particular, neuronal development and synaptic ion transport are significantly altered at the gene expression and metabolite accumulation levels for both generations, which could act as messengers and enhancers of fluoride-induced systemic neuronal injury. Choline and arachidonic acid metabolism, which highlighted in the integrative analysis, exhibited different regulatory patterns between the two generations, particularly for synaptic vesicle formation and inflammatory factor transport. It may suggest that choline and arachidonic acid metabolism play important roles in developmental neurotoxic responses for offspring mice. Our study provides comprehensive insights into the metabolomic and transcriptomic regulation of fluoride stress responses in the mechanistic explanation of fluoride-induced developmental neurotoxicity.

Targeting Mitochondrial Apoptotic Priming State to Personalize Therapy for Relapsed Acute Myeloid Leukemia

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivo acquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured via dynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models. Using unbiased whole transcriptomic analysis of naïve and resistant PDXs (n=78 samples), we found common pathways of resistance where positive enrichment was observed for cytokine-cytokine receptor interactions, xenobiotics biodegradation, metabolic pathways, Hippo signaling, and Ras signaling in resistant compared to treatment-naïve PDXs. Conversely, mechanisms relating to DNA repair and replication, such as base excision repair, mismatch repair, and homologous recombination, were negatively enriched. Moreover, pathways associated with specific drug resistance were also observed. One of the known multi-drug resistance mechanisms to chemotherapy drugs in AML is increased expression of efflux pumps from the superfamily of ATP binding cassette. We found enrichment in ABC transporter family pathway signatures in all 3 PDX models, DFAM-61786, DFAM-15354, and DFAM-61345, resistant to quizartinib, birinapant, and JQ-1 and validated higher drug-efflux activity in quizartinib and birinapant resistant model using calcein-AM efflux assay. We finally tested whether DBP could predict in vivo response of patient myeloblasts in PDX models. We tested in vivo sensitivity to 5 drugs of disparate mechanisms of action: birinapant and LCL-161 (SMAC mimetics), JQ-1 (BRD-4 inhibitor), venetoclax (BCL-2 antagonist), and quizartinib (FLT-3 inhibitor) in 4-9 different PDX models each. Using ex vivo DBP assay, we identified drugs that prime not only treatment naïve PDXs but also R/R PDXs. We identified persistent in vivo activity in a drug-specific manner, with BH3 mimetics and HDAC inhibitors showing overlapping activity across different drug-resistant models. Overall, we demonstrate that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Further, we find that, even in the context of a multiply-resistant PDX model, DBP can still identify therapeutic vulnerabilities that can be efficaciously exploited in vivo.

Cord blood DNA methylation signatures associated with preeclampsia are enriched for cardiovascular pathways: insights from the VDAART trial

Preeclampsia has been associated with maternal epigenetic changes, in particular DNA methylation changes in the placenta. It has been suggested that preeclampsia could also cause DNA methylation changes in the neonate. We examined DNA methylation in relation to gene expression in the cord blood of offspring born to mothers with preeclampsia. This study included 128 mother-child pairs who participated in the Vitamin D Antenatal Asthma Reduction Trial (VDAART), where assessment of preeclampsia served as secondary outcome. We performed an epigenome-wide association study of preeclampsia and cord blood DNA methylation (Illumina 450K chip). We then examined gene expression of the same subjects for validation and replicated the gene signatures in independent DNA methylation datasets. Lastly, we applied functional enrichment and network analyses to identify biological pathways that could potentially be involved in preeclampsia. In the cord blood samples (n=128), 263 CpGs were differentially methylated (FDR <0.10) in preeclampsia (n=16), of which 217 were annotated. Top pathways in the functional enrichment analysis included apelin signaling pathway and other endothelial and cardiovascular pathways. Of the 217 genes, 13 showed differential expression (p's<0.001) in preeclampsia and 11 had been previously related to preeclampsia (p's<0.0001). These genes were linked to apelin, cGMP and Notch signaling pathways, all having a role in angiogenic process and cardiovascular function. Preeclampsia is related to differential cord blood DNA methylation signatures of cardiovascular pathways, including the apelin signaling pathway. The association of these cord blood DNA methylation signatures with offspring's long-term morbidities due to preeclampsia should be further investigated. VDAART is funded by National Heart, Lung, and Blood Institute grants of R01HL091528 and UH3OD023268. HMK is supported by Jane and Aatos Erkko Foundation, Paulo Foundation, and the Pediatric Research Foundation. HM is supported by K01 award from NHLBI (1K01HL146977-01A1). PK is supported by K99HL159234 from NIH/NHLBI.

Reactome pathway analysis from whole-blood transcriptome reveals unique characteristics of systemic sclerosis patients at the preclinical stage.

This study aims to characterize differential expressed pathways (DEP) in subjects with preclinical systemic sclerosis (PreSSc) characterized uniquely by Raynaud phenomenon, specific autoantibodies, and/or capillaroscopy positive for scleroderma pattern. Whole-blood samples from 33 PreSSc with clinical prospective data (baseline and after 4 years of follow-up) and 16 matched healthy controls (HC) were analyzed for global gene expression transcriptome analysis via RNA sequencing. Functional Analysis of Individual Microarray Expression method annotated Reactome individualized pathways. ANOVA analysis identified DEP whose predictive capability were tested in logistic regression models after extensive internal validation. At 4 years, 42.4% subjects progressed (evolving PreSSc), while the others kept stable PreSSc clinical features (stable PreSSc). At baseline, out of 831 pathways, 541 DEP were significant at a false discovery rate <0.05, differentiating PreSSc versus HC with an AUROC = 0.792 ± 0.242 in regression models. Four clinical groups were identified via unsupervised clustering (HC, HC and PreSSc with HC-like features, PreSSc and HC with PreSSc-like features, and PreSSc). Biological signatures changed with disease progression while remaining unchanged in stable subjects. The magnitude of change was related to the baseline cluster, yet no DEP at baseline was predictive of progression. Disease progression was mostly related to changes in signal transduction pathways especially linked to calcium-related events and inositol 1,4,5-triphosphate metabolism. PreSSc had distinguished Reactome pathway signatures compared to HC. Progression to definite SSc was characterized by a shift in biological fingertips. Calcium-related events promoting endothelial damage and vasculopathy may be relevant to disease progression.

Epigenetic Control of B Cell Autoimmunity By Jmjd1c

Emerging evidence underscores the importance of e pigenetic modifications in controlling B-cell development and function. While Jmjd1c, a member of the l ysine - specific histone demethylase 3 subfamily, is known to regulate the self-renewal of normal hematopoietic stem cells and leukemia stem cells, its impact on B cell biology is unknown. To explore Jmjd1c's role in B cells, we developed B-cell-specific Jmjd1c-deficient mice through crossbreeding Jmjd1c “floxed” mice with B cell-specific Mb1-Cre mice. The absence of Jmjd1c did not impact B-cell development, but it did lead to significant alterations in basal B cell antibody production. Specifically, we observed skewed isotypes from IgG2c and IgG3 to IgG1 and IgG2b. Additionally, Jmjd1c deficiency affected antigen-specific immune responses upon T-dependent and T-independent antigen challenges, implying a crucial role of Jmjd1c in B cell function. Furthermore, auto-antibody array assays revealed increased self-reactive antibody production in Jmjd1c-deficient animals, indicating the role of Jmjd1c in regulating self-reactive B cells. At a cellular level, Jmjd1c-deficient B cells showed elevated expression of CD86, a canonical marker of B cell activation. Moreover, these B cells exhibited increased BCR-induced cell proliferation, suggesting hyperactivity to antigens, including self-antigens. Notably, Jmjd1c deficiency induced systemic autoimmune disorder in a BM12-induced systemic lupus erythematosus (SLE) animal model, resulting in amplified auto-antibody production and CD86 expression. To investigate the underlying molecular mechanisms, RNA sequencing analysis demonstrated upregulation of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes in Jmjd1c-deficient B cells upon anti-IgM stimulation, consistent with their hyperresponsiveness. Of particular interest, we observed upregulation of genes encoding the 26S proteasome complex, a pivotal regulator of diverse cellular processes including NF-κB pathway activation and self-antigen presentation. To explore the epigenetic aspect of these genes, we employed CUT&amp;Tag sequencing and found that Jmjd1c deficiency correlated with increased levels of H3K36me1, a marker of active chromatin and gene transcription. Moreover, the H3K36me1 levels of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes were significantly augmented in Jmjd1c-deficient B cells upon anti-IgM stimulation. Notably, 26S proteasome subunit genes that were upregulated in Jmjd1c-deficient B cells exhibited higher H3K36me1 modification at the promoter regions. Collectively, our study provides valuable insights into the epigenetic control of B cell autoimmunity through the regulation of H3K36 mono-methylation by Jmjd1c. This mechanism plays a critical role in B cell activation and function, including the regulation of key genes such as those encoding the 26S proteasome complex.

Genomic Characterization of Low and High Grade Cervical Intraepithelial Neoplasia in Comparison to Cervical Cancer

Screening reduces incidence of cervical cancer (CC) through identification and treatment of cervical intraepithelial neoplasia (CIN). Triage of CIN must balance between overtreatment versus progression as invasive therapies increase the risk of side effects including obstetric complications. The majority of CIN 1-2 lesions regress and a subset of CIN 3 lesions progress, however, due to inability to differentiate lesions likely to progress, the majority of CIN 2-3 lesions are treated with resection. Improved genomic and molecular identifiers of disease represent an urgent unmet clinical need. Using next generation sequencing of a targeted exome panel of 1109 genes (previously validated), we characterized somatic mutations in 36 CIN (14 CIN 1, 11 CIN 2, 11 CIN 3) and 13 CC samples. Sequencing of CIN samples was performed on exfoliated cervical cells. CIN diagnosis and grade was confirmed on biopsy. Mutation profiles between CIN grades and CC were compared to identify genomic patterns that distinguish these groups. Across the 49 samples sequenced, we identified a total of 5142 somatic mutations, including 2178 missense, 2522 synonymous, 171 nonsense, 62 splice site, 135 inframe indels, and 74 frameshift mutations. The mutation frequency was significantly higher in CC vs CIN1-3 (Table), p<0.01 (Wilcoxon signed-rank test). The difference in mutation frequency and type (Table) was not significant between CIN grades 1, 2 and 3 (p = 0.07, Kruskal-Wallis test). Cancer related pathway signatures were analyzed for the percentage of CC vs CIN samples with at least one altered variant as follows: RTK-RAS (92% vs 31%), PI3K (92% vs 17%), NOTCH (100% vs 39%), WNT (85% vs 14%) and cell cycle (53% vs 3%). Ninety percent of CC samples versus only 30% of CINs had nonsynonymous variants in the RTK-RAS and NOTCH pathways as well as the PI3K pathway which is implicated as a late event in cervical carcinogenesis. We observed recurrent missense variants in ABL1, IGF1R, TSC2 in the CIN2, CIN3 and CC samples, particularly in genes that belong to the RTK-RAS, PI3K, NOTCH pathway signatures. Potential driver mutations in EGFR, PIK3CA, ERBB4, MTOR, CSF1R genes were exclusive to CC samples. Our results show that there is a clear distinction of mutational burden and type between cervical cancer and CIN1-3. However, the pathologic grading of CIN is not consistently associated with dynamic changes in overall mutation burden, type, or specific genes with escalating CIN grade. This data supports the hypothesis that late genetic events in CIN accompany a transition to invasive cervical cancer.