Many functional analysis tools have been developed to extract functional and mechanistic insight from bulk transcriptome data. With the advent of single-cell RNA sequencing (scRNA-seq), it is in ...principle possible to do such an analysis for single cells. However, scRNA-seq data has characteristics such as drop-out events and low library sizes. It is thus not clear if functional TF and pathway analysis tools established for bulk sequencing can be applied to scRNA-seq in a meaningful way.
To address this question, we perform benchmark studies on simulated and real scRNA-seq data. We include the bulk-RNA tools PROGENy, GO enrichment, and DoRothEA that estimate pathway and transcription factor (TF) activities, respectively, and compare them against the tools SCENIC/AUCell and metaVIPER, designed for scRNA-seq. For the in silico study, we simulate single cells from TF/pathway perturbation bulk RNA-seq experiments. We complement the simulated data with real scRNA-seq data upon CRISPR-mediated knock-out. Our benchmarks on simulated and real data reveal comparable performance to the original bulk data. Additionally, we show that the TF and pathway activities preserve cell type-specific variability by analyzing a mixture sample sequenced with 13 scRNA-seq protocols. We also provide the benchmark data for further use by the community.
Our analyses suggest that bulk-based functional analysis tools that use manually curated footprint gene sets can be applied to scRNA-seq data, partially outperforming dedicated single-cell tools. Furthermore, we find that the performance of functional analysis tools is more sensitive to the gene sets than to the statistic used.
Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be ...modulated by anticancer agents, altering immunity. However, understanding the peptide repertoire's response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking normalization controls. We describe an experimental platform that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform immunotherapy design. Applying this platform in melanoma cell lines to profile the immunopeptidome response to CDK4/6 inhibition and interferon-γ - known modulators of antigen presentation - uncovers treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.
Acidosis is a fundamental feature of the tumor microenvironment, which directly regulates tumor cell invasion by affecting immune cell function, clonal cell evolution, and drug resistance. Despite ...the important association of tumor microenvironment acidosis with tumor cell invasion, relatively little is known regarding which areas within a tumor are acidic and how acidosis influences gene expression to promote invasion. Here, we injected a labeled pH-responsive peptide to mark acidic regions within tumors. Surprisingly, acidic regions were not restricted to hypoxic areas and overlapped with highly proliferative, invasive regions at the tumor-stroma interface, which were marked by increased expression of matrix metalloproteinases and degradation of the basement membrane. RNA-seq analysis of cells exposed to low pH conditions revealed a general rewiring of the transcriptome that involved RNA splicing and enriched for targets of RNA binding proteins with specificity for AU-rich motifs. Alternative splicing of Mena and CD44, which play important isoform-specific roles in metastasis and drug resistance, respectively, was sensitive to histone acetylation status. Strikingly, this program of alternative splicing was reversed
and
through neutralization experiments that mitigated acidic conditions. These findings highlight a previously underappreciated role for localized acidification of tumor microenvironment in the expression of an alternative splicing-dependent tumor invasion program. SIGNIFICANCE: This study expands our understanding of acidosis within the tumor microenvironment and indicates that acidosis induces potentially therapeutically actionable changes to alternative splicing.
Although elevated levels of reactive oxygen species (ROS) have been observed in cancer cells and cancer cells aberrantly proliferate, it is not known whether the level of reactive oxygen species and ...the accumulation of oxidative damage to macromolecules vary across the cell cycle. Here, we measure the prevalence of reactive oxygen species and of biomolecule oxidation across the cell cycle in freely cycling cancer cells. We report that reactive oxygen species vary during the cell cycle and peak in mitosis, resulting in mitotic accumulation of oxidized protein cysteine residues. Prolonged mitotic arrest further increased the levels of ROS and the abundance of oxidatively damaged biomolecules, including cysteine-sulfenic-acid-containing proteins and 8-oxoguanine. These finding suggest that mitotic arrest agents may enhance the effects of ROS-dependent anticancer therapies.
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•ROS varies with the cell cycle in freely cycling cancer cells•ROS levels peak in G2 and mitosis•Oxidation of biomolecules is maximal in mitosis•Mitotic arrest further enhances ROS and oxidative damage to proteins and nucleotides
We studied connections between ROS and the cell cycle in unsynchronized cancer cells and using multiple independent assays found that ROS and oxidative damage to biomolecules are highest in mitosis and can be further enhanced by mitotic arrest.
Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in ...mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO
)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO
Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO
-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD.
Accumulating public health and epidemiological literature support the hypothesis that arsenic in drinking water or food affects the brain adversely.
Experiments on the consequences of nitric oxide ...(NO) formation in neuronal cell culture and mouse brain were conducted to probe the mechanistic pathways of nitrosative damage following arsenic exposure.
After exposure of mouse embryonic neuronal cells to low doses of sodium arsenite (SA), we found that Ca
was released leading to the formation of large amounts of NO and apoptosis. Inhibition of NO synthase prevented neuronal apoptosis. Further, SA led to concerted S-nitrosylation of proteins significantly associated with synaptic vesicle recycling and acetyl-CoA homeostasis. Our findings show that low-dose chronic exposure (0.1-1 ppm) to SA in the drinking water of mice led to S-nitrosylation of proteomic cysteines. Subsequent removal of arsenic from the drinking water reversed the biochemical alterations.
This work develops a mechanistic understanding of the role of NO in arsenic-mediated toxicity in the brain, incorporating Ca
release and S-nitrosylation as important modifiers of neuronal protein function.
Alzheimer’s disease (AD) is characterized by the appearance of amyloid‐β plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have ...quantified the phosphoproteome of the CK‐p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec‐F which was upregulated on a subset of reactive microglia. The human paralog Siglec‐8 was also upregulated on microglia in AD. Siglec‐F and Siglec‐8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV‐2 cell line and human stem cell‐derived microglia models. Siglec‐F overexpression activates an endocytic and pyroptotic inflammatory response in BV‐2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine‐based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV‐2 cells. Collectively, our results point to an important role for mouse Siglec‐F and human Siglec‐8 in regulating microglial activation during neurodegeneration.
SYNOPSIS
Phosphoproteomics analyses of CK‐p25, 5XFAD, and Tau P301S mouse models of neurodegeneration identify dysregulated signaling networks associated with Alzheimer’s disease pathologies.
Phosphorylation sites on Siglec‐F and Inpp5d are upregulated across three mouse models of Alzheimer’s disease.
Expression of Siglec‐F and its human paralog Siglec‐8 is increased in reactive microglia.
Siglec‐F and Siglec‐8 can be upregulated by interferon gamma in BV‐2 cell line and human stem cell‐derived microglia models.
Overexpression of Siglec‐F and Siglec‐8 in BV‐2 cells drives an endocytic and pyroptotic inflammatory response.
Phosphoproteomics analyses of CK‐p25, 5XFAD, and Tau P301S mouse models of neurodegeneration identify dysregulated
signaling networks associated with Alzheimer’s disease pathologies.
Although immune checkpoint blockade (ICB) has strong clinical benefit for treating some tumor types, it fails in others, indicating a need for additional modalities to enhance the ICB effect. Here, ...we identified one such modality by using DNA damage to create a live, injured tumor cell adjuvant. Using an optimized ex vivo coculture system, we found that treating tumor cells with specific concentrations of etoposide, mitoxantrone, or doxorubicin markedly enhanced dendritic cell–mediated T cell activation. These immune-enhancing effects of DNA damage did not correlate with immunogenic cell death markers or with the extent of apoptosis or necroptosis; instead, these effects were mediated by live injured cells with activation of the DNA-PK, ATR, NF-κB, p38 MAPK, and RIPK1 signaling pathways. In mice, intratumoral injection of ex vivo etoposide–treated tumor cells in combination with systemic ICB (by anti-PD-1 and anti-CTLA4 antibodies) increased the number of intratumoral CD103
dendritic cells and circulating tumor-antigen–specific CD8
T cells, decreased tumor growth, and improved survival. These effects were absent in
mice and in mice in which the DNA-damaging drug was injected directly into the tumor, due to DNA damage in the immune cells. The combination treatment induced complete tumor regression in a subset of mice that were then able to reject tumor rechallenge, indicating that the injured cell adjuvant treatment induced durable antitumor immunological memory. These results provide a strategy for enhancing the efficacy of immune checkpoint inhibition in tumor types that do not respond to this treatment modality by itself.
RNA-binding proteins (RBPs) play critical roles in regulating gene expression by modulating splicing, RNA stability, and protein translation. Stimulus-induced alterations in RBP function contribute ...to global changes in gene expression, but identifying which RBPs are responsible for the observed changes remains an unmet need. Here, we present Transite, a computational approach that systematically infers RBPs influencing gene expression through changes in RNA stability and degradation. As a proof of principle, we apply Transite to RNA expression data from human patients with non-small-cell lung cancer whose tumors were sampled at diagnosis or after recurrence following treatment with platinum-based chemotherapy. Transite implicates known RBP regulators of the DNA damage response and identifies hnRNPC as a new modulator of chemotherapeutic resistance, which we subsequently validated experimentally. Transite serves as a framework for the identification of RBPs that drive cell-state transitions and adds additional value to the vast collection of publicly available gene expression datasets.
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•Transite nominates RBPs that influence gene expression from RNA expression datasets•Transite identifies enriched and depleted k-mers in differentially expressed genes•Transite identifies hnRNPC as a modulator of cisplatin resistance in human lung cancer•Transite is available online at https://transite.mit.edu and as an R package
Krismer et al. present a computational approach to identify RNA-binding proteins (RBPs) that modulate post-transcriptional control of gene expression using RNA expression data as inputs. By applying this approach to publicly available patient datasets, they identify and experimentally confirm that the RBP hnRNPC contributes to chemotherapy resistance in lung cancer.
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