Aerobic glycolysis (the Warburg effect) is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses, including expression f of the proinflammatory ...cytokine interferon-γ (IFN-γ), via 3 untranslated region (3'UTR)-mediated mechanisms. Here, we show that lactate dehydrogenase A (LDHA) is induced in activated T cells to support aerobic glycolysis but promotes IFN-γ expression independently of its 3'UTR. Instead, LDHA maintains high concentrations of acetyl-coenzyme A to enhance histone acetylation and transcription of Ifng. Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells. These findings reveal an epigenetic mechanism by which aerobic glycolysis promotes effector T cell differentiation and suggest that LDHA may be targeted therapeutically in autoinflammatory diseases.
Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human ...leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.
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•Affinity-tagging protocol enables proteomic profiling of individual HLA-II alleles•Even in “hot” tumors, professional APCs—not cancer cells—drive HLA-II expression•Cellular localization influences which phagocytosed cancer proteins get presented•Machine-learning models for binding and processing improve HLA-II prediction
Despite their role in directing T cell responses, HLA-II epitopes remain difficult to predict, hindering their therapeutic potential. Abelin et al. develop proteomic strategies that resolve diverse HLA-II motifs and pinpoint tumor epitopes that are presented by professional APCs. These data enable improved HLA-II epitope prediction and therapeutic targeting.
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) ...for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various ...mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4
Ldha
mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4
Ldha
mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.
Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown
. ...Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression.
The initial next-generation sequencing technologies produced reads of 25 or 36 bp, and only from a single-end of the library sequence. Currently, it is possible to reliably produce 300 bp paired-end ...sequences for RNA expression analysis. While read lengths have consistently increased, people have assumed that longer reads are more informative and that paired-end reads produce better results than single-end reads. We used paired-end 101 bp reads and trimmed them to simulate different read lengths, and also separated the pairs to produce single-end reads. For each read length and paired status, we evaluated differential expression levels between two standard samples and compared the results to those obtained by qPCR.
We found that, with the exception of 25 bp reads, there is little difference for the detection of differential expression regardless of the read length. Once single-end reads are at a length of 50 bp, the results do not change substantially for any level up to, and including, 100 bp paired-end. However, splice junction detection significantly improves as the read length increases with 100 bp paired-end showing the best performance. We performed the same analysis on two ENCODE samples and found consistent results confirming that our conclusions have broad application.
A researcher could save substantial resources by using 50 bp single-end reads for differential expression analysis instead of using longer reads. However, splicing detection is unquestionably improved by paired-end and longer reads. Therefore, an appropriate read length should be used based on the final goal of the study.
Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER
) breast cancer. We identify that ...ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER
breast cancer. An epigenome CRISPR-CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the ER degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER-FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER
breast cancer.
High-accuracy mass spectrometry enables near comprehensive quantification of the components of the cellular proteomes, increasingly including their chemically modified variants. Likewise, large-scale ...libraries of quantified synthetic peptides are becoming available, enabling absolute quantification of chemically modified proteoforms, and therefore systems-level analyses of changes of their absolute abundance and stoichiometry. Existing computational methods provide advanced tools for mass spectral analysis and statistical inference, but lack integrated functions for quantitative analysis of post-translationally modified proteins and their modification stoichiometry.
Here, we develop ProteoModlR, a program for quantitative analysis of abundance and stoichiometry of post-translational chemical modifications across temporal and steady-state biological states. While ProteoModlR is intended for the analysis of experiments using isotopically labeled reference peptides for absolute quantitation, it also supports the analysis of labeled and label-free data, acquired in both data-dependent and data-independent modes for relative quantitation. Moreover, ProteoModlR enables functional analysis of sparsely sampled quantitative mass spectrometry experiments by inferring the missing values from the available measurements, without imputation. The implemented architecture includes parsing and normalization functions to control for common sources of technical variation. Finally, ProteoModlR's modular design and interchangeable format are optimally suited for integration with existing computational proteomics tools, thereby facilitating comprehensive quantitative analysis of cellular signaling.
ProteoModlR and its documentation are available for download at http://github.com/kentsisresearchgroup/ProteoModlR as a stand-alone R package.
The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we ...demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.
Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals ...temporal changes due to BRAF
. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAF
. Adult zebrafish expressing BRAF
in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAF
and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAF
-mediated transformation.
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