Genomic profiling can provide prognostic and predictive information to guide clinical care. Biomarkers that reliably predict patient response to chemotherapy and immune checkpoint inhibition in ...gastric cancer are lacking. In this retrospective analysis, we use our machine learning algorithm NTriPath to identify a gastric-cancer specific 32-gene signature. Using unsupervised clustering on expression levels of these 32 genes in tumors from 567 patients, we identify four molecular subtypes that are prognostic for survival. We then built a support vector machine with linear kernel to generate a risk score that is prognostic for five-year overall survival and validate the risk score using three independent datasets. We also find that the molecular subtypes predict response to adjuvant 5-fluorouracil and platinum therapy after gastrectomy and to immune checkpoint inhibitors in patients with metastatic or recurrent disease. In sum, we show that the 32-gene signature is a promising prognostic and predictive biomarker to guide the clinical care of gastric cancer patients and should be validated using large patient cohorts in a prospective manner.
Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by ...pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.
Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation ...defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined.
We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry.
We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation.
Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.
Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA ...methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.
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•DNA methylation regulates mRNA alternative cleavage and polyadenylation•CTCF binds unmethylated CpG islands downstream of proximal poly(A) sites•CTCF subsequently recruits cohesin complex to form chromatin loops•Chromatin loops promote usage of proximal poly(A) sites in vitro and in vivo
The functions of DNA methylation outside of gene promoters are not fully understood. Nanavaty et al. found gene-body methylation to modulate transcriptome diversity through alternative cleavage and polyadenylation. In the absence of DNA methylation, CTCF and the cohesin complex orchestrate chromatin loop formation and promote proximal polyadenylation isoform expressions.
Abstract
The space radiation environment consists of multiple species of charged particles, including
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Si ions, that may impact brain function during and following missions. To develop biomarkers ...of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with
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Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following
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Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following
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Si ion irradiation could be used for the development of biomarker of the space radiation response.
BackgroundThe CAR T cell therapy initiated as a treatment for CD19+ malignant B cell has rapidly expanded to many other cancers including solid tumors. Among multiple key factors to improve efficacy ...while minimizing adverse effects, it is critical to understand the mechanisms of T cell exhaustion and harness it. Upregulation of TIGIT is a hallmark of exhausted T cells in non-responders among non-Hodgkin’s lymphoma (NHL) patients under CD19+ CAR T therapy.1 However, we have not paid attention to PBMC that can (in)directly interact with CAR+T cells and its synergistic role in the treatment result. Furthermore, it is desirable to identify transcriptional factors (TFs) facilitating the unexpected T cell status. Ultimately, we can target them with TIGIT for better outcomes.MethodsWe evaluate 27 scRNA-seq data for CAR+T (n=12) and its matched PBMC (n=15) in two sequential time points after the infusion from 8 relapsed/refractory NHL patients; 6 responders (R) and 2 non-responders (NR).1 Results TIGIT is upregulated in post-infusion PBMC samples. The TIGIT is overexpressed across CD8+T, NK, and most significantly Treg population. DEG analysis reveals that more NR cells activate TIGIT whereas more R cells activate CD226. Furthermore, NECTIN2 is mostly expressed in a monocyte subpopulation dominated by the NR group. CellphoneDB analysis3 demonstrates that KLRC1 in an inflammatory T population in CAR+T interacts with HLA-E detected across PBMC cells (potentially originated from malignant B cells). These suggest that more prevalent immune checkpoints in NR trigger immune evasion.4–7 Next, we investigate how the dysfunctional T state was acquired. A gene set analysis uncovers that it is due to chronic antigen exposure (CAE).2 NK-like T cell populations resemble dysfunctional tumor-infiltrated lymphocytes (TILs) under CAE. Among monocyte populations, the NR-dominant monocytes upregulate TFs represented into CAE TILs. A comprehensive TF analysis reveals that TSC22D3, ZFP36, and DUSP1 known for anti-inflammatory response8 are strikingly overexpressed in the NR group. A higher interaction of TSC22D3 with AP-1 proteins results in a lack of AP-1 to form a binding with NFAT required for T cell effectors. Even worse, NR patients upregulate NR4A2 expression. The gene potentially interferes with the binding of NFAT/AP-1 and remodels chromatin accessibility to facilitate T cell exhaustion.9 ConclusionsIn conclusion, dysregulated TSC22D3 and NR4A2 compromise the effector function of CAR+T, hijack AP-1 proteins, and impede eradicating the antigens, and thus CAR+T cells transit to exhausted T cells represented by overexpressed TIGIT.ReferencesJackson Z, Hong C, Schauner R, Dropulic B, Caimi PF, de Lima M, Giraudo MF, Gupta K, Reese JS, Hwang TH, Wald DN. Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma. Cancer Discov. 2022 Aug 5;12(8):1886–1903. doi: 10.1158/2159–8290.CD-21–1586.Good CR, Aznar MA, Kuramitsu S, Samareh P, Agarwal S, Donahue G, Ishiyama K, Wellhausen N, Rennels AK, Ma Y, Tian L, Guedan S, Alexander KA, Zhang Z, Rommel PC, Singh N, Glastad KM, Richardson MW, Watanabe K, Tanyi JL, O’Hara MH, Ruella M, Lacey SF, Moon EK, Schuster SJ, Albelda SM, Lanier LL, Young RM, Berger SL, June CH. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell. 2021 Dec 9;184(25):6081–6100.e26. doi: 10.1016/j.cell.2021.11.016. Epub 2021 Dec 2.Efremova M, Vento-Tormo M, Teichmann SA, Vento-Tormo R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat Protoc. 2020 Apr;15(4):1484–1506. doi: 10.1038/s41596–020-0292-x. Epub 2020 Feb 26.Chiang EY, Mellman I. TIGIT-CD226-PVR axis: advancing immune checkpoint blockade for cancer immunotherapy. J Immunother Cancer. 2022 Apr;10(4):e004711. doi: 10.1136/jitc-2022–004711.Borst L, van der Burg SH, van Hall T. The NKG2A-HLA-E Axis as a Novel Checkpoint in the Tumor Microenvironment. Clin Cancer Res. 2020 Nov 1;26(21):5549–5556. doi: 10.1158/1078–0432.CCR-19–2095. Epub 2020 May 14.Odagiu L, May J, Boulet S, Baldwin TA, Labrecque N. Role of the Orphan Nuclear Receptor NR4A Family in T-Cell Biology. Front Endocrinol (Lausanne). 2021 Feb 1;11:624122. doi: 10.3389/fendo.2020.624122.Mognol GP, Spreafico R, Wong V, Scott-Browne JP, Togher S, Hoffmann A, Hogan PG, Rao A, Trifari S. Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells. Proc Natl Acad Sci U S A. 2017 Mar 28;114(13):E2776-E2785. doi: 10.1073/pnas.1620498114. Epub 2017 Mar 10.Bereshchenko O, Migliorati G, Bruscoli S, Riccardi C. Glucocorticoid-Induced Leucine Zipper: A Novel Anti-inflammatory Molecule. Front Pharmacol. 2019 Mar 27;10:308. doi: 10.3389/fphar.2019.00308.Chen J, López-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, Yoshimura A, Scott-Browne JP, Rao A. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019 Mar;567(7749):530–534. doi: 10.1038/s41586–019-0985-x. Epub 2019 Feb 27.
To date, there has been no multi-omic analysis characterizing the intricate relationships between the intragastric microbiome and gastric mucosal gene expression in gastric carcinogenesis. Using ...multi-omic approaches, we provide a comprehensive view of the connections between the microbiome and host gene expression in distinct stages of gastric carcinogenesis (i.e., healthy, gastritis, cancer). Our integrative analysis uncovers various associations specific to disease states. For example, uniquely in gastritis, Helicobacteraceae is highly correlated with the expression of FAM3D, which has been previously implicated in gastrointestinal inflammation. In addition, in gastric cancer but not in adjacent gastritis, Lachnospiraceae is highly correlated with the expression of UBD, which regulates mitosis and cell cycle time. Furthermore, lower abundances of B cell signatures in gastric cancer compared to gastritis may suggest a previously unidentified immune evasion process in gastric carcinogenesis. Our study provides the most comprehensive description of microbial, host transcriptomic, and immune cell factors of the gastric carcinogenesis pathway.
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•Multi-omics finds genetic, microbial, and immunological links in gastric cancer•Helicobacteraceae was highly associated with the expression of inflammation genes•Pasteurellaceae and Lachnospiraceae were associated with cancer-related genes•B cell infiltration was prominent in gastritis tissues but not in gastric cancer
Cellular physiology; Microbiome; Omics
Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of ...dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.
Lenvatinib is a multitargeted tyrosine kinase inhibitor that is being tested in combination with immune checkpoint inhibitors to treat advanced gastric cancer; however, little data exists regarding ...the efficacy of lenvatinib monotherapy. Patient-derived xenografts (PDX) are established by engrafting human tumors into immunodeficient mice. The generation of PDXs may be hampered by growth of lymphomas. In this study, we compared the use of mice with different degrees of immunodeficiency to establish PDXs from a diverse cohort of Western gastric cancer patients. We then tested the efficacy of lenvatinib in this system.
PDXs were established by implanting gastric cancer tissue into NOD.Cg-Prkdc
Il2rg
/SzJ (NSG) or Foxn1
(nude) mice. Tumors from multiple passages from each PDX line were compared histologically and transcriptomically. PDX-bearing mice were randomized to receive the drug delivery vehicle or lenvatinib. After 21 days, the percent tumor volume change (%Δv
) was calculated.
23 PDX models were established from Black, non-Hispanic White, Hispanic, and Asian gastric cancer patients. The engraftment rate was 17% (23/139). Tumors implanted into NSG (16%; 18/115) and nude (21%; 5/24) mice had a similar engraftment rate. The rate of lymphoma formation in nude mice (0%; 0/24) was lower than in NSG mice (20%; 23/115; p < 0.05). PDXs derived using both strains maintained histologic and gene expression profiles across passages. Lenvatinib treatment (mean %Δv
: -33%) significantly reduced tumor growth as compared to vehicle treatment (mean %Δv
: 190%; p < 0.0001).
Nude mice are a superior platform than NSG mice for generating PDXs from gastric cancer patients. Lenvatinib showed promising antitumor activity in PDXs established from a diverse Western patient population and warrants further investigation in gastric cancer.
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