Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin.

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.

Exhausted CD4+ T Cells during Malaria Exhibit Reduced mTORc1 Activity Correlated with Loss of T-bet Expression.

CD4+ T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4+ T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (Ag-exp) CD4+ T cell exhaustion during Plasmodium yoelii nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4+ T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted Ag-expCD4+ T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4+ T cells. Consistent with this, Ag-expTh1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) Ag-expT cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4+ T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4+ T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in Ag-expCD4+ T cells but that reduction in mTOR activity may not directly underpin Ag-expTh1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4+ T cells during malaria infection and other chronic conditions.

Physico-chemical fingerprinting of RNA genes.

We advance here a novel concept for characterizing different classes of RNA genes on the basis of physico-chemical properties of DNA sequences. As knowledge-based approaches could yield unsatisfactory outcomes due to limitations of training on available experimental data sets, alternative approaches that utilize properties intrinsic to DNA are needed to supplement training based methods and to eventually provide molecular insights into genome organization. Based on a comprehensive series of molecular dynamics simulations of Ascona B-DNA consortium, we extracted hydrogen bonding, stacking and solvation energies of all combinations of DNA sequences at the dinucleotide level and calculated these properties for different types of RNA genes. Considering ∼7.3 million mRNA, 255 524 tRNA, 40 649 rRNA (different subunits) and 5250 miRNA, 3747 snRNA, gene sequences from 9282 complete genome chromosomes of all prokaryotes and eukaryotes available at NCBI, we observed that physico-chemical properties of different functional units on genomic DNA differ in their signatures.

A statistical thermodynamic model for investigating the stability of DNA sequences from oligonucleotides to genomes.

We describe the development and testing of a simple statistical mechanics methodology for duplex DNA applicable to sequences of any composition and extensible to genomes. The microstates of a DNA sequence are modeled in terms of blocks of basepairs that are assumed to be fully closed (paired) or open. This approach generates an ensemble of bubblelike microstates that are used to calculate the corresponding partition function. The energies of the microstates are calculated as additive contributions from hydrogen bonding, basepair stacking, and solvation terms parameterized from a comprehensive series of molecular dynamics simulations including solvent and ions. Thermodynamic properties and nucleotide stability constants for DNA sequences follow directly from the partition function. The methodology was tested by comparing computed free energies per basepair with the experimental melting temperatures of 60 oligonucleotides, yielding a correlation coefficient of -0.96. The thermodynamic stability of genic/nongenic regions was tested in terms of nucleotide stability constants versus sequence for the Escherichia coli K-12 genome. It showed clear differentiation of the genes from promoters and captures genic regions with a sensitivity of 0.94. The statistical thermodynamic model presented here provides a seemingly new handle on the challenging problem of interpreting genomic sequences.

Longitudinal surveillance of serum titanium ion levels in patients with indigenous 3D printed total temporomandibular joint replacement.

The purpose of this longitudinal study was to surveil the serum titanium ion levels at various time intervals in patients with indigenous 3D-printed total temporomandibular joint replacement (TMJ TJR). The study was conducted on 11 patients (male: 8; female: 3) who had undergone unilateral or bilateral TMJ TJR. Blood samples were drawn preoperatively (T0), 3 months (T1), 6 months (T2), and 1 year (T3) postoperatively. Data were analyzed and a p value of < 0.05 was considered statistically significant. The mean serum titanium ion levels at T0, T1, T2, and T3 was 9.34 ± 8.70 µg/L (mcg/L), 35.97 ± 20.27 mcg/L, 31.68 ± 17.03 mcg/L, and 47.91 ± 15.47 mcg/L respectively. The mean serum titanium ion levels increased significantly at T1 (p = 0.009), T2 (p = 0.032), and T3 (p = 0.00) interval. There was no significant difference between unilateral and bilateral groups. Serum titanium ion continued to show increased levels till the last follow-up of 1 year. These initial serum titanium ion levels increase is due to the initial wear phase of the prosthesis which manifests over 1 year. Further studies with large sample sizes and long-term follow-ups are required to see the deleterious effect if any on the TMJ TJR.

Cross-Sectional Study of Serum Metal Ions in Patients With Metal Implants in the Maxillofacial Region.

Purpose: The purpose of the present study was to measure the serum metal ion levels (titanium, cobalt, chromium) in patients who have metal implants in the maxillofacial region. Methods: The investigators implemented a cross sectional study on patients treated with procedures requiring metal implants for management of maxillofacial trauma, fixation for orthognathic surgery, and total temporomandibular joint replacement (TJR). Inductively coupled plasma mass spectrometry was used as an analytical method to detect metal ions in serum samples. Results: The study comprised of 50 patients who were divided into 4 groups- group I- total TJR (n = 18), group II- orthognathic (n = 8), group III- trauma (n = 8), and group IV- control (n = 16). The mean values of metal ions level were raised than the control group. Conclusion: The present study's results suggest a rise in serum metal ion levels after the metal implantation in maxillofacial region. None of the patients had any abnormal signs and symptoms due to raised metal levels. Further studies are warranted to correlate the serum metal ion levels and their clinical relevance.

DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain gene repression and is essential for cell differentiation. In low-grade endometrial stromal sarcoma (LG-ESS), the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. We show that JAZF1-SUZ12 dysregulates PRC2 composition, genome occupancy, histone modification, gene expression, and cell differentiation. Loss of the SUZ12 N terminus in the fusion protein abrogates interaction with specific PRC2 accessory factors, reduces occupancy at PRC2 target genes, and diminishes H3K27me3. Fusion to JAZF1 increases H4Kac at PRC2 target genes and triggers recruitment to JAZF1 binding sites during cell differentiation. In human endometrial stromal cells, JAZF1-SUZ12 upregulated PRC2 target genes normally activated during decidualization while repressing genes associated with immune clearance, and JAZF1-SUZ12-induced genes were also overexpressed in LG-ESS. These results reveal defects in chromatin regulation, gene expression, and cell differentiation caused by JAZF1-SUZ12 that may underlie its role in oncogenesis.

Increased Immune-Regulatory Receptor Expression on Effector T Cells as Early Indicators of Relapse Following Autologous Stem Cell Transplantation for Multiple Myeloma.

The benefit of autologous stem cell transplantation (ASCT) in newly diagnosed myeloma patients, apart from supporting high dose chemotherapy, may include effects on T cell function in the bone marrow (BM). We report our exploratory findings on marrow infiltrating T cells early post-ASCT (day+100), examining phenotype and T cell receptor (TCR) repertoire, seeking correlations with timing of relapse. Compared to healthy donors (HD), we observed an increase in regulatory T cells (CD4+FoxP3+, Tregs) with reduction in CD4 T cells, leading to lower CD4:8 ratios. Compared to paired pre-treatment marrow, both CD4 and CD8 compartments showed a reduction in naïve, and increase in effector memory subsets, suggestive of a more differentiated phenotype. This was supported by increased levels of several immune-regulatory and activation proteins (ICOS, PD-1, LAG-3, CTLA-4 and GzmB) when compared with HD. Unsupervised analysis identified a patient subgroup with shorter PFS (p=0.031) whose BM contained increased Tregs, and higher immune-regulatory markers (ICOS, PD-1, LAG-3) on effector T cells. Using single feature analysis, higher frequencies of marrow PD-1+ on CD4+FoxP3- cells and Ki67+ on CD8 cells were independently associated with early relapse. Finally, studying paired pre-treatment and post-ASCT BM (n=5), we note reduced abundance of TCR sequences at day+100, with a greater proportion of expanded sequences indicating a more focused persistent TCR repertoire. Our findings indicate that, following induction chemotherapy and ASCT, marrow T cells demonstrate increased activation and differentiation, with TCR repertoire focusing. Pending confirmation in larger series, higher levels of immune-regulatory proteins on T cell effectors at day+100 may indicate early relapse.

Inhibition of Histone H3K27 Demethylases Inactivates Brachyury (TBXT) and Promotes Chordoma Cell Death.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

Brain microenvironment-driven resistance to immune and targeted therapies in acral melanoma.

BACKGROUND: Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies. METHODS: We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. RESULTS: Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. CONCLUSION: Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments.

Ex vivo culture of cells derived from circulating tumour cell xenograft to support small cell lung cancer research and experimental therapeutics.

BACKGROUND AND PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with median survival of <2 years. Tumour biopsies for research are scarce, especially from extensive-stage patients, with repeat sampling at disease progression rarely performed. We overcame this limitation for relevant preclinical models by developing SCLC circulating tumour cell derived explants (CDX), which mimic the donor tumour pathology and chemotherapy response. To facilitate compound screening and identification of clinically relevant biomarkers, we developed short-term ex vivo cultures of CDX tumour cells. EXPERIMENTAL APPROACH: CDX tumours were disaggregated, and the human tumour cells derived were cultured for a maximum of 5 weeks. Phenotypic, transcriptomic and pharmacological characterization of these cells was performed. KEY RESULTS: CDX cultures maintained a neuroendocrine phenotype, and most changes in the expression of protein-coding genes observed in cultures, for up to 4 weeks, were reversible when the cells were re-implanted in vivo. Moreover, the CDX cultures exhibited a similar sensitivity to chemotherapy compared to the corresponding CDX tumour in vivo and were able to predict in vivo responses to therapeutic candidates. CONCLUSIONS AND IMPLICATIONS: Short-term cultures of CDX provide a tractable platform to screen new treatments, identify predictive and pharmacodynamic biomarkers and investigate mechanisms of resistance to better understand the progression of this recalcitrant tumour.

G-tract RNA removes Polycomb repressive complex 2 from genes.

Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.

Author Correction: G-tract RNA removes Polycomb repressive complex 2 from genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer.

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient-derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC.Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression.Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted.Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell-based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153-64. ©2018 AACR.

Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models.

Patient-derived xenograft (PDX) and circulating tumor cell-derived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNA- and RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX- and CDX-derived whole-exome and RNA-Seq data.Implications: This study describes a sensitive method to identify contaminating host reads in xenograft and explant DNA- and RNA-Seq data and is applicable to other forms of deep sequencing. Mol Cancer Res; 15(8); 1012-6. ©2017 AACR.

Disinfecting efficacy of three chemical disinfectants on contaminated diagnostic instruments: A randomized trial.

CONTEXT: Cross infection remains one of the major challenges in the dental profession, especially in field settings. Transmission of hepatitis B, hepatitis C, and human immunodeficiency virus have raised a major concern for patients and dental staff. These risks can be eliminated by effective sterilization and disinfection techniques. AIM: The aim was to compare the disinfecting efficacy of three chemical disinfectants on contaminated diagnostic instruments. SETTINGS AND DESIGN: This was a randomized, cross over trial conducted among three participants selected from a research laboratory, Bhopal, Madhya Pradesh, India. MATERIALS AND METHODS: The study participants were examined 4 times on different days. Each time, the coded mouth mirrors of different make were used, and the disinfection was accomplished using coded disinfectants. The reduction in total viable count was compared between the three groups (2% glutaraldehyde, 6% hydrogen peroxide (H2O2) and 99.9% ethyl alcohol) with distilled water as negative control and autoclaving as a positive control. Furthermore, the predisinfection count was compared between the instruments of different make. STATISTICAL ANALYSIS USED: Statistical analysis was performed using paired t-test and One-way ANOVA. The statistical significance was fixed at 0.05. RESULTS: Autoclaved instruments resulted in complete elimination of viable micro-organisms. Maximum reduction in microbial load was observed after disinfection with H2O2 followed by glutaraldehyde, ethyl alcohol and distilled water in descending order. Furthermore, maximum microbial contamination was recorded on locally manufactured mirrors, while standard plain mirrors showed least contamination. CONCLUSIONS: Although, a significant reduction in total viable count was observed with all the disinfectants evaluated in the present study, none of the disinfectants was successful in completely eliminating the viable micro-organisms.

DNA-energetics-based analyses suggest additional genes in prokaryotes.

We present here a novel methodology for predicting new genes in prokaryotic genomes on the basis of inherent energetics of DNA. Regions of higher thermodynamic stability were identified, which were filtered based on already known annotations to yield a set of potentially new genes. These were then processed for their compatibility with the stereo-chemical properties of proteins and tripeptide frequencies of proteins in Swissprot data, which results in a reliable set of new genes in a genome. Quite surprisingly, the methodology identifies new genes even in well-annotated genomes. Also, the methodology can handle genomes of any GC-content, size and number of annotated genes.

A phenomenological model for predicting melting temperatures of DNA sequences.

We report here a novel method for predicting melting temperatures of DNA sequences based on a molecular-level hypothesis on the phenomena underlying the thermal denaturation of DNA. The model presented here attempts to quantify the energetic components stabilizing the structure of DNA such as base pairing, stacking, and ionic environment which are partially disrupted during the process of thermal denaturation. The model gives a Pearson product-moment correlation coefficient (r) of approximately 0.98 between experimental and predicted melting temperatures for over 300 sequences of varying lengths ranging from 15-mers to genomic level and at different salt concentrations. The approach is implemented as a web tool (www.scfbio-iitd.res.in/chemgenome/Tm_predictor.jsp) for the prediction of melting temperatures of DNA sequences.