The approach to annotating a genome critically affects the number and accuracy of genes identified in the genome sequence. Genome annotation based on stringent gene identification is prone to ...underestimate the complement of genes encoded in a genome. In contrast, over-prediction of putative genes followed by exhaustive computational sequence, motif and structural homology search will find rarely expressed, possibly unique, new genes at the risk of including non-functional genes. We developed a two-stage approach that combines the merits of stringent genome annotation with the benefits of over-prediction. First we identify plausible genes regardless of matches with EST, cDNA or protein sequences from the organism (stage 1). In the second stage, proteins predicted from the plausible genes are compared at the protein level with EST, cDNA and protein sequences, and protein structures from other organisms (stage 2). Remote but biologically meaningful protein sequence or structure homologies provide supporting evidence for genuine genes. The method, applied to the Drosophila melanogaster genome, validated 1,042 novel candidate genes after filtering 19,410 plausible genes, of which 12,124 matched the original 13,601 annotated genes. This annotation strategy is applicable to genomes of all organisms, including human.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Background: Inhibition of immune-checkpoint targets including PD1 is clinically effective in a variety of cancers. However, only a subset of patients respond and complete response remains ...uncommon. To understand the mechanisms of response and resistance, recent studies have focused on neoantigens, copy-number alterations, and transcriptional signatures of tumor tissues collected from patients treated with immune-checkpoint inhibitors. Given the known role of metabolites in modulating immunity, we sought to understand how individual patients' metabolic activities adapt to PD1 immune checkpoint blockade and how they associate with therapeutic benefits. Methods: We profiled 106-202 metabolites in pre- and multiple on-treatment patient serum samples from three independent immunotherapy trials using liquid chromatography-mass spectrometry. These metabolites are involved in the metabolism of amino acids, nucleotides, nitrogen, and lipids, among others. Our study consisted of two Phase 1 trials (CA209-038 and -009) which included 78 patients with advanced melanoma and 91 patients with metastatic renal cell carcinoma (RCC) treated with nivolumab. RNASeq was performed on matched pre- and on-treatment tumor biopsies from the melanoma cohort. To investigate the generalizability of our results, we also performed metabolomics and serum specimens from a large randomized Phase 3 trial (CheckMate 025) with 743 RCC patients, among which 394 received nivolumab and 349 received everolimus. Results: During treatment with nivolumab, kynurenine, a product of tryptophan catabolism and the IDO/TDO genes, was the most significantly changed metabolite at week 4 and at week 6 compared to pre-treatment levels among melanoma patients (37% and 34% increase on average, q=1 × 10-10 and q=1 × 10-8 respectively, paired t-test). By using Kyn/Trp (Kynurenine/Tryptophan) as a metric indicating tryptophan-kynurenine conversion, we found that this ratio falls in a range spanning approximately 8-fold among these patients, suggesting prominent individual-to-individual differences. Specifically, 78% patients had any increases and 26.5% patients had increases above 50% at week 4. We confirmed this significant kynurenine up-regulation following nivolumab treatment in RCC patients in the phase 1 and phase 3 trials. In particular, the phase 3 cohort showed 23% and 24% increase on average at week 4 and week 8 respectively (q=1 × 10-10 and q=1 × 10-12, paired t-test). Additionally, 69.4% and 8.2% patients had Kyn/Trp increases above zero and 50% respectively at week 4. Notably, patients receiving everolimus control treatment had a decrease in kynurenine (q=1 × 10-5, t-test). Kynurenine is synthesized during tryptophan catabolism by indoleamine-2,3-dioxygenase (IDO) or tryptophan dioxygenase (TDO), and has been shown to suppress anti-tumor immune responses. To explore whether the circulating Kyn/Trp correlates with immune-suppression in the tumor microenvironment, we analyzed RNAseq data of matched tumor biopsies from the CA209-038 melanoma trial. We found a significant correlation between the Kyn/Trp ratio and PD-L1 expression, 4 weeks after starting nivolumab treatment (Pearson, p=0.01). We also discovered a correlation between Kyn/Trp and IDO1 but not TDO mRNA levels at the same time point. In contrast, Kyn/Trp was not associated with prior anti-CTLA4 treatment, or tumor mutational load. Moreover, compared to all other metabolites, increases of the Kyn/Trp ratio in the melanoma cohort (week 4 vs baseline) were consistently associated with greater risks for death (p=1.2*10-4, HR=2.71, 95% CI, 1.63-4.51). In particular, patients with a >50% increase in Kyn/Trp had a median OS of 15.7 months while those with decreases had a median survival time of > 38 months (p = 6.0*10-5, log-rank test). In contrast, the baseline Kyn/Trp ratio did not significantly associate with the melanoma patients' overall survival. To confirm this result, the association between Kyn/Trp ratios and overall survival in the larger phase 3 trial in RCC (CheckMate 025) was evaluated using serum samples collected at different time points. We found that at baseline, higher Kyn/Trp ratios associated with shorter overall survival both for the nivolumab- and the everolimus-treated patients (p = 3.6*10-4, HR=1.79, 95% CI, 1.30-2.47; p = 1.7*10-5, HR=2.06, 95% CI, 1.48-2.85; Cox model). However, at week 4, Kyn/Trp significantly associated with overall survival only in the nivolumab arm (p = 4.7*10-4, HR=2.81, 95% CI, 1.57-5.01; Cox model) but not in the everolimus arm (p = 0.53, HR=0.76, 95% CI, 0.32-1.78; Cox model). For nivolumab-treated RCC patients, those with a >50% increases of Kyn/Trp had a median survival of 16.7 months while those with any Kyn/Trp decreases had a median survival of 31.3 months (p = 4.3*10-4, log-rank test).Conclusions: We identified increased tryptophan to kynurenine conversion in response to PD1 blockade in a subset of melanoma and RCC patients. By using independent cohorts, we showed that Kyn/Trp temporal alterations robustly correlated with overall survival of patients receiving nivolumab. Our findings illustrate that checkpoint blockade in combination with IDO/TDO inhibitors might only benefit a selected group of patients with checkpoint-inhibition-triggered kynurenine pathway activation. Given the lack of improved therapeutic outcomes with PD1 and selective IDO1 inhibition among unselected patient populations in a recent phase 3 trial, our findings highlight the need and feasibility of patient stratification by monitoring serum Kyn/Trp alterations, show that kynurenine signaling may still be a relevant therapeutic target and more generally point to the relevance of metabolic adaptations in cancer immunotherapy. Our findings highlight the need and feasibility of patient stratification by monitoring serum Kyn/Trp alterations and point to the relevance of metabolic adaptations in cancer immunotherapy.
Citation Format: Haoxin Li, Kevin Bullock, Carino Gurjao, David Braun, Sachet A. Shukla, Dominick Bosse, Aly-Khan A. Lalani, Shuba Gopal, Chelsea Jin, Christine Horak, Megan Wind-Rotolo, Sabina Signoretti, David F. McDermott, Gordon J. Freeman, Eliezer M. Van Allen, Stuart L. Schreiber, Frank Stephen Hodi, William R. Sellers, Levi A. Garraway, Clary B. Clish, Toni K. Choueiri, Marios Giannakis. Metabolomic adaptations and correlates of survival to immune checkpoint blockade abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG13.
Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an ...image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state.
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•An image-based screen identifies regulators of glioblastoma tumor-initiating cells•ZFHX4 is required for the glioblastoma tumor-initiating cell state•ZFHX4 interacts with and regulates CHD4, a core member of the NuRD complex•ZFHX4 and CHD4 colocalize throughout the genome and coregulate gene expression
Glioblastoma (GBM), the most common and aggressive primary brain tumor, contains a subpopulation of stem cell-like, therapy-resistant tumor-initiating cells (TICs). Sabatini, Hahn, and colleagues performed an image-based RNAi screen in order to identify candidate regulators of GBM TIC functions. ZFHX4, identified in this screen, is essential for the stem cell-like state and tumorigenicity of TICs. Additionally, ZFHX4 interacts with CHD4, a core member of the chromatin regulatory NuRD complex, and drives CHD4-dependent gene expression programs.
Abstract only
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Background: Immune-checkpoint inhibition has been shown to be effective in a variety of cancers, including renal cell carcinoma (RCC) and melanoma. However, only a subset of ...patients with RCC and melanoma respond to anti-PD1 therapy. Given the importance of metabolism in the tumor immune microenvironment, we performed serum metabolomics in nivolumab-treated patients towards identifying novel non-invasive correlates of response and progression-free survival in immunotherapy-treated patients. Methods: We performed liquid chromatography-mass spectrometry on pre- and on-treatment serum samples from 79 patients with advanced melanoma (CA209-038 study) and 82 patients with metastatic RCC (CA209-009 study) receiving nivolumab. We precisely measured more than one-hundred named metabolites at baseline (prior to starting nivolumab), at 4 weeks and at 6 (melanoma) or 9 weeks (RCC) after initiation of treatment and correlated these with best overall response as well as progression-free survival (PFS). Results: In melanoma patients treated with nivolumab, the difference in mean levels of kynurenine (the product of IDO / TDO activity in tryptophan catabolism) between weeks 4 and 6 compared to baseline was significantly different between responders and non-responders (t-test with unequal variance p-value = 0.043 and p-value = 0.044 respectively). In RCC patients, we observed that patients with no response to nivolumab had significantly higher adenosine levels, than those who responded, at baseline and at 4 weeks after initiation of treatment (158% and 138% higher, t-test p-value = 0.0019 and p-value = 0.0011 respectively). RCC nivolumab-treated patients with higher (top quartile) baseline adenosine levels also had a significantly worse PFS (log rank test p-value = 0.004). Conclusions: In this first-of-its kind metabolomic analysis of peripheral blood from nivolumab-treated patients, we find that the change in kynurenine levels in melanoma patients correlates to response. In addition, higher baseline levels of adenosine in RCC patients are associated with worse PFS and lack of response to nivolumab. These results suggest a possible role for serum metabolites as biomarkers of benefit to PD1 inhibition.
Abstract only Background: Prior studies of metabolomic profiles and coronary heart disease (CHD) have been limited by relatively small case numbers and scant data in women. Methods: The discovery set ...examined 371 metabolites in 400 confirmed, incident CHD cases and 400 controls (frequency matched on age, race/ethnicity, hysterectomy status and time of enrollment) in the Women’s Health Initiative Observational Study (WHI-OS). All selected metabolites were validated in a separate set of 394 cases and 397 matched controls drawn from the placebo arms of the WHI Hormone Therapy trials and the WHI-OS. Discovery used 4 methods: false-discovery rate (FDR) adjusted logistic regression for individual metabolites, permutation corrected least absolute shrinkage and selection operator (LASSO) algorithms, sparse partial least squares discriminant analysis (PLS-DA) algorithms, and random forest algorithms. Each method was performed with matching factors only and with matching plus both medication use (aspirin, statins, anti-diabetics and anti-hypertensives) and traditional CHD risk factors (smoking, systolic blood pressure, diabetes, total and HDL cholesterol). Replication in the validation set was defined as a logistic regression coefficient of p<0.05 for the metabolites selected by 3 or 4 methods (tier 1), or a FDR adjusted p<0.05 for metabolites selected by only 1 or 2 methods (tier 2). Results: Sixty-seven metabolites were selected in the discovery data set (30 tier 1 and 37 tier 2). Twenty-six successfully replicated in the validation data set (21 tier 1 and 5 tier 2), with 25 significant with adjusting for matching factors only and 11 significant after additionally adjusting for medications and CHD risk factors. Validated metabolites included amino acids, sugars, nucleosides, eicosanoids, plasmologens, polyunsaturated phospholipids and highly saturated triglycerides. These include novel metabolites as well as metabolites such as glutamate/glutamine, which have been shown in other populations. Conclusions: Multiple metabolites in important physiological pathways with robust associations for risk of CHD in women were identified and replicated. These results may offer insights into biological mechanisms of CHD as well as identify potential markers of risk.
BackgroundAdvances in lipidomics allow profiling of hundreds of lipid compounds, but prior studies have been limited by relatively small case numbers and scant data in women.MethodsFour hundred ...confirmed, incident cases of MI and fatal CHD in the Women’s Health Initiative (WHI-OS) were frequency matched to 400 controls by age, race, hysterectomy, and time-period (mean age 62.7 years; no hormone use). Lipidomic analysis of 196 lipid species from 13 different lipid classes including cholesterol esters (CE), diacylglycerols (DAG),and triacylglycerols TAG) was performed by liquid chromatography mass spectroscopy. Two independent modeling approaches were used1) Individual lipids were studied in logistic regression models and a false-discovery rate (FDR) correction was used to adjust for multiple comparisons (FDR corrected p-value of < 0.05); 2) Lipids were simultaneously considered using a least absolute shrinkage and selection operator (LASSO) algorithm. Metabolites selected by both approaches were included in combined multivariable logistic regression models.ResultsIn multivariable-adjusted logistic models, 17 lipid metabolites were individually and significantly associated with CHD controlling for multiple comparisons, while in mutually adjusted LASSO regression, 8 lipids were significantly associated with CHD. Five lipids were associated with CHD by both methods (Table). When modeled together, only C18.1 CE and C34.3 DAG were associated with risk in fully adjusted models. Results will be validated in separate replication dataset of women from the WHI (396 cases/ 396 controls; to be presented).ConclusionsThree long chain highly unsaturated triacylglycerols and one diacylglycerol were associated with decreased risk, while a short chain monounsaturated lipid CE (18.1) was associated with increased risk of CHD in women. Lipidomics may offer insights into biological mechanisms of CHD as well as identify potential markers of risk.
Summary
A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene‐specific barcoded ...deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non‐essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out‐compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose‐ or sucrose‐containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co‐cultures of deletion strains were grown either in vitro in a chemostat to steady‐state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.
Towards patient-based cancer therapeutics Schreiber, Stuart L; Shamji, Alykhan F; Clemons, Paul A ...
Nature biotechnology,
201009, 2010-Sep, 2010-9-00, 20100901, Letnik:
28, Številka:
9
Journal Article
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Orienting cancer drug discovery to the patient requires relating the genetic features of tumors to acquired gene and pathway dependencies and identifying small-molecule therapeutics that target them. ...PUBLICATION ABSTRACT
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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