ORCID Profile
0000-0003-2953-7728
Current Organisation
The University of Hong Kong
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Biochemistry and Cell Biology | Bioinformatics | Gene Expression (incl. Microarray and other genome-wide approaches) | Virology | Biological Physics
Expanding Knowledge in the Biological Sciences | Diagnostic Methods | Expanding Knowledge in the Physical Sciences | Cancer and Related Disorders | Application Software Packages (excl. Computer Games) |
Publisher: American Association for Cancer Research (AACR)
Date: 30-04-2017
DOI: 10.1158/0008-5472.CAN-16-1663
Abstract: Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene– lified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN- lified neuroblastoma. Cancer Res 77(9) 2522–33. ©2017 AACR.
Publisher: Wiley
Date: 03-01-2019
DOI: 10.1002/IJC.32064
Abstract: Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.
Publisher: Springer Science and Business Media LLC
Date: 17-07-2021
DOI: 10.1007/S11154-021-09675-9
Abstract: Developed as an antidiabetic drug, recent evidence suggests that several sodium-glucose co-transporter 2 inhibitors (SGLT2i), especially canagliflozin and dapagliflozin, may exhibit in vitro and in vivo anticancer activities in selected cancer types, including an inhibition of tumor growth and induction of cell death. When used in combination with chemotherapy or radiotherapy, SGLT2i may offer possible synergistic effects in enhancing their treatment efficacy while alleviating associated side effects. Potential mechanisms include a reduction of glucose uptake into cancer cells, systemic glucose restriction, modulation of multiple signaling pathways, and regulation of different gene and protein expression. Furthermore, preliminary clinical findings have reported potential anticancer properties of canagliflozin and dapagliflozin in patients with liver and colon cancers respectively, with reference to decreases in their tumor marker levels. Given its general tolerability and routine use in diabetes management, SGLT2i may be a good candidate for drug repurposing in cancer treatment and as adjunct to conventional therapies. While current evidence reveals that only certain SGLT2i appear to be effective against selected cancer types, further studies are needed to explore the antitumor abilities of each SGLT2i in various cancers. Moreover, clinical trials are called for to evaluate the safety and feasibility of introducing SGLT2i in the treatment regimen of patients with specific cancers, and to identify the preferred route of drug administration for targeted delivery to selected tumor sites.
Publisher: American Chemical Society (ACS)
Date: 22-02-2003
DOI: 10.1021/AC026400H
Abstract: The interaction between ribonuclease (RNase) S-protein and S-peptide is examined by studying their limited oxidation within the RNase-S complex and free forms using radicals. The limited oxidation of the RNase-S complex and each component is effected through their reaction with a high flux of oxygen-based radicals generated by an electrical discharge within an electrospray ion source. Their exposure to radicals occurs on short millisecond time scales and has been consistently found not to cause any measurable structural damage or conformational change to proteins in a number of published reports. Consistent with these studies, S-peptide is preferentially protected from reactions with radicals under conditions in which it is bound to S-protein. Conversely, a region of S-protein comprising residues 96-100 constitutes the S-peptide binding domain based on its diminished reactivity with radicals within the RNase-S complex over the free S-protein. The results, for the first time, demonstrate the use of radicals generated by an electrical discharge to study protein complexes.
Publisher: Elsevier BV
Date: 07-2023
Publisher: The Company of Biologists
Date: 09-2004
DOI: 10.1242/JEB.01155
Abstract: Recent work has revealed that monkeys as well as pigeons are able to categorise complex visual objects. We show here that the ability to group similar, natural, visual images together extends to an invertebrate - the honeybee. Bees can be trained to distinguish between different types of naturally occurring scenes in a rather general way, and to group them into four distinct categories: landscapes, plant stems and two different kinds of flowers. They exhibit the same response to novel visual objects that differ greatly in their in idual, low-level features, but belong to one of the four categories. We exclude the possibility that they might be using single,low-level features as a cue to categorise these natural visual images and suggest that the categorisation is based on a combination of low-level features and configurational cues.
Publisher: American Society of Hematology
Date: 13-10-2016
DOI: 10.1182/BLOOD-2016-02-697870
Abstract: ENG regulatory elements target hemogenic mesoderm and hemogenic endothelium. Hemogenic progenitors can be enriched using these elements as molecular probes to discover novel regulators of hematopoiesis.
Publisher: American Society of Hematology
Date: 08-01-2015
DOI: 10.1182/BLOOD-2014-05-576470
Abstract: The glucocorticoid receptor coordinately regulates the antiapoptotic BCL2 and proapoptotic BIM genes in pediatric ALL cells in vivo. GR binding at a novel intronic region is associated with BIM transcription and dexamethasone sensitivity in pediatric ALL cells in vivo.
Publisher: Impact Journals, LLC
Date: 23-11-2016
Publisher: Impact Journals, LLC
Date: 02-2017
Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-01-2021
Abstract: Human adipocytes are a source of tissue-regenerative multipotent stem cells.
Publisher: Oxford University Press (OUP)
Date: 30-07-2010
Abstract: Disulfide bonds play critical roles in protein stability and function. They are generally considered to be strongly conserved among species. Although there is compelling evidence in the literature for this conservation on a case-by-case basis, comparative genomic analyses of disulfide conservation have in the past been limited. By analyzing the conservation of all structurally validated disulfide bonds from the Protein Data Bank across 29 completely sequenced eukaryotic genomes, we observe elevated conservation of disulfide-bonded cysteines (half-cystines) compared with unpaired cysteines and other amino acids. Remarkably, half-cystines are even more conserved than tryptophan--the most conserved amino acid. Overall, once disulfide bonds are acquired in proteins, they are rarely lost. Moreover, the acquisition of disulfide bonds shows a strong positive correlation (R(2) = 0.74) with organismal complexity. Although the correlation weakens (R(2) = 0.59) when yeast is excluded from the analysis, this trend is still apparent when compared with the slightly negative correlation of unpaired cysteine acquisition with organismal complexity. The accrual of disulfide bonds is likely to reflect the demand for greater sophistication in protein function in complex species. Our findings provide further support for the increasing usage of cysteines in modern proteomes and suggest that there has been positive selection for disulfide bonds through eukaryotic evolution. Finally, we show that the acquisition of the functionally relevant disulfide bond in domain 2 of the CD4 protein occurred independently in primates and rodents.
Publisher: American Society of Hematology
Date: 16-11-2012
DOI: 10.1182/BLOOD.V120.21.112.112
Abstract: Abstract 112 Alternative splicing of RNA is a fundamental biological process that affects almost all multi-exonic genes to promote protein ersity. Of the 3 modes of RNA splicing that also include exon skipping and alternative splice site usage, intron retention (IR) is the least abundant and least understood. Despite isolated instances of IR-associated biological function, IR has been widely regarded as a failure in the splicing machinery to excise intronic sequences from pre-messenger RNAs. Since an overall role for IR is unknown, we systematically examined the impact of IR in normal primary myeloid cells during differentiation. Using messenger RNA sequencing (mRNA-seq) and a novel algorithm we termed IRFinder, we determined intron-retaining genes that were differentially regulated in FACS-purified cells at three progressive stages of mouse granulopoiesis CD34+Kit+Gr-1low promyelocytes, CD34−Kit−Gr-1mid myelocytes and CD34−Kit−Gr-1high granulocytes. We demonstrated that IR exhibits a specific pattern of dynamic regulation in 86 genes during granulocytic differentiation. Figure 1, intron retaining transcripts were predominantly accumulated during differentiation, with low levels in promyelocytes, increasing through myelocytes to the highest levels in granulocytes. These genes include those with functions specific to granulocytes (Lyz2 and MMP8) and those governing the nuclear architecture (Lmnb1, Lmnb2, Lbr and Npm1), consistent with the unique change of nuclear morphology from promyelocytes to granulocytes. Figure 2, IR was significantly associated with nuclear localisation and functions involving the nuclear periphery. Subsequent mRNA-seq and IRFinder analysis of FACS purified human granulocytes displayed significant overlap of intron retaining genes between human and mouse (71/86 genes, P=2.85E-22, hypergeometric test), showing that IR is conserved between these two species. Inhibition of nonsense-mediated decay (NMD) in primary granulocytes using caffeine coupled with actinomycin D resulted in marked accumulation of 39/86 intron retaining transcripts (P .05, RUV procedure with Holm-Bonferroni correction), indicating that intron retaining transcripts are subjected to degradation by NMD. Mass spectometry analysis detected proteins encoded by 21 NMD-regulated intron-retaining genes. There was a strong negative correlation of protein expression with levels of IR (P=0.0015, binomial test). In mouse bone marrow reconstitution experiments, we showed that enforced re-expression of the Lmnb1 gene, which displayed the highest levels of endogenous IR led to decreased granulocyte cell count, increased nuclear volume by 30% and altered nuclear morphology. We conclude that IR coupled with NMD is a conserved physiological mechanism that may provide an energetically favourable level of gene expression control during granulopoiesis. Our findings establish a foundation to examine the role of IR- coupled NMD in normal haemopoiesis as well as in hemopoietic diseases now known to be affected by mutations of splicing factors. Figure 1,. intron retaining transcripts were predominantly accumulated during differentiation, with low levels in promyelocytes, increasing through myelocytes to the highest levels in granulocytes. These genes include those with functions specific to granulocytes (Lyz2 and MMP8) and those governing the nuclear architecture (Lmnb1, Lmnb2, Lbr and Npm1), consistent with the unique change of nuclear morphology from promyelocytes to granulocytes. Figure 2, IR was significantly associated with nuclear localisation and functions involving the nuclear periphery. Subsequent mRNA-seq and IRFinder analysis of FACS purified human granulocytes displayed significant overlap of intron retaining genes between human and mouse (71/86 genes, P=2.85E-22, hypergeometric test), showing that IR is conserved between these two species. Inhibition of nonsense-mediated decay (NMD) in primary granulocytes using caffeine coupled with actinomycin D resulted in marked accumulation of 39/86 intron retaining transcripts (P .05, RUV procedure with Holm-Bonferroni correction), indicating that intron retaining transcripts are subjected to degradation by NMD. Mass spectometry analysis detected proteins encoded by 21 NMD-regulated intron-retaining genes. There was a strong negative correlation of protein expression with levels of IR (P=0.0015, binomial test). In mouse bone marrow reconstitution experiments, we showed that enforced re-expression of the Lmnb1 gene, which displayed the highest levels of endogenous IR led to decreased granulocyte cell count, increased nuclear volume by 30% and altered nuclear morphology. We conclude that IR coupled with NMD is a conserved physiological mechanism that may provide an energetically favourable level of gene expression control during granulopoiesis. Our findings establish a foundation to examine the role of IR- coupled NMD in normal haemopoiesis as well as in hemopoietic diseases now known to be affected by mutations of splicing factors. Rasko: Genea Ltd: Employment Rarecyte: Consultancy.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2009
Publisher: Informa UK Limited
Date: 03-10-2014
Publisher: Elsevier BV
Date: 08-2010
Publisher: Springer Science and Business Media LLC
Date: 20-08-2012
Abstract: Influenza is one of the oldest and deadliest infectious diseases known to man. Reassorted strains of the virus pose the greatest risk to both human and animal health and have been associated with all pandemics of the past century, with the possible exception of the 1918 pandemic, resulting in tens of millions of deaths. We have developed and tested new computer algorithms, FluShuffle and FluResort, which enable reassorted viruses to be identified by the most rapid and direct means possible. These algorithms enable reassorted influenza, and other, viruses to be rapidly identified to allow prevention strategies and treatments to be more efficiently implemented. The FluShuffle and FluResort algorithms were tested with both experimental and simulated mass spectra of whole virus digests. FluShuffle considers different combinations of viral protein identities that match the mass spectral data using a Gibbs s ling algorithm employing a mixed protein Markov chain Monte Carlo (MCMC) method. FluResort utilizes those identities to calculate the weighted distance of each across two or more different phylogenetic trees constructed through viral protein sequence alignments. Each weighted mean distance value is normalized by conversion to a Z-score to establish a reassorted strain. The new FluShuffle and FluResort algorithms can correctly identify the origins of influenza viral proteins and the number of reassortment events required to produce the strains from the high resolution mass spectral data of whole virus proteolytic digestions. This has been demonstrated in the case of constructed vaccine strains as well as common human seasonal strains of the virus. The algorithms significantly improve the capability of the proteotyping approach to identify reassorted viruses that pose the greatest pandemic risk.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 17-02-2023
Abstract: Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo–electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.22545465.V1
Abstract: Supplementary Figure 2 shows transposable element methylation (A) and expression (B) at baseline, week 9 and week 22
Publisher: Elsevier BV
Date: 12-2018
DOI: 10.1016/J.FREERADBIOMED.2018.09.034
Abstract: Complement Factor H (CFH) is an important inhibitor of the alternate complement pathway in Bruch's membrane (BM), located between the choriocapillaris and the retinal pigment epithelium. Furthermore dysfunction of its activity as occurs with certain polymorphisms is associated with an increased risk of age related macular degeneration (AMD). The retina is a site of high generation of reactive oxygen species (ROS) and dysfunction of redox homeostasis in this milieu also contributes to AMD pathogenesis. In this study we wanted to explore if CFH exists in distinct redox forms and whether these species have unique protective biological functions. CFH can be reduced by the naturally occurring thioredoxin - 1 in CFH domains 1-4, 17-20. We found a duality of function between the oxidised and reduced forms of CFH. The oxidised form was more efficient in binding to C3b and lipid peroxidation by-products that are known to accumulate in the retinae and activate the alternate complement pathway. Oxidised CFH enhances Factor I mediated cleavage of C3 and C3b whereas the reduced form loses this activity. In the setting of oxidative stress (hydrogen peroxide)-mediated death of human retinal pigment epithelial cells as can occur in AMD, the free thiol form of CFH offers a protective function compared to the oxidised form. We found for the first time using a novel ELISA system we have developed for free thiol CFH, that both redox forms of CFH are found in the human plasma. Furthermore there is a distinct ratio of these redox forms in plasma depending if an in idual has early or late AMD, with in iduals with early AMD having higher levels of the free thiol form compared to late AMD.
Publisher: Informa UK Limited
Date: 04-2016
DOI: 10.1128/MCB.01035-15
Publisher: Impact Journals, LLC
Date: 20-08-2015
Publisher: American Chemical Society (ACS)
Date: 26-12-2014
DOI: 10.1021/BI401518B
Abstract: The vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors play a pivotal role in angiogenesis and lymphangiogenesis during development and in pathologies such as tumor growth. The VEGFs function as disulfide-linked antiparallel homodimers. The lymphangiogenic factors, VEGF-C and VEGF-D, exist as monomers and dimers, and dimerization is regulated by a unique unpaired cysteine. In this study, we have characterized the redox state of this unpaired cysteine in a recombinant mature monomeric and dimeric VEGF-C by mass spectrometry. Our findings indicate that the unpaired cysteine regulates dimerization via thiol-disulfide exchange involving the interdimer disulfide bond.
Publisher: American Society of Hematology
Date: 21-03-2013
DOI: 10.1182/BLOOD-2012-07-446120
Abstract: The ERG stem cell enhancer is active in acute myeloid leukemia and is regulated by a heptad of transcription factors. Expression signatures derived from ERG promoter–enhancer activity and heptad expression are associated with clinical outcome.
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1016/J.CELREP.2016.11.055
Abstract: CTCF binding sites are frequently mutated in cancer, but how these mutations accumulate and whether they broadly perturb CTCF binding are not well understood. Here, we report that skin cancers exhibit a highly specific asymmetric mutation pattern within CTCF motifs attributable to ultraviolet irradiation and differential nucleotide excision repair (NER). CTCF binding site mutations form independently of replication timing and are enriched at sites of CTCF/cohesin complex binding, suggesting a role for cohesin in stabilizing CTCF-DNA binding and impairing NER. Performing CTCF ChIP-seq in a melanoma cell line, we show CTCF binding site mutations to be functional by demonstrating allele-specific reduction of CTCF binding to mutant alleles. While topologically associating domains with mutated CTCF anchors in melanoma contain differentially expressed cancer-associated genes, CTCF motif mutations appear generally under neutral selection. However, the frequency and potential functional impact of such mutations in melanoma highlights the need to consider their impact on cellular phenotype in in idual genomes.
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B912234F
Abstract: The use of high resolution mass spectrometry to record the accurate mass of signature peptides within proteolytic digests of the nucleoprotein antigen, and whole influenza virus, is shown to be able to rapidly type and subtype the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of those for the nucleoprotein across all influenza types and subtypes. Peptides with unique theoretical masses from those generated in silico for all influenza antigen sequences, and from those proteins known to contaminate virus preparations in laboratory grown s les, were identified using a purpose built algorithm (FluGest). The frequency of occurrence of such conserved peptide signatures was assessed across all nucleoprotein sequences to subsequently type and subtype human strains of the virus. The application of the approach is illustrated for both type A H1N1 and H3N2, and type B strains of human influenza virus.
Publisher: Springer Science and Business Media LLC
Date: 08-03-2016
DOI: 10.1038/LEU.2016.55
Publisher: Cold Spring Harbor Laboratory
Date: 23-03-2023
DOI: 10.1101/2023.03.21.533716
Abstract: Endogenous transposable elements (TEs) are implicated in human diseases due to their propensity to compromise genome integrity. Although short-read sequencing is now frequently used to examine TE expression, the highly repetitive nature of TEs limits their accurate quantification at the locus-specific level. We have developed LocusMasterTE, an improved method that integrates information from long-read RNA sequencing to enhance TE quantification. The fractional transcript per million (TPM) from long reads serves as a prior distribution during the Expectation-Maximization (EM) model in short-read TE quantification, thereby enabling the reassignment of multi-mapped reads to correct expression values. Using simulated short-reads, our results indicate that LocusMasterTE outperforms existing quantitative approaches and is especially favorable for quantifying evolutionarily younger TEs. Using matched cell line RNA-seq data, we further demonstrate improved locus-specific TE quantification by LocusMasterTE with stronger enrichment in active, and depletion at repressive, histone marks. Finally, by integrating colorectal cancer cell line long-read sequencing data with short read RNA-seq data from The Cancer Genome Atlas colorectal cancer cohort, we demonstrate LocusMasterTE’s ability to identify survival-related TEs and uncover new expression associations between locus-specific TEs and neighboring genes. By providing more accurate quantification, LocusMasterTE offers the potential to reveal novel functions of TE transcripts.
Publisher: Oxford University Press (OUP)
Date: 31-10-2013
DOI: 10.1093/NAR/GKT1036
Publisher: Wiley
Date: 22-01-2021
DOI: 10.1111/BJH.17316
Abstract: Clonal haematopoiesis of indeterminant potential (CHIP) increases in frequency with age. The effect of CHIP on the mobilization of autologous CD34+ peripheral blood stem cells (PBSC) has not been reported. This study uses a DNA‐based targeted candidate gene approach to identify the presence of somatic mutations in ASXL1, DNMT3A, JAK2, SF3B1, TET2 and TP53 in CD34+ haematopoietic progenitor cell‐apheresis products of 96 patients who undergo PBSC mobilization for autologous stem cell transplantation (ASCT). Variants were identified in a significantly greater proportion of patients who experience poor CD34+ PBSC mobilization. A DNA‐based targeted candidate gene array is able to predict poor CD34+ PBSC mobilization and may be deployed pre‐emptively to minimize mobilization and graft failures.
Publisher: Springer Science and Business Media LLC
Date: 10-2014
Publisher: American Chemical Society (ACS)
Date: 15-02-2011
DOI: 10.1021/JA110226Y
Publisher: Elsevier BV
Date: 12-2010
Publisher: Springer Singapore
Date: 2019
Publisher: Wiley
Date: 25-04-2003
DOI: 10.1002/JMS.474
Abstract: A new algorithm has been designed and tested to identify protein, or any other macromolecular, complexes that have been widely reported in mass spectral data. The program takes advantage of the appearance of multiply charged ions that are common to both electrospray ionization and, to a lesser extent, matrix-assisted laser desorption/ionization (MALDI) mass spectra. The algorithm, known as COMPLX for the COMposition of Protein-Ligand compleXes, is capable of identifying complexes for any protein or macromolecule with a binding partner of molecular mass up to 100 000 Da. It does so by identifying ion pairs present in a mass spectrum that, when they share a common charge, have an m/z value difference that is an integer fraction of a ligand or binding partner molecular mass. Several additional criteria must be met in order for the result to be ranked in the output file including that all m/z values for ions of the protein or complex have progressively lower values as their assigned charge increases, the difference between the m/z values for adjacent charge states (z, z + 1) decrease as the assigned charge state increases, and the ratio of any two m/z values assigned to a protein or complex is equal to the inverse ratio of their charge. The entries that satisfy these criteria are then ranked according to the appearance of ions in the mass spectrum associated with the binding partner, the length of a continuous series of charges across any set of ions for a protein and complex and the lowest error recorded for the molecular mass of the ligand or binding partner. A erse range of hypothetical and experimental mass spectral data were used to implement and test the program, including those recorded for antibody-peptide, protein-peptide and protein-heme complexes. Spectra of increasing complexity, in terms of the number of ions input, were also successfully analysed in which the number of input m/z values far exceeds the few associated with a macromolecular complex. Thus the program will be of value in a future goal of proteomics, where mass spectrometry already plays a central role, for the direct analysis of protein and other associations within biological extracts.
Publisher: Informa UK Limited
Date: 16-05-2014
DOI: 10.4161/EPI.29222
Publisher: Elsevier BV
Date: 11-2021
Publisher: Oxford University Press (OUP)
Date: 17-02-2012
DOI: 10.1093/BIOINFORMATICS/BTS085
Abstract: Motivation: Chromatin structure, including post-translational modifications of histones, regulates gene expression, alternative splicing and cell identity. ChIP-seq is an increasingly used assay to study chromatin function. However, tools for downstream bioinformatics analysis are limited and are only based on the evaluation of signal intensities. We reasoned that new methods taking into account other signal characteristics such as peak shape, location and frequencies might reveal new insights into chromatin function, particularly in situation where differences in read intensities are subtle. Results: We introduced an analysis pipeline, based on linear predictive coding (LPC), which allows the capture and comparison of ChIP-seq histone profiles. First, we show that the modeled signal profiles distinguish differentially expressed genes with comparable accuracy to signal intensities. The method was robust against parameter variations and performed well up to a signal-to-noise ratio of 0.55. Additionally, we show that LPC profiles of activating and repressive histone marks cluster into distinct groups and can be used to predict their function. Availability and implementation: www.cancerresearch.unsw.edu.au/crcweb.nsf age/LPCHP A Matlab implementation along with usage instructions and an ex le input file are available from: www.cancerresearch.unsw.edu.au/crcweb.nsf age/LPCHP Contact: d.beck@student.unsw.edu.au jpimanda@unsw.edu.au jason.wong@unsw.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.
Publisher: Cold Spring Harbor Laboratory
Date: 08-02-2017
DOI: 10.1101/106872
Abstract: Cytosine methylation (5mC) is vital for cellular function, and yet 5mC sites are also commonly mutated in the genome. In this study, we analyse the genomes of over 900 cancer s les, together with tissue type-specific methylation and replication timing data. We describe a strong mutation-methylation association in colorectal cancers with microsatellite instability (MSI) or with Polymerase epsilon (POLE) exonuclease domain mutation. We describe a potential role for mismatch repair in the correction of mismatches resulting from deamination of 5mC, and propose a mutator phenotype to exist in POLE -mutant cancers specifically at 5mC sites. We also associate POLE -mutant hotspot coding mutations in APC and TP53 with CpG methylation. Analysing mutations across additional cancer types, we identify nucleotide excision repair- and AID/APOBEC-induced processes to underlie differential mutation-methylation associations in certain cancer subtypes. This study reveals differential associations vital for accurately mapping regional variation in mutation density and pinpointing driver mutations in cancer.
Publisher: American Association for Cancer Research (AACR)
Date: 30-11-2015
DOI: 10.1158/0008-5472.CAN-15-0423
Abstract: MYCN gene lification in neuroblastoma drives a gene expression program that correlates strongly with aggressive disease. Mechanistically, trimethylation of histone H3 lysine 4 (H3K4) at target gene promoters is a strict prerequisite for this transcriptional program to be enacted. WDR5 is a histone H3K4 presenter that has been found to have an essential role in H3K4 trimethylation. For this reason, in this study, we investigated the relationship between WDR5-mediated H3K4 trimethylation and N-Myc transcriptional programs in neuroblastoma cells. N-Myc upregulated WDR5 expression in neuroblastoma cells. Gene expression analysis revealed that WDR5 target genes included those with MYC-binding elements at promoters such as MDM2. We showed that WDR5 could form a protein complex at the MDM2 promoter with N-Myc, but not p53, leading to histone H3K4 trimethylation and activation of MDM2 transcription. RNAi-mediated attenuation of WDR5 upregulated expression of wild-type but not mutant p53, an effect associated with growth inhibition and apoptosis. Similarly, a small-molecule antagonist of WDR5 reduced N-Myc/WDR5 complex formation, N-Myc target gene expression, and cell growth in neuroblastoma cells. In MYCN-transgenic mice, WDR5 was overexpressed in precancerous ganglion and neuroblastoma cells compared with normal ganglion cells. Clinically, elevated levels of WDR5 in neuroblastoma specimens were an independent predictor of poor overall survival. Overall, our results identify WDR5 as a key cofactor for N-Myc–regulated transcriptional activation and tumorigenesis and as a novel therapeutic target for MYCN- lified neuroblastomas. Cancer Res 75(23) 5143–54. ©2015 AACR.
Publisher: Elsevier BV
Date: 08-2021
DOI: 10.1016/J.JAUT.2021.102675
Abstract: Β
Publisher: American Society of Hematology
Date: 10-11-2011
DOI: 10.1182/BLOOD-2011-06-360297
Abstract: VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis.
Publisher: American Society of Hematology
Date: 21-10-2021
Abstract: Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other’s, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.
Publisher: The Royal Society
Date: 02-2018
DOI: 10.1098/RSOS.171058
Abstract: Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecules of a protein crystal but absent in others, or present in some structures of a protein but absent in others. We reasoned that the labile nature of these disulfides signifies a propensity for cleavage and so possible allosteric regulation of the protein in which the bond resides. A total of 511 labile disulfide bonds were identified. The labile disulfides are more stressed than the average bond, being characterized by high average torsional strain and stretching of the sulfur–sulfur bond and neighbouring bond angles. This pre-stress likely underpins their susceptibility to cleavage. The coagulation, complement and oxygen-sensing hypoxia inducible factor-1 pathways, which are known or have been suggested to be regulated by allosteric disulfides, are enriched in proteins containing labile disulfides. The identification of labile disulfide bonds will facilitate the study of this post-translational modification.
Publisher: Mary Ann Liebert Inc
Date: 2011
Abstract: Protein disulfide bonds are the links between the sulfur atoms of two cysteine amino acids. All the known life forms appear to make this bond. Most disulfide bonds perform a structural role by stabilizing the tertiary and quaternary structures. Some perform a functional role and can be characterized as either catalytic or allosteric disulfides. Catalytic disulfides/dithiols transfer electrons between proteins, whereas the allosteric bonds control the function of the protein in which they reside when they undergo redox change. There are currently five clear ex les of allosteric disulfide bonds and a number of potential allosteric disulfides at various stages of characterization. The features of these bonds and how they control the activity of the respective proteins are discussed. A common aspect of the allosteric disulfides identified to date is that they all link β-strands or β-loops.
Publisher: Springer Science and Business Media LLC
Date: 13-04-2016
DOI: 10.1038/NATURE17437
Abstract: Promoters are DNA sequences that have an essential role in controlling gene expression. While recent whole cancer genome analyses have identified numerous hotspots of somatic point mutations within promoters, many have not yet been shown to perturb gene expression or drive cancer development. As such, positive selection alone may not adequately explain the frequency of promoter point mutations in cancer genomes. Here we show that increased mutation density at gene promoters can be linked to promoter activity and differential nucleotide excision repair (NER). By analysing 1,161 human cancer genomes across 14 cancer types, we find evidence for increased local density of somatic point mutations within the centres of DNase I-hypersensitive sites (DHSs) in gene promoters. Mutated DHSs were strongly associated with transcription initiation activity, in which active promoters but not enhancers of equal DNase I hypersensitivity were most mutated relative to their flanking regions. Notably, analysis of genome-wide maps of NER shows that NER is impaired within the DHS centre of active gene promoters, while XPC-deficient skin cancers do not show increased promoter mutation density, pinpointing differential NER as the underlying cause of these mutation hotspots. Consistent with this finding, we observe that melanomas with an ultraviolet-induced DNA damage mutation signature show greatest enrichment of promoter mutations, whereas cancers that are not highly dependent on NER, such as colon cancer, show no sign of such enrichment. Taken together, our analysis has uncovered the presence of a previously unknown mechanism linking transcription initiation and NER as a major contributor of somatic point mutation hotspots at active gene promoters in cancer genomes.
Publisher: The Royal Society
Date: 02-2013
DOI: 10.1098/RSOB.120148
Abstract: Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4OB02133A
Abstract: A bioorthogonal method of transforming 3-nitrotyrosine to 3-azidotyrosine is described, providing new opportunities to study 3-nitrotyrosine in biological s les.
Publisher: Springer Science and Business Media LLC
Date: 13-10-2014
DOI: 10.1038/LEU.2014.299
Abstract: High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.
Publisher: Elsevier BV
Date: 06-2010
Publisher: Cold Spring Harbor Laboratory
Date: 26-10-2020
DOI: 10.1101/2020.10.26.353797
Abstract: Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesised that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, RUNX1) bind key hematopoietic genes in human CD34+ haematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other’s, regulatory elements. However, their mutual regulation during normal haematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. Here, we integrated bulk and single cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and in leukemic cells. The heptad factors GATA2, TAL1 and ERG formed an integrated sub-circuit that regulates stem cell to erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits could be harnessed to promote specific cell type transitions and overcome dysregulated haematopoiesis.
Publisher: SAGE Publications
Date: 02-2010
DOI: 10.1255/EJMS.1056
Abstract: The application of high-resolution mass spectrometry to type and subtype strains of the influenza virus within recent recommended vaccine formulations is described. Proteolytic digests of whole virus or separated hemagglutinin antigen generate conserved signature peptides of unique mass that can be used to characterise each component virus in a rapid and direct manner by the detection of their ions alone. The approach is demonstrated for two type A strains and one type B strain of human influenza viruses present in recommended seasonal vaccines in the northern and southern hemispheres from 2007 through 2010.
Publisher: Springer Science and Business Media LLC
Date: 31-10-2022
DOI: 10.1186/S13073-022-01131-W
Abstract: Ganciclovir (GCV) is widely used in solid organ and haematopoietic stem cell transplant patients for prophylaxis and treatment of cytomegalovirus. It has long been considered a mutagen and carcinogen. However, the contribution of GCV to cancer incidence and other factors that influence its mutagenicity remains unknown. This retrospective cohort study analysed genomics data for 121,771 patients who had undergone targeted sequencing compiled by the Genomics Evidence Neoplasia Information Exchange (GENIE) or Foundation Medicine (FM). A statistical approach was developed to identify patients with GCV-associated mutational signature (GCV sig ) from targeted sequenced data of tumour s les. Cell line exposure models were further used to quantify mutation burden and DNA damage caused by GCV and other antiviral and immunosuppressive drugs. Mutational profiles from 22 of 121,771 patient s les in the GENIE and FM cohorts showed evidence of GCV sig . A erse range of cancers was represented. All patients with detailed clinical history available had previously undergone solid organ transplantation and received GCV and mycophenolate treatment. RAS hotspot mutations associated with GCV sig were present in 9 of the 22 s les, with all s les harbouring multiple GCV-associated protein-altering mutations in cancer driver genes. In vitro testing in cell lines showed that elevated DNA damage response and GCV sig are uniquely associated with GCV but not acyclovir, a structurally similar antiviral. Combination treatment of GCV with the immunosuppressant, mycophenolate mofetil (MMF), increased the misincorporation of GCV in genomic DNA and mutations attributed to GCV sig in cell lines and organoids. In summary, GCV can cause a erse range of cancers. Its mutagenicity may be potentiated by other therapies, such as mycophenolate, commonly co-prescribed with GCV for post-transplant patients. Further investigation of the optimal use of these drugs could help reduce GCV-associated mutagenesis in post-transplant patients.
Publisher: Portland Press Ltd.
Date: 27-07-2011
DOI: 10.1042/BJ20110566
Abstract: Current lipidomic profiling methods rely mainly on MS to identify unknown lipids within a complex s le. We describe a new approach, involving LC×MS/MS (liquid chromatography×tandem MS) analysis of sphingolipids based on both mass and hydrophobicity, and use this method to characterize the SM (sphingomyelin), ceramide and GalCer (galactosylceramide) content of hippoc us from AD (Alzheimer's disease) and control subjects. Using a mathematical relationship we exclude the influence of sphingolipid mass on retention time, and generate two-dimensional plots that facilitate accurate visualization and characterization of the different ceramide moieties within a given sphingolipid class, because related molecules align horizontally or vertically on the plots. Major brain GalCer species that differ in mass by only 0.04 Da were easily differentiated on the basis of their hydrophobicity. The importance of our method's capacity to define all of the major GalCer species in the brain s les is illustrated by the novel observation that the proportion of GalCer with hydroxylated fatty acids increased approximately 2-fold in the hippoc us of AD patients, compared with age- and gender-matched controls. This suggests activation of fatty acid hydroxylase in AD. Our method greatly improves the clarity of data obtained in a lipid profiling experiment and can be expanded to other lipid classes.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.22545471.V1
Abstract: Supplementary Figure 1 shows Whole blood cell counts at baseline, week 9 and week 22
Publisher: Elsevier BV
Date: 05-2014
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.GENE.2016.03.031
Abstract: Aberrant transcription read-through of a gene promoter as a result of genetic structural rearrangements can cause the epigenetic inactivation of a neighbouring gene. All reported cases have involved copy number alterations that remove the 3' poly(A) transcription terminator sequence of a gene leading to transcription read-through (TRT) and methylation of the gene promoter of a downstream gene. We aimed to determine whether deletion of poly (A) transcription terminator sequences was associated with the methylation of neighbouring genes in a CRC with extensive copy number alterations. We performed a high resolution CGH array and methylation analysis on a CRC specimen to identify such alterations. Analysis of the CRC using high-resolution CGH identified 6 genes with deletions in the 3' part of the gene that encompassed the poly(A) transcription terminator sequence. Bisulphite sequencing of the promoter region of neighbouring (affected) genes at these six regions showed all candidate genes were unmethylated. Considering the fact that six TRT affected genes in a CRC with multiple deletions show no signs of hypermethylated promoters, it would be fairly appropriate to suggest that epigenetic inactivation by TRT might be a rare phenomenon in sporadic CRCs.
Publisher: American Society of Hematology
Date: 06-12-2014
DOI: 10.1182/BLOOD.V124.21.483.483
Abstract: Background: Long noncoding RNAs (lncRNAs) are regulators of cell identify and their aberrant expression has been associated with the development of cancer. Several studies have shown that lncRNAs are required for normal hematopoiesis and also function as oncogenic drivers in acute leukemia. However, the association of lncRNA expression with AML subtypes and impact on prognosis is not known. Methods: LincRNAs are transcribed from the intergenic part of the genome and their transcripts are typically capped, polyadenylated and often spliced. Given their mRNA like features their expression levels can be detected using standard expression profiling assays. Tiling arrays have been used to profile gene expression levels of large patient cohorts and are currently the largest existing resource to study leukemia genomics. We have repurposed intragenic probes of the Affymetrics HG-133 Plus 2 (HG-133P2) to estimate the expression levels of 1664 known lincRNA genes (Figure 1a). Results: To estimate lincRNA levels in AML, we first analyzed a dataset of 159 s les from the Cancer Genome Atlas (TCGA) that were profiled using the HG-133P2 chip and RNA-seq. In all but one case, the expression levels obtained from both technologies were significantly correlated (r .6 Pearson correlation p .001 Figure 1b,c) suggesting that lincRNA levels can be accurately estimated from microarray data. Expression analysis of lincRNAs was then carried out in three datasets totaling 737 patient s les for which HG-133P2 data was available. S les included those from the Netherlands (NL, n=419), USA (TCGA, n= 179) and Germany (GER, n=139). To evaluate whether lincRNA expression was associated with AML subgroups, hierarchical clustering was performed on the NL and TCGA sets (Figure 2a). Patients with t(8:21) and t(15 )/FAB M3 sub-groups and those with mutations in CEBPA, NPM1 and/or FLT3-ITD associated with distinct lincRNA profiles in both cohorts. In addition, we found associations that were unique to one or the other dataset i.e. FAB M2 and M5 in the NL and inv (16) in the TCGA sets. TP53 mutations were only available in the TCGA set and patients with these mutations showed a distinct lincRNA expression profile. Taken together, these data suggest that specific lincRNA expression profiles, similar to gene expression profiles, are associated with known AML subgroups. The 1664 lincRNAs were further analyzed using non-negative matrix factorization clustering in the NL and TCGA sets. The NL cohort optimally separated into four groups (Figure 2b) that were associated (p .01 Fisher exact test) with either good or poor prognostic subtypes. For ex le, cluster one was associated with patients of poor cytogenetics and those having re-arrangements of chromosome 11q32, while cluster three was associated with patients in FAB M3/ t(15:17), FABM4 and NPM1 mutations. Similarly, the TCGA cohort was optimally separated into five groups (Figure 2c), including cluster three which was associated (p .01) with patients of complex karyotype, those in FAB M0 and carrying mutations in RUNX1 and TP53 while cluster five was associated with patients with chromosomal translocation in t(8,21) and t(15 )/FAB M3 and mutations in CEBPA. These data suggest that lincRNA profiles segregate with subgroups of overlapping characteristics that are enriched for either good or poor prognostics. The NL, TCGA and GER cohorts were also analyzed for overall survival using the cox-regression model. In total we found that 78 (NL), 92 (TCGA) and 60 (GER) lincRNAs were significantly associated with overall survival (p 0.05). An integrative approach including a meta-analysis of the cox-regression p-values (Souffers method p .01) revealed a survival signature of 17 lincRNAS (linc-sig) across the three sets. The prognostic power was maintained in the NL (p .001), TCGA (p .001) and GER cohorts (p=0.1) using Kaplan–Meier statistics (Figure 3). Importantly, the linc-sig remained an independent prognostic factor when accounted for age, sex, WBC and CEBPA. Conclusions We investigated the role of 1664 lincRNAs across three AML patient cohorts. The data presented shows for the first time that distinct lincRNA expression profiles are associated with recognized cytogenetic and mutational subgroups that demonstrate good or poor characteristics and that a signature of 17 lincRNAs predicts overall survival in AML. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. No relevant conflicts of interest to declare.
Publisher: Cold Spring Harbor Laboratory
Date: 16-01-2018
DOI: 10.1101/249300
Abstract: Large-scale whole cancer-genome sequencing projects have led to the identification of a handful of cis-regulatory driver mutations in cancer genomes. However, recent studies have demonstrated that very large cancer cohorts will be required in order to identify low frequency non-coding drivers. To further this endeavour, in this study, we performed highdepth sequencing across 95 colorectal cancers and matched normal s les using a unique target capture sequencing (TCS) assay focusing on over 35 megabases of gene regulatory elements. We first assessed coverage and variant detection capability from our TCS data, and compared this with a s le that was additionally whole-genome sequenced (WGS). TCS enabled substantially deeper sequencing and thus we detected 51% more somatic single nucleotide variants ( n = 2,457) and 144% more somatic insertions and deletions ( n = 39) by TCS than WGS. Variants obtained from TCS data were suitable for somatic mutational signature detection, enabling us to define the signatures associated with germline deleterious variants in MSH6 and MUTYH in s les within our cohort. Finally, we surveyed regulatory mutations to find putative drivers by assessing variant recurrence and function, identifying some regulatory variants that may influence oncogenesis. Our study demonstrates TCS to be a sequencing-efficient alternative to traditional WGS, enabling improved coverage and variant detection when seeking to identify variants at specific loci among larger cohorts. Interestingly, we found no candidate variants that have a clear driver function, suggesting that regulatory drivers may be rare in a colorectal cancer cohort of this size. In recent years, some cancer research focus has turned towards the role of somatic mutations in the 98% of the genome that is non-coding. To investigate such mutations, we performed deep sequencing of regulatory regions and a selection of coding genes across 95 colorectal cancer and matched-normal s les. To determine the ability of our targeted deep sequencing methodology to accurately detect variants, we compared our results with those from a s le that was additionally whole-genome sequenced. We found target capture sequencing to enable greater sequencing depth, allowing the detection of 51% and 144% more somatic single nucleotide and insertion/deletion mutations, respectively. Our study here demonstrates target capture sequencing to be a useful approach for researchers seeking to identify variants at specific loci among larger cohorts. Our results also enabled the generation of mutational signatures, implicating deleterious germline single nucleotide variants in coding exons of MSH6 and MUTYH in s les within our cohort. Finally, we surveyed regulatory elements in search of somatic cancer driver mutations. We identified some regulatory variants that may influence oncogenesis, but found no candidate variants with clear driver function. These findings suggest that regulatory driver mutations may be rare in a colorectal cancer cohort of this size.
Publisher: Wiley
Date: 04-2022
Publisher: Springer Science and Business Media LLC
Date: 06-04-2017
DOI: 10.1038/S41598-017-00739-Y
Abstract: Cancer is a multifactorial disease driven by a combination of genetic and environmental factors. Many cancer driver mutations have been characterised in protein-coding regions of the genome. However, mutations in noncoding regions associated with cancer have been less investigated. G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in the regulatory regions. In this study, we used published whole cancer genome sequence data to find mutations in cancer patients that overlap potential RNA G4-forming sequences in 5′ UTRs. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5′ UTRs. Of the 217 identified mutations, we found that 33 are predicted to destabilise and 21 predicted to stabilise potential RNA G4s. We experimentally validated the effect of destabilising mutations in the 5′ UTRs of BCL2 and CXCL14 and one stabilising mutation in the 5′ UTR of TAOK2 . These mutations resulted in an increase or a decrease in translation of these mRNAs, respectively. These findings suggest that mutations that modulate the G4 stability in the noncoding regions could act as cancer driver mutations, which present an opportunity for early cancer diagnosis using in idual sequencing information.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.C.6550950
Abstract: Purpose: Drug repurposing offers the opportunity for chemotherapy to be used to reestablish sensitivity to immune checkpoint blockade (ICB) therapy. Here we investigated the clinical and translational aspects of an early phase II study of azacitidine and carboplatin priming for anti-PDL1 immunotherapy (avelumab) in patients with advanced ICB-resistant melanoma. Experimental Design: A total of 20 participants with ICB-resistant metastatic melanoma received 2 × 4-week cycles of azacitidine and carboplatin followed by ICB rechallenge with anti-PD-L1 avelumab. The primary objective was overall response rate after priming and ICB rechallenge. Secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). Translational correlation analysis of HLA-A and PD-L1 expression, RNA sequencing, and reduced representation bisulfite sequencing of biopsies at baseline, after priming and after six cycles of avelmuab was performed. Results: The overall response rate (ORR) determined after azacitidine and carboplatin priming was 10% (2/20) with two partial responses (PR). The ORR determined after priming followed by six cycles of avelumab (week 22) was 10%, with 2 of 20 participants achieving immune partial response (iPR). The CBR for azacitidine and carboplatin priming was 65% (13/20) and after priming followed by six cycles of avelumab CBR was 35% ( i n /i = 7/20). The median PFS was 18.0 weeks [95% confidence interval (CI): 14.87–21.13 weeks] and the median OS was 47.86 weeks (95% CI: 9.67–86.06 weeks). Translational correlation analysis confirmed HLA-A generally increased after priming with azacitidine and carboplatin, particularly if it was absent at the start of treatment. Average methylation of CpGs across the HLA-A locus was decreased after priming and T cells, in particular CD8 sup + /sup , showed the greatest increase in infiltration. Conclusions: Priming with azacitidine and carboplatin can induce disease stabilization and resensitization to ICB for metastatic melanoma. Significance: There are limited treatments for melanoma once resistance to ICB occurs. Chemotherapy induces immune-related responses and may be repurposed to reinstate the response to ICB. This study provides the first evidence that chemotherapy can provide clinical benefit and increase OS for ICB-resistant melanoma. /
Publisher: American Society of Hematology
Date: 15-11-2022
Publisher: American Society of Clinical Oncology (ASCO)
Date: 06-2023
DOI: 10.1200/PO.22.00649
Abstract: Next-generation sequencing comprehensive genomic panels (NGS CGPs) have enabled the delivery of tailor-made therapeutic approaches to improve survival outcomes in patients with cancer. Within the China Greater Bay Area (GBA), territorial differences in clinical practices and health care systems and strengthening collaboration warrant a regional consensus to consolidate the development and integration of precision oncology (PO). Therefore, the Precision Oncology Working Group (POWG) formulated standardized principles for the clinical application of molecular profiling, interpretation of genomic alterations, and alignment of actionable mutations with sequence-directed therapy to deliver clinical services of excellence and evidence-based care to patients with cancer in the China GBA. Thirty experts used a modified Delphi method. The evidence extracted to support the statements was graded according to the GRADE system and reported according to the Revised Standards for Quality Improvement Reporting Excellence guidelines, version 2.0. The POWG reached consensus in six key statements: harmonization of reporting and quality assurance of NGS molecular tumor board and clinical decision support systems for PO education and training research and real-world data collection, patient engagement, regulations, and financial reimbursement of PO treatment strategies and clinical recommendations and implementation of PO in clinical practice. POWG consensus statements standardize the clinical application of NGS CGPs, streamline the interpretation of clinically significant genomic alterations, and align actionable mutations with sequence-directed therapies. The POWG consensus statements may harmonize the utility and delivery of PO in China's GBA.
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.CELL.2013.06.052
Abstract: Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.
Publisher: Wiley
Date: 16-08-2005
DOI: 10.1002/JMS.894
Abstract: The performance of the algorithm COMPLX for detecting protein-ligand or other macromolecular complexes has been tested for highly complex data sets. These data contain m/z values for ions of proteins of the SWISS-PROT database within simulated biological mixtures where each component shares a similar molecular weight and/or isoelectric point (pI). As many as 1600 ion signals were entered to challenge the algorithm to identify ion signals associated with a single protein complex that has been ionised and detected within a mass spectrometer. Despite the complexity of such data sets, the algorithm is shown to be able to identify the presence of in idual bimolecular complexes. The output data can be re-evaluated by the user as necessary in light of any additional information that is known concerning the nature of predicted associations, as well as the quality of the data-set in terms of errors in m/z values as a direct consequence of the mass calibration or resolution achieved. The data presented illustrates that the best results are obtained when output results are ranked according to the largest continuous series of ion pairs detected for a protein or macromolecule and its complex for which the ligand mass is assigned the lowest mass error.
Publisher: Springer Science and Business Media LLC
Date: 04-11-2022
DOI: 10.1186/S40168-022-01366-0
Abstract: Gut microbiota (GM) dysregulation, known as dysbiosis, has been proposed as a crucial driver of obesity associated with “Western” diet (WD) consumption. Gut dysbiosis is associated with increased gut permeability, inflammation, and insulin resistance. However, host metabolic pathways implicated in the pathophysiology of gut dysbiosis are still elusive. Exchange protein directly activated by cAMP (Epac) plays a critical role in cell-cell junction formation and insulin secretion. Here, we used homozygous Epac1-knockout (Epac1 –/– ), Epac2-knockout (Epac2 –/– ), and wild-type (WT) mice to investigate the role of Epac proteins in mediating gut dysbiosis, gut permeability, and inflammation after WD feeding. The 16S rRNA gene sequencing of fecal DNA showed that the baseline GM of Epac2 –/– , but not Epac1 –/– , mice was represented by a significantly higher Firmicutes to Bacteroidetes ratio and significant alterations in several taxa compared to WT mice, suggesting that Epac2 –/– mice had gut dysbiosis under physiological conditions. However, an 8-week WD led to a similar gut microbiome imbalance in mice regardless of genotype. While Epac1 deficiency modestly exacerbated the WD-induced GM dysbiosis, the WD-fed Epac2 –/– mice had a more significant increase in gut permeability than corresponding WT mice. After WD feeding, Epac1 –/– , but not Epac2 –/– , mice had significantly higher mRNA levels of tumor necrosis factor-alpha (TNF-α) and F4/80 in the epididymal white adipose tissue (EWAT), increased circulating lipocalin-2 protein and more severe glucose intolerance, suggesting greater inflammation and insulin resistance in WD-fed Epac1 –/– mice than corresponding WT mice. Consistently, Epac1 protein expression was significantly reduced in the EWAT of WD-fed WT and Epac2 –/– mice. Despite significantly dysregulated baseline GM and a more pronounced increase in gut permeability upon WD feeding, WD-fed Epac2 –/– mice did not exhibit more severe inflammation and glucose intolerance than corresponding WT mice. These findings suggest that the role of gut dysbiosis in mediating WD-associated obesity may be context-dependent. On the contrary, we demonstrate that deficiency of host signaling protein, Epac1, drives inflammation and glucose intolerance which are the hallmarks of WD-induced obesity.
Publisher: Elsevier BV
Date: 10-2013
DOI: 10.1016/J.BBRC.2013.09.035
Abstract: Methionyl aminopeptidase 2 (MetAP2) plays an important role in the regulation of angiogenesis. This study examined whether nitration of MetAP2 alters its enzymatic activity in vitro. The activity of unmodified, nitrated and oxidised MetAP2 was assessed and it was found that nitration significantly reduced its ability to cleave a chromogenic substrate. Mass spectrometry analysis identified Tyr336 as a nitrated residue in MetAP2. Structural and evolutionary analysis indicate that this is an important residue for MetAP2 activity. Combined, the results show that the activity of MetAP2 is reduced by nitration and raise the possibility that nitration of MetAP2 is a mechanism contributing to endothelial dysfunction.
Publisher: Springer Science and Business Media LLC
Date: 09-08-2017
DOI: 10.1038/S41467-017-00212-4
Abstract: Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs—such as LINC00173 in granulocytes — and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia s les into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.
Publisher: Oxford University Press (OUP)
Date: 28-09-2007
DOI: 10.1093/BIB/BBM046
Abstract: Liquid chromatography (LC) coupled to electrospray mass spectrometry (MS) is well established in high-throughput proteomics. The technology enables rapid identification of large numbers of proteins in a relatively short time. Comparative quantification of identified proteins from different s les is often regarded as the next step in proteomics experiments enabling the comparison of protein expression in different proteomes. Differential labeling of s les using stable isotope incorporation or conjugation is commonly used to compare protein levels between s les but these procedures are difficult to carry out in the laboratory and for large numbers of s les. Recently, comparative quantification of label-free LC(n)-MS proteomics data has emerged as an alternative approach. In this review, we discuss different computational approaches for extracting comparative quantitative information from label-free LC(n)-MS proteomics data. The procedure for computationally recovering the quantitative information is described. Furthermore, statistical tests used to evaluate the relevance of results will also be discussed.
Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2021
DOI: 10.1101/2021.12.19.473333
Abstract: Metabolic reprogramming is a hallmark of cancer characterized by global changes in metabolite levels. However, compared with the study of gene expression, profiling of metabolites in cancer s les remains relatively understudied. We obtained metabolomic profiling and gene expression data from 454 human solid cancer cell lines across 24 cancer types from the Cancer Cell Line Encyclopedia (CCLE) database, to evaluate the feasibility of inferring metabolite levels from gene expression data. For each metabolite, we trained multivariable LASSO regression models to identify gene sets that are most predictive of the level of each metabolite profiled. Even when accounting for cell culture conditions or cell lineage in the model, few metabolites could be accurately predicted. In some cases, the inclusion of the upstream and downstream metabolites improved prediction accuracy, suggesting that gene expression is a poor predictor of steady-state metabolite levels. Our analysis uncovered a single robust relationship between the expression of nicotinamide N-methyltransferase ( NNMT ) and 1-methylnicotinamide (MNA), however, this relationship could only be validated in cancer s les with high purity, as NNMT is not expressed in immune cells. Together, our findings reveal the challenge of inferring metabolite levels from metabolic enzyme levels and suggest that direct metabolomic profiling is necessary to study metabolism in cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 18-11-2020
DOI: 10.1101/2020.11.18.388108
Abstract: DNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer 1 . MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell ision 2 . Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures 3 , suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro 4,5 and in vivo 6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2) complexes 7 . Cancers with deficiency of MutSα exhibit mutational signature contributions distinct from those deficient of MutLα. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutSα, our results suggest that MutSα is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non- iding cells.
Publisher: American Chemical Society (ACS)
Date: 05-05-2010
DOI: 10.1021/AC100594J
Abstract: Signature peptides of the neuraminidase antigen across all common circulating human subtypes of type A and B influenza are identified through the bioinformatic alignment of translated gene sequences. The detection of these peptides within the high-resolution mass spectra of whole antigen, virus, and vaccine digests enables the strains to be rapidly and directly typed and subtyped. Importantly, unique signature peptides for pandemic (H1N1) 2009 influenza are identified and detected that enable pandemic strains to be rapidly and directly differentiated from seasonal type A (H1N1) influenza strains. The detection of these peptides can enable the origins of the neuraminidase gene to be monitored in the case of reassorted strains.
Publisher: American Association for Cancer Research (AACR)
Date: 04-2012
DOI: 10.1158/1538-7445.AM2012-990
Abstract: Promoter CpG island (CGI) hypermethylation is an established mechanism of silencing gene expression in cancer. Although the silenced state is thought to be consolidated by the recruitment of nucleosomes across the promoter, this has been observed in only a limited number of genes. Here we performed genome-wide MBD-Seq, MNase-Seq and RNA-Seq to investigate the relationship between CGI methylation, nucleosome occupancy around the transcription start site (TSS) and gene expression in a premalignant colorectal adenoma and matched normal mucosa. Analysis of RNA-seq data identified 1987 silent genes that were not expressed in the normal or adenoma tissues. Of these 1987 genes, 1401 (70.51%) showed either methylation in both the normal and adenoma tissues or nucleosome occupancy across the TSS, with 524 (26.37%) of genes showing both, suggestive of a consolidated silenced state. Our RNA-Seq data also revealed that 2010 genes were downregulated & -fold in the adenoma when compared to paired normal mucosa. Of these 2010 genes, only 67 (3.33%) genes showed aberrant CGI hypermethylation, and of these 67 genes, only six (8.96%) showed nucleosome occupancy across the TSS. Therefore, the presence of both CGI hypermethylation and nucleosome occupancy across the TSS of downregulated genes is significantly lower than at silent genes (p& .0001). Interestingly, 272 of the remaining 1966 (13.84%) genes that were downregulated in the absence of aberrant CGI hypermethylation showed clear evidence of nucleosome occupancy across the TSS, including the important cancer-related genes CDH1 and CDKN2B. Only seven of these 272 genes (2.57%) have previously been described as frequent targets of hypermethylation in cancer, suggesting nucleosome recruitment represents an alternative mechanism of inactivation of these genes. In summary, we identified some of the earliest epigenetic events in colorectal neoplasia by profiling the transcriptome and epigenetic landscape of a premalignant colorectal adenoma. Our data show that for the majority of genes showing adenoma-specific downregulation and aberrant CGI hypermethylation, nucleosomes are not recruited to the promoter. Moreover, our data reveals a potential novel mechanism of epigenetic gene inactivation in the absence of promoter hypermethylation, through physical occlusion of transcription by nucleosome occupancy at the TSS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research 2012 Mar 31-Apr 4 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2012 (8 Suppl):Abstract nr 990. doi:1538-7445.AM2012-990
Publisher: American Association for Cancer Research (AACR)
Date: 08-2015
DOI: 10.1158/1541-7786.MCR-15-0146
Abstract: With the recent discovery of recurrent mutations in the TERT promoter in melanoma, identification of other somatic causal promoter mutations is of considerable interest. Yet, the impact of sequence variation on the regulatory potential of gene promoters has not been systematically evaluated. This study assesses the impact of promoter mutations on promoter activity in the whole-genome sequenced malignant melanoma cell line COLO-829. Combining somatic mutation calls from COLO-829 with genome-wide chromatin accessibility and histone modification data revealed mutations within promoter elements. Interestingly, a high number of potential promoter mutations (n = 23) were found, a result mirrored in subsequent analysis of TCGA whole-melanoma genomes. The impact of wild-type and mutant promoter sequences were evaluated by subcloning into luciferase reporter vectors and testing their transcriptional activity in COLO-829 cells. Of the 23 promoter regions tested, four mutations significantly altered reporter activity relative to wild-type sequences. These data were then subjected to multiple computational algorithms that score the cis-regulatory altering potential of mutations. These analyses identified one mutation, located within the promoter region of NDUFB9, which encodes the mitochondrial NADH dehydrogenase (ubiquinone) 1 beta subcomplex 9, to be recurrent in 4.4% (19 of 432) of TCGA whole-melanoma exomes. The mutation is predicted to disrupt a highly conserved SP1/KLF transcription factor binding motif and its frequent co-occurrence with mutations in the coding sequence of NF1 supports a pathologic role for this mutation in melanoma. Taken together, these data show the relatively high prevalence of promoter mutations in the COLO-829 melanoma genome, and indicate that a proportion of these significantly alter the regulatory potential of gene promoters. Implications: Genomic-based screening within gene promoter regions suggests that functional cis-regulatory mutations may be common in melanoma genomes, highlighting the need to examine their role in tumorigenesis. Mol Cancer Res 13(8) 1218–26. ©2015 AACR.
Publisher: American Chemical Society (ACS)
Date: 07-04-2009
DOI: 10.1021/AC900026F
Abstract: High resolution, high mass accuracy mass spectra of hemagglutinin and whole virus digests of influenza are shown to be able to be used to type and subtype the major circulating forms of the virus in humans. Conserved residues and peptide segments of the hemagglutinin antigen have been identified across type A and B strains, and for type B strains of the Yamagata 16/88 and Victoria 2/87 lineages. The theoretical masses for the protonated peptide ions for tryptic peptides of conserved sequence were subsequently shown to be unique in mass when compared to in silico generated peptides from all influenza viral protein sequences and those proteins known to contaminate virus preparations. The approach represents a more rapid and direct approach with which to type and subtype the virus that is of critical need to prepare strategies and treatments in the event of a local epidemic or global pandemic.
Publisher: Elsevier BV
Date: 12-2018
DOI: 10.1016/J.STEM.2018.09.016
Abstract: Gastric cancer displays marked molecular heterogeneity with aggressive behavior and treatment resistance. Therefore, good in vitro models that encompass unique subtypes are urgently needed for precision medicine development. Here, we have established a primary gastric cancer organoid (GCO) biobank that comprises normal, dysplastic, cancer, and lymph node metastases (n = 63) from 34 patients, including detailed whole-exome and transcriptome analysis. The cohort encompasses most known molecular subtypes (including EBV, MSI, intestinal/CIN, and diffuse/GS, with CLDN18-ARHGAP6 or CTNND1-ARHGAP26 fusions or RHOA mutations), capturing regional heterogeneity and subclonal architecture, while their morphology, transcriptome, and genomic profiles remain closely similar to in vivo tumors, even after long-term culture. Large-scale drug screening revealed sensitivity to unexpected drugs that were recently approved or in clinical trials, including Napabucasin, Abemaciclib, and the ATR inhibitor VE-822. Overall, this new GCO biobank, with linked genomic data, provides a useful resource for studying both cancer cell biology and precision cancer therapy.
Publisher: American Society of Hematology
Date: 06-12-2014
DOI: 10.1182/BLOOD.V124.21.4601.4601
Abstract: Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA) is a DNA demethylating agent that is effective in treating MDS and CMML. However, response rates are less than 50% and the basis for poor response is currently unknown. A patient's potential to respond cannot be currently determined until after multiple cycles of AZA treatment and alternative treatment options for poor responders are limited. To address these fundamental questions, we enrolled patients on a compassionate access program prior to the listing of AZA on the pharmaceuticals benefit scheme in Australia. We have collected bone marrow from 18 patients (10 MDS, 8 CMML) at seven different stages of treatment, starting from before treatment until after six cycles of AZA treatment, and isolated high-purity CD34+ HSPCs at each stage. 10 of these patients (5 MDS and 5 CMML) responded completely to AZA while 8 did not achieve complete response. We performed next-generation sequencing (RNA-seq) of these HSPCs to identify the basis of poor response to AZA therapy. Analysis of the RNA-seq data from pre-treatment HSPCs has revealed a striking differential expression of 1148 genes between patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA therapy (Figure 1A). Using a Fluidigm nanofluidic system, we have validated the differential expression of a subset of these genes between CR and non-CR patients in two independent cohorts, totalling 67 patients, from the U.K. and Sweden. We have additionally confirmed that our gene signature does not simply segregate patients based on disease severity or poor overall survival, but rather uniquely prognosticates best AZA response. Pathway analyses of the differentially expressed genes indicates that the HSPCs of non-CR patients have decreased cell cycle progression and DNA damage pathways, while concomitantly possessing increased signalling through integrin and mTOR/AKT pathways. Using computational methods, we have determined that the expression of 15 genes (within the 1148 gene set) is sufficient to separate CRs from non-CRs across independent cohorts (Figure 1B). We have also developed a predictive AZA response algorithm that utilises the expression of these genes to identify potential complete and non-complete responders to AZA with high specificity and sensitivity (Figure 1C). Furthermore, we have identified statistically significant correlations between recurrent DNA mutations in MDS and our prognostic gene signature (SF3B1 & TET2 with CR, STAG2 and NUP98 with non-CR, p .05). We have used these findings to first, develop a clinically useful method to predict the likelihood of AZA response and second, use targeted therapies to promote AZA response in likely poor responders. To predict AZA response, we assess cell cycle progression of MDS/CMML CD34+ subsets by flow cytometry using unfractionated bone marrow aspirates. To improve drug response in predicted non-CR patients, we have performed combinatorial drug testing experiments with AZA using primary MDS/CMML CD34+ HSPCs, in a co-culture system using MS5 stromal cells, targeting up-regulated pathways identified from our RNA-seq data. (Figures 1D, 1E). Our findings have immediate clinical utility to both prospectively identify CR and non-CR patients prior to AZA therapy and to improve AZA response in the latter by using combination therapy targeting specific pathways. Fig 1. A.) Differential expression of 1148 genes in pre-treatment HSPCs of patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA. B.) The differential expression of a subset of 15 genes is sufficient to separate the two groups. C.) A predictive AZA response algorithm that utilises gene expression data to prospectively identify patients. D.) Representative images of CFU colonies illustrating improved colony formation following combination drug treatment. E.) Improved CFU colony counts following combination drug treatment. * p .05 Fig 1. A.) Differential expression of 1148 genes in pre-treatment HSPCs of patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA. B.) The differential expression of a subset of 15 genes is sufficient to separate the two groups. C.) A predictive AZA response algorithm that utilises gene expression data to prospectively identify patients. D.) Representative images of CFU colonies illustrating improved colony formation following combination drug treatment. E.) Improved CFU colony counts following combination drug treatment. * p .05 Lynch: Celgene Pty Ltd: Employment, Equity Ownership. Mufti:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pimanda:Celgene Pty Ltd: Research Funding.
Publisher: Oxford University Press (OUP)
Date: 22-05-2017
DOI: 10.1093/NAR/GKX463
Publisher: Springer Science and Business Media LLC
Date: 08-2004
DOI: 10.1039/B315904C
Abstract: The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin-melittin protein-peptide complexes are presented to illustrate the approach.
Publisher: BMJ
Date: 26-03-2020
DOI: 10.1136/GUTJNL-2019-320019
Abstract: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. We observed a striking ersity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2 , but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.
Publisher: American Society of Hematology
Date: 03-10-2013
DOI: 10.1182/BLOOD-2013-03-490425
Abstract: Genome-wide binding profiles of FLI1, ERG, GATA2, RUNX1, SCL, LMO2, and LYL1 in human HSPCs reveals patterns of combinatorial TF binding. Integrative analysis of transcription factor binding reveals a densely interconnected network of coding and noncoding genes in human HSPCs.
Publisher: Springer New York
Date: 15-12-2016
DOI: 10.1007/978-1-4939-6740-7_11
Abstract: Through advances in molecular biology, comparative analysis of DNA sequences is currently the cornerstone in the study of molecular evolution and phylogenetics. Nevertheless, protein mass spectrometry offers some unique opportunities to enable phylogenetic analyses in organisms where DNA may be difficult or costly to obtain. To date, the methods of phylogenetic analysis using protein mass spectrometry can be classified into three categories: (1) de novo protein sequencing followed by classical phylogenetic reconstruction, (2) direct phylogenetic reconstruction using proteolytic peptide mass maps, and (3) mapping of mass spectral data onto classical phylogenetic trees. In this chapter, we provide a brief description of the three methods and the protocol for each method along with relevant tools and algorithms.
Publisher: Springer Science and Business Media LLC
Date: 25-07-2019
DOI: 10.1038/S41467-019-11132-W
Abstract: Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.
Publisher: Hindawi Limited
Date: 17-04-2015
DOI: 10.1002/HUMU.22785
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9187-7_2
Abstract: Studying the evolutionary conservation of proteins can be a valuable tool for understanding its function. At the sequence level, the conservation of each residue can be used to infer the importance of the particular regions of proteins. In the case of protein disulphide bonds, the conservation of the cysteines involved can be used to infer the conservation of the disulphide bond itself. In this chapter, bioinformatics methods are described that can be used to assess the conservation of a protein disulphide bond with a focus on the study of human proteins. Conservation will be assessed at the species and at the human population level. The methods described make use of publicly available databases and can be applied by any researcher using a standard desktop computer with Internet access.
Publisher: Informa UK Limited
Date: 20-04-2021
Publisher: Impact Journals, LLC
Date: 27-09-2017
Publisher: Springer Science and Business Media LLC
Date: 12-2018
Publisher: Oxford University Press (OUP)
Date: 06-09-2016
DOI: 10.1093/NAR/GKW804
Publisher: American Chemical Society (ACS)
Date: 02-2005
DOI: 10.1016/J.JASMS.2004.11.009
Abstract: The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.
Publisher: Public Library of Science (PLoS)
Date: 03-02-2020
Publisher: Springer Science and Business Media LLC
Date: 02-04-2019
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.JAUT.2012.05.005
Abstract: Factor XI (FXI), a disulfide-linked covalent homodimer, circulates in plasma, and upon activation initiates the intrinsic/consolidation phase of coagulation. We present evidence that disulfide bonds in FXI are reduced to free thiols by oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). We identified that Cys362-Cys482 and Cys118-Cys147 disulfide bonds are reduced by TRX-1. The activation of TRX-1-treated FXI by thrombin, FXIIa or FXIa was significantly increased compared to non-reduced FXI, indicating that the reduced factor is more efficiently activated than the oxidized protein. Using a novel ELISA system, we compared the amount of reduced FXI in antiphospholipid syndrome (APS) thrombosis patients with levels in healthy controls, and found that APS patients have higher levels of reduced FXI. This may have implication for understanding the contribution of FXI to APS thrombosis, and the predisposition to thrombosis in patients with elevated plasma levels of reduced FXI.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.C.6550950.V1
Abstract: Purpose: Drug repurposing offers the opportunity for chemotherapy to be used to reestablish sensitivity to immune checkpoint blockade (ICB) therapy. Here we investigated the clinical and translational aspects of an early phase II study of azacitidine and carboplatin priming for anti-PDL1 immunotherapy (avelumab) in patients with advanced ICB-resistant melanoma. Experimental Design: A total of 20 participants with ICB-resistant metastatic melanoma received 2 × 4-week cycles of azacitidine and carboplatin followed by ICB rechallenge with anti-PD-L1 avelumab. The primary objective was overall response rate after priming and ICB rechallenge. Secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). Translational correlation analysis of HLA-A and PD-L1 expression, RNA sequencing, and reduced representation bisulfite sequencing of biopsies at baseline, after priming and after six cycles of avelmuab was performed. Results: The overall response rate (ORR) determined after azacitidine and carboplatin priming was 10% (2/20) with two partial responses (PR). The ORR determined after priming followed by six cycles of avelumab (week 22) was 10%, with 2 of 20 participants achieving immune partial response (iPR). The CBR for azacitidine and carboplatin priming was 65% (13/20) and after priming followed by six cycles of avelumab CBR was 35% ( i n /i = 7/20). The median PFS was 18.0 weeks [95% confidence interval (CI): 14.87–21.13 weeks] and the median OS was 47.86 weeks (95% CI: 9.67–86.06 weeks). Translational correlation analysis confirmed HLA-A generally increased after priming with azacitidine and carboplatin, particularly if it was absent at the start of treatment. Average methylation of CpGs across the HLA-A locus was decreased after priming and T cells, in particular CD8 sup + /sup , showed the greatest increase in infiltration. Conclusions: Priming with azacitidine and carboplatin can induce disease stabilization and resensitization to ICB for metastatic melanoma. Significance: There are limited treatments for melanoma once resistance to ICB occurs. Chemotherapy induces immune-related responses and may be repurposed to reinstate the response to ICB. This study provides the first evidence that chemotherapy can provide clinical benefit and increase OS for ICB-resistant melanoma. /
Publisher: Cold Spring Harbor Laboratory
Date: 24-06-2020
DOI: 10.1101/681767
Abstract: Cancer genomes with mutations in the exonuclease domain of Polymerase Epsilon (POLE) present with an extraordinarily high somatic mutation burden. In vitro studies have shown that distinct POLE mutants exhibit different polymerase activity and yet, how these POLE mutants generate mutations across cancer genomes and influence driver events remains poorly understood. Here we analyzed 7,345 colorectal cancer s les, including nine whole genome sequenced s les harboring POLE mutations. Our analysis identified differential mutation spectra across the mutants including methylation-independent enrichment of C T mutations in POLE V411L. In contrast, analysis of other genomic regions showed similar mutation profiles across the different POLE mutants. Notably, we found that POLE mutants with the TP53 R213* mutation, caused by a TT[C T]GA substitution, have significantly higher relative frequency of this mutational context compared with s les without this mutation. This finding demonstrates that variations in underlying mutation spectra can increase the likelihood of specific driver mutation formation.
Publisher: American Association for Cancer Research (AACR)
Date: 17-08-2022
DOI: 10.1158/2767-9764.CRC-22-0128
Abstract: Drug repurposing offers the opportunity for chemotherapy to be used to reestablish sensitivity to immune checkpoint blockade (ICB) therapy. Here we investigated the clinical and translational aspects of an early phase II study of azacitidine and carboplatin priming for anti-PDL1 immunotherapy (avelumab) in patients with advanced ICB-resistant melanoma. A total of 20 participants with ICB-resistant metastatic melanoma received 2 × 4-week cycles of azacitidine and carboplatin followed by ICB rechallenge with anti-PD-L1 avelumab. The primary objective was overall response rate after priming and ICB rechallenge. Secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). Translational correlation analysis of HLA-A and PD-L1 expression, RNA sequencing, and reduced representation bisulfite sequencing of biopsies at baseline, after priming and after six cycles of avelmuab was performed. The overall response rate (ORR) determined after azacitidine and carboplatin priming was 10% (2/20) with two partial responses (PR). The ORR determined after priming followed by six cycles of avelumab (week 22) was 10%, with 2 of 20 participants achieving immune partial response (iPR). The CBR for azacitidine and carboplatin priming was 65% (13/20) and after priming followed by six cycles of avelumab CBR was 35% (n = 7/20). The median PFS was 18.0 weeks [95% confidence interval (CI): 14.87–21.13 weeks] and the median OS was 47.86 weeks (95% CI: 9.67–86.06 weeks). Translational correlation analysis confirmed HLA-A generally increased after priming with azacitidine and carboplatin, particularly if it was absent at the start of treatment. Average methylation of CpGs across the HLA-A locus was decreased after priming and T cells, in particular CD8+, showed the greatest increase in infiltration. Priming with azacitidine and carboplatin can induce disease stabilization and resensitization to ICB for metastatic melanoma. There are limited treatments for melanoma once resistance to ICB occurs. Chemotherapy induces immune-related responses and may be repurposed to reinstate the response to ICB. This study provides the first evidence that chemotherapy can provide clinical benefit and increase OS for ICB-resistant melanoma.
Publisher: Oxford University Press (OUP)
Date: 23-10-2023
DOI: 10.1093/NAR/GKAD918
Publisher: Springer Science and Business Media LLC
Date: 12-02-2021
DOI: 10.1038/S41698-021-00142-X
Abstract: Studies have shown that tumor microenvironment (TME) might affect drug sensitivity and the classification of colorectal cancer (CRC). Using TME-specific gene signature to identify CRC subtypes with distinctive clinical relevance has not yet been tested. A total of 18 “bulk” RNA-seq datasets (total n = 2269) and four single-cell RNA-seq datasets were included in this study. We constructed a “Signature associated with FOLFIRI resistant and Microenvironment” (SFM) that could discriminate both TME and drug sensitivity. Further, SFM subtypes were identified using K -means clustering and verified in three independent cohorts. Nearest template prediction algorithm was used to predict drug response. TME estimation was performed by CIBERSORT and microenvironment cell populations-counter (MCP-counter) methods. We identified six SFM subtypes based on SFM signature that discriminated both TME and drug sensitivity. The SFM subtypes were associated with distinct clinicopathological, molecular and phenotypic characteristics, specific enrichments of gene signatures, signaling pathways, prognosis, gut microbiome patterns, and tumor lymphocytes infiltration. Among them, SFM-C and -F were immune suppressive. SFM-F had higher stromal fraction with epithelial-to-mesenchymal transition phenotype, while SFM-C was characterized as microsatellite instability phenotype which was responsive to immunotherapy. SFM-D, -E, and -F were sensitive to FOLFIRI and FOLFOX, while SFM-A, -B, and -C were responsive to EGFR inhibitors. Finally, SFM subtypes had strong prognostic value in which SFM-E and -F had worse survival than other subtypes. SFM subtypes enable the stratification of CRC with potential chemotherapy response thereby providing more precise therapeutic options for these patients.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.22545471
Abstract: Supplementary Figure 1 shows Whole blood cell counts at baseline, week 9 and week 22
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.CELREP.2017.06.067
Abstract: Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking in idual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.
Publisher: American Society of Hematology
Date: 03-12-2015
DOI: 10.1182/BLOOD.V126.23.710.710
Abstract: Background: Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. There are few alternative therapies for the non-responders. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse. The in vivo effect of AZA therapy on dysplastic cells in responders is unclear and there are no predictive markers for impending relapse in responders. Methods: To address these fundamental questions, we enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points - before treatment through 6 cycles of treatment and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes (and validated by Fluidigm-based PCR) and deduced the clonal evolution in the bone marrow in response to AZA therapy (by whole exome-sequencing, followed by targeted capture resequencing, and genotyping of in idual CFU colonies). Our findings were validated in an independent cohort of 57 patients. We used flow cytometry to develop a clinically relevant prognostic assay for AZA resistance and developed a novel stromal co-culture based functional drug testing platform to rationally discover combinational drug therapies to overcome AZA resistance. Results: We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data strikingly revealed the differential expression of 1148 genes between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle and DNA damage response pathways were relatively up-regulated in responders compared to non-responders, indicating that HSPCs of non-responders are more quiescent compared to responders (Figure C). We validated these gene expression differences in independent patient cohorts from the U.K. and Sweden (n=57 27 responders, 30 non-responders). We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders across all cohorts (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, and make them more susceptive to AZA, we leveraged our RNA-seq discoveries to target pathways that were relatively up-regulated in non-responders. Using a stromal co-culture drug testing platform that we developed, we discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). Additionally, dysplastic cells were particularly sensitive to the inhibition of the mTOR pathway. To trace the fate of dysplastic cells as patients undergo AZA therapy, and thereby understand the basis of eventual relapse in responders, we performed whole exome sequencing of all patients (Figure F). Using the mutations as "molecular barcodes", we deduced the clonal architecture in each in idual and observed the clonal evolution that occurred in response to AZA treatment. Combined with genotyping of CFU colonies grown in vitro, we have discovered that clonal haematopoiesis originating from resistant multipotent cells bearing mutations persists even upon complete response and forms the basis for eventual relapse (Figure G). Conclusions: Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments. Figure 1. Figure 1. Kulasekararaj: Alexion: Consultancy. Lynch:Celgene: Employment, Equity Ownership. C bell:14M genomics: Other: Co-founder and consultant.
Publisher: Oxford University Press (OUP)
Date: 16-04-2019
Abstract: Genetic testing of cancer s les primarily focuses on protein-coding regions, despite most mutations arising in noncoding DNA. Noncoding mutations can be pathogenic if they disrupt gene regulation, but the benefits of assessing promoter mutations in driver genes by panel testing has not yet been established. This is especially the case in colorectal cancer, for which few putative driver variants at regulatory elements have been reported. We designed a unique target capture sequencing panel of 39 colorectal cancer driver genes and their promoters, together with more than 35 megabases of regulatory elements focusing on gene promoters. Using this panel, we sequenced 95 colorectal cancer and matched normal s les at high depth, averaging 170× and 82× coverage, respectively. Our target capture sequencing design enabled improved coverage and variant detection across captured regions. We found cases with hereditary defects in mismatch and base excision repair due to deleterious germline coding variants, and we identified mutational spectra consistent with these repair deficiencies. Focusing on gene promoters and other regulatory regions, we found little evidence for base or region-specific recurrence of functional somatic mutations. Promoter elements, including TERT, harbored few mutations, with none showing strong functional evidence. Recurrent regulatory mutations were rare in our sequenced regions in colorectal cancer, though we highlight some candidate mutations for future functional studies. Our study supports recent findings that regulatory driver mutations are rare in many cancer types and suggests that the inclusion of promoter regions into cancer panel testing is currently likely to have limited clinical utility in colorectal cancer.
Publisher: Elsevier BV
Date: 05-2010
DOI: 10.1016/J.JVIROMET.2010.01.015
Abstract: The use of high resolution mass spectrometry to detect signature peptides within proteolytic digests of the isolated matrix M1 protein, and whole virus digests, for both human and animal strains of influenza is shown to be able to rapidly and reliably type the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of matrix M1 protein sequences across all human, avian and swine strains of the influenza virus. Peptides with unique masses, when compared with those from the in silico digestion of all influenza antigens and those proteins known to contaminate egg grown strains, were identified using the purpose built FluGest algorithm. Their frequency of occurrence within the matrix M1 protein across all type A and type B strains was established with the FluAlign algorithm. The subsequent detection of the signature peptides of matrix M1 protein within proteolytic digests of type A and type B human and avian strains has been demonstrated.
Publisher: American Association for Cancer Research (AACR)
Date: 12-2016
DOI: 10.1158/1541-7786.MCR-16-0175
Abstract: Laterally spreading tumors (LST) are colorectal adenomas that develop into extremely large lesions with predominantly slow progression to cancer, depending on lesion subtype. Comparing and contrasting the molecular profiles of LSTs and colorectal cancers offers an opportunity to delineate key molecular alterations that drive malignant transformation in the colorectum. In a discovery cohort of 11 LSTs and paired normal mucosa, we performed a comprehensive and unbiased screen of the genome, epigenome, and transcriptome followed by bioinformatics integration of these data and validation in an additional 84 large, benign colorectal lesions. Mutation rates in LSTs were comparable with microsatellite-stable colorectal cancers (2.4 vs. 2.6 mutations per megabase) however, copy number alterations were infrequent (averaging only 1.5 per LST). Frequent genetic, epigenetic, and transcriptional alterations were identified in genes not previously implicated in colorectal neoplasia (ANO5, MED12L, EPB41L4A, RGMB, SLITRK1, SLITRK5, NRXN1, ANK2). Alterations to pathways commonly mutated in colorectal cancers, namely, the p53, PI3K, and TGFβ pathways, were rare. Instead, LST-altered genes converged on axonal guidance, Wnt, and actin cytoskeleton signaling. These integrated omics data identify molecular features associated with noncancerous LSTs and highlight that mutation load, which is relatively high in LSTs, is a poor predictor of invasive potential. Implications: The novel genetic, epigenetic, and transcriptional changes associated with LST development reveal important insights into why some adenomas do not progress to cancer. The finding that LSTs exhibit a mutational load similar to colorectal carcinomas has implications for the validity of molecular biomarkers for assessing cancer risk. Mol Cancer Res 14(12) 1217–28. ©2016 AACR.
Publisher: American Society of Hematology
Date: 16-09-2010
Publisher: Wiley
Date: 30-12-2008
Abstract: Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large-scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide-binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide-binding proteins was indeed taking place. Finally, by employing label-free MS-based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide-binding proteome.
Publisher: Cold Spring Harbor Laboratory
Date: 04-02-2022
DOI: 10.1101/2022.02.03.22270286
Abstract: Drug repurposing offers the opportunity for approved chemotherapy agents to be used to re-establish sensitivity to immune checkpoint blockade (ICB) therapy. Here we investigated the clinical and translational aspects of an early phase II study of azacitidine and carboplatin priming for anti-PDL1 immunotherapy (Avelumab) in patients with advanced ICB-resistant melanoma. 20 participants with ICB resistant metastatic melanoma received 2 × 4-week cycles of azacitidine and carboplatin followed by ICB re-challenge with anti-PD-L1 avelumab. The overall response rate (ORR) determined after 2 × 4-week cycles of azacitidine and carboplatin priming was 10% (2/20) with 2 partial responses (PR). The ORR determined after priming followed by 6 cycles of avelumab (week 22) was 10%, with 2/20 participants achieving iPR. The clinical benefit rate (CBR) for azacitidine and carboplatin priming was 65% (13/20) and after priming followed by 6 cycles of avelumab CBR was 35% (n = 7/20). The median PFS was 18.0 weeks (95% CI: 14.87 – 21.13 weeks) and the median OS was 47.86 weeks (95% CI: 9.67 – 86.06 weeks). Translational correlation analysis of tumour biopsies at baseline, after priming and after 6 cycles of avelmuab confirmed HLA-A generally increased after priming with azacitidine and carboplatin, particularly if it was absent at the start of treatment. Average methylation of CpGs across the HLA-A locus showed a consistent decrease in methylation after priming and T-cells, in particular CD8+, showed the greatest increase in infiltration. Priming with azacitidine and carboplatin can induce disease stabilization and re-sensitisation to ICB for metastatic melanoma. Sequential azacitidine and carboplatin stabilises disease burden and re-establishes sensitivity to checkpoint immune blockade immunotherapy.
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.ACA.2015.09.004
Abstract: A novel computer algorithm FluClass has been developed to facilitate the phylogenetic classification of influenza virus using mass spectral data. FluClass accepts a DNA or protein-based phylogenetic tree as input and generates theoretical peptide mass lists for each node. An experimental mass spectrum from an influenza virus protein digest is then placed onto the phylogenetic tree using a novel random res ling function (Z-score) that allows the scoring of spectrum against both internal and leaf nodes. Testing of the algorithm using hemagglutinin protein sequences from human-host influenza viruses showed that the Z-score performs comparably to the Profound scoring method for the scoring of leaf nodes and is substantially better at scoring internal nodes. Scoring of internal nodes allows colorizations of nodes of the phylogenetic tree enabling the classification of the query spectrum to be rapidly visualized. Finally we demonstrate the utility of FluClass on experimental spectra from six strains. Given that mass spectrometry data can be generated rapidly for influenza virus proteins, FluClass provides a fast and direct method for phylogenetic analysis of influenza proteins.
Publisher: American Society of Hematology
Date: 30-06-2011
DOI: 10.1182/BLOOD-2010-12-317990
Abstract: The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit+ cells, adult bone marrow stem rogenitors and early CD4−CD8− double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.
Publisher: Elsevier BV
Date: 06-2023
Publisher: American Association for Cancer Research (AACR)
Date: 07-2018
DOI: 10.1158/1538-7445.AM2018-3173
Abstract: Glucocorticoids are well known for their immunosuppressive activity, and play a critical role in the treatment of lymphoid malignancies. However, the development of resistance remains a significant barrier to cure and the mechanisms are poorly defined. Moreover, glucocorticoids are rarely efficacious in treating myeloid and other non-lymphoid malignancies, and the mechanisms of lymphocyte-specific efficacy are unclear. To address these issues, we first carried out a global analysis of DNase I hypersensitive sites in 18 lymphoid and 64 non-lymphoid cell types to map lymphocyte-specific open chromatin domains (LSOs). We then integrated these domains with genome-wide glucocorticoid-induced gene transcription and epigenetic modulation in an in vivo patient-derived xenograft (PDX) model of acute lymphoblastic leukemia (ALL). Performing chromatin immunoprecipitation and assays for transposase-accessible chromatin (ATAC), we determined a strong correlation between glucocorticoid receptor (GR) binding and chromatin accessibility, acetylated histone marks and binding of a DNA structural protein (CTCF), and identified 1,536 GR bound LSOs. We next analyzed RNA-seq data in glucocorticoid sensitive and resistant ALL PDXs for expression changes in genes located within 100 kb of the LSOs after glucocorticoid treatment in vivo, and identified four groups comprising 389 genes that were significantly differentially expressed. Of the 198 up-regulated genes, 143 showed increased H3K27Ac enrichments at 177 LSOs. Forty-two LSOs showed the increase only in glucocorticoid sensitive but not resistant ALLs. Applying this to an extended panel of ALL PDXs, basal DNA methylation at the 42 LSOs was significantly higher in resistant ALLs despite no difference within the gene bodies, whereas the basal chromatin accessibility indicated by ATAC abundance was diminished. Pathway analysis indicated that the LSO regulated genes were involved in repressing B- and T- cell receptor signaling pathways and activating the apoptotic pathway. One such LSO was at the pro-apoptotic BIM gene locus, where CTCF binding was found only in lymphocytes but not in other cell types. The GR cooperated with CTCF to mediate interactions between the promoter and the BIM LSO to direct DNA looping, thus triggering BIM transcription. Importantly, this LSO was heavily methylated in resistant ALLs as well as non-lymphoid cells. Azacitidine, a DNA demethylating drug that is routinely used in the clinic, could partially reverse these changes and restore glucocorticoid sensitivity. Taken together, this study demonstrated for the first time that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell type. Reversal of these epigenetic changes may lead to improvements in the use of glucocorticoids in the clinic. Citation Format: Duohui Jing, Yizhou Huang, Xiaoyun Liu, Keith Sia, Rebecca C. Poulos, Miriam Span, Chao Zhang, Jianqing Mi, Jason WH Wong, Dominik Beck, John E. Pimanda, Richard B. Lock. Lymphocyte-specific chromatin accessibility predetermines glucocorticoid resistance in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018 2018 Apr 14-18 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2018 (13 Suppl):Abstract nr 3173.
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2022
DOI: 10.1101/2022.01.21.477237
Abstract: Xeroderma pigmentosum group D (XPD) is a DNA helicase involved in transcription initiation and nucleotide excision repair. Missense mutations in XPD are putative drivers in bladder cancer (BLCA) and are associated with a specific single base substitution mutational signature. However, the impact of XPD on the genome-wide distribution of somatic mutations remains unexplored. We analysed somatic mutation distribution in whole-genome sequenced (WGS) BLCA s les with (XPD mutant) and without XPD mutations (WT). XPD genotype had a large impact on the distribution of somatic mutations. XPD mutant s les had increased mutation density at open chromatin, including striking mutation hotspots at CTCF-cohesin binding sites (CBS). We validate these findings in additional WGS cohorts and BLCA exomes. Analysis of XPD occupancy and CBS hotspot mutations in other cancer types suggest that XPD protects CBS from DNA damage. Our study implicates XPD in genomic integrity maintenance at topologically-associating domain boundaries marked by CTCF-cohesin binding.
Publisher: Elsevier BV
Date: 03-2023
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2767-9764.22545465
Abstract: Supplementary Figure 2 shows transposable element methylation (A) and expression (B) at baseline, week 9 and week 22
Publisher: American Society of Hematology
Date: 27-03-2014
DOI: 10.1182/BLOOD-2014-01-549816
Abstract: Most proteins in nature are chemically modified after they are made to control how, when, and where they function. The 3 core features of proteins are posttranslationally modified: amino acid side chains can be modified, peptide bonds can be cleaved or isomerized, and disulfide bonds can be cleaved. Cleavage of peptide bonds is a major mechanism of protein control in the circulation, as exemplified by activation of the blood coagulation and complement zymogens. Cleavage of disulfide bonds is emerging as another important mechanism of protein control in the circulation. Recent advances in our understanding of control of soluble blood proteins and blood cell receptors by functional disulfide bonds is discussed as is how these bonds are being identified and studied.
Publisher: American Association for Cancer Research (AACR)
Date: 15-07-2020
DOI: 10.1158/1078-0432.CCR-20-0184
Abstract: RNA splicing is a fundamental biological process that generates protein ersity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematologic cancers but are relatively uncommon in acute myeloid leukemia (AML, & 20% of patients). We examined whether RNA splicing differences exist in AML, even in the absence of splicing factor mutations. We developed a bioinformatics pipeline to study alternative RNA splicing in RNA-sequencing data from large cohorts of patients with AML. We have identified recurrent differential alternative splicing between patients with poor and good prognosis. These splicing events occurred even in patients without any discernible splicing factor mutations. Alternative splicing recurrently occurred in genes with specific molecular functions, primarily related to protein translation. Developing tools to predict the functional impact of alternative splicing on the translated protein, we discovered that approximately 45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced and the splicing of their target transcripts were altered. Studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Finally, using machine learning techniques, we identified a splicing signature of four genes which refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance. See related commentary by Bowman, p. 3503
Publisher: Public Library of Science (PLoS)
Date: 09-11-2018
Publisher: Cold Spring Harbor Laboratory
Date: 11-01-2020
DOI: 10.1101/2020.01.10.895714
Abstract: RNA splicing is a fundamental biological process that generates protein ersity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are relatively uncommon in Acute Myeloid Leukemia (AML, 20%). We examined whether RNA splicing differences exist in AML even in the absence of splicing factor mutations. Analyzing RNA-seq data from two independent cohorts of AML patients, we identified recurrent differential alternative splicing between patients with poor and good prognosis. These alternative splicing events occurred even in patients without any discernible splicing factor mutations. The alternative splicing events recurrently occurred in genes involved in specific molecular functions, primarily related to protein translation. Developing informatics tools to predict the functional impact of alternative splicing on the translated protein, we discovered that ~45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced in patients, and the splicing of their target transcripts were also altered. By studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and up- regulation of inflammation-related genes. Lastly, using machine learning techniques, we identified a set of four genes whose alternative splicing can refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance.
Publisher: Springer Science and Business Media LLC
Date: 04-07-2018
DOI: 10.1038/LEU.2017.210
Abstract: Prognostic gene expression signatures have been proposed as clinical tools to clarify therapeutic options in acute myeloid leukemia (AML). However, these signatures rely on measuring large numbers of genes and often perform poorly when applied to independent cohorts or those with older patients. Long intergenic non-coding RNAs (lincRNAs) are emerging as important regulators of cell identity and oncogenesis, but knowledge of their utility as prognostic markers in AML is limited. Here we analyze transcriptomic data from multiple cohorts of clinically annotated AML patients and report that (i) microarrays designed for coding gene expression can be repurposed to yield robust lincRNA expression data, (ii) some lincRNA genes are located in close proximity to hematopoietic coding genes and show strong expression correlations in AML, (iii) lincRNA gene expression patterns distinguish cytogenetic and molecular subtypes of AML, (iv) lincRNA signatures composed of three or four genes are independent predictors of clinical outcome and further dichotomize survival in European Leukemia Net (ELN) risk groups and (v) an analytical tool based on logistic regression analysis of quantitative PCR measurement of four lincRNA genes (LINC4) can be used to determine risk in AML.
Publisher: Wiley
Date: 27-02-2015
DOI: 10.1002/GCC.22243
Abstract: The progression of benign colorectal adenomas into cancer is associated with the accumulation of chromosomal aberrations. Even though patterns and frequencies of chromosomal aberrations have been well established in colorectal carcinomas, corresponding patterns of aberrations in adenomas are less well documented. The aim of this study was to profile chromosomal aberrations across colorectal adenomas and carcinomas to provide a better insight into key changes during tumor initiation and progression. Single nucleotide polymorphism array analysis was performed on 216 colorectal tumor/normal matched pairs, comprising 60 adenomas and 156 carcinomas. While many chromosomal aberrations were specific to carcinomas, those with the highest frequency in carcinomas ( lification of chromosome 7, 13q, and 20q deletion of 17p and chromosome 18 LOH of 1p, chromosome 4, 5q, 8p, 17p, chromosome 18, and 20p) were also identified in adenomas. Hierarchical clustering using chromosomal aberrations revealed three distinct subtypes. Interestingly, these subtypes were only partially dependent on tumor staging. A cluster of colorectal cancer patients with frequent chromosomal deletions had the least favorable prognosis, and a number of adenomas (n = 9) were also present in the cluster suggesting that, at least in some tumors, the chromosomal aberration pattern is determined at a very early stage of tumor formation. Finally, analysis of LOH events revealed that copy-neutral/gain LOH (CN/G-LOH) is frequent (>10%) in carcinomas at 5q, 11q, 15q, 17p, chromosome 18, 20p, and 22q. Deletion of the corresponding region is sometimes present in adenomas, suggesting that LOH at these loci may play an important role in tumor initiation.
Publisher: American Chemical Society (ACS)
Date: 21-05-2013
DOI: 10.1021/AC4005875
Abstract: A new phylogenetics approach and algorithm with which to chart the evolutionary history of organisms is presented. It utilizes mass spectral data produced from the proteolytic digestion of proteins, rather than partial or complete gene or translated gene sequences. The concept and validity of the approach is demonstrated herein using both theoretical and experimental mass data, together with the translated gene sequences of the hemagglutinin protein of the influenza virus. A comparison of the mass trees with conventional sequenced-based phylogenetic trees, using two separate tree comparison algorithms, reveals a high degree of similarity and congruence among the trees. Given that the mass map data can be generated more rapidly than gene sequences, even when next generation parallel sequencing is employed, mass trees offer new opportunities and advantages for phylogenetic analysis.
Publisher: Cold Spring Harbor Laboratory
Date: 17-07-2020
DOI: 10.1101/2020.07.14.20129031
Abstract: The immune system plays a key role in protecting against cancer. Increased immune infiltration in tumor tissue is usually associated with improved clinical outcome, but in colorectal cancer (CRC), excessive immune infiltration has also been shown to lead to worst prognosis. The factors underlying this immune overdrive phenotype remains unknown. Using RNA sequencing data from The Cancer Genome Atlas, the expression of over 1,000 transposable element (TE) subfamilies were quantified using the “REdiscoverTE” pipeline. Candidate prognostic and immunogenic TEs were screened by survival and correlation analysis, respectively. Based on these candidates, a TE expression score was developed and CRC patients were clustered using the “kaps” algorithm. In CRC, we found that the TE expression score stratified patients into four clusters each with distinctive prognosis. Those with the highest TE expression were associated with immune overdrive and had the poorest outcomes. Importantly, this association was independent of microsatellite instability status and tumor mutation burden. To link TE overexpression to the immune overdrive phenotype, we showed that cell lines treated with DNA methyltransferase inhibitors also had a high TE expression score and activation of cellular innate immune response pathways. Finally, a pan-cancer survey of TE expression identified a subset of kidney renal clear cell carcinoma with a similar adverse immune overdrive phenotype with poor prognosis. Our findings reveal that TE expression is associated with immune overdrive in cancer and is an independent predictor of immune infiltration and prognosis in CRC patients. 1. Cancers with high immune infiltration generally have better prognosis, but it is unknown why a subset of colorectal cancers (CRC) with high immune infiltration have the poorest outcomes. Transposable element (TE) expression has been shown to be strongly associated with immune infiltration in cancers but its role in patient prognosis is unclear. TEs can be reactivated by DNA hypomethylation in cancers, resulting in immune response via viral mimicry. 2. A TE expression score has been developed that is predicative of prognosis in CRC patients where those who have the highest TE score show an immune overdrive phenotype and have the worst prognosis. The TE expression score predicts prognostic and immune infiltration independent of microsatellite instability and tumor mutation burden (TMB). Immune response pathways and infiltrate profiles of high TE expression CRC recapitulates those of DNA methyltransferase inhibitor treated cells where TEs are reactivated, suggesting that TE overexpression may drive immune infiltration in CRC. A pan-cancer analysis found that kidney renal clear cell carcinoma shares are a similar TE expression associated immune overdrive phenotype with adverse prognosis. 3. Our work highlights the importance of TE expression in evaluating CRC patient prognosis. The association of TE expression with the immune overdrive phenotype independent of MSI and TMB status suggests that by considering TE expression, there may be new opportunities to identify MSS CRC patients for immunotherapy and develop new strategies to harness TE driven immune response.
Publisher: American Society of Hematology
Date: 05-2014
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2023
DOI: 10.1101/2023.04.18.537282
Abstract: Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts and TF binding profiles in HSPC subsets (HSC, CMP, GMP, MEP) and found that heptad occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. These findings suggest that specific heptad-TF combinations play critical roles in regulating hematopoietic differentiation and provide a valuable resource for development of targeted therapies to manipulate specific HSPC subsets.
Publisher: American Association for Cancer Research (AACR)
Date: 14-09-2018
DOI: 10.1158/1078-0432.CCR-17-3678
Abstract: Purpose: MLH1 is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified. Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9–mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter. Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9–mediated deletion of a core binding region impairs endogenous MLH1 expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G & A rs143969848 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells. Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Clin Cancer Res 24(18) 4602–11. ©2018 AACR.
Publisher: Springer Science and Business Media LLC
Date: 19-05-2010
Publisher: American Society of Hematology
Date: 26-10-2023
Abstract: Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (the Heptad, PU.1, CTCF, and STAG2) in HSPC subsets (HSC-MPP, CMP, GMP, MEP) and found that TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs such as PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of specific TF complexes was at least partially regulated by features encoded in specific DNA sequence motifs. Taken together, this study provides a comprehensive characterisation of the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying datasets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analysing aberrant regulatory networks in leukemic cells.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-11-2021
Abstract: DNA mismatch repair is essential for protecting the human genome from damage induced by 5-methylcytosine deamination.
Publisher: American Chemical Society (ACS)
Date: 13-12-2011
DOI: 10.1021/AC2026578
Abstract: Selected reaction monitoring (SRM) is a mass spectrometry-based approach commonly used to increase analytical sensitivity and selectively for specific compounds in complex metabolomic s les. While the goal of well-designed SRM methods is to monitor for unique precursor-product ion pairs, in practice this is not always possible due to the ersity of the metabome and the resolution limits of mass spectrometers that are capable of SRM. Isobaric or near-isobaric precursor ions with different chromatographic properties but identical product ions often arise in complex s les. Without analytical standards, such metabolites will go undetected by conventional data analysis methods. Furthermore, a single SRM method may include simultaneous monitoring of tens to hundreds of different metabolites across multiple s les making quantification of all detected ions a challenging task. To facilitate the analysis of SRM data from complex metabolomic s les, we have developed the Metabolite Mass Spectrometry Analysis Tool (MMSAT). MMSAT is a web-based tool that objectively quantifies every metabolite peak detected in a set of s les and aligns peaks across multiple s les to enable quantitative comparison of each metabolite between s les. The analysis incorporates quantification of multiple peaks/ions that have different chromatographic retention times but are detected within a single SRM transition. We compare the performance of MMSAT against existing tools using a human glioblastoma tissue extract and illustrate its ability to automatically quantify multiple precursors within each of three different transitions. The Web-interface and source code is avaliable at www.cancerresearch.unsw.edu.au/crcweb.nsf age/MMSAT .
Publisher: Cold Spring Harbor Laboratory
Date: 05-06-2020
DOI: 10.1101/2020.06.04.130872
Abstract: Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor–AB and 5-Azacytidine into multipotent stem cells with stromal cell characteristics. To generate a product that is amenable for therapeutic application, we have modified and optimised culture conditions to reprogram human adipocytes into induced multipotent stem cells (iMS) and expand them in vitro . The basal transcriptomes of adipocyte-derived iMS cells and matched adipose-tissue-derived mesenchymal stem cells were remarkably similar. However, there were distinct changes in histone modifications and CpG methylation at cis- regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to new muscle, bone, cartilage and blood vessels with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibres without ectopic tissue formation. Taken together, human adipocyte derived iMS cells regenerate tissues in a context dependent manner without ectopic or neoplastic growth.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 02-2012
End Date: 12-2016
Amount: $275,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2014
End Date: 03-2018
Amount: $705,585.00
Funder: Australian Research Council
View Funded Activity