Inhibition of AlkB Nucleic Acid Demethylases: Promising New Epigenetic Targets

The AlkB family of nucleic acid demethylases is currently of intense chemical, biological, and medical interest because of its critical roles in several key cellular processes, including epigenetic gene regulation, RNA metabolism, and DNA repair. Emerging evidence suggests that dysregulation of AlkB demethylases may underlie the pathogenesis of several human diseases, particularly obesity, diabetes, and cancer. Hence there is strong interest in developing selective inhibitors for these enzymes to facilitate their mechanistic and functional studies and to validate their therapeutic potential. Herein we review the remarkable advances made over the past 20 years in AlkB demethylase inhibition research. We discuss the rational design of reported inhibitors, their mode-of-binding, selectivity, cellular activity, and therapeutic opportunities. We further discuss unexplored structural elements of the AlkB subfamilies and propose potential strategies to enable subfamily selectivity. It is hoped that this perspective will inspire novel inhibitor design and advance drug discovery research in this field

Functional characterization of DNA dealkylation repair protein AlkB from Escherichia coli and use of indenone derivatives as inhibitors of AlkB and human AlkB homologue 3 (AlkBH3)

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli (E coli) AlkB is involved in protecting DNA from alkylation damage. Previous studies have shown that AlkB preferentially removes alkyl DNA adducts from single-stranded DNA (ssDNA). Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Therefore, AlkB-mediated DNA repair using SSBbound DNA as substrate is studied in the present thesis. By using in vitro repair reaction, it is found that AlkB can efficiently remove N3-methyl Cytosine (N3meC) adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N3meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction is found to be abated compared to SSB-bound DNA substrate of identical length. It is also observed here that truncated SSB containing only the DNA binding domain can support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70- mer oligonucleotide containing single N3meC, it is demonstrated that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts. Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. E. coli single-stranded DNA binding protein SSB binds and stabilizes ssDNA. The N-terminal of SSB contains characteristic oligonucleotide/oligosaccharide-binding (OB) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with large number proteins via the C-terminal interaction domain consisting mostly of acidic amino xi acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an intrinsically disordered region (IDR) and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Therefore, AlkB-SSB interaction is characterized in detail in this thesis. It is demonstrated by in vitro pull-down and yeast two-hybrid analysis that SSB binds to AlkB via the IDR. The site of contact is found to be the residues 152-169 of SSB. The present study also reveals that unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction. The human homologue of AlkB, AlkB homologue 3 (AlkBH3), is also a member of the dioxygenase family of enzymes involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. The present thesis also reports screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkB and AlkBH3 DNA repair activity. The indenone-derived AlkB and AlkBH3 inhibitors are found to display specific binding and competitive mode of inhibition. It is demonstrated that AlkB inhibitor has the ability to sensitize cells to methyl methane sulfonate (MMS) that mainly produce DNA alkylation damage and AlkBH3 inhibitor can prevent the proliferation of lung cancer cell line and enhance sensitivity to MMS

Molecular and computational approach to the link between nutrition and cancer

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 22-11-201

Molecular profiling of cutaneous squamous cell carcinoma

PhDCutaneous squamous cell carcinoma (cSCC) is the second most common form of non-melanoma skin cancer and accounts for the majority of deaths from this disease. Its incidence is increasing rapidly, contributing significant morbidity to patients and a burden on healthcare resources. The molecular events underlying cSCC development remain largely uncharacterised, despite the well established role of ultraviolet radiation as a principal carcinogen. Genomewide analyses of the genetic changes underlying cSCC development have shown they are subject to large chromosomal aberrations, which often involve whole chromosome arms. Many of these events occur in a high proportion of tumours, yet the genes they target are unknown. In this study, genomewide expression microarray data has been obtained from a series of cSCC and integrated with single nucleotide polymorphism (SNP) microarray data, to provide a comprehensive analysis of the events associated with tumour development. In total, 222 genes were identified as differentially expressed in cSCC, of which, 21% were concordant with copy number changes. Previous genomewide SNP data of cSCC had identified microdeletions within the PTPRD gene in a subset of tumours (Purdie et al., 2009). This was investigated in further detail and revealed microdeletions in this gene were significantly associated with metastatic cSCC. Sequencing analysis showed 37% of cSCC had a mutation at this locus, which suggests PTPRD is aberrant in a significant proportion of tumours. Decreased expression levels of PTPRD were correspondingly found in moderately and poorly differentiated tumours. The role of PTPRD in skin biology is not known and further functional work is required to elucidate its role in skin cancer. Taken together, these data provide a valuable insight into the genetic background against which cSCC develop. Furthermore, the association of PTPRD disruption with aggressive tumours may potentially be of future benefit as a prognostic biomarker and therapeutic target

Investigating USP2 as a mediator of therapy resistance in lethal prostate cancer

The growth of prostate cancer (PCa) is dependent on male sex hormones, termed androgens, and the androgen receptor (AR). Therefore, implementing strategies to inhibit AR activity, collectively referred to as androgen deprivation therapy (ADT), is the key therapeutic strategy for metastatic prostate cancer. Unfortunately, ADT is never curative, and patients eventually develop a lethal therapy resistant form of the disease termed castration-resistant prostate cancer (CRPC). The AR signalling pathway is altered in CRPC and a subset of CRPC tumours may evade inhibition by ADT by progressing to a state in which tumour growth is independent of this pathway. One such AR-independent CRPC subtype is termed neuroendocrine PCa (NEPC). Understanding how tumours transition to therapy-resistant, AR-independent states is crucial for the development of new and more effective therapies. We recently undertook transcriptomic profiling of patient tumours treated ex vivo with a clinical AR antagonist, enzalutamide, as a strategy to identify therapy-mediated adaptive changes. This study identified Ubiquitin specific protease 2 (USP2) as being increased in response to enzalutamide. The role of USP2 is to remove ubiquitin groups from proteins that causes therapy resistance and cancer progression, thereby preventing these proteins from degradation and increasing their stability in cancer cells. Thus, we hypothesised that USP2 can mediate resistance to ADT by stabilising key oncoproteins, a concept that was tested in my PhD project. Increased USP2 expression in response to ADT and in therapy-resistant states was validated in multiple prostate cancer cell line models, clinical transcriptomic cohorts, and additional patient tumours. Growing androgen-dependent cell lines in the presence of enzalutamide resulted in increased USP2 expression; conversely, androgen treatment resulted in the repression of USP2 expression. Clinical datasets also revealed that USP2 expression is elevated in AR-low/negative CRPC tumours, particularly those classified as neuroendocrine PCa. These observations suggest that USP2 is consistently upregulated in response to AR-targeted therapies and may represent a previously unknown resistance factor. Supporting this, targeting USP2 in multiple castrate-resistant prostate cancer models, either pharmacologically with a USP2-specific inhibitor (ML364) or by genetic knockdown, resulted in reduced cell viability and increased cell death. In contrast, overexpression of USP2 drove the development of an aggressive, therapy-resistant neuroendocrine phenotype and conferred partial resistance to enzalutamide and a growth advantage in androgen-depleted growth conditions. Importantly, ML364 was also potently active in an in vivo model of aggressive, AR-negative prostate cancer. Mechanistically, we found that USP2 stabilises the levels of oncogenic proteins including Aurora kinase A (AURKA), Cyclin D1 and Fatty acid synthase (FAS). Interrogation of the proteome and transcriptome of PCa cells overexpressing USP2 revealed positive enrichment of neuroendocrine-associated signalling, lipid metabolism and cell cycle, and negative enrichment of interferon signalling. All these enriched pathways are important in driving growth, survival, and progression of cancer. Overall, the findings herein revealed that USP2 can promote the development of neuroendocrine prostate cancer and acts to confer resistance to standard-of-care therapies, revealing it as a bona fide therapeutic target.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 202

Establishment of a cell-based anti-prion compound screen and analysis of host response to prion infection in cerebellar organotypic slice cultures

Prions are unconventional infectious agents that cause always fatal neurodegenerative diseases termed prion disease or transmissible spongiform encephalopathies in mammals. Prion diseases are caused by an accumulation of the misfolded, aggregated host encoded prion protein (PrP). The normal, cellular, α-helix rich isoform (PrPC) is converted into the disease-associated β-sheet rich pathogenic isoform (PrPSc). PrPSc can adopt multiple conformations that likely encipher prion strain characteristics. Currently, prion therapeutic clinical trials lack success and there is an urgent need for novel therapeutics. The aim of this study was to develop a cell-based assay for high content screening of large compound libraries with an automated microscope to identify compounds that might impair prion replication. Furthermore, identified compounds should be tested on prion infected organotypic slice cultures to test whether in vitro detected anti-prion compounds are also effective in a more complex neuronal environment. Additionally, two promising compounds, FeTMPyP and PIM-B31, identified by our collaboration partner Emiliano Biasini (University of Trento), were tested ex vivo. Beside this a comparative study of host response between ex vivo and in vivo should evaluate the transferability between the two systems, as this has not been was not shown until now. In the established screen 152 compounds were tested, 84 had an inhibitory effect on PrPSc accumulation in persistently infected N2a22L cells and the seven strongest inhibitors were further validated by western blot analysis. The most promising candidate, PHA665752, was tested ex vivo and showed a reduction of PrPSc accumulation that was however not significant. FeTMPyP showed strong toxicity and PIM-B31 showed inconsistent results that depended on different concentration and strain-specificity. Beside this, pathway analysis of ex vivo and in vivo infected mouse cerebella with different strains at various time points was performed with DAVID 6.8, an online bioinformatics resource. Analysis of the 250 most significant differentially expressed genes revealed that several comparable pathways were changed due to prion infection in brain slices and brains. The calcium signaling pathways and neuroactive ligand-receptor pathways were deregulated the most by prion infection ex vivo as well as in vivo

Funktionelle Relevanz epigenetischer Modifikationen in chronisch-entzündlichen Darmerkrankungen

The etiology of ulcerative colitis (UC), a chronic inflammatory bowel disease, is still not fully understood. One of the current hypotheses is, that genetic and environmental factors modulate the epigenetic landscape and thus contribute to susceptibility, manifestation and progression of the disease. In the presented thesis, a three-layer epigenome-wide association study (EWAS) is reported, using intestinal biopsies from ten monozygotic twin pairs discordant for the manifestation of UC (n=20). The primary result is a map of links between disease-associated transcripts and epigenetic modifications. These candidates of cis-linked gene expression and DNA methylation (DNAm) were validated in two larger independent patient populations (n=185) by quantitative real-time PCR and bisulfite-pyrosequencing, resulting high DNAm/RNA correlations, indicating a potential impact of DNAm on transcription. Many of the identified candidate genes have been functionally implicated in inflammatory processes. In contrast to the observed hypomethylation of several candidate loci in UC, patients with acute inflammation showed no altered methylation. To validate an alternative epigenetic modification in these patients, hydroxymethylation was quantified at selected loci, indicating that this mechanism might play a role in acute inflammation when compared to chronic inflammation. In conclusion, this study represents the first replicated EWAS in affected tissue of UC patients, integrated with transcriptional signatures. The results indicate a potential role of epigenetic modification in disease manifestation and progression of UC.Die Ätiologie von colitis Ulcerosa (UC), einer chronisch entzündlichen Darmerkrankung, ist bisher noch nicht vollständig aufgeklärt. Eine der gängigen Hypothesen geht davon aus, daß genetische Faktoren und Umwelteinflüsse das epigenetische Profil verändern und somit zu Suszeptibilität, Manifestation und Progression der Erkrankung beitragen. In der vorliegenden Dissertation wird eine epigenomweite Assoziationsstudie (EWAS) vorgestellt, die auf der Verwendung von intestinalen Biopsien von 10 eineiigen Zwillingspaaren basiert, die diskordant für colitis Ulcerosa (UC) sind (n=20). Als primäres Ergebnis wurden krankheitsassoziierte Transkripte identifiziert, die von epigenetische Modifikationen begleitet werden. Diese Kandidaten von Genexpression und DNA-Methylierung (DNAm) in direkter Nachbarschaft („in cis“) wurden in zwei größeren Patientengruppen (n=185) mittels quantitativer Real-Time PCR und Bisulfid-Pyrosequenzierung validiert. Die resultierende hohe Korrelation zwischen DNAm und mRNA deutet auf den potentiellen Einfluss der DNAm auf das Transkriptom in UC hin. Viele der identifizierten Kandidatengene stehen in funktionellem Zusammenhang mit Entzündungsprozessen. Im Gegensatz zur beobachteten Hypomethylierung vieler Kandidatengene in UC zeigten Patienten mit akuten Darmentzündungen keine solchen Veränderungen. Um alternative epigenetische Modifikationen zu prüfen, wurde in diesen Patienten die Hydroxymethylierung quantifiziert. Die Ergebnisse hieraus sprechen für eine mögliche Rolle der Hydroxymethylierung bei akuter Entzündung im Vergleich zur chronischen Entzündung. Zusammenfassend stellt die vorgestellte Arbeit die erste replizierte EWAS in entzündetem Gewebe von UC Patienten dar, die mit dem Transkriptom verknüpft wurde. Die Ergebnisse deuten auf eine mögliche Rolle der epigenetischen Modifikationen bei Manifestation und Progression von UC hin

Technologies to study protein oxidation in ageing Investigating the effect of protein oxidation on protein function

Protein oxidation can cause aggregation, fragmentation, and affect enzymatic activity and binding partner interactions. Protein oxidation is implicated in a range of agerelated pathologies including neurodegeneration and cancer. The VHR and PTEN phosphatases studied are sensitive to oxidation and regulated by protein-protein interactions. PTEN acts by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, negatively regulating the Akt pathway as part of a signalling control network that can protect against apoptosis, and is involved in the regulation of cell fate regulation and cancer. VHR is involved in neural development and cancer. A technology workflow for detecting protein oxidation and to correlate oxidative modifications to enzymatic activity and protein-protein interaction was developed; which may contribute towards the advancement of fundamental science as well as potential therapeutic and biomarker target identification in proteins. The technology platform consists of the mass spectrometric technique MS2 to detect, validate, map and quantify oxidative modifications. The technology workflow consists of enzymatic activity assays to correlate modification with changes in activity, targeted MS2 and statistical analysis. The fundamental and distinct contribution to knowledge in this thesis is a systematic mapping of protein oxidative modifications over a range of oxidants and concentrations of hypochlorous acid (HOCl), 3-morpholino-sydnonimine (sin-1) and tetranitromethane for VHR (vaccinia H1 related) and PTEN (phosphatase and tensin homolog on chromosome 10), including modification identification including active site residues and putative binding domain, mapping the relative abundances of those modification and statistically correlating them to changes in enzymatic activity. Additional contributions to knowledge have been i) the nonspecificity and complexity of oxidation profiles and oxidant damage of nitrating agents, that have largely been proposed to be specific without substantial oxidative capacity and ii) expanding the known interactome of VHR (vaccinia H1 related) through array and co-immunoprecipitation