Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients.

No abstract available

Microsporidia are an abundant group of obligate intracellular parasites of other eukaryotes, including immunocompromised humans, but the molecular basis of their intracellular lifestyle and pathobiology are poorly understood. New genomes from a taxonomically broad range of microsporidians, complemented by published expression data, provide an opportunity for comparative analyses to identify conserved and lineage-specific patterns of microsporidian genome evolution that have underpinned this success. In this study, we infer that a dramatic bottleneck in the last common microsporidian ancestor (LCMA) left a small conserved core of genes that was subsequently embellished by gene family expansion driven by gene acquisition in different lineages. Novel expressed protein families represent a substantial fraction of sequenced microsporidian genomes and are significantly enriched for signals consistent with secretion or membrane location. Further evidence of selection is inferred from the gain and reciprocal loss of functional domains between paralogous genes, for example, affecting transport proteins. Gene expansions among transporter families preferentially affect those that are located on the plasma membrane of model organisms, consistent with recruitment to plug conserved gaps in microsporidian biosynthesis and metabolism. Core microsporidian genes shared with other eukaryotes are enriched in orthologs that, in yeast, are highly expressed, highly connected, and often essential, consistent with strong negative selection against further reduction of the conserved gene set since the LCMA. Our study reveals that microsporidian genome evolution is a highly dynamic process that has balanced constraint, reductive evolution, and genome expansion during adaptation to an extraordinarily successful obligate intracellular lifestyle.

The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

Most phylogenetic models assume that the evolutionary process is stationary and reversible. In addition to being biologically improbable, these assumptions also impair inference by generating models under which the likelihood does not depend on the position of the root. Consequently, the root of the tree cannot be inferred as part of the analysis. Yet identifying the root position is a key component of phylogenetic inference because it provides a point of reference for polarizing ancestor-descendant relationships and therefore interpreting the tree. In this paper, we investigate the effect of relaxing the unrealistic reversibility assumption and allowing the position of the root to be another unknown. We propose two hierarchical models that are centered on a reversible model but perturbed to allow nonreversibility. The models differ in the degree of structure imposed on the perturbations. The analysis is performed in the Bayesian framework using Markov chain Monte Carlo methods for which software is provided. We illustrate the performance of the two nonreversible models in analyses of simulated data using two types of topological priors. We then apply the models to a real biological data set, the radiation of polyploid yeasts, for which there is robust biological opinion about the root position. Finally, we apply the models to a second biological alignment for which the rooted tree is controversial: the ribosomal tree of life. We compare the two nonreversible models and conclude that both are useful in inferring the position of the root from real biological data.

The Androgen Receptor (AR) is a key molecule in the development, maintenance and progression of prostate cancer (PC). However, the relationship between the AR and co-regulatory proteins that facilitate AR activity in castrate resistant settings remain understudied. Here we show that protein phosphatase 1 regulatory subunits, identified from a phosphatase RNAi screen, direct PP1 catalytic subunits to a varied yet significant response in AR function. As such, we have characterised the PP1β holoenzyme, myosin phosphatase (MLCP), as a novel ligand independent regulator of the AR. Sustained MLCP activity through down-regulation of the MLCP inhibitory subunit, PPP1R14C, results in impaired AR nuclear translocation, protein stability and transcriptional activity in distinct models of PC progression, culminating in restoration of a non-malignant prostate genotype. Phenotypically, a marked reduction in cell proliferation and migration, characterised by G1 cell cycle arrest is observed, confirming PP1 holoenzyme disruption as a novel treatment approach in PC.

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11-13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes.

Chronic lymphocytic leukaemia (CLL) patients display a highly variable clinical course, with progressive acquisition of drug resistance. We sought to identify aberrant epigenetic traits that are enriched following exposure to treatment that could impact patient response to therapy.

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC-even in the absence of progressive injury and fibrosis.

The loss of key biosynthetic pathways is a common feature of important parasitic protists, making them heavily dependent on scavenging nutrients from their hosts. This is often mediated by specialized transporter proteins that ensure the nutritional requirements of the parasite are met. Over the past decade, the completion of several parasite genome projects has facilitated the identification of parasite transporter proteins. This has been complemented by functional characterization of individual transporters along with investigations into their importance for parasite survival. In this review, we summarize the current knowledge on transporters from parasitic protists and highlight commonalities and differences in the transporter repertoires of different parasitic species, with particular focus on characterized transporters that act at the host-pathogen interface.

Archaeplastida (=Kingdom Plantae) are primary plastid-bearing organisms that evolved via the endosymbiotic association of a heterotrophic eukaryote host cell and a cyanobacterial endosymbiont approximately 1,400 Ma. Here, we present analyses of cyanobacterial and plastid genomes that show strongly conflicting phylogenies based on 75 plastid (or nuclear plastid-targeted) protein-coding genes and their direct translations to proteins. The conflict between genes and proteins is largely robust to the use of sophisticated data- and tree-heterogeneous composition models. However, by using nucleotide ambiguity codes to eliminate synonymous substitutions due to codon-degeneracy, we identify a composition bias, and dependent codon-usage bias, resulting from synonymous substitutions at all third codon positions and first codon positions of leucine and arginine, as the main cause for the conflicting phylogenetic signals. We argue that the protein-coding gene data analyses are likely misleading due to artifacts induced by convergent composition biases at first codon positions of leucine and arginine and at all third codon positions. Our analyses corroborate previous studies based on gene sequence analysis that suggest Cyanobacteria evolved by the early paraphyletic splitting of Gloeobacter and a specific Synechococcus strain (JA33Ab), with all other remaining cyanobacterial groups, including both unicellular and filamentous species, forming the sister-group to the Archaeplastida lineage. In addition, our analyses using better-fitting models suggest (but without statistically strong support) an early divergence of Glaucophyta within Archaeplastida, with the Rhodophyta (red algae), and Viridiplantae (green algae and land plants) forming a separate lineage.

Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis.

Metastatic castration resistant prostate cancer is one of the main causes of male cancer associated deaths worldwide. Development of resistance is inevitable in patients treated with anti-androgen therapies. This highlights a need for novel therapeutic strategies that would be aimed upstream of the androgen receptor (AR). Here we report that the novel small molecule anti-androgen, galeterone targets USP12 and USP46, two highly homologous deubiquitinating enzymes that control the AR-AKT-MDM2-P53 signalling pathway. Consequently, galeterone is effective in multiple models of prostate cancer including both castrate resistant and AR-negative prostate cancer. However, we have observed that USP12 and USP46 selectively regulate full length AR protein but not the AR variants. This is the first report of deubiquitinating enzyme targeting as a strategy in prostate cancer treatment which we show to be effective in multiple, currently incurable models of this disease.

Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family.

Hypotheses about the origin of eukaryotic cells are classically framed within the context of a universal 'tree of life' based on conserved core genes. Vigorous ongoing debate about eukaryote origins is based on assertions that the topology of the tree of life depends on the taxa included and the choice and quality of genomic data analysed. Here we have reanalysed the evidence underpinning those claims and apply more data to the question by using supertree and coalescent methods to interrogate >3,000 gene families in archaea and eukaryotes. We find that eukaryotes consistently originate from within the archaea in a two-domains tree when due consideration is given to the fit between model and data. Our analyses support a close relationship between eukaryotes and Asgard archaea and identify the Heimdallarchaeota as the current best candidate for the closest archaeal relatives of the eukaryotic nuclear lineage.

There is an expectation that analyses of molecular sequences might be able to distinguish between alternative hypotheses for ancient relationships, but the phylogenetic methods used and types of data analyzed are of critical importance in any attempt to recover historical signal. Here, we discuss some common issues that can influence the topology of trees obtained when using overly simple models to analyze molecular data that often display complicated patterns of sequence heterogeneity. To illustrate our discussion, we have used three examples of inferred relationships which have changed radically as models and methods of analysis have improved. In two of these examples, the sister-group relationship between thermophilic Thermus and mesophilic Deinococcus, and the position of long-branch Microsporidia among eukaryotes, we show that recovering what is now generally considered to be the correct tree is critically dependent on the fit between model and data. In the third example, the position of eukaryotes in the tree of life, the hypothesis that is currently supported by the best available methods is fundamentally different from the classical view of relationships between major cellular domains. Since heterogeneity appears to be pervasive and varied among all molecular sequence data, and even the best available models can still struggle to deal with some problems, the issues we discuss are generally relevant to phylogenetic analyses. It remains essential to maintain a critical attitude to all trees as hypotheses of relationship that may change with more data and better methods.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.

A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, has very poor outcomes. Current therapies often have low efficacy and significant toxicities. Thus, there is a critical need for the development of novel therapeutic approaches for HCC. We have developed a novel bioinformatics pipeline, which integrates genome-wide DNA methylation and gene expression data, to identify genes required for the survival of specific molecular cancer subgroups but not normal cells. Targeting these genes may induce cancer-specific "synthetic lethality". Initially, five potential HCC molecular subgroups were identified based on global DNA methylation patterns. Subgroup-2 exhibited the most unique methylation profile and two candidate subtype-specific vulnerability or SL-like genes were identified for this subgroup, including TIAM1, a guanine nucleotide exchange factor encoding gene known to activate Rac1 signalling. siRNA targeting TIAM1 inhibited cell proliferation in TIAM1-positive (subgroup-2) HCC cell lines but had no effect on the normal hepatocyte HHL5 cell line. Furthermore, TIAM1-positive/subgroup-2 cell lines were significantly more sensitive to the TIAM1/RAC1 inhibitor NSC23766 compared with TIAM1-negative HCC lines or the normal HHL5 cell line. The results are consistent with a synthetic lethal role for TIAM1 in a methylation-defined HCC subgroup and suggest it may be a viable therapeutic target in this subset of HCC patients.

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.