Elucidating the anti-cancer mechanisms for transition-state structure inhibitors of nucleoside phosphorylases methylthioadenosine-DADMe-immucillin A and forodesine

<p>Transition-state structure analogues are among the most powerful chemical inhibitors discovered to date with picomolar efficacy for enzymes. The nucleoside analogue methylthioadenosine-DADMe-immucillin A (MTDIA) is an inhibitor of the enzyme methylthioadenosine phosphorylase (MTAP) in polyamine biosynthesis. The recently approved forodesine (Mundesine®) is an inhibitor of purine nucleoside phosphorylase (PNP) and purine synthesis. Although the targets of these drugs were known at the time of drug design, it is important to know the compendium of cellular perturbations resulting from use of these inhibitors. Several suspected mechanisms of MTDIA and forodesine in progression of apoptotic cell death have been identified but the underlying mechanisms initiating apoptosis remain elusive. We hypothesize that numerous cellular processes are affected in MTDIA and forodesine treatments given the importance of polyamine and purine synthesis in cancer cells. To elucidate the unsuspected mechanisms mediating anti-cancer activity, unbiased genomic analyses were employed using Saccharomyces cerevisiae. First, gene-gene interactions with MEU1 (the MTAP orthologue in yeast) were determined using Synthetic Genetic Array methodology followed by assessment of drug-gene interactions with MTDIA treatment under a MEU1 essential condition with MTA as the sole source of sulphur. Disruptions to suspected mechanisms of amino acid metabolism, carbohydrate metabolism, response to starvation, vesicle-mediated transport, vacuole fusion, lipid homeostasis, chromatin organisation, transcription, and translation were implicated well as unsuspected mechanisms of NAD+ dependent cellular processes, multi-vesicular body formation, endosomal transport, ion homeostasis, mitochondrion organisation, and cell cycle progression. Induction of autophagy was subsequently confirmed with MTDIA to validate the disruptions to vesicle-mediated transport, response to starvation, multi-vesicular body formation and vacuolar fusion. Reduction in ergosterol levels and disruptions to ergosterol biosynthetic proteins were confirmed with MTDIA and meu1Δ to validate disruptions to lipid homeostasis. To complement the genetic analyses, the abundance and localisation of proteins were evaluated in response to MTDIA or MEU1-deficiency. Disruptions to proteins implicated in carbohydrate metabolism, methionine salvage, transcription, translation, transmembrane transport, lipid homeostasis, cell cycle and DNA repair were identified with meu1Δ and MTDIA. Key findings from the analysis of protein abundance and localization were the relocalisation of plasma membrane proteins and disruptions to vesicle mediated transport proteins consistent with the induction of autophagy and disruptions to proteins in homeostasis of all major lipid classes, further corroborating the findings of screening gene deletion mutants for elucidating drug mechanisms. To investigate the mechanisms of forodesine toxicity, genetic interactions with PNP1 (the PNP orthologue in yeast) were determined using Synthetic Genetic Array methodology. Disruptions to amino acid metabolism, starvation responsive genes, vacuolar organisation and vesicle mediated transport, carbohydrate metabolism, lipid homeostasis, chromatin organisation, chromosome segregation, transcription, and translation were identified in response to PNP1-deficency. Despite the introduction of several human genes and supplementation of metabolites required for forodesine bioactivity in humans, forodesine was not sufficiently bioactive in yeast to evaluate sensitivity of gene deletion mutants to forodesine. Overall, chemical genomic analyses in yeast with transition-state structure analogues MTDIA and forodesine effectively highlight the vast number of cellular processes affected by inhibition of a single target. Moreover, genome-wide pre-screening should be carried out in yeast to identify side-effects and secondary effects from drug target inhibition prior to assessing desired and undesired outcomes of highly specific drugs in human cells.</p>

Elucidating the anti-cancer mechanisms for transition-state structure inhibitors of nucleoside phosphorylases methylthioadenosine-DADMe-immucillin A and forodesine

<p>Transition-state structure analogues are among the most powerful chemical inhibitors discovered to date with picomolar efficacy for enzymes. The nucleoside analogue methylthioadenosine-DADMe-immucillin A (MTDIA) is an inhibitor of the enzyme methylthioadenosine phosphorylase (MTAP) in polyamine biosynthesis. The recently approved forodesine (Mundesine®) is an inhibitor of purine nucleoside phosphorylase (PNP) and purine synthesis. Although the targets of these drugs were known at the time of drug design, it is important to know the compendium of cellular perturbations resulting from use of these inhibitors. Several suspected mechanisms of MTDIA and forodesine in progression of apoptotic cell death have been identified but the underlying mechanisms initiating apoptosis remain elusive. We hypothesize that numerous cellular processes are affected in MTDIA and forodesine treatments given the importance of polyamine and purine synthesis in cancer cells. To elucidate the unsuspected mechanisms mediating anti-cancer activity, unbiased genomic analyses were employed using Saccharomyces cerevisiae. First, gene-gene interactions with MEU1 (the MTAP orthologue in yeast) were determined using Synthetic Genetic Array methodology followed by assessment of drug-gene interactions with MTDIA treatment under a MEU1 essential condition with MTA as the sole source of sulphur. Disruptions to suspected mechanisms of amino acid metabolism, carbohydrate metabolism, response to starvation, vesicle-mediated transport, vacuole fusion, lipid homeostasis, chromatin organisation, transcription, and translation were implicated well as unsuspected mechanisms of NAD+ dependent cellular processes, multi-vesicular body formation, endosomal transport, ion homeostasis, mitochondrion organisation, and cell cycle progression. Induction of autophagy was subsequently confirmed with MTDIA to validate the disruptions to vesicle-mediated transport, response to starvation, multi-vesicular body formation and vacuolar fusion. Reduction in ergosterol levels and disruptions to ergosterol biosynthetic proteins were confirmed with MTDIA and meu1Δ to validate disruptions to lipid homeostasis. To complement the genetic analyses, the abundance and localisation of proteins were evaluated in response to MTDIA or MEU1-deficiency. Disruptions to proteins implicated in carbohydrate metabolism, methionine salvage, transcription, translation, transmembrane transport, lipid homeostasis, cell cycle and DNA repair were identified with meu1Δ and MTDIA. Key findings from the analysis of protein abundance and localization were the relocalisation of plasma membrane proteins and disruptions to vesicle mediated transport proteins consistent with the induction of autophagy and disruptions to proteins in homeostasis of all major lipid classes, further corroborating the findings of screening gene deletion mutants for elucidating drug mechanisms. To investigate the mechanisms of forodesine toxicity, genetic interactions with PNP1 (the PNP orthologue in yeast) were determined using Synthetic Genetic Array methodology. Disruptions to amino acid metabolism, starvation responsive genes, vacuolar organisation and vesicle mediated transport, carbohydrate metabolism, lipid homeostasis, chromatin organisation, chromosome segregation, transcription, and translation were identified in response to PNP1-deficency. Despite the introduction of several human genes and supplementation of metabolites required for forodesine bioactivity in humans, forodesine was not sufficiently bioactive in yeast to evaluate sensitivity of gene deletion mutants to forodesine. Overall, chemical genomic analyses in yeast with transition-state structure analogues MTDIA and forodesine effectively highlight the vast number of cellular processes affected by inhibition of a single target. Moreover, genome-wide pre-screening should be carried out in yeast to identify side-effects and secondary effects from drug target inhibition prior to assessing desired and undesired outcomes of highly specific drugs in human cells.</p>

A single molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters and optical aberrations. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organisation and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

Emerging Single-Cell Technological Approaches to Investigate Chromatin Dynamics and Centromere Regulation in Human Health and Disease

Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, we compare standard and emerging single-cell techniques and their relevance for investigating human diseases. Finally, we describe emerging methodologies that investigate centromeric chromatin specification and neocentromere formation.

A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing

Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally, through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organization, induces intestinal autophagy through transcriptional regulation, prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

Cryptochromes and the Circadian Clock: The Story of a Very Complex Relationship in a Spinning World

Cryptochromes are flavin-containing blue light photoreceptors, present in most kingdoms, including archaea, bacteria, plants, animals and fungi. They are structurally similar to photolyases, a class of flavoproteins involved in light-dependent repair of UV-damaged DNA. Cryptochromes were first discovered in Arabidopsis thaliana in which they control many light-regulated physiological processes like seed germination, de-etiolation, photoperiodic control of the flowering time, cotyledon opening and expansion, anthocyanin accumulation, chloroplast development and root growth. They also regulate the entrainment of plant circadian clock to the phase of light–dark daily cycles. Here, we review the molecular mechanisms by which plant cryptochromes control the synchronisation of the clock with the environmental light. Furthermore, we summarise the circadian clock-mediated changes in cell cycle regulation and chromatin organisation and, finally, we discuss a putative role for plant cryptochromes in the epigenetic regulation of genes.

The role of insulators and transcription in 3D chromatin organisation of flies

The DNA in many organisms, including humans, is shown to be organised in topologically associating domains (TADs). InDrosophila, several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. Here, we show that depletion of BEAF-32, Cp190, Chro and Dref leads to changes in TAD organisation and chromatin loops. Their depletion predominantly affects TAD borders located in heterochromatin, while TAD borders located in euchromatin are resilient to these mutants. Furthermore, transcriptomic data has revealed hundreds of genes displaying differential expression in these mutants and showed that the majority of differentially expressed genes are located within reorganised TADs. Our work identifies a novel and functional role for architectural proteins at TAD borders inDrosophilaand a link between TAD reorganisation and subsequent changes in gene expression.

Widespread reorganisation of pluripotent factor binding and gene regulatory interactions between human pluripotent states

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.