Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Errors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates a spectrum of simple and complex genomic rearrangements that are associated with human cancers and disorders. How specific DSB repair pathways recognize and process these lesions remains poorly understood. Here we used CRISPR/Cas9 to systematically inactivate distinct DSB processing or repair pathways and interrogated the rearrangement landscape of fragmented chromosomes from micronuclei. Deletion of canonical non-homologous end joining (NHEJ) components, including DNA-PKcs, LIG4, and XLF, substantially reduced the formation of complex rearrangements and shifted the rearrangement landscape toward simple alterations without the characteristic patterns of cancer-associated chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within micronuclei bodies (MN bodies) and undergo successful ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments were rarely engaged by polymerase theta-mediated alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics and persistent 53BP1-labeled MN bodies in the interphase nucleus. Prolonged DNA damage signaling from unrepaired fragments ultimately triggered cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements following chromothripsis from mitotic errors.

Mitotic clustering of pulverized chromosomes from micronuclei.

Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis1-5. As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis6. Here we tracked micronucleated chromosomes with live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for asymmetric inheritance by a single daughter cell. Mechanistically, the CIP2A-TOPBP1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises pulverized chromosomes for clustering upon mitotic entry. Inactivation of CIP2A-TOPBP1 caused acentric fragments to disperse throughout the mitotic cytoplasm, stochastically partition into the nucleus of both daughter cells and aberrantly misaccumulate as cytoplasmic DNA. Mitotic clustering facilitates the reassembly of acentric fragments into rearranged chromosomes lacking the extensive DNA copy-number losses that are characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer genomes revealed clusters of DNA copy-number-neutral rearrangements-termed balanced chromothripsis-across diverse tumour types resulting in the acquisition of known cancer driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial clustering of pulverized chromosomes from micronuclei.

Mechanistic origins of diverse genome rearrangements in cancer.

Cancer genomes frequently harbor structural chromosomal rearrangements that disrupt the linear DNA sequence order and copy number. To date, diverse classes of structural variants have been identified across multiple cancer types. These aberrations span a wide spectrum of complexity, ranging from simple translocations to intricate patterns of rearrangements involving multiple chromosomes. Although most somatic rearrangements are acquired gradually throughout tumorigenesis, recent interrogation of cancer genomes have uncovered novel categories of complex rearrangements that arises rapidly through a one-off catastrophic event, including chromothripsis and chromoplexy. Here we review the cellular and molecular mechanisms contributing to the formation of diverse structural rearrangement classes during cancer development. Genotoxic stress from a myriad of extrinsic and intrinsic sources can trigger DNA double-strand breaks that are subjected to DNA repair with potentially mutagenic outcomes. We also highlight how aberrant nuclear structures generated through mitotic cell division errors, such as rupture-prone micronuclei and chromosome bridges, can instigate massive DNA damage and the formation of complex rearrangements in cancer genomes.

Discovery of Harmaline as a Potent Inhibitor of Sphingosine Kinase-1: A Chemopreventive Role in Lung Cancer.

The sphingosine kinase-1/sphingosine-1-phosphate pathway is linked with the cancer progression and survival of the chemotherapy-challenged cells. Sphingosine kinase-1 (SphK1) has emerged as an attractive drug target, but their inhibitors from natural sources are limited. In this study, we have chosen harmaline, one of the ß-carboline alkaloids, and report its mechanism of binding to SphK1 and subsequent inhibition. Molecular docking combined with fluorescence binding studies revealed that harmaline binds to the substrate-binding pocket of SphK1 with an appreciable binding affinity and significantly inhibits the kinase activity of SphK1 with an IC50 value in the micromolar range. The cytotoxic effect of harmaline on non-small-cell lung cancer cells by MTT assay was found to be higher for H1299 compared to A549. Harmaline induces apoptosis in non-small-cell lung carcinoma cells (H1299 and A549), possibly via the intrinsic pathway. Our findings suggest that harmaline could be implicated as a scaffold for designing potent anticancer molecules with SphK1 inhibitory potential.

Insights into the Conserved Regulatory Mechanisms of Human and Yeast Aging.

Aging represents a significant biological process having strong associations with cancer, diabetes, and neurodegenerative and cardiovascular disorders, which leads to progressive loss of cellular functions and viability. Astonishingly, age-related disorders share several genetic and molecular mechanisms with the normal aging process. Over the last three decades, budding yeast Saccharomyces cerevisiae has emerged as a powerful yet simple model organism for aging research. Genetic approaches using yeast RLS have led to the identification of hundreds of genes impacting lifespan in higher eukaryotes. Numerous interventions to extend yeast lifespan showed an analogous outcome in multi-cellular eukaryotes like fruit flies, nematodes, rodents, and humans. We collected and analyzed a multitude of observations from published literature and provide the contribution of yeast in the understanding of aging hallmarks most applicable to humans. Here, we discuss key pathways and molecular mechanisms that underpin the evolutionarily conserved aging process and summarize the current understanding and clinical applicability of its trajectories. Gathering critical information on aging biology would pave the way for future investigation targeted at the discovery of aging interventions.

Effect of pH on the structure and function of pyruvate dehydrogenase kinase 3: Combined spectroscopic and MD simulation studies.

Pyruvate dehydrogenase kinase-3 (PDK3) plays important role in the glucose metabolism and is associated with cancer progression, and thus being considered as an attractive target for cancer therapy. In this study, we employed spectroscopic techniques to study the structural and conformational changes in the PDK3 at varying pH conditions ranging from pH 2.0 to 12.0. UV/Vis, fluorescence and circular dichroism spectroscopic measurements revealed that PDK3 maintains its native-like structure (both secondary and tertiary) in the alkaline conditions (pH 7.0-12.0). However, a significant loss in the structure was observed under acidic conditions (pH 2.0-6.0). The propensity of aggregate formation at pH 4.0 was estimated by thioflavin T fluorescence measurements. To further complement structural data, kinase activity assay was performed, and maximum activity of PDK3 was observed at pH 7.0-8.0 range; whereas, its activity was lost under acidic pH. To further see conformational changes at atomistic level we have performed all-atom molecular dynamics at different pH conditions for 150 ns. A well defined correlation was observed between experimental and computational studies. This work highlights the significance of structural dependence of pH for wide implications in protein-protein interaction, biological function and drug design procedures.

Functional implications of pH-induced conformational changes in the Sphingosine kinase 1.

Sphingosine kinase 1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1-phosphate that acts as a bioactive signalling molecule, and regulates various cellular processes including lymphocyte trafficking, angiogenesis and response to apoptotic stimuli. Abnormal expression of SphK1 has been observed in a wide range of cancers highlighting their role in tumour growth and metastasis. This enzyme also plays a critical role in metabolic and inflammatory diseases, including pulmonary fibrosis, diabetic neuropathy and Alzheimer's disease. In the present study, we have investigated the structural and conformational changes in SphK1 at varying pH using various spectroscopic techniques. Consistent results were observed with the function of SphK1 at corresponding pH values. SphK1 maintains its secondary and tertiary structure in the pH range of 7.5-10.0. However, protein aggregation was observed in the acidic pH range (4.0-6.5). At pH 2.0, the SphK1 exists in the molten-globule state. Kinase assay also shows that SphK1 activity was optimal in the pH range of 7.5-8.5. To complement in vitro results, we have performed 100 ns molecular dynamics simulation to examine the effect of pH on the structural stability of SphK1 at molecular level. SphK1 maintains its native conformation in the alkaline pH range with some residual fluctuations detected at acidic pH. A considerable correlation was noticed between spectroscopic, enzymatic activity and MD simulation studies. pH dependent structural changes can be further implicated to understand its association with disease condition, and cellular homeostasis with respect to protein function under variable pH conditions.

Evaluation of binding and inhibition mechanism of dietary phytochemicals with sphingosine kinase 1: Towards targeted anticancer therapy.

Sphingosine kinase 1 (SphK1) has recently gained attention as a potential drug target for its association with cancer and other inflammatory diseases. Here, we have investigated the binding affinity of dietary phytochemicals viz., ursolic acid, capsaicin, DL-α tocopherol acetate, quercetin, vanillin, citral, limonin and simvastatin with the SphK1. Docking studies revealed that all these compounds bind to the SphK1 with varying affinities. Fluorescence binding and isothermal titration calorimetric measurements suggested that quercetin and capsaicin bind to SphK1 with an excellent affinity, and significantly inhibits its activity with an admirable IC50 values. The binding mechanism of quercetin was assessed by docking and molecular dynamics simulation studies for 100 ns in detail. We found that quercetin acts as a lipid substrate competitive inhibitor, and it interacts with important residues of active-site pocket through hydrogen bonds and other non-covalent interactions. Quercetin forms a stable complex with SphK1 without inducing any significant conformational changes in the protein structure. In conclusion, we infer that quercetin and capsaicin provide a chemical scaffold to develop potent and selective inhibitors of SphK1 after required modifications for the clinical management of cancer.

Identification of High-Affinity Inhibitors of Cyclin-Dependent Kinase 2 Towards Anticancer Therapy.

Cyclin-dependent kinase 2 (CDK2) is an essential protein kinase involved in the cell cycle regulation. The abnormal activity of CDK2 is associated with cancer progression and metastasis. Here, we have performed structure-based virtual screening of the PubChem database to identify potent CDK2 inhibitors. First, we retrieved all compounds from the PubChem database having at least 90% structural similarity with the known CDK2 inhibitors. The selected compounds were subjected to structure-based molecular docking studies to investigate their pattern of interaction and estimate their binding affinities with CDK2. Selected compounds were further filtered out based on their physicochemical and ADMET properties. Detailed interaction analysis revealed that selected compounds interact with the functionally important residues of the active site pocket of CDK2. All-atom molecular dynamics simulation was performed to evaluate conformational changes, stability and the interaction mechanism of CDK2 in-complex with the selected compound. We found that binding of 6-N,6-N-dimethyl-9-(2-phenylethyl)purine-2,6-diamine stabilizes the structure of CDK2 and causes minimal conformational change. Finally, we suggest that the compound (PubChem ID 101874157) would be a promising scaffold to be further exploited as a potential inhibitor of CDK2 for therapeutic management of cancer after required validation.

Investigation of inhibitory potential of quercetin to the pyruvate dehydrogenase kinase 3: Towards implications in anticancer therapy.

Pyruvate dehydrogenase kinase 3 (PDK3) is a mitochondrial protein, has recently been considered as a potential pharmacological target for varying types of cancer. Here, we report the binding mechanism of quercetin to the PDK3 by using molecular docking, simulation, fluorescence spectroscopy and isothermal titration calorimetric assays. Molecular docking along with simulation provided an in-depth analysis of protein-ligand interactions. We have observed that quercetin interacts to the important residues of active site cavity of PDK3 and shows a well-ordered conformational fitting. The stability of quercetin-PDK3 complex is maintained by several non-covalent interactions throughout the simulation. To complement in silico findings with the experiments, we have successfully expressed and purified human PDK3. Both fluorescence and isothermal titration calorimetric experiments showed excellent binding affinity of quercetin to the PDK3. Kinase inhibition assay further revealed a significant inhibitory potential of quercetin to the PDK3 with the IC50 values in µM range. Quercetin is non-toxic to HEK293, and significantly inhibits the proliferation of cancer (HepG2 and A549) cell lines. All these observations clearly indicate that quercetin may be further evaluated as promising therapeutic molecule for PDK3 with required modifications and in vivo validation.

Investigating the structural features of chromodomain proteins in the human genome and predictive impacts of their mutations in cancers.

Epigenetic readers are specific proteins which recognize histone marks and represents the underlying mechanism for chromatin regulation. Histone H3 lysine methylation is a potential epigenetic code for the chromatin organization and transcriptional control. Recognition of histone methylation is achieved by evolutionary conserved reader modules known as chromodomain, identified in several proteins, and is involved in transcriptional silencing and chromatin remodelling. Genetic perturbations within the structurally conserved chromodomain could potentially mistarget the reader protein and impair their regulatory pathways, ultimately leading to cellular chaos by setting the stage for tumor development and progression. Here, we report the structural conservations associated with diverse functions, prognostic significance and functional consequences of mutations within chromodomain of human proteins in distinct cancers. We have extensively analysed chromodomain containing human proteins in terms of their structural-functional ability to act as a molecular switch in the recognition of methyl-lysine recognition. We further investigated the combinatorial potential, target promiscuity and binding specificity associated with their underlying mechanisms. Indeed, the molecular mechanism of epigenetic silencing significantly underlies a newer cancer therapy approach. We hope that a critical understanding of chromodomains will pave the way for novel paths of research providing newer insights into the designing of effective anti-cancer therapies.

Molecular interaction studies on ellagic acid for its anticancer potential targeting pyruvate dehydrogenase kinase 3.

Pyruvate dehydrogenase kinase 3 (PDK3) plays a central role in the cancer metabolic switch through the reversible phosphorylation of pyruvate dehydrogenase complex thereby blocking the entry of pyruvate for its catabolism into the TCA cycle, and thus it is considered as an important drug target for various types of cancers. We have successfully expressed full length human PDK3 and investigated its interaction mechanism with dietary polyphenols in the search for potential inhibitors. Molecular docking analysis revealed that the selected compounds preferentially bind to the ATP-binding pocket of PDK3 and interact with functionally important residues. In silico observations were further complemented by experimental measurements of the fluorescence quenching of PDK3 and confirmed with the isothermal titration calorimetry measurements. Ellagic acid (EA) significantly binds and inhibits the kinase activity of PDK3. In vitro cytotoxicity and the anti-proliferative properties of EA were evaluated by MTT assay. Conformational dynamics of the EA-PDK3 complex during molecular dynamics simulation revealed that a stable complex was maintained by a significant number of hydrogen bonds throughout the 100 ns trajectories. In conclusion, EA may be considered as a promising molecule for PDK3 inhibition and could be exploited as a lead molecule against PDK3 associated diseases.

Mutational analysis of TAF6 revealed the essential requirement of the histone-fold domain and the HEAT repeat domain for transcriptional activation.

TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex in vivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.

The TAF9 C-terminal conserved region domain is required for SAGA and TFIID promoter occupancy to promote transcriptional activation.

A common function of the TFIID and SAGA complexes, which are recruited by transcriptional activators, is to deliver TBP to promoters to stimulate transcription. Neither the relative contributions of the five shared TBP-associated factor (TAF) subunits in TFIID and SAGA nor the requirement for different domains in shared TAFs for transcriptional activation is well understood. In this study, we uncovered the essential requirement for the highly conserved C-terminal region (CRD) of Taf9, a shared TAF, for transcriptional activation in yeast. Transcriptome profiling performed under Gcn4-activating conditions showed that the Taf9 CRD is required for induced expression of ∼9% of the yeast genome. The CRD was not essential for the Taf9-Taf6 interaction, TFIID or SAGA integrity, or Gcn4 interaction with SAGA in cell extracts. Microarray profiling of a SAGA mutant (spt20Δ) yielded a common set of genes induced by Spt20 and the Taf9 CRD. Chromatin immunoprecipitation (ChIP) assays showed that, although the Taf9 CRD mutation did not impair Gcn4 occupancy, the occupancies of TFIID, SAGA, and the preinitiation complex were severely impaired at several promoters. These results suggest a crucial role for the Taf9 CRD in genome-wide transcription and highlight the importance of conserved domains, other than histone fold domains, as a common determinant for TFIID and SAGA functions.