An ATM D-compartmentalization in DNA damage response.

How chromatin configuration impacts DNA repair is an emerging question. A recent study by Arnould et al. shows that ATM orchestrates a new chromatin compartment (D compartment) following DNA double-strand breaks and establishes that this compartment enhances cellular response to such breaks but also introduces a risk to genome integrity.

Repurposing Ceritinib Induces DNA Damage and Enhances PARP Inhibitor Responses in High-Grade Serous Ovarian Carcinoma.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

ßIII-tubulin overexpression in cancer: Causes, consequences, and potential therapies.

Class III ß-tubulin (ßIII-tubulin) is frequently overexpressed in human tumors and is associated with resistance to microtubule-targeting agents, tumor aggressiveness, and poor patient outcome. Understanding the mechanisms regulating ßIII-tubulin expression and the varied functions ßIII-tubulin may have in different cancers is vital to assess the prognostic value of this protein and to develop strategies to enhance therapeutic benefits in ßIII-tubulin overexpressing tumors. Here we gather all the available evidence regarding the clinical implications of ßIII-tubulin overexpression in cancer, describe factors that regulate ßIII-tubulin expression, and discuss current understanding of the mechanisms underlying ßIII-tubulin-mediated resistance to microtubule-targeting agents and tumor aggressiveness. Finally, we provide an overview of emerging therapeutic strategies to target tumors that overexpress ßIII-tubulin.

VLX600 Disrupts Homologous Recombination and Synergizes with PARP Inhibitors and Cisplatin by Inhibiting Histone Lysine Demethylases.

Tumors with defective homologous recombination (HR) DNA repair are more sensitive to chemotherapies that induce lesions repaired by HR as well as PARP inhibitors (PARPis). However, these therapies have limited activity in HR-proficient cells. Accordingly, agents that disrupt HR may be a means to augment the activities of these therapies in HR-proficient tumors. Here we show that VLX600, a small molecule that has been in a phase I clinical trial, disrupts HR and synergizes with PARPis and platinum compounds in ovarian cancer cells. We further found that VLX600 and other iron chelators disrupt HR, in part, by inhibiting iron-dependent histone lysine demethylases (KDM) family members, thus blocking recruitment of HR repair proteins, including RAD51, to double-strand DNA breaks. Collectively, these findings suggest that pharmacologically targeting KDM family members with VLX600 may be a potential novel strategy to therapeutically induce HR defects in ovarian cancers and correspondingly sensitize them to platinum agents and PARPis, two standard-of-care therapies for ovarian cancer.

Beyond the Paclitaxel and Vinca Alkaloids: Next Generation of Plant-Derived Microtubule-Targeting Agents with Potential Anticancer Activity.

Plants are an important source of chemically diverse natural products that target microtubules, one of the most successful targets in cancer therapy. Colchicine, paclitaxel, and vinca alkaloids are the earliest plant-derived microtubule-targeting agents (MTAs), and paclitaxel and vinca alkaloids are currently important drugs used in the treatment of cancer. Several additional plant-derived compounds that act on microtubules with improved anticancer activity are at varying stages of development. Here, we move beyond the well-discussed paclitaxel and vinca alkaloids to present other promising plant-derived MTAs with potential for development as anticancer agents. Various biological and biochemical aspects are discussed. We hope that the review will provide guidance for further exploration and identification of more effective, novel MTAs derived from plant sources.

BRCA1 Deficiency Upregulates NNMT, Which Reprograms Metabolism and Sensitizes Ovarian Cancer Cells to Mitochondrial Metabolic Targeting Agents.

BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. SIGNIFICANCE: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer.

Reduced BRCA1 expression causes homologous recombination (HR) repair defects in high-grade serous ovarian cancers (HGSOCs). Here, we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 is a DNA-binding protein that interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with ZC3H18 role in activating BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes cells to DNA crosslinkers and poly(ADP-ribose) polymerase inhibitors. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 and E2F4 mRNA levels are positively correlated with BRCA1 mRNA levels, further supporting ZC3H18 role in regulating BRCA1. Given that ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC, these findings suggest that ZC3H18 copy number losses could contribute to HR defects in HGSOC.

Marine Invertebrate Natural Products that Target Microtubules.

Marine natural products as secondary metabolites are a potential major source of new drugs for treating disease. In some cases, cytotoxic marine metabolites target the microtubules of the eukaryote cytoskeleton for reasons that will be discussed. This review covers the microtubule-targeting agents reported from sponges, corals, tunicates, and molluscs and the evidence that many of these secondary metabolites are produced by bacterial symbionts. The review finishes by discussing the directions for future development and production of clinically relevant amounts of these natural products and their analogues through aquaculture, chemical synthesis, and biosynthesis by bacterial symbionts.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding-dependent role that ensures adequate repair of common replication errors.

Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes.

Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis, we engineered mice lacking the PDZ-binding domain (PDZ-BD). Here, we show that the PDZ-BD regulates centrosome movement and that its heterozygous or homozygous deletion promotes aneuploidy and tumour formation. We found that Pten is recruited to pre-mitotic centrosomes in a Plk1-dependent fashion to create a docking site for protein complexes containing the PDZ-domain-containing protein Dlg1 (also known as Sap97) and Eg5 (also known as Kif11), a kinesin essential for centrosome movement and bipolar spindle formation. Docking of Dlg1-Eg5 complexes to Pten depended on Eg5 phosphorylation by the Nek9-Nek6 mitotic kinase cascade and Cdk1. PDZ-BD deletion or Dlg1 ablation impaired loading of Eg5 onto centrosomes and spindle pole motility, yielding asymmetrical spindles that are prone to chromosome missegregation. Collectively, these data demonstrate that Pten, through the Dlg1-binding ability of its PDZ-BD, accumulates phosphorylated Eg5 at duplicated centrosomes to establish symmetrical bipolar spindles that properly segregate chromosomes, and suggest that this function contributes to tumour suppression.

Microtubule-stabilizing properties of the avocado-derived toxins (+)-(R)-persin and (+)-(R)-tetrahydropersin in cancer cells and activity of related synthetic analogs.

The avocado toxin (+)-R-persin (persin) is active at low micromolar concentrations against breast cancer cells and synergizes with the estrogen receptor modulator 4-hydroxytamoxifen. Previous studies in the estrogen receptor-positive breast cancer cell line MCF-7 indicate that persin acts as a microtubule-stabilizing agent. In the present study, we further characterize the properties of persin and several new synthetic analogues in human ovarian cancer cells. Persin and tetrahydropersin cause G2M cell cycle arrest and increase intracellular microtubule polymerization. One analog (4-nitrophenyl)-deshydroxypersin prevents cell proliferation and blocks cells in G1 of the cell cycle rather than G2M, suggesting an additional mode of action of these compounds independent of microtubules. Persin can synergize with other microtubule-stabilizing agents, and is active against cancer cells that overexpress the P-glycoprotein drug efflux pump. Evidence from Flutax-1 competition experiments suggests that while the persin binding site on ß-tubulin overlaps the classical taxoid site where paclitaxel and epothilone bind, persin retains activity in cell lines with single amino acid mutations that affect these other taxoid site ligands. This implies the existence of a unique binding location for persin at the taxoid site.

Peloruside A: a lead non-taxoid-site microtubule-stabilizing agent with potential activity against cancer, neurodegeneration, and autoimmune disease.

Covering: 2000 up to 2016Peloruside A, a macrocyclic secondary metabolite from a New Zealand marine sponge, Mycale hentscheli, has shown potent antiproliferative activity in cultured cancer cells as well as inhibitory effects on tumor growth in mouse models. The compound also has promising effects against cell models of neurodegenerative and autoimmune diseases. In mechanistic studies, peloruside A shares with paclitaxel (Taxol®) the ability to stabilize microtubules by binding to ß-tubulin. Peloruside A, however, occupies a unique external site on ß-tubulin that does not overlap the classical taxoid site that is located on the inside of the microtubule. As such, peloruside A has been of central importance in defining a new microtubule-stabilizer binding site localized on the exterior surface of the microtubule that has led to increased interest in the design of an upscaled total synthesis of the natural product and its analogues. Here, we review advances in the biochemical and biological validation of peloruside A as an attractive therapeutic candidate for the treatment of cancer, neurodegeneration, and autoimmune disease.

ßI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug-tubulin interactions and microtubule stability.

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to ß-tubulin at a site that is different from the classical taxoid site. Multiple ßI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M ßI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian ßI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug-tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on ß-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

Potential role of tubulin tyrosine ligase-like enzymes in tumorigenesis and cancer cell resistance.

Polyglutamylation of tubulin and other non-tubulin substrates is a reversible posttranslational modification brought about by tubulin tyrosine-like ligases. Altered polyglutamylation is linked to tumorigenesis and resistance to chemotherapeutic drugs that target the microtubule, and therefore is a potential pharmacological target in cancer therapy. Despite the large amount of research focused on the development of anticancer agents, only a small number of well-characterized inhibitors of polyglutamylases have been identified, including the phosphinic acid-based inhibitors of Ttll7. In this minireview, we summarize the role of polyglutamylation in cancer, and draw attention to the largely unexplored area of polyglutamylase inhibition in the treatment of cancer.

Microtubule-targeting agents are clinically successful due to both mitotic and interphase impairment of microtubule function.

Microtubules undergo continual dynamic changes in mitotic cells as the mitotic spindle forms and is broken down and in interphase cells where they play a central role in intracellular trafficking, cell signaling, cell migration, and angiogenesis. Compounds that target the microtubule have been hugely successful in the clinic as chemotherapeutics, and this success is likely due to their ability to target cells regardless of their cell cycle stage. Additionally, new generation antibody-conjugated microtubule-targeting agents are improving the targeting of these drugs to tumors. Microtubule-targeting agents have been shown to have anti-angiogenic and vascular-disrupting properties as well as effects on cellular migration, intracellular trafficking, and cell secretion. There are a number of these compounds in development that target the vasculature, and different formulations of clinically used drugs are being developed to take advantage of these anti-angiogenic properties. Microtubule-targeting agents have also been shown to have the potential to treat neurodegenerative diseases, such as Alzheimer's disease. Thus, drugs that target the microtubule will continue to have a major impact in oncology not only as anti-mitotics but also as potent inhibitors of interphase functions, and in future may also prove to be effective in reducing the consequences of neurodegenerative disease.

Resistance to peloruside A and laulimalide: functional significance of acquired ßI-tubulin mutations at sites important for drug-tubulin binding.

Cancer cell lines selected for resistance to microtubule targeting agents (MTA) often have acquired mutations in the ß-tubulin binding sites for these agents. Despite strong correlational evidence, the functional and quantitative significance of such mutations in the resistance to MTA remains unknown. We recently showed that peloruside A (PLA) and laulimalide (LAU)-resistant cancer cell lines, 1A9-R1 (R1) and 1A9-L4 (L4), generated through multi-step selection of human 1A9 ovarian cancer cells with high concentrations of either PLA (for R1) or LAU (for L4) have single distinct mutations in their ßI-tubulin gene. The R1 cells have a mutation at amino acid position 296 (A296T), and the L4 cells have a mutation at position 306 (R306H/C), both of which lie at the putative binding sites of PLA and LAU. To gain insights on the functional role of these mutations in the resistance phenotype, R1 and L4 cells were transfected with wild type ßI-tubulin. MTT cell proliferation assays revealed that restoration of wild type ßI-tubulin expression partially sensitized the R1 and L4 cells to PLA and LAU. Cell cycle analysis and intracellular tubulin polymerization assays demonstrated that the increased sensitivity was correlated with an increased ability of PLA and LAU to induce G2-M arrest and tubulin polymerization in the cells. Unlike paclitaxel-selected clones of 1A9 cells, both R1 and L4 cells exhibited a functional p53 gene, and the abundance of the mismatch repair gene hMSH2 (human mutS homolog 2) was comparable to the parental 1A9 cells. This study provides the first direct evidence that A296 and R306 of ßI-tubulin are important determinants of the PLA and LAU response in cancer cells.

MicroRNAs: novel mediators of resistance to microtubule-targeting agents.

MicroRNAs (miRNAs) are small, non-coding RNAs that act as expression regulators of genes involved in diverse cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal expression of miRNAs can have profound effects on cellular function, and miRNAs are implicated in tumourigenesis either as oncogenes or tumour suppressors. Deregulated miRNAs have been shown to have a role in the resistance of cancer cells to microtubule-targeting agents (MTAs). Here, we discuss how altered expression of miRNAs mediates resistance to clinically useful MTAs, such as paclitaxel and vincristine. Understanding the molecular role of miRNAs in drug resistance would help improve therapeutic efficacy of these agents by helping circumvent the problem of drug resistance.

Cytoskeletal alterations that confer resistance to anti-tubulin chemotherapeutics.

Drugs that target microtubules are a successful class of anti-cancer agents that have been in clinical use for over two decades. Acquired resistance to these drugs, however, remains a serious problem. Microtubule alterations, such as tubulin mutations and altered ß- tubulin isotype expression, are prominent factors in development of resistance. Changes in actin and intermediate filament proteins can also mediate sensitivity to microtubule-targeting drugs. This review focuses on the mechanisms by which alterations in cytoskeletal proteins lead to drug resistance. This information will be helpful for improving the targeting of microtubule toxins.

Zampanolide, a potent new microtubule-stabilizing agent, covalently reacts with the taxane luminal site in tubulin α,ß-heterodimers and microtubules.

Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to ß-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,ß-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates ß-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

Acquired resistance to peloruside A and laulimalide is associated with downregulation of vimentin in human ovarian carcinoma cells.

PURPOSE: Acquired ß-tubulin alterations in human ovarian carcinoma 1A9 cells were previously shown to confer resistance to the microtubule stabilizing agents peloruside A (PLA) and laulimalide (LAU). We examined the proteome of resistant cells to see what other protein changes occurred as a result of the acquired drug resistance. METHODS: Two-dimensional differential in-gel electrophoresis was performed to explore differentially expressed proteins in the resistant 1A9-R1 (R1) and 1A9-L4 (L4) cells. The proteins on the gels were identified by MALDI-TOF MS, and altered protein abundance was confirmed by Western blotting and immunocytochemistry. Vimentin expression was restored in vimentin-deficient L4 cells by transfecting a full-length human vimentin cDNA, and sensitivity to PLA and LAU were tested using an MTT cell proliferation assay. RESULTS: Proteomic analysis identified several proteins that were significantly altered in the resistant cells relative to the parental 1A9 cells. Using Western blotting and immunocytochemistry, a decreased vimentin abundance in the L4 cells was validated. Vimentin levels were unchanged in PLA-resistant R1 cells and paclitaxel/epothilone-resistant derivatives of 1A9 cells. Vimentin cDNA transfection into L4 cells partially restored PLA and LAU sensitivity. CONCLUSIONS: Downregulation of vimentin contributes to the resistance of 1A9 cells to the microtubule stabilizing agents, PLA and LAU.