Systematic Analysis of Compounds Specifically Targeting Telomeres and Telomerase for Clinical Implications in Cancer Therapy.

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene; compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds, Cu-ttpy and Pt-ttpy, induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (ultrafine bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.Significance: An assay provides a unique opportunity to screen thousands of chemical compounds for their ability to inactivate replication of telomeric ends in cancer cells and holds potential to lay the foundation for the discovery of new treatments for cancer. Cancer Res; 78(21); 6282-96. ©2018 AACR.

Publisher Correction: A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage.

The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.

A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage.

The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.

The Dawn of Aurora Kinase Research: From Fly Genetics to the Clinic.

Aurora kinases comprise a family of highly conserved serine-threonine protein kinases that play a pivotal role in the regulation of cell cycle. Aurora kinases are not only involved in the control of multiple processes during cell division but also coordinate chromosomal and cytoskeletal events, contributing to the regulation of checkpoints and ensuring the smooth progression of the cell cycle. Because of their fundamental contribution to cell cycle regulation, Aurora kinases were originally identified in independent genetic screens designed to find genes involved in the regulation of cell division. The first aurora mutant was part of a collection of mutants isolated in C. Nusslein-Volhard's laboratory. This collection was screened in D. M. Glover's laboratory in search for mutations disrupting the centrosome cycle in embryos derived from homozygous mutant mothers. The mutants identified were given names related to the "polar regions," and included not only aurora but also the equally famous polo. Ipl1, the only Aurora in yeast, was identified in a genetic screen looking for mutations that caused chromosome segregation defects. The discovery of a second Aurora-like kinase in mammals opened a new chapter in the research of Aurora kinases. The rat kinase AIM was found to be highly homologous to the fly and yeast proteins, but localized at the midzone and midbody and was proposed to have a role in cytokinesis. Homologs of the equatorial Aurora (Aurora B) were identified in metazoans ranging from flies to humans. Xenopus Aurora B was found to be in a complex with the chromosomal passenger INCENP, and both proteins were shown to be essential in flies for chromosome structure, segregation, central spindle formation and cytokinesis. Fifteen years on, Aurora kinase research is an active field of research. After the successful introduction of the first anti-mitotic agents in cancer therapy, both Auroras have become the focus of attention as targets for the development of new anti-cancer drugs. In this review we will aim to give a historical overview of the research on Aurora kinases, highlighting the most relevant milestones in the advance of the field.

Polo-like kinase-activating kinases: Aurora A, Aurora B and what else?

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G(2), and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.

The chromosomal passenger complex: one for all and all for one.

The chromosomal passenger complex-composed of Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin-modulates multiple events during mitosis. In this issue, Jeyaprakash et al. (2007) report the crystal structure of the regulatory subunit complex and reveal how interactions between these proteins promote the targeting and function of the chromosomal passenger complex during mitosis.

Chromosomal passengers: conducting cell division.

Mitosis and meiosis are remarkable processes during which cells undergo profound changes in their structure and physiology. These events are orchestrated with a precision that is worthy of a classical symphony, with different activities being switched on and off at precise times and locations throughout the cell. One essential 'conductor' of this symphony is the chromosomal passenger complex (CPC), which comprises Aurora-B protein kinase, the inner centromere protein INCENP, survivin and borealin (also known as Dasra-B). Studies of the CPC are providing insights into its functions, which range from chromosome-microtubule interactions to sister chromatid cohesion to cytokinesis, and constitute one of the most dynamic areas of ongoing mitosis and meiosis research.

Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system.

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, we have performed a genetic screen looking for new alleles and interactors of Drosophila Incenp. We have isolated a new null allele of Incenp that has allowed us for the first time to study the functions of the chromosomal passengers during development. Homozygous incenp(EC3747) embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process.

RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate.

DNA topoisomerase II (Topo II) is a major component of mitotic chromosomes and an important drug target in cancer chemotherapy, however, its role in chromosome structure and dynamics remains controversial. We have used RNAi to deplete Topo II in Drosophila S2 cells in order to carry out a detailed functional analysis of the role of the protein during mitosis. We find that Topo II is not required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it is essential for anaphase sister chromatid separation. In response to a long-running controversy, we show that Topo II does have some role in mitotic chromatin condensation. Chromosomes formed in its absence have a 2.5-fold decrease in the level of chromatin compaction, and are morphologically abnormal. However, it is clear that the overall programme of mitotic chromosome condensation can proceed without Topo II. Surprisingly, in metaphase cells depleted of Topo II, one or more chromosome arms frequently stretch out from the metaphase plate to the vicinity of the spindle pole. This is not kinetochore-based movement, as the centromere of the affected chromosome is located on the plate. This observation raises the possibility that further unexpected functions for Topo II may remain to be discovered.

Survivin is required for stable checkpoint activation in taxol-treated HeLa cells.

Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension.

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity.

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.