Automated classification of mitotic catastrophe by use of the centromere fragmentation morphology.

Mitotic catastrophe is a common mode of tumor cell death. Cancer cells with a defective cell-cycle checkpoint often enter mitosis with damaged or under replicated chromosomes following genotoxic treatment. Premature condensation of the under-replicated (or damaged) chromosomes results in double-stranded DNA breaks at the centromere (centromere fragmentation). Centromere fragmentation is a morphological marker of mitotic catastrophe and is distinguished by the clustering of centromeres away from the chromosomes. We present an automated 2-step system for segmentation of cells exhibiting centromere fragmentation. The first step segments individual cells from clumps. We added two new terms, weighted local repelling term (WLRt) and weighted gradient term (WGt), in the energy functional of the traditional Chan-Vese based level set method. WLRt was used to generate a repelling force when contours of adjacent cells merged and then penalized the overlap. WGt enhances gradients between overlapping cells. The second step consists of a new algorithm, SBaN (shape-based analysis of each nucleus), which extracts features like circularity, major-axis length, minor-axis length, area, and eccentricity from each chromosome to identify cells with centromere fragmentation. The performance of SBaN algorithm for centromere fragmentation detection was statistically evaluated and the results were robust.

Thyroid hormone receptor interacting protein 13 (TRIP13) AAA-ATPase is a novel mitotic checkpoint-silencing protein.

The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochore-microtubule attachment. The target of the checkpoint is the E3 ubiquitin ligase anaphase-promoting complex/cyclosome. Once all chromosomes are properly attached and bioriented at the metaphase plate, the checkpoint needs to be silenced. Previously, we and others have reported that TRIP13 AAA-ATPase binds to the mitotic checkpoint-silencing protein p31(comet). Here we show that endogenous TRIP13 localizes to kinetochores. TRIP13 knockdown delays metaphase-to-anaphase transition. The delay is caused by prolonged presence of the effector for the checkpoint, the mitotic checkpoint complex, and its association and inhibition of the anaphase-promoting complex/cyclosome. These results suggest that TRIP13 is a novel mitotic checkpoint-silencing protein. The ATPase activity of TRIP13 is essential for its checkpoint function, and interference with TRIP13 abolished p31(comet)-mediated mitotic checkpoint silencing. TRIP13 overexpression is a hallmark of cancer cells showing chromosomal instability, particularly in certain breast cancers with poor prognosis. We suggest that premature mitotic checkpoint silencing triggered by TRIP13 overexpression may promote cancer development.

Human mitochondrial Fis1 links to cell cycle regulators at G2/M transition.

We have previously shown that prolonged mitochondrial elongation triggers cellular senescence. Here, we report that enforced mitochondrial elongation by hFis1 depletion caused a severe defect in cell cycle progression through G2/M phase (~3-fold reduction in mitotic index; p < 0.01). Reintroduction of Myc-hFis1 to these cells induced mitochondrial fragmentation and restored the cell cycle, indicating that morphodynamic changes of mitochondria closely link to the cell cycle. In hFis1-knockdown cells, cell cycle regulators governing the G2/M phase, including cyclin A, cyclin B1, cyclin-dependent kinase1 (Cdk1), polo-like kinase1 (Plk1), aurora kinase A and Mad2, were significantly suppressed (2- to 10-fold). Notably, however, when mitochondrial fragmentation was induced by double knockdown of hFis1 and Opa1, the cells regained their ability to enter mitosis, and cell cycle regulators were rebounded. Reconstitution of the cyclin B1/Cdk1 complex, a major regulator of the G2/M transition, failed to restore mitotic entry in hFis1-depleted cells. In contrast, expression of Plk1, an upstream regulator of the cyclin B1/Cdk1 complex, or FoxM1 (forkhead box M1), a master transcriptional factor for the cell cycle regulators of G2/M phase, restored the cell cycle in these cells. Our findings suggest that mitochondrial fission molecule hFis1 ensures the proper cell division by interplay with the cell cycle machinery.

Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors.

Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments.

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.

hZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10-interaction domain.

Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein-dynactin and Mad1-Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

Cell Synchronization Techniques for Studying Mitosis.

Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.

Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor.

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3'-phosphatase and DNA 5'-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.

Aurora B kinase-dependent recruitment of hZW10 and hROD to tensionless kinetochores.

The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores (reviewed in [1, 2]). Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. Here, we report that hZW10 and hROD, two components of the evolutionarily conserved RZZ complex, accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, we showed that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. We also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, we show that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. We therefore conclude that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest.

Kinetochore structure and function.

The vertebrate kinetochore is a complex structure that specifies the attachments between the chromosomes and microtubules of the spindle and is thus essential for accurate chromosome segregation. Kinetochores are assembled on centromeric chromatin through complex pathways that are coordinated with the cell cycle. In the light of recent discoveries on how proteins assemble onto kinetochores and interact with each other, we review these findings in this article (which is part of the Chromosome Segregation and Aneuploidy series), and discuss their implications for the current mitotic checkpoint models - the template model and the two-step model. The template model proposes that Mad1-Mad2 at kinetochores acts as a template to change the conformation of another binding molecule of Mad2. This templated change in conformation is postulated as a mechanism for the amplification of the 'anaphase wait' signal. The two-step model proposes that the mitotic checkpoint complex (MCC) is the kinetochore-independent anaphase inhibitor, and the role of the unaligned kinetochore is to sensitize the anaphase-promoting complex/cyclosome (APC/C) to MCC-mediated inhibition.

Inhibiting Wee1 and ATR kinases produces tumor-selective synthetic lethality and suppresses metastasis.

We used the cancer-intrinsic property of oncogene-induced DNA damage as the base for a conditional synthetic lethality approach. To target mechanisms important for cancer cell adaptation to genotoxic stress and thereby to achieve cancer cell-specific killing, we combined inhibition of the kinases ATR and Wee1. Wee1 regulates cell cycle progression, whereas ATR is an apical kinase in the DNA-damage response. In an orthotopic breast cancer model, tumor-selective synthetic lethality of the combination of bioavailable ATR and Wee1 inhibitors led to tumor remission and inhibited metastasis with minimal side effects. ATR and Wee1 inhibition had a higher synergistic effect in cancer stem cells than in bulk cancer cells, compensating for the lower sensitivity of cancer stem cells to the individual drugs. Mechanistically, the combination treatment caused cells with unrepaired or under-replicated DNA to enter mitosis leading to mitotic catastrophe. As these inhibitors of ATR and Wee1 are already in phase I/II clinical trials, this knowledge could soon be translated into the clinic, especially as we showed that the combination treatment targets a wide range of tumor cells. Particularly, the antimetastatic effect of combined Wee1/ATR inhibition and the low toxicity of ATR inhibitors compared with Chk1 inhibitors have great clinical potential.

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition.

Adavosertib (also known as AZD1775 or MK1775) is a small-molecule inhibitor of the protein kinase Wee1, with single-agent activity in multiple solid tumors, including sarcoma, glioblastoma, and head and neck cancer. Adavosertib also shows promising results in combination with genotoxic agents such as ionizing radiation or chemotherapy. Previous studies have investigated molecular mechanisms of primary resistance to Wee1 inhibition. Here, we investigated mechanisms of acquired resistance to Wee1 inhibition, focusing on the role of the Wee1-related kinase Myt1. Myt1 and Wee1 kinases were both capable of phosphorylating and inhibiting Cdk1/cyclin B, the key enzymatic complex required for mitosis, demonstrating their functional redundancy. Ectopic activation of Cdk1 induced aberrant mitosis and cell death by mitotic catastrophe. Cancer cells with intrinsic adavosertib resistance had higher levels of Myt1 compared with sensitive cells. Furthermore, cancer cells that acquired resistance following short-term adavosertib treatment had higher levels of Myt1 compared with mock-treated cells. Downregulating Myt1 enhanced ectopic Cdk1 activity and restored sensitivity to adavosertib. These data demonstrate that upregulating Myt1 is a mechanism by which cancer cells acquire resistance to adavosertib. SIGNIFICANCE: Myt1 is a candidate predictive biomarker of acquired resistance to the Wee1 kinase inhibitor adavosertib.

Epigenetics regulate centromere formation and kinetochore function.

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.

Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores.

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.

Mitotic aberration coupled with centrosome amplification is induced by hepatitis B virus X oncoprotein via the Ras-mitogen-activated protein/extracellular signal-regulated kinase-mitogen-activated protein pathway.

Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n >or=3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.

A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores.

Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein-dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein-protein interaction.

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization.

Laulimalide is a natural product that has strong taxoid-like properties but binds to a distinct site on ß-tubulin in the microtubule (MT) lattice. At elevated concentrations, it generates MTs that are resistant to depolymerization, and it induces a conformational state indistinguishable from taxoid-treated MTs. In this study, we describe the effect of low-dose laulimalide on various stages of the cell cycle and compare these effects to docetaxel as a representative of taxoid stabilizers. No evidence of MT bundling in interphase was observed with laulimalide, in spite of the fact that MTs are stabilized at low dose. Cells treated with laulimalide enter mitosis but arrest at prometaphase by generating multiple asters that coalesce into supernumerary poles and interfere with the integrity of the metaphase plate. Cells with a preformed bipolar spindle exist under heightened tension under laulimalide treatment, and chromosomes rapidly shear from the plate, even though the bipolar spindle is well-preserved. Docetaxel generates a similar phenotype for HeLa cells entering mitosis, but when treated at metaphase, cells undergo chromosomal fragmentation and demonstrate reduced centromere dynamics, as expected for a taxoid. Our results suggest that laulimalide represents a new class of molecular probe for investigating MT-mediated events, such as kinetochore-MT interactions, which may reflect the location of the ligand binding site within the interprotofilament groove.