Spindle Assembly Checkpoint Research Articles

Abstract P3-07-10: CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC)

Abstract CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) Philippe L. Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton-Marovac, Pierre-Olivier Gaudreau Background: CFI-402257 is a selective oral inhibitor of TTK protein kinase, a critical regulator of the mitotic spindle assembly checkpoint overexpressed in breast cancer (BC). CFI-402257 monotherapy has anti-proliferative and cytotoxic activity and enhances antitumor activity of paclitaxel in BC xenograft models. Material and methods: Primary objectives were to establish safety and Recommended Phase 2 dose (RP2D) of CFI-402257 combined with weekly paclitaxel (Phase 1b) and Overall Response Rate (ORR) as per RECIST 1.1 (Phase 2). Patients with HER2- aBC with adequate organ function, PS 0-1, previously treated with >1 non-taxane chemotherapy, were eligible. A 3+3 design was used for Phase 1b, with dose limiting toxicities (DLTs) assessed during cycle 1 (28 days). Starting dose CFI-402257 was 84mg (DL1 = 84mg, DL2 = 112mg, DL3 = 168mg, DL4 = 210mg and DL5 = 252mg) on a 2-day on, 5-day off schedule with paclitaxel 80mg/m2 day 1, 8, 15. Safety assessments were performed weekly (CTCAE v5.0) and response every 2 cycles. A Simon 2-stage design was used for Phase 2 (stage 2 required ≥4 responses in 17 evaluable patients from stage 1). Results: 37 patients received a total of 260 cycles including all 5 dose levels. Median age was 59; 92% ER+/HER2-; 49% PS1; 22% 3 prior chemotherapy lines; 41% 4 sites of metastatic disease, and 81% had received prior CDK4/6 inhibitors. Grade 3 hematological adverse events (AEs, all dose levels) were neutropenia (70%), lymphopenia (41%) and anemia (14%). Six DLTs occurred: 5 dose-related grade 4 neutropenia and 1 febrile neutropenia. Three DLTs occurred at DL3, two at DL4, and one at DL5. Three serious AEs (two at DL3, and one at DL4) at least possibly related to treatment were seen: 2 febrile neutropenia and 1 skin infection (all grade 3). Frequent AEs (˃5%; all dose levels) considered at least possibly related to treatment were: diarrhea (38%), nausea (30%), fatigue (27%), vomiting (16%), anorexia (14%), maculo-papular rash (14%), oral mucositis (11%), alopecia (11%) and pruritus (8%). DL3 (168mg) was selected as RP2D. ORR was 3/36=8% and 1/17=5.9% in all vs Phase 2 evaluable patients, respectively. Clinical Benefit Rate (CBR; defined as complete response, partial response or stable disease ˃16 weeks in duration) was 18/33=54.6% and 10/17=58.8% in all vs Phase 2 evaluable patients, respectively. During Phase 2, the 17 evaluable patients from stage 1 did not meet pre-specified threshold for anti-tumor activity to proceed to stage 2. Conclusions: CFI-402257 and paclitaxel was well tolerated, with neutropenia as the main toxicity. DL3 (168mg) was selected as RP2D. Phase 2 ORR and CBR was 5.9% and 58.8%, respectively; during Phase 2, the 17 evaluable patients from stage 1 did not meet the pre-specified threshold for anti-tumor activity to proceed to stage 2 and the trial was closed to accrual on April 7, 2022. Final analysis and correlative analyses are ongoing. Acknowledgements: Coordinated by the CCTG. Funding supported by SU2C Canada - Canadian Cancer Society Breast Cancer Dream Team Research Funding (SU2C-AACR-DT-18-15) and OICR. CFI-402257 provided by Treadwell Therapeutics. Citation Format: Philippe Bedard, Mihaela Mates, John Hilton, Nathalie Levasseur, Arif Awan, Amirrtha Srikanthan, David W. Cescon, Karen Gelmon, Andrew Robinson, Nancy Drummond-Ivars, Irene Li, Laleh Rastgou, Jackie Edwards, Linda Hagerman, Siwei Zhang, Mark Bray, Lesley Seymour, Moira Rushton, Pierre-Olivier Gaudreau. CCTG IND.236: A Phase 1b trial of combined CFI-402257 and weekly paclitaxel in patients with HER2-negative (HER2-) advanced breast cancer (aBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-10.

Abstract P6-10-13: An update to a Phase I trial of CFI-402257, an oral TTK inhibitor, in patients with advanced solid tumors with HER2-negative breast cancer expansion cohorts

Abstract Background: TTK (also known as MPS1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint, chromosome alignment, and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, to result in chromosomal missegregation, aneuploidy, and cell death. CFI-402257 is a potent and highly selective inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. CFI-402257 has previously exhibited monotherapy and combination efficacy with a tolerable safety profile in ER+/Her2- Breast cancer patients in an ongoing clinical study which is updated here. Methods: This is an ongoing phase I, multi-center, dose escalation study (3+3 design) to determine the safety, tolerability, and maximum tolerated dose of CFI-402257 and to evaluate anti-tumor activity at the recommended phase 2 dose (RP2D). CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5 mg. Dose escalation included patients (pts) with advanced solid tumors. Dose expansion at the RP2D included pts with advanced solid tumors (Cohort A), advanced Her2-negative (ER+ or TNBC) with 1-4 prior lines of chemotherapy for metastatic disease (Cohort B), and ER+/Her2- breast cancer in combination with Fulvestrant (500 mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (≥ 3 months) and ≤1 prior chemotherapy for metastatic disease (Cohort C). Results: At data cutoff of May 2, 2022, 87 pts were enrolled. 86 pts (66 pts receiving monotherapy and 20 patients receiving combination therapy received ≥1 dose of study therapy and were analyzed for safety. One pt was not dosed due to elevated liver enzymes prior to first dose. The median age for mono was 61 years (range, 35-81) and for combo was 54 (range, 31-70). The median number of prior regimens mono and combo was 5 (range, 0-17; and 1-9, respectively). Tumor types in mono were breast cancer (27 pts), ovarian cancer (7 pts), GI cancer (3 pts), pancreatic cancer (3 pts), and other (30 pts). 20 breast cancer pts were enrolled in the combo. To date, 11 dose levels have been studied (range: 5 to 294 mg) in mono. There were 4 dose limiting toxicities (neutropenia >7days at 168mg/day, febrile neutropenia at 210mg/day, and neutropenia and colitis at 294mg/day, all grade 3). The RP2D of 168 mg was established. 3 pts in mono (biliary obstruction, febrile neutropenia, and pancytopenia) and 0 pts in combo discontinued study due to adverse events (AEs).Treatment emergent adverse events (TEAE’s) occurring in ≥30% of pts were fatigue (31 pts; 47%), nausea (30 pts; 46%), decreased appetite (22 pts; 33%), and diarrhea (21 pts; 32%) in mono; and nausea (11 pts; 55%) and diarrhea (7 pts; 35%) in combo. 35% of mono and 39% of combo AEs were considered related to CFI-402257 by the investigators. Grade ≥3 AEs and serious AEs were reported in 25 pts (38%) and 17 pts (26%), respectively in mono; and 5 pts (25%) and 3 pts (15%) in combo. The investigator assessed best overall response rate (partial response [PR] or better within the efficacy population) of 6% (PR; hepatocellular carcinoma n=1, breast cancer n=2 from n=47) in mono and 18% (PR; n=2 breast cancer) in combo (n=11), with additional patients still to be assessed. Conclusion: CFI-402257 is well tolerated as mono and combination with fulvestrant. Efficacy signals are emerging with pts in the combo cohort demonstrating anti-tumor activity. Additional efficacy will be updated at the time of the presentation. Citation Format: John Hilton, Daniel Renouf, David W. Cescon, Aaron Hansen, Alibiruni Abdul Razak, Lee-Anne Stayner, Trisha A. Denny, Emily Roberts-Thomson, Dih-Yih Chen, Mark Bray, Philippe Bedard. An update to a Phase I trial of CFI-402257, an oral TTK inhibitor, in patients with advanced solid tumors with HER2-negative breast cancer expansion cohorts [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-13.

Comprehensive analysis of BUBs gene family in lung adenocarcinoma with immunological analysis.

Lung adenocarcinoma (LUAD) is one of the most commonly malignant tumors, and major challenges remain in the treatment of LUAD. Budding uninhibited by benzimidazole 1/3 (BUB1/3) play significant roles in the process of spindle-assembly checkpoint (SAC) during mitosis. However, their roles in LUAD have not been established. Here, we performed an immunological analysis of BUB1/3 in LUAD using a comprehensive bioinformatics approach, quantitative real-time-PCR and Western blotting technique. Our results indicated that the expression levels of BUB1 and BUB3 in LUAD samples were higher than the expression levels in the control groups and were associated with some clinicopathologic parameters in patients with LUAD. BUB1/3 and their related genes were enriched in cell immune, and the immune infiltration analysis revealed that the BUB1/3 expression profile was significantly correlated with characteristics of immune cell infiltration. Survival analysis showed that the disease-free survival and overall survival of patients with LUAD decreased with an increase in the BUB1/3 expression levels. The mRNA and protein expression levels of BUB1 and BUB3 in each of the LUAD cell lines were upregulated to varying degrees. BUB1 and BUB3 are the potential immunological therapeutic intervention targets for patients with LUAD.

Identification of potential biomarkers for colorectal cancer by clinical database analysis and Kaplan-Meier curves analysis.

This study aimed to explore critical genes as potential biomarkers for the diagnosis and prognosis of colorectal cancer (CRC) for clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded microarray datasets GSE89076, GSE73360, and GSE32323 from the GEO database identified differentially expressed genes (DEGs), and performed a functional enrichment analysis. A protein-protein interaction network was constructed, and correlated module analysis was performed using STRING and Cytoscape. The Kaplan-Meier survival curve shows the survival of the hub genes. The expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), and PCNA in tissues and changes in tumor grade were analyzed. A total of 329 DEGs were identified, including 264 upregulated and 65 downregulated genes. The functions and pathways of DEGs include the mitotic cell cycle, poly(A) RNA binding replication, ATP binding, DNA replication, ribosome biogenesis in eukaryotes, and RNA transport. Forty-seven Hub genes were identified, and biological process analysis showed that these genes were mainly enriched in cell cycle and DNA replication. Patients with mutations in CDK1, PCNA, and CCNB1 had poorer survival rates. CDK1, PCNA, and CCNB1 were significantly overexpressed in the tumor tissues. The expression of CDK1 and CCNB1 gradually decreased with increasing tumor grade. CDK1, CCNB1, and PCNA can be used as potential markers for the diagnosis and prognosis of CRC. These genes are overexpressed in colon cancer tissues and are associated with low survival rates in CRC patients.

CCNB2 as a potential biomarker of bladder cancer via the high throughput technology.

Bladder cancer and oral squamous cell carcinoma (OSCC) seriously affect people's health. However, the relationship between bladder cancer and OSCC remains unclear. Got GSE138206, GSE146483, GSE184616, and bladder cancer datasets GSE65635, GSE100926 from Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the significant module. Functional enrichment analysis was performed via the Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes. Furthermore, the Gene Set Enrichment Analysis was also used to complete the enrichment analysis. Comparative Toxicogenomics Database found most relevant diseases to core genes. TargetScan is used to forecast analysis of microRNA and target genes. In Gene Ontology analysis, differentially expressed genes were mostly concentrated in cell differentiation, extrallular region, structural molecule activity, and actin binding. In Kyoto Encyclopedia of Genes and Genomes analysis, the differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, pathway in cancer, and extracellular matrix-receptor interaction. Seven hub genes (cyclin B2 [CCNB2], TK1, CDC20, PCNA, CKS1B, CDCA5, MCM4) were obtained. Hub genes (CCNB2, CDC20) are highly expressed in OSCC and bladder cancer samples. CCNB2 was one common oncogene of bladder cancer and OSCC.

The correlation between the expression of ubiquitin-conjugating enzyme 2C and prostate cancer prognosis.

Ubiquitin-conjugating enzyme E2 C (UBE2C) is known to show a causal relationship with cancer development and advancement. The role of UBE2C is to control the mitotic spindle checkpoint. Excess UBE2C has been identified in patients with advanced prostate cancer. The objective of the present study was to examine positive connections between the expression of UBE2C and prognostic factors for prostate cancer. Prostate cancer patients' clinical data were analysed. Tissue microarrays (TMAs) were also performed for human prostate cancer tissues (n = 335) and adjacent non-neoplastic tissues (n = 22). TMA slides were incubated with antibodies against UBE2C. Cores were scored by a pathologist who was blind to cancer results. Of 335 prostate cancer patients, 200 could be assessed for biochemical recurrence, clinical recurrence, and overall survival. Human prostate cancer tissues showed higher expression of UBE2C than adjacent non-neoplastic tissues. High expression level of UBE2C showed a strong positive relationship with a high prostate-specific antigen (PSA), Gleason's score, and pathological stage of prostate cancer. Patients with a higher UBE2C grade demonstrated greater lymphatic engagement of prostate cancer than those with a lower UBE2C grade. The expression of UBE2C has positive correlations with several prognostic factors for prostate cancer. Thus, investigating the expression level of UBE2C staining is a promising tool for predicting prostate cancer prognosis.

Loss of tRNA methyltransferase 9 and DNA damage response genes in yeast confers sensitivity to aminoglycosides.

tRNA methyltransferase 9 (Trm9)-catalysed tRNA modifications have been shown to translationally enhance the DNA damage response (DDR). Here, we show that Saccharomyces cerevisiae trm9Δ, distinct DNA repair and spindle assembly checkpoint (SAC) mutants are differentially sensitive to the aminoglycosides tobramycin, gentamicin and amikacin, indicating DDR and SAC activation might rely on translation fidelity, under aminoglycoside stress. Further, we report that the DNA damage induced by aminoglycosides in the base excision repair mutants ogg1Δ and apn1Δ is mediated by reactive oxygen species, which induce the DNA adduct 8-hydroxy deoxyguanosine. Finally, the synergistic effect of tobramycin and the DNA-damaging agent bleomycin to sensitize trm9Δ and the DDR mutants mlh1Δ, rad51Δ, mre11Δ and sgs1Δ at significantly lower concentrations compared with wild-type suggests that cells with tRNA modification dysregulation and DNA repair gene defects can be selectively sensitized using a combination of translation inhibitors and DNA-damaging agents.

Functional tug of war between kinases, phosphatases, and the Gcn5 acetyltransferase in chromatin and cell cycle checkpoint controls.

Covalent modifications of chromatin regulate genomic structure and accessibility in diverse biological processes such as transcriptional regulation, cell cycle progression, and DNA damage repair. Many histone modifications have been characterized, yet understanding the interactions between these and their combinatorial effects remains an active area of investigation, including dissecting functional interactions between enzymes mediating these modifications. In budding yeast, the histone acetyltransferase Gcn5 interacts with Rts1, a regulatory subunit of protein phosphatase 2A (PP2A). Implicated in the interaction is the potential for the dynamic phosphorylation of conserved residues on histone H2B and the Cse4 centromere-specific histone H3 variant. To probe these dynamics, we sought to identify kinases which contribute to the phosphorylated state. In a directed screen beginning with in silico analysis of the 127 members of yeast kinome, we have now identified 16 kinases with genetic interactions with GCN5 and specifically found distinct roles for the Hog1 stress-activated protein kinase. Deletion of HOG1 (hog1Δ) rescues gcn5Δ sensitivity to the microtubule poison nocodazole and the lethality of the gcn5Δ rts1Δ double mutant. The Hog1-Gcn5 interaction requires the conserved H2B-T91 residue, which is phosphorylated in vertebrate species. Furthermore, deletion of HOG1 decreases aneuploidy and apoptotic populations in gcn5Δ cells. Together, these results introduce Hog1 as a kinase that functionally opposes Gcn5 and Rts1 in the context of the spindle assembly checkpoint and suggest further kinases may also influence GCN5's functions.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

Unique progerin C-terminal peptide ameliorates Hutchinson–Gilford progeria syndrome phenotype by rescuing BUBR1

An accumulating body of evidence indicates an association between mitotic defects and the aging process in Hutchinson–Gilford progeria syndrome (HGPS), which is a premature aging disease caused by progerin accumulation. Here, we found that BUBR1, a core component of the spindle assembly checkpoint, was downregulated during HGPS cellular senescence. The remaining BUBR1 was anchored to the nuclear membrane by binding with the C terminus of progerin, thus further limiting the function of BUBR1. Based on this, we established a unique progerin C-terminal peptide (UPCP) that effectively blocked the binding of progerin and BUBR1 and enhanced the expression of BUBR1 by interfering with the interaction between PTBP1 and progerin. Finally, UPCP significantly inhibited HGPS cellular senescence and ameliorated progeroid phenotypes, extending the lifespan of LmnaG609G/G609G mice. Our findings reveal an essential role for the progerin-PTBP1-BUBR1 axis in HGPS. Therapeutics designed around UPCP may be a beneficial strategy for HGPS treatment.

USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy

The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.

The HIPK2/CDC14B-MeCP2 axis enhances the spindle assembly checkpoint block by promoting cyclin B translation.

Mitotic perturbations activate the spindle assembly checkpoint (SAC) that keeps cells in prometaphase with high CDK1 activity. Prolonged mitotic arrest is eventually bypassed by gradual cyclin B decline followed by slippage of cells into G1 without chromosome segregation, a process that promotes cell transformation and drug resistance. Hitherto, the cyclin B1 decay is exclusively defined by mechanisms that involve its proteasomal degradation. Here, we report that hyperphosphorylated HIPK2 kinase accumulates in mitotic cells and phosphorylates the Rett syndrome protein MeCP2 at Ser92, a regulation that is counteracted by CDC14B phosphatase. MeCP2S92 phosphorylation leads to the enhanced translation of cyclin B1, which is important for cells with persistent SAC activation to counteract the proteolytic decline of cyclin B1 and therefore to suspend mitotic slippage. Hence, the HIPK2/CDC14B-MeCP2 axis functions as an enhancer of the SAC-induced mitotic block. Collectively, our study revises the prevailing view of how cells confer a sustainable SAC.

Integrated transcriptome and proteome revealed that the declined expression of cell cycle-related genes associated with follicular atresia in geese

BackgroundGeese exhibit relatively low reproductive performance, and follicular atresia is an important factor that restricts the egg production of geese. Systematic analysis of the regulation of follicle atresia in geese through transcriptome and proteome levels could provide meaningful information on clarifying the mechanism of follicle atresia in poultry.ResultThe granulosa cell layer was loose, disintegrated and showed apoptosis in atretic follicles and remained intact in normal follicles. The hormone levels of FSH and LH were significantly decreased in the atresia follicles compared to the normal follicles (P < 0.05). A total of 954 differentially expressed genes (DEGs, 315 increased and 639 decreased) and 161 differentially expressed proteins (DEPs, 61 increased and 100 decreased) were obtained in atresia follicles compared to normal follicles, of which, 15 genes were differentially expressed in both transcriptome and proteome. The DEGs were mainly enriched in sodium transmembrane transport, plasma membrane, and transmembrane transporter activity based on the GO enrichment analysis and in the cell cycle pathway based on the KEGG enrichment analysis. The DEPs were mainly enriched in localization, lysosome, and phospholipid-binding based on the GO enrichment analysis. Candidate genes Smad2/3, Smad4, Annexin A1 (ANXA1), Stromelysin-1 (MMP3), Serine/threonine-protein kinase (CHK1), DNA replication licensing factor (MCM3), Cyclin-A2 (CCNA2), mitotic spindle assembly checkpoint protein (MAD2), Cyclin-dependent kinase 1 (CDK1), fibroblast growth factor 12 (FGF12), and G1/S-specific cyclin-D1 (CCND1) were possibly responsible for the regulation of atresia.ConclusionThe cell cycle is an important pathway for the regulation of follicular atresia. Sodium outflow and high expression of MMP3 and MMP9 could be responsible for structural destruction and apoptosis of follicular cells.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.

First person – Yanze Jian

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Yanze Jian is first author on ‘ The fission yeast kinetochore complex Mhf1–Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation’, published in JCS. Yanze is a PhD student in the lab of Chuanhai Fu at the University of Science and Technology of China, Hefei, China, investigating checkpoint signalling in mitosis and meiosis.

RANBP1 (RAN Binding Protein 1): The Missing Genetic Piece in Cancer Pathophysiology and Other Complex Diseases

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus-cytoplasmic gradient, thus making nuclear import-export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implicated in chemo-hormone and radio-resistance. A key role of this small-GTPases related protein has also been demonstrated in alterations of axonal flow and neuronal plasticity, as well as in viral and bacterial metabolism and in embryological maturation. In conclusion, RANBP1 appears not only to be an interesting factor in several pathological conditions but also a putative target of clinical interest.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168–333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168–333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168–333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

CDCA3 is a prognostic biomarker for cutaneous melanoma and is connected with immune infiltration.

Dysregulation of cell cycle progression (CCP) is a trait that distinguishes cancer from other diseases. In several cancer types, CCP-related genes serve as the primary risk factor for prognosis, but their role in cutaneous melanoma remains unclear. Data from cutaneous melanoma patients were acquired from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Using a Wilcoxon test, the level of CCP-related gene expression in cutaneous melanoma patient tissues was compared to that in normal skin tissues. Logistic analysis was then utilized to calculate the connection between the CCP-related genes and clinicopathological variables. The important functions of the CCP-related genes were further investigated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and single-sample Gene Set Enrichment Analysis (ssGSEA). Univariate and multivariate Cox analyses and Kaplan-Meier analysis were used to estimate the association between CCP-related genes and prognosis. In addition, using Cox multivariate analysis, a nomogram was constructed to forecast the influence of CCP-related genes on survival rates. High expression of CCP-related genes was associated with TNM stage, age, pathological grade, and Breslow depth (P < 0.05). Multivariate analysis demonstrated that CCP-related genes were an independent factor in overall survival and disease-specific survival. High levels of gene expression originating from CCP were shown by GSEA to trigger DNA replication, the G1-S specific transcription factor, the mitotic spindle checkpoint, and the cell cycle. There was a negative association between CCP-related genes and the abundance of innate immune cells. Finally, we revealed that knockdown of cell division cycle-associated gene 3 (CDCA3) significantly suppressed the proliferation and migration ability of cutaneous melanoma cells. According to this study, CCP-related genes could serve as potential biomarkers to assess the prognosis of cutaneous melanoma patients and are crucial immune response regulators.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Acquisition of the spindle assembly checkpoint and its modulation by cell fate and cell size in a chordate embryo.

The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.