Multipolar Spindles Research Articles

SRSF1 Is Crucial for Maintaining Satellite Cell Homeostasis During Skeletal Muscle Growth and Regeneration.

The splicing factor SRSF1 emerges as a mater regulator of cell proliferation, displaying high expression in actively proliferative satellite cells (SCs). In SRSF1 knockout mice (KO) generated via MyoD-Cre, early mortality and muscle atrophy are observed during postnatal muscle growth. Despite these findings, the precise mechanisms through which SRSF1 loss influences SCs' functions and its role in muscle regeneration remain to be elucidated. To unravel the exact mechanisms underlying the impact of SRSF1 deficiency SC functions, we employed single-cell RNA sequencing (scRNA-seq) on a mononuclear cell suspension isolated from the newborn diaphragm of KO and control mice. Concurrently, we subjected diaphragm muscles to RNA-seq analysis to identify dysregulated splicing events associated with SRSF1 deletion. For the analysis of the effect of SRSF1 deletion on muscle regeneration, we generated mice with inducible SC-specific Srsf1 ablation through Pax7-CreER. SRSF1 ablation was induced by intraperitoneal injection of tamoxifen. Using cardiotoxin-induced muscle injury, we examined the consequences of SRSF1 depletion on SC function through HE staining, immunostaining and EdU incorporation assay. C2C12 myoblasts and isolated myoblasts were employed to assess stem cell function and senescence. Utilizing scRNA-seq analysis, we observed a noteworthy increase in activated and proliferating myoblasts when SRSF1 was absent. This increase was substantial, with the proportion rising from 28.68% in the control group to 77.06% in the knockout group. However, these myoblasts experienced mitotic abnormalities in the absence of SRSF1, resulting in cell cycle arrest and the onset of cellular senescence. In the knockout mice, the proportion of Pax7+ cells within improper niche positioning increased significantly to 25% compared to 12% in the control cells (n ≥ 10, p < 0.001). Furthermore, there was an observation of persistent cell cycle exit specifically in the Pax7+ cells deficient in SRSF1 (n = 6, p < 0.001). SRSF1 plays a pivotal role in regulating the splicing of Fgfr1op2, favouring the full-length isoform crucial for mitotic spindle organization. Disrupting SRSF1 in C2C12 and primary myoblasts results in multipolar spindle formation (p < 0.001) and dysregulated splicing of Fgfr1op2 and triggers cellular senescence. Consequently, adult SCs lacking SRSF1 initially activate upon injury but face substantial challenge in proliferation (n = 4, p < 0.001), leading to a failure in muscle regeneration. SRSF1 plays a critical role in SCs by ensuring proper splicing, maintaining mitotic progression and preventing premature senescence. These findings underscore the significant role of SRSF1 in controlling SC proliferation during skeletal muscle growth and regeneration.

Kinesins regulate the heterogeneity in centrosome clustering after whole-genome duplication.

After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

Identification of KIFC1 as a putative vulnerability in lung cancers with centrosome amplification

Centrosome amplification (CA), an abnormal increase in the number of centrosomes in the cell, is a recurrent phenomenon in lung and other malignancies. Although CA promotes tumor development and progression by inducing genomic instability (GIN), it also induces mitotic stress that jeopardizes cellular integrity. CA leads to the formation of multipolar mitotic spindles that can cause lethal chromosome segregation errors. To sustain the benefits of CA by mitigating its consequences, malignant cells are dependent on adaptive mechanisms that represent therapeutic vulnerabilities. We aimed to discover genetic dependencies associated with CA in lung cancer. Combining a CRISPR/Cas9 functional genomics screen with tumor genomic analyses, we identified the motor protein KIFC1, also known as HSET, as a putative vulnerability specifically in lung adenocarcinoma (LUAD) with CA. KIFC1 expression was positively correlated with CA in LUAD and associated with worse patient outcomes, smoking history, and indicators of GIN. KIFC1 loss-of-function sensitized LUAD cells with high basal KIFC1 expression to potentiation of CA, which was associated with a diminished ability to cluster extra centrosomes into pseudo-bipolar mitotic spindles. Our work suggests that KIFC1 inhibition represents a novel approach for potentiating GIN to lethal levels in LUAD with CA by forcing cells to divide with multipolar spindles, rationalizing further studies to investigate its therapeutic potential.

B-type plexins regulate mitosis via RanGTPase.

Aberrant mitosis can result in aneuploidy and cancer. The small GTPase, Ran, is a key regulator of mitosis. B-type Plexins regulate Ran activity by acting as RanGTPase activating proteins (GAPs) and have been implicated in cancer progression. However, whether B-type plexins have a role in mitosis has not so far been investigated. We show here that PlexinB1 functions in the control of mitosis. Depletion of PlexinB1 affects mitotic spindle assembly, significantly delaying anaphase. This leads to mitotic catastrophe in some cells, and prolonged application of the spindle assembly checkpoint. PlexinB1 depletion also promoted acentrosomal microtubule nucleation and defects in spindle pole refocussing and increased the number of cells with multipolar or aberrant mitotic spindles. An increase in lagging chromosomes or chromosomal bridges at anaphase was also found upon PlexinB1 depletion. PlexinB1 localises to the mitotic spindle in dividing cells. The mitotic defects observed upon PlexinB1 depletion were rescued by an RCC1 inhibitor, indicating that PlexinB1 signals, via Ran, to affect mitosis. These errors in mitosis generated multinucleate cells, and nuclei of altered morphology and abnormal karyotype. Furthermore, Semaphorin4D-treatment increased the percentage of cells with micronuclei, precursors of chromothripsis. Implications: Defects in B-type plexins may contribute to the well-established role of plexins in cancer progression by inducing chromosomal instability.

Targeting YES1 Disrupts Mitotic Fidelity and Potentiates the Response to Taxanes in Triple-Negative Breast Cancer.

Clinical trials examining broad-spectrum Src family kinase (SFK) inhibitors revealed significant dose-limiting toxicities, preventing advancement for solid tumors. SFKs are functionally heterogeneous, thus targeting individual members is a potential strategy to elicit anti-tumor efficacy while avoiding toxicity. Here, we identified that YES1 is the most highly overexpressed SFK in triple negative breast cancer (TNBC) and is associated with poor patient outcomes. Disrupting YES1, genetically or pharmacologically, induced aberrant mitosis, centrosome amplification, multi-polar spindles, and chromosomal instability (CIN). Mechanistically, YES1 sustained FOXM1 protein levels and elevated expression of FOXM1 target genes that control centrosome function and are essential for effective and accurate mitotic progression. In both in vitro and in vivo TNBC models, YES1 suppression potentiated the efficacy of taxanes, cornerstone drugs for TNBC that require elevated CIN for efficacy. Clinically, elevated expression of YES1 was associated with worse overall survival of TNBC patients treated with taxane and anthracycline combination regimens. Together, this study demonstrates that YES1 is an essential regulator of genome stability in TNBC that can be leveraged to improve taxane efficacy.

Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

Ultra-high static magnetic fields altered the embryonic division and development in Caenorhabditis elegans via multipolar spindles

IntroductionUltra-high static magnetic fields (SMFs) have unique advantages in improving medical and academic research. However, the research on the early embryo exposure of ultra-high SMFs is minimal, extensive exploration is indispensable in living organisms. ObjectivesThe present study was aimed to study the effects of ultra-high SMFs on the early embryonic division and development of Caenorhabditis elegans (C. elegans). MethodsEarly adult parents containing fertilized eggs in vivo were exposed to SMFs at intensities ranging from 4 T to 27 T. The number of mitotic cells in the reproductive glands of the P0 worms, early embryonic cell spindle localization, embryo hatching and the reproductive as well as developmental indicators of F1 and F2 nematodes were examined as endpoints. ResultsOur results indicated that ultra-high SMFs has no obvious effect on the germ cell cycle, while 14 T and 27 T SMFs significantly increased the proportion of multi-polar spindle formation in early embryonic cells, and reduced the developmental rate and lifespan of C. elegans exposed at the embryonic stage. Spindle abnormalities of early embryonic cells, as well as the down-regulation of genes related to asymmetric embryonic division and the abnormal expression of the non-muscle myosin NMY-2 in the division grooves played a critical role in the slowing down of embryonic development induced by ultra-high SMFs. ConclusionsThis study provided novel information and a new sight for evaluating the biosafety assessment by exposure to ultra-high SMFs at the early embryonic stage in vivo.

Di(2-ethylexyl) phthalate and chromosomal damage: Insight on aneugenicity from the cytochalasin-block micronucleus assay

Bis(2-ethylhexyl) phthalate is the most abundant phthalate used as plasticizer to soften plastics and polymers included in medical devices. Human and environmental exposure may occur because DEHP is not chemically bound to plastics and can easily leach out of the materials. This phthalate is classified as reproductive toxicant and possible carcinogen to humans. The genotoxic potential has still to be clarified, but there are indications suggesting that DEHP may have aneugenic effects. To further investigate DEHP genotoxicity, the cytochalasin-block micronucleus assay was applied and combined with the CREST staining to characterise micronucleus content and gain insights on its genotoxic mode of action. Chromosomal damage was also analysed in metaphase and ana-telophase cells and the morphology of the mitotic spindle was investigated to evaluate the possible involvement of this cellular apparatus as a target of DEHP.Our findings indicated that DEHP induced a statistically significant increase in the frequency of micronuclei as well as in the frequency of CREST-positive micronuclei. Consistently, disturbance of chromosome segregation and induction of numerical chromosome changes were observed together with changes in spindle morphology, formation of multipolar spindles and alteration of the microtubule network. Experiments performed without metabolic activation demonstrated a direct action of DEHP on chromosome segregation not mediated by its metabolites.In conclusion, there is consistent evidence for an aneugenic activity of DEHP. A thresholded genotoxic activity was identified for DEHP, disclosing possible implications for risk assessment.

INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

Mutations in the human INF2 gene cause autosomal dominant focal segmental glomerulosclerosis (FSGS)—a condition characterized by podocyte loss, scarring, and subsequent kidney degeneration. To understand INF2-linked pathogenicity, we examined the effect of pathogenic INF2 on renal epithelial cell lines and human primary podocytes. Our study revealed an increased incidence of mitotic cells with surplus microtubule-organizing centers fostering multipolar spindle assembly, leading to nuclear abnormalities, particularly multi-micronucleation. The levels of expression of exogenous pathogenic INF2 were similar to those of endogenous INF2. The aberrant nuclear phenotypes were observed regardless of the expression method used (retrovirus infection or plasmid transfection) or the promoter (LTR or CMV) used, and were absent with exogenous wild type INF2 expression. This indicates that the effect of pathogenic INF2 is not due to overexpression or experimental cell manipulation, but instead to the intrinsic properties of pathogenic INF2. Inactivation of the INF2 catalytic domain prevented aberrant nuclei formation. Pathogenic INF2 triggered the translocation of the transcriptional cofactor MRTF into the nucleus. RNA sequencing revealed a profound alteration in the transcriptome that could be primarily attributed to the sustained activation of the MRTF-SRF transcriptional complex. Cells eventually underwent mitotic catastrophe and death. Reducing MRTF-SRF activation mitigated multi-micronucleation, reducing the extent of cell death. Our results, if validated in animal models, could provide insights into the mechanism driving glomerular degeneration in INF2-linked FSGS and may suggest potential therapeutic strategies for impeding FSGS progression.

Deoxynivalenol triggers mitotic catastrophe and apoptosis in C2C12 myoblasts

Deoxynivalenol (DON), commonly known as vomitoxin, is a mycotoxin produced by fungi and is frequently found as a contaminant in various cereal-based food worldwide. While the harmful effects of DON have been extensively studied in different tissues, its specific impact on the proliferation of skeletal muscle cells remains unclear. In this study, we utilized murine C2C12 myoblasts as a model to explore the influence of DON on their proliferation. Our observations indicated that DON exhibits dose-dependent toxicity, significantly inhibiting the proliferation of C2C12 cells. Through the application of RNA-seq analysis combined with gene set enrichment analysis, we identified a noteworthy downregulation of genes linked to the extracellular matrix (ECM) and condensed chromosome. Concurrently with the reduced expression of ECM genes, immunostaining analysis revealed notable changes in the distribution of fibronectin, a vital ECM component, condensing into clusters and punctate formations. Remarkably, the exposure to DON induced the formation of multipolar spindles, leading to the disruption of the normal cell cycle. This, in turn, activated the p53-p21 signaling pathway and ultimately resulted in apoptosis. These findings contribute significant insights into the mechanisms through which DON induces toxicity within skeletal muscle cells.

Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.

Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.

CENP-E Inhibition Induces Chromosomal Instability and Synergizes with Diverse Microtubule-Targeting Agents in Breast Cancer.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E. Significance: The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.

Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73 h in fresh IVM, 17.33 h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47 µm vs 11.23 µm in IVM and 11.40 µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37 µm vs 8.12 µm in IVM and 8.16 µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

A survey of chromosomal instability measures across mechanistic models

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Abstract 4565: Reckless mitotic entry as a chemosensitization approach for alkylating agents

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome scale CRISPR/Cas9 chemogenomic screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Analysis on patient cohort demonstrated that PKMYT1 is not only highly presented in malignancy, but also predisposes an unfavorable disease outcome. Genetic depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James A. Cornwell, Karambir Kaur, Mirit I. Aladjem, Michael Aregger, Steven D. Cappell, Chunzhang Yang. Reckless mitotic entry as a chemosensitization approach for alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4565.

Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis

Abstract The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α. Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1638.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 527.

Abstract 4478: Development and characterization of kinase sparing TPX2-tethering Aurora kinase A ligands for cancer therapy

Abstract A major limitation in the treatment of cancer is the limited therapeutic index of currently available chemotherapies or molecularly targeted therapies. Drug related toxicities often necessitate restricted drug dosing regimens, resulting in subtotal tumor remissions, which represent an important cause for tumor recurrences and the development of therapy resistance. Following up on previous work suggesting Aurora kinase A (AURKA) as a promising therapeutic target for the treatment of TP53 altered liver carcinomas (Dauch D. et al., Nat. Med., 2016) and TP53; RB1 altered small cell lung carcinomas (Gong X. et al., Cancer Discovery, 2019), we here report on the development and characterization of first in class kinase sparing AURKA ligands, which selectively kill TP53 deficient liver cancer cells and TP53; RB1 deficient small cell lung cancer cells by tethering AURKA to its binding partner TPX2 in mitotic cells. Small molecule mediated stabilization of AURKA/TPX2 complexes resulted in formation of multipolar spindles and subsequent cell death through mitotic catastrophe. Mechanistically, we were able to show that the hypersensitivity of TP53 deficient liver cancer cells and TP53; Rb1 deficient small cell lung cancer cells towards AURKA ligands is due to low expression levels of the antiapoptotic protein MCL1, which is necessary for survival of cancer cells in prolonged mitosis. Our novel AURKA ligands do not affect the kinase function of AURKA and thus, in contrast to conventional AURKA kinase inhibitors, can be administered continuously to mice without inducing toxicity related to inhibition of kinase related non-canonical (non-mitotic) functions of AURKA in normal cells or TP53 and RB1 wildtype cancer cells. Preclinical therapy studies in cell-line derived xenograft (CDX) tumor models of small cell lung cancer (SCLC) and orthotopic models of TP53 altered hepatocellular carcinoma and TP53;Rb1 altered SCLC demonstrated marked monotherapeutic efficacy of AURKA ligands. Conceptually, our herein provided data suggest that small molecules designed to stabilize protein-protein interactions represent powerful anti-cancer drugs with exceptional therapeutic window and thus open a venue towards the development of novel cancer therapies. Citation Format: Athina Anastasia Moschopoulou, Melanie Henning, Benedikt Wagner, Dirk Flötgen, Juliander Reiner, Yulia Skliarenko, Stefan Zwirner, Clemens Hinterleitner, Marco Seehawer, Luana dArtista, Elke Rist, Tatu Pantsar, Thales Kronenberger, Daniel Dauch, Corinna Gehring-Khav, Antti Poso, Liudmila Andreeva, Matthias Schwab, Michael Forster, Wolfgang Albrecht, Stefan Laufer, Lars Zender. Development and characterization of kinase sparing TPX2-tethering Aurora kinase A ligands for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4478.

Nek2A prevents centrosome clustering and induces cell death in cancer cells via KIF2C interaction

Unlike normal cells, cancer cells frequently exhibit supernumerary centrosomes, leading to formation of multipolar spindles that can trigger cell death. Nevertheless, cancer cells with supernumerary centrosomes escape the deadly consequences of unequal segregation of genomic material by coalescing their centrosomes into two poles. This unique trait of cancer cells presents a promising target for cancer therapy, focusing on selectively attacking cells with supernumerary centrosomes. Nek2A is a kinase involved in mitotic regulation, including the centrosome cycle, where it phosphorylates linker proteins to separate centrosomes. In this study, we investigated if Nek2A also prevents clustering of supernumerary centrosomes, akin to its separation function. Reduction of Nek2A activity, achieved through knockout, silencing, or inhibition, promotes centrosome clustering, whereas its overexpression results in inhibition of clustering. Significantly, prevention of centrosome clustering induces cell death, but only in cancer cells with supernumerary centrosomes, both in vitro and in vivo. Notably, none of the known centrosomal (e.g., CNAP1, Rootletin, Gas2L1) or non-centrosomal (e.g., TRF1, HEC1) Nek2A targets were implicated in this machinery. Additionally, Nek2A operated via a pathway distinct from other proteins involved in centrosome clustering mechanisms, like HSET and NuMA. Through TurboID proximity labeling analysis, we identified novel proteins associated with the centrosome or microtubules, expanding the known interaction partners of Nek2A. KIF2C, in particular, emerged as a novel interactor, confirmed through coimmunoprecipitation and localization analysis. The silencing of KIF2C diminished the impact of Nek2A on centrosome clustering and rescued cell viability. Additionally, elevated Nek2A levels were indicative of better patient outcomes, specifically in those predicted to have excess centrosomes. Therefore, while Nek2A is a proposed target, its use must be specifically adapted to the broader cellular context, especially considering centrosome amplification. Discovering partners such as KIF2C offers fresh insights into cancer biology and new possibilities for targeted treatment.