Renal cell carcinoma (RCC) is a highly heterogeneous malignancy with complex molecular features. Mitotic catastrophe (MC), a key regulator of cell fate during aberrant mitosis, contributes to tumor progression and therapeutic response; however, its prognostic relevance in RCC remains unclear. We integrated multi-omics, clinical, and imaging data from TCGA and multiple external cohorts. Prognostic mitotic catastrophe-related genes were identified to construct a mitotic catastrophe-related score (MCRS) using CoxBoost and random survival forest models. Associations with clinicopathologic features, immune contexture, and mutational profiles were analyzed. Single-cell RNA sequencing characterized Cyclin F (CCNF) expression and its association with immunotherapy-treated cohort outcomes, and these findings were further validated in vitro and in murine xenograft models. The nine-gene MCRG signature stratified patients into high- and low-risk groups with strong prognostic performance (TCGA 1-, 3-, 5-year AUCs > 0.9). High MCRS correlated with advanced stage, adverse CT features, angiogenesis, inflammatory signaling, regulatory T-cell infiltration, and elevated LAG3/TIGIT expression. MTOR mutations were enriched in high-risk tumors. SurvSHAP(t) analysis identified CCNF as the top prognostic contributor. CCNF was upregulated in tumor tissues and enriched in proliferating cells and epithelial cells. High CCNF expression was associated with worse survival and inferior outcomes in immunotherapy-treated cohorts. Overexpression of CCNF promoted RCC cell proliferation, migration, invasion, and in vivo tumor growth, partly through mTOR pathway activation. We established a robust MC-based prognostic model for RCC and identified CCNF as a key regulator of tumor progression and the immune microenvironment, characterized by increased Treg abundance and checkpoint expression. CCNF may represent a candidate biomarker and therapeutic target in RCC, although its relevance to outcomes in immunotherapy-treated cohorts requires further validation.

The RAW264.7/THP1 cells have been extensively employed as macrophage models in inflammation research. However, comprehensive comparisons of their expression profiles remain largely unexplored. In this study, we conducted a systematic bioinformatics analysis to characterize the whole-cell proteome, phosphoproteome, acetylome, and ubiquitinome profiles of lipopolysaccharide-stimulated RAW264.7/THP1 cells. Through comparative temporal analysis of differentially regulated proteins (classified as rapid, persistent, or slow dysregulation patterns), we identified IGF2, COPZ1, and GTF2B, exhibiting persistent downregulation in both cell models. Additionally, we observed that nearly all dysregulated proteins within each modification type exhibited significant enrichment in the other two modification types, forming a highly interconnected protein-protein interaction network. Notably, VIMENTIN demonstrated significant downregulation across three modification types (phosphorylation at Ser56/214, acetylation at Lys235, and ubiquitination at Lys139), with coordinated downregulation observed in both cell lines. It was also shown that VIMENTIN mutation at Ser56, Lys235, and Lys139 dramatically reduces the expression of CDK1, which may help the inflammation response by averting mitotic catastrophe and changing the macrophage cell cycle. These findings provide valuable molecular insights into species-specific macrophage responses and establish an important reference data set for discovering novel inflammatory proteins and investigating macrophage-associated diseases using these experimental models. Notably, our work delineates vimentin-centered regulatory networks that may offer novel diagnostic biomarkers and therapeutic targets for inflammatory diseases.

The protein kinase PKMYT1 regulates a key cell cycle checkpoint as part of the cell's DNA-damage repair response, but in cancer, this function can promote tumor cell survival through avoiding mitotic catastrophe. PKMYT1 has been linked to a variety of cancer types, including breast, gastric, and nonsmall cell lung cancers, as well as kidney renal clear cell carcinoma, making it an important therapeutic target. However, potent and selective small-molecule inhibitors of PKMYT1 are scarce, and its specific biological role in tumor proliferation remains understudied. Here, we report the discovery and characterization of a novel PKMYT1 inhibitor, P29, bound to a previously unknown allosteric site. Structural and kinetic data reveal that P29 induces a conformational rearrangement of the P-loop and inhibits PKMYT1 through a mixed ATP competitive and noncompetitive mechanism. A closely related analogue, P32, exhibits selectivity and enhanced potency and engages PKMYT1 in cells. Surprisingly, however, it binds in the ATP binding pocket, demonstrating that subtle chemical modifications can shift binding mode and mechanism of inhibition. Furthermore, computational analysis using structural modeling methods, including AlphaFold2, AlphaFold3, Boltz-2, as well as unbiased MD simulations, indicates that these approaches are limited in their ability to capture this inhibitor-induced cryptic binding site and conformational change. Our study identifies an underexplored allosteric site in PKMYT1 and establishes a new avenue for the rational design of selective kinase inhibitors targeting a cryptic binding site in this emerging drug target. These findings also reveal intrinsic challenges in the computational discovery of noncanonical kinase binding sites and underscore the necessity of integrating computational modeling with experimental testing using structural and functional approaches.

Discovery of furan-trimethoxyphenyl hybrids as novel colchicine-binding site inhibitors that induce mitotic catastrophe with potent anti-acute myeloid leukemia effect.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer mortality worldwide. Tumors carrying activating epidermal growth factor receptor (EGFR) mutations initially respond to tyrosine kinase inhibitors (TKIs), with Osimertinib representing the current standard of care. However, acquired resistance inevitably develops, involving both genetic and non-genetic mechanisms, the latter playing a major role in sustaining cellular plasticity and promoting tumor aggressiveness. Here, we show that Osimertinib-resistant H1975 cells acquire a more aggressive phenotype than their parental (Par) counterparts, characterized by enhanced migratory behavior and transcriptional enrichment of BMI1 target genes, as well as mitotic defects and concomitant alterations in expression of mitotic cell-cycle pathways. Despite unchanged proliferation, resistant cells display increased mitotic activity and frequent cytokinetic defects, revealing a dependency on mitotic machinery and microtubule integrity, an Achilles' heel created by adaptive resistance. Functionally, BMI1 overexpression in Par cells recapitulates both resistance and enhanced migration, highlighting its central role in driving the resistant phenotype. Exploiting these vulnerabilities, Unesbulin (PTC596), a tubulin-binding agent with BMI1 inhibitory activity, triggers mitotic catastrophe, mechanistically induces apoptosis in vitro and drives regression of resistant xenografts in vivo. Our findings establish BMI1 as a key mediator of Osimertinib resistance and aggressiveness, uncovering a mutation-context-dependent mitotic vulnerability that can be therapeutically exploited, providing a rationale for targeting BMI1 and mitotic abnormalities to overcome resistance in T790M/L858R backgrounds.

Tetraploidy in cancer: Diagnostic and therapeutic perspectives.

ARID1A (AT-rich interaction domain 1A), a key subunit of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, is frequently mutated in cancers. However, effective clinical treatments for patients with this mutation are limited, highlighting a need for therapeutic strategies. Here, we identify Werner syndrome adenosine 5′-triphosphate–dependent helicase (WRN) as a critical vulnerability in ARID1A-mutated cancers. Upon genetic and pharmacological inhibition of WRN, ARID1A-mutated cells had defective checkpoint kinase 1 (Chk1)-mediated DNA damage signaling, resulting in compensatory checkpoint kinase 2 (Chk2) activation, leading to G1 phase arrest and apoptosis, whereas ARID1A-proficient cells underwent Chk1-dependent G2-M arrest. Additional p21 inhibition in the context of WRN suppression promoted cell cycle reentry of G1-arrested ARID1A-mutated cells, resulting in enhanced cytotoxicity through mitotic catastrophe. The antitumor efficacy of WRN inhibition alone and in combination with p21 inhibition was validated using cell line–based xenograft and patient-derived xenograft mouse models. Our findings define WRN as a selective therapeutic target in ARID1A-mutated cancers and suggest a combinatorial strategy of WRN and p21 inhibition as a therapeutic approach.

PKMYT1 is a Targetable Vulnerability in del(17p) High-Risk Multiple Myeloma.

PurposeGlioblastoma (GBM) is characterized by poor clinical outcomes and marked resistance to radiotherapy. Because effective radiosensitizing strategies for GBM remain limited, we investigated whether inhibition of KRAS/RAS signaling could enhance radiation response in GBM. In particular, we evaluated the radiosensitizing potential of RMC-6236, an RAS(ON) multiselective inhibitor that suppresses active RAS signaling across multiple RAS-dependent states.Experimental DesignHuman GBM cell lines (U251, LN-18, ACPK1, and OSU61) were treated with radiation, with or without genetic or pharmacological KRAS inhibition. KRAS signaling was suppressed by siRNA-mediated knockdown or RMC-6236 treatment. Radiation-induced KRAS activation and downstream MAPK signaling were assessed by Raf-RBD pull-down assays and immunoblotting. Radiosensitivity was evaluated using clonogenic survival assay. DNA damage persistence, cell cycle distribution, and mitotic catastrophe were analyzed by γH2AX immunofluorescence, flow cytometry, and nuclear morphology assessment, respectively. In vivo therapeutic efficacy was examined in an orthotopic U251 xenograft model.ResultsRadiation-induced transient activation and increased KRAS protein expression of KRAS, accompanied by activation of ERK, JNK, and p38 signaling in GBM cells. siKRAS suppressed radiation-induced KRAS and MAPK activation, and significantly enhanced radiosensitivity in all four GBM cell lines. Similarly, RMC-6236 inhibited radiation-induced KRAS activation and attenuated downstream MAPK signaling without reducing the total KRAS protein expression. RMC-6236 significantly increased the radiosensitivity across all GBM cell lines, with dose enhancement factors ranging from 1.33 1.46. Mechanistically, combined treatment with RMC-6236 and radiation increased persistent γH2AX foci and enhanced mitotic catastrophe without producing consistent redistribution of cells into radiosensitive cell cycle phases. In an orthotopic GBM model, the combination of RMC-6236 and radiation significantly prolonged survival compared to that of the control and radiation alone.ConclusionsThese findings indicate that radiation-induced KRAS signaling is a functionally important mediator of radioresistance in GBM and demonstrate that inhibition of KRAS/RAS signaling enhances the radiation responsein vitroandin vivo. RMC-6236 may represent a promising radiosensitizing strategy for GBM by suppressing adaptive RAS/MAPK signaling and promoting persistent DNA damage and mitotic catastrophe following irradiation. However, clinical trials of this combination are warranted.

Dual inhibition of PP2A and WEE1 induces apoptosis and mitotic catastrophe in cancer cells.

RNA polymerase I inhibitor CX-5461 suppresses cervical cancer cell growth by inducing DNA damage and mitotic catastrophe and enhances cisplatin sensitivity.

Cervical cancers remain a significant health burden. Limitations on cervical cancer chemotherapeutic intervention caused by toxic side effects are a persistent barrier to care. In this study, we show that the human papillomavirus oncogenes that cause most cervical cancers also increase the levels of a cancer-associated isoform of proliferating cell nuclear antigen (PCNA) known as caPCNA. The abundance of caPCNA is specifically elevated in cervical cancer. Similar to observations in other cancers, we found that a small-molecule inhibitor of caPCNA (AOH1996) selectively killed cell line, organoid, and xenograft models of cervical cancer. Our subsequent molecular analysis identified a novel ability of AOH1996 to induce cell death by disrupting the interaction between PCNA and γ-tubulin, resulting in mitotic arrest. We show AOH1996 selectively induces mitotic death in transformed cells, because these cells attempt to progress through mitosis, rather than decondensing their chromosomes and reforming their nuclear membranes like untransformed control cells. Furthermore, we show that these differences allow AOH1996 to specifically sensitize cervical cancer cells to cisplatin, a frontline chemotherapeutic used to treat cervical cancer. We found that subtherapeutic doses of AOH1996 and cisplatin could reduce cervical cancer xenograft growth and improve survival, similarly to a therapeutic dose of cisplatin without the cisplatin-induced toxicity that restricts care. To our knowledge, this study provides the first evidence that AOH1996 can function as a cisplatin-sensitizing agent in cervical cancer models.Significance:We identify a novel mechanism by which the small-molecule inhibitor AOH1996 targets cancer-associated PCNA to induce mitotic death in cervical cancer cells. By disrupting PCNA:γ-tubulin interactions, AOH1996 selectively sensitizes tumors to a lower dose of cisplatin, enabling effective therapy with reduced toxicity and suggesting a potential strategy to reduce treatment-associated toxicity.

High-altitude hypoxia (HAH) can cause adverse reactions, such as tinnitus and barotrauma, but the role of mitotic catastrophe (MC) in HAH remains unreported. This study investigated MC-associated biomarkers in HAH. HAH-related datasets and MC-related genes (MC-RGs) were retrieved from public databases. Differentially expressed genes (DEGs) between the HAH and control groups were screened, and overlapping genes between DEGs and MC-RGs were defined as candidates. Biomarkers were identified using three machine learning methods, receiver operating characteristic curve analysis, and expression validation. A nomogram was constructed for diagnostic assessment. Functional enrichment, immune infiltration, regulatory network, and drug prediction analyses were performed, with biomarker expression verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). DIAPH1 and MDM4 were confirmed as MC-associated biomarkers in HAH, and the nomogram displayed excellent diagnostic ability (area under the curve = 0.929). These biomarkers were coenriched in pathways such as oxidative phosphorylation. DIAPH1 was positively correlated with the infiltration of resting NK cells, whereas MDM4 was negatively correlated with that of M2 macrophages. A transcription factor-mRNA-miRNA network was established, with USF2 and GATA2 jointly targeting both biomarkers. Drugs such as rescinnamine targeted MDM4. RT-qPCR verified that USF2 and GATA2 were significantly downregulated in HAH (both P < 0.01), consistent with the bioinformatics results. DIAPH1 and MDM4 are valid MC-associated biomarkers for HAH. This study provides novel insights into HAH prevention and treatment.

Lung adenocarcinoma (LUAD) exhibits significant molecular and immune heterogeneity, limiting the prognostic and therapeutic value of single biomarkers. Mitotic catastrophe (MC) is a key biological axis, but externally validated MC-related gene (MCRG) signatures integrating immune and pharmacologic annotations remain lacking. We obtained datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differentially expressed genes overlapping with mitotic catastrophe-related genes (MCRGs) were screened. GSVA-derived MCRG scores guided WGCNA to identify associated modules; after KEGG/GO-BP enrichment and mutation analysis, a six-gene RiskScore model was constructed via LASSO-Cox and multivariate Cox regression. Immune microenvironment was analyzed by ESTIMATE and MCPcounter; ICI responsiveness by TIDE; drug sensitivity by pRRophetic. Wet-lab validation included qPCR, CCK-8, and siRNA-mediated GNPNAT1 knockdown assays. A total of 2,290 upregulated and 1,959 downregulated genes were identified, with 266 overlapping MCRGs. The turquoise module showed the highest correlation with MCRG scores, and 940 overlapping genes were enriched in pathways like p53 signaling. The six-gene model (GNPNAT1, etc.) effectively stratified OS, PFI, and DSS in TCGA and was validated in GSE31210. High-risk tumors had lower immune scores, depleted key immune lineages, poor ICI response, and correlated with various drug IC50 values. Wet-lab assays confirmed abnormal expression of target genes in LUAD cells, and GNPNAT1 silencing inhibited proliferation, migration, and invasion. The MC-related molecular axis integrates tumor proliferation-metabolism-immunity networks, providing actionable guidance for perioperative and translational therapeutic stratification. We proposed an MC-anchored six-gene signature integrating prognostic, immune, and therapeutic dimensions in LUAD. This exploratory study has biological plausibility, but prospective multi-center studies are required before clinical application.

VIC-1911 (formerly TAS-119) is a next-generation, ATP-competitive aurora kinase A (AURKA) inhibitor with a favorable biosafety profile. However, it has not been evaluated in prostate cancer (PCa), wherein AURKA is highly expressed in advanced stages and represents a critical therapeutic target. Here, we demonstrate that VIC-1911 potently inhibits AURKA activity with high selectivity over AURKB/C across diverse PCa cell lines. Treatment with VIC-1911, even at nanomolar concentrations, substantially inhibits the growth of both androgen receptor–positive (AR-positive) and AR-negative PCa cells. VIC-1911 triggers mitotic failure, induces DNA double-strand breaks (DSBs), and activates the p53 pathway, halting cell division and inducing cell death. Notably, VIC-1911 showed synergistic effects in inhibiting PCa cell growth in vitro and xenograft tumor growth in vivo with poly (ADP-ribose) polymerase inhibitors, which have proven effective in PCa with a deficiency in homologous recombination (HR) repair. Mechanistically, VIC-1911 disabled HR-mediated repair of DSBs in otherwise HR-proficient PCa cells, leading to a “BRCAness” phenotype and pronounced accumulation of DNA damage and mitotic catastrophe. In summary, our study uncovers what we believe is a novel mechanism to induce functional BRCAness through mitotic arrest and highlights VIC-1911 as a promising therapeutic agent for advanced PCa, either as a single agent or in combination, sensitizing HR-proficient tumors to PARP inhibitors.

ATRX Knockdown Enhances Irradiation-Induced Mitotic Catastrophe in p53-Deficient Cancer Cells.

Chemotherapy-induced alopecia (CIA) remains one of the most distressing adverse effects of cancer therapy. Yet, no therapy is available to selectively protect healthy hair follicles (HFs) and their epithelial stem cells (eHFSCs) from chemotherapy-induced damage without awarding potential survival benefits to cancer cells. Here, we report how human HFs can be protected against 2 lead CIA-inducing chemotherapeutics by inducing selective transient cell cycle arrest. Pretreating scalp HFs before chemotherapy exposure ex vivo with ALRN-6924, a clinical-stage "stapled peptide" drug that binds with high affinity to key endogenous inhibitors of p53, selectively activated p53 signaling only in cells with wild-type TP53 genotype and upregulated p21. This led to temporary cell cycle arrest in healthy tissues without protecting TP53-mutant cancer cells and mitigated chemotherapy-induced HF damage on multiple levels, including excessive hair matrix apoptosis, premature catagen, pigmentary abnormalities, "mitotic catastrophe," and micronucleation. It also protected eHFSCs against DNA damage, apoptosis, and pathological epithelial-mesenchymal transition. Notably, even topically applied ALRN-6924 afforded relative chemotherapy protection ex vivo. These results provide proof of principle for a strategy to selectively protect rapidly proliferating healthy epithelial tissues and their stem cells in patients with TP53-mutant cancers, which promises to protect against acute and permanent CIA.

Migrasome-mediated clearance of excess PLK4 defines a targetable vulnerability.

ADP-ribosylation is a versatile post-translational modification that governs fundamental processes, including DNA repair, transcription, and stress adaptation. Its homeostasis relies on the dynamic interplay between poly(ADP-ribose) polymerases (PARPs), which assemble mono- or poly-ADP-ribose (PAR) chains on target macromolecules, and ADP-ribosyl hydrolases, which dismantle them. Disruption of this balance leads to the accumulation of toxic PAR and cell death, revealing vulnerabilities that can be therapeutically exploited. PARP inhibitors (PARPis) have revolutionised the treatment of homologous recombination-deficient cancers via synthetic lethality. Yet, emerging resistance limits their long-term efficacy, underscoring the need for novel targets within ADP-ribose signalling. The poly(ADP-ribose) glycohydrolase (PARG), the principal enzyme involved in hydrolysing PAR, has emerged as a compelling candidate: its inhibition amplifies replication stress, drives mitotic catastrophe, and selectively kills cancer cells, particularly those reliant on PAR turnover for survival. Elevated PARG expression correlates with aggressive tumours and poor prognosis, positioning it as both a prognostic biomarker and therapeutic target. This review integrates recent structural and biochemical insights into PARG, highlighting the mechanisms of PAR reversal, regulatory control, and potential synthetic lethal interactions. We also discuss the discovery and development of selective PARG inhibitors, which promise to expand the therapeutic landscape, overcome PARPis resistance, and exploit vulnerabilities in replication-stressed cancers. By bridging mechanistic understanding with translational potential, targeting PARG represents a frontier in precision cancer therapy.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal malignancies, with limited treatment options and poor clinical outcomes. KU-57788, a selective inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has shown promise in cancer therapy when combined with radiotherapy and chemotherapy. However, its therapeutic potential and underlying mechanisms in ATC remain unclear. In this study, we demonstrate that KU-57788 exerts potent anti-ATC activity both in vitro and in vivo by inducing DNA damage and triggering mitotic catastrophe. Unexpectedly, we identify a novel mechanism whereby KU-57788 directly binds to and activates dynamin-related protein 1 (DRP1), leading to excessive mitochondrial fission and fragmentation. This process is accompanied by the protective activation of the NRF2/SLC7A11/GSH axis, which mitigates the cytotoxic effects of KU-57788. Notably, pharmacological induction of ferroptosis or cystine depletion effectively synergizes with ATC cells to KU-57788, overcoming resistance and promoting ferroptosis. Collectively, our findings highlight the therapeutic potential of KU-57788 in ATC while revealing an intrinsic resistance mechanism mediated by DRP1 activation and the potential involvement of the NRF2/SLC7A11/GSH axis. More importantly, we provide strong evidence that combining KU-57788 with ferroptosis inducers significantly enhances its anticancer efficacy, offering a promising therapeutic strategy for ATC.