Centrosome amplification primes ovarian cancer cells for apoptosis and potentiates the response to chemotherapy.

Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here, we show that centrosome amplification potentiates responses to conventional chemotherapy in addition to its effect on multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death can occur independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved treatment responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature.

CDK4 is co-amplified with either TP53 promoter gene fusions or MDM2 through distinct mechanisms in osteosarcoma.

Amplification of the MDM2 and CDK4 genes on chromosome 12 is commonly associated with low-grade osteosarcomas. In this study, we conducted high-resolution genomic and transcriptomic analyses on 33 samples from 25 osteosarcomas, encompassing both high- and low-grade cases with MDM2 and/or CDK4 amplification. We discerned four major subgroups, ranging from nearly intact genomes to heavily rearranged ones, each harbouring CDK4 and MDM2 amplification or CDK4 amplification with TP53 structural alterations. While amplicons involving MDM2 exhibited signs of an initial chromothripsis event, no evidence of chromothripsis was found in TP53-rearranged cases. Instead, the initial disruption of the TP53 locus led to co-amplification of the CDK4 locus. Additionally, we observed recurring promoter swapping events involving the regulatory regions of the FRS2, PLEKHA5, and TP53 genes. These events resulted in ectopic expression of partner genes, with the ELF1 gene being upregulated by the FRS2 and TP53 promoter regions in two distinct cases.

Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study.

Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. A pathologic response (primary objective) was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. A partial radiologic response (secondary objective) was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on magnetic resonance imaging. All patients completed treatment without severe toxicity (exploratory objective). At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. In-depth analysis of the loco-regional and systemic immune response (predefined exploratory objective) showed that monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints, efficacy in terms of pathologic response as primary endpoint, radiologic response as secondary outcome and safety and tolerability as exploratory endpoint, were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment.

Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.

Single-nucleus multi-omic profiling of polyploid heart nuclei identifies fusion-derived cardiomyocytes in the human heart.

Understanding the mechanisms of polyploidization in cardiomyocytes is crucial for advancing strategies to stimulate myocardial regeneration. Although endoreplication has long been considered the primary source of polyploid human cardiomyocytes, recent animal work suggests the potential for cardiomyocyte fusion. Moreover, the effects of polyploidization on the genomic-transcriptomic repertoire of human cardiomyocytes have not been studied previously. We applied single-nuclei whole genome sequencing, single nuclei RNA sequencing, and multiome ATAC + gene expression (from the same nuclei) techniques to nuclei isolated from 11 healthy hearts. Utilizing post-zygotic non-inherited somatic mutations occurring during development as "endogenous barcodes," to reconstruct lineage relationships of polyploid cardiomyocytes. Of 482 cardiomyocytes from multiple healthy donor hearts 75.7% can be sorted into several developmental clades marked by one or more somatic single-nucleotide variants (SNVs). At least ~10% of tetraploid cardiomyocytes contain cells from distinct clades, indicating fusion of lineally distinct cells, whereas 60% of higher-ploidy cardiomyocytes contain fused cells from distinct clades. Combined snRNA-seq and snATAC-seq revealed transcriptome and chromatin landscapes of polyploid cardiomyocytes distinct from diploid cardiomyocytes, and show some higher-ploidy cardiomyocytes with transcriptional signatures suggesting fusion between cardiomyocytes and endothelial and fibroblast cells. These observations provide the first evidence for cell and nuclear fusion of human cardiomyocytes, raising the possibility that cell fusion may contribute to developing or maintaining polyploid cardiomyocytes in the human heart.

MDM2 amplification in rod-shaped chromosomes provides clues to early stages of circularized gene amplification in liposarcoma.

Well-differentiated liposarcoma (WDLS) displays amplification of genes on chromosome 12 (Chr12) in supernumerary ring or giant marker chromosomes. These structures have been suggested to develop through chromothripsis, followed by circularization and breakage-fusion-bridge (BFB) cycles. To test this hypothesis, we compared WDLSs with Chr12 amplification in rod-shaped chromosomes with WDLSs with rings. Both types of amplicons share the same spectrum of structural variants (SVs), show higher SV frequencies in Chr12 than in co-amplified segments, have SVs that fuse the telomeric ends of co-amplified chromosomes, and lack interspersed deletions. Combined with the finding of cells with transient rod-shaped structures in tumors with ring chromosomes, this suggests a stepwise process starting with the gain of Chr12 material that, after remodeling which does not fit with classical chromothripsis, forms a dicentric structure with other chromosomes. Depending on if and when telomeres from other chromosomes are captured, circularized or linear gain of 12q sequences will predominate.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Extra centrosomes delay DNA damage-driven tumorigenesis.

Deregulated centrosome numbers are frequently found in human cancer and can promote malignancies in model organisms. Current research aims to clarify if extra centrosomes are cause or consequence of malignant transformation, and if their biogenesis can be targeted for therapy. Here, we show that oncogene-driven blood cancer is inert to genetic manipulation of centrosome numbers, whereas the formation of DNA damage-induced malignancies is delayed. We provide first evidence that this unexpected phenomenon is connected to extra centrosomes eliciting a pro-death signal engaging the apoptotic machinery. Apoptosis induction requires the PIDDosome multi-protein complex, as it can be abrogated by loss of any of its three components, Caspase-2, Raidd/Cradd, or Pidd1. BCL2 overexpression equally blocks cell death, documenting for the first time induction of mitochondrial apoptosis downstream of extra centrosomes. Our findings demonstrate context-dependent effects of centrosome amplification during transformation and ask to adjust current belief that extra centrosomes are intrinsically pro-tumorigenic.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

Drug-resilient Cancer Cell Phenotype Is Acquired via Polyploidization Associated with Early Stress Response Coupled to HIF2α Transcriptional Regulation.

Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole-genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF2α. We found altered chromatin accessibility, ablated expression of retinoblastoma protein (RB1), and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF2α in stress response by polyploidy suggests a novel avenue for tackling chemotherapy-induced resistance in cancer. SIGNIFICANCE: In response to cisplatin treatment, some surviving cancer cells undergo whole-genome duplications without mitosis, which represents a mechanism of drug resistance. This study presents mechanistic data to implicate AP-1 and HIF2α signaling in the formation of this surviving cell phenotype. The results open a new avenue for targeting drug-resistant cells.

Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition.

The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.

Chromosomal instability in aneuploid acute lymphoblastic leukemia associates with disease progression.

Chromosomal instability (CIN) lies at the core of cancer development leading to aneuploidy, chromosomal copy-number heterogeneity (chr-CNH) and ultimately, unfavorable clinical outcomes. Despite its ubiquity in cancer, the presence of CIN in childhood B-cell acute lymphoblastic leukemia (cB-ALL), the most frequent pediatric cancer showing high frequencies of aneuploidy, remains unknown. Here, we elucidate the presence of CIN in aneuploid cB-ALL subtypes using single-cell whole-genome sequencing of primary cB-ALL samples and by generating and functionally characterizing patient-derived xenograft models (cB-ALL-PDX). We report higher rates of CIN across aneuploid than in euploid cB-ALL that strongly correlate with intraclonal chr-CNH and overall survival in mice. This association was further supported by in silico mathematical modeling. Moreover, mass-spectrometry analyses of cB-ALL-PDX revealed a "CIN signature" enriched in mitotic-spindle regulatory pathways, which was confirmed by RNA-sequencing of a large cohort of cB-ALL samples. The link between the presence of CIN in aneuploid cB-ALL and disease progression opens new possibilities for patient stratification and offers a promising new avenue as a therapeutic target in cB-ALL treatment.

Characterizing chromosomal instability-driven cancer evolution and cell fitness at a glance.

Chromosomal instability (CIN), an increased rate of chromosome segregation errors during mitosis, is a hallmark of cancer cells. CIN leads to karyotype differences between cells and thus large-scale heterogeneity among individual cancer cells; therefore, it plays an important role in cancer evolution. Studying CIN and its consequences is technically challenging, but various technologies have been developed to track karyotype dynamics during tumorigenesis, trace clonal lineages and link genomic changes to cancer phenotypes at single-cell resolution. These methods provide valuable insight not only into the role of CIN in cancer progression, but also into cancer cell fitness. In this Cell Science at a Glance article and the accompanying poster, we discuss the relationship between CIN, cancer cell fitness and evolution, and highlight techniques that can be used to study the relationship between these factors. To that end, we explore methods of assessing cancer cell fitness, particularly for chromosomally unstable cancer.

Disruption of the TP53 locus in osteosarcoma leads to TP53 promoter gene fusions and restoration of parts of the TP53 signalling pathway.

TP53 is the most frequently mutated gene in human cancer. This gene shows not only loss-of-function mutations but also recurrent missense mutations with gain-of-function activity. We have studied the primary bone malignancy osteosarcoma, which harbours one of the most rearranged genomes of all cancers. This is odd since it primarily affects children and adolescents who have not lived the long life thought necessary to accumulate massive numbers of mutations. In osteosarcoma, TP53 is often disrupted by structural variants. Here, we show through combined whole-genome and transcriptome analyses of 148 osteosarcomas that TP53 structural variants commonly result in loss of coding parts of the gene while simultaneously preserving and relocating the promoter region. The transferred TP53 promoter region is fused to genes previously implicated in cancer development. Paradoxically, these erroneously upregulated genes are significantly associated with the TP53 signalling pathway itself. This suggests that while the classical tumour suppressor activities of TP53 are lost, certain parts of the TP53 signalling pathway that are necessary for cancer cell survival and proliferation are retained. In line with this, our data suggest that transposition of the TP53 promoter is an early event that allows for a new normal state of genome-wide rearrangements in osteosarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

IFNγ protects motor neurons from oxidative stress via enhanced global protein synthesis in FUS-associated amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis type 6 (ALS6) is a familial subtype of ALS linked to Fused in Sarcoma (FUS) gene mutation. FUS mutations lead to decreased global protein synthesis, but the mechanism that drives this has not been established. Here, we used ALS6 patient-derived induced pluripotent stem cells (hIPSCs) to study the effect of the ALS6 FUSR521H mutation on the translation machinery in motor neurons (MNs). We find, in agreement with findings of others, that protein synthesis is decreased in FUSR521H MNs. Furthermore, FUSR521H MNs are more sensitive to oxidative stress and display reduced expression of TGF-ß and mTORC gene pathways when stressed. Finally, we show that IFNγ treatment reduces apoptosis of FUSR521H MNs exposed to oxidative stress and partially restores the translation rates in FUSR521H MNs. Overall, these findings suggest that a functional IFNγ response is important for FUS-mediated protein synthesis, possibly by FUS nuclear translocation in ALS6.

Chromosomal Instability-Driven Cancer Progression: Interplay with the Tumour Microenvironment and Therapeutic Strategies.

Chromosomal instability (CIN) is a prevalent characteristic of solid tumours and haematological malignancies. CIN results in an increased frequency of chromosome mis-segregation events, thus yielding numerical and structural copy number alterations, a state also known as aneuploidy. CIN is associated with increased chances of tumour recurrence, metastasis, and acquisition of resistance to therapeutic interventions, and this is a dismal prognosis. In this review, we delve into the interplay between CIN and cancer, with a focus on its impact on the tumour microenvironment-a driving force behind metastasis. We discuss the potential therapeutic avenues that have resulted from these insights and underscore their crucial role in shaping innovative strategies for cancer treatment.

Neurodegeneration-associated protein VAPB regulates proliferation in medulloblastoma.

VAMP (Vesicle-associated membrane protein)-associated protein B and C (VAPB) has been widely studied in neurodegenerative diseases such as ALS, but little is known about its role in cancer. Medulloblastoma is a common brain malignancy in children and arises from undifferentiated cells during neuronal development. Therefore, medulloblastoma is an interesting model to investigate the possible relationship between VAPB and tumorigenesis. Here we demonstrate that high VAPB expression in medulloblastoma correlates with decreased overall patient survival. Consistent with this clinical correlation, we find that VAPB is required for normal proliferation rates of medulloblastoma cells in vitro and in vivo. Knockout of VAPB (VAPBKO) delayed cell cycle progression. Furthermore, transcript levels of WNT-related proteins were decreased in the VAPBKO. We conclude that VAPB is required for proliferation of medulloblastoma cells, thus revealing VAPB as a potential therapeutic target for medulloblastoma treatment.

Taking the STING out of CIN.

Chromosomal instability (CIN), a hallmark of cancer, promotes cell-intrinsic inflammatory signaling. Although inflammation is generally considered tumor-suppressive, this relationship is more complex in cancers with CIN. We discuss new findings by Li et al. that can explain how cancer cells with CIN tolerate, adopt, and rewire the CIN-induced inflammatory response to fuel tumorigenesis.

Chromosomal instability and inflammation: a catch-22 for cancer cells.

Chromosomal instability (CIN), an increased rate of chromosomal segregation abnormalities, drives intratumor heterogeneity and affects most human cancers. In addition to chromosome copy number alterations, CIN results in chromosome(s) (fragments) being mislocalized into the cytoplasm in the form of micronuclei. Micronuclei can be detected by cGAS, a double-strand nucleic acid sensor, which will lead to the production of the second messenger 2'3'-cGAMP, activation of an inflammatory response, and downstream immune cell activation. However, the molecular network underlying the CIN-induced inflammatory response is still poorly understood. Furthermore, there is emerging evidence that cancers that display CIN circumvent this CIN-induced inflammatory response, and thus immune surveillance. The STAT1, STAT3, and NF-κB signaling cascades appear to play an important role in the CIN-induced inflammatory response. In this review, we discuss how these pathways are involved in signaling CIN in cells and how they are intertwined. A better understanding of how CIN is being signaled in cells and how cancer cells circumvent this is of the utmost importance for better and more selective cancer treatment.

Deletion of SERF2 in mice delays embryonic development and alters amyloid deposit structure in the brain.

In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation-prone and form amyloid-like deposits. Depletion of SERF proteins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full-body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-ß aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possibilities for polymorphism-based interventions.