Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents.

Aneuploidy is a condition in which cells have an abnormal number of chromosomes that is not a multiple of the haploid complement. It is known that aneuploidy has detrimental consequences on cell physiology, such as genome instability, metabolic and proteotoxic stress and decreased cellular fitness. Importantly, aneuploidy is a hallmark of tumors and it is associated with resistance to chemotherapeutic agents and poor clinical outcome. To shed light into how aneuploidy contributes to chemoresistance, we induced chromosome mis-segregation in human cancer cell lines, then treated them with several chemotherapeutic agents and evaluated the emergence of chemoresistance. By doing so, we found that elevation of chromosome mis-segregation promotes resistance to chemotherapeutic agents through the expansion of aneuploid karyotypes and subsequent selection of specific aneuploidies essential for cellular viability under those stressful conditions. Here, we describe a method to generate aneuploid cell populations and to evaluate their resistance to anti-cancer agents. This protocol has been already successfully employed and can be further utilized to accelerate the exploration of the role of aneuploidy in chemoresistance.

KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Short-term molecular consequences of chromosome mis-segregation for genome stability.

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

Profilin 1 deficiency drives mitotic defects and reduces genome stability.

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

Regulation of protein complex partners as a compensatory mechanism in aneuploid tumors.

Aneuploidy, a state of chromosome imbalance, is a hallmark of human tumors, but its role in cancer still remains to be fully elucidated. To understand the consequences of whole-chromosome-level aneuploidies on the proteome, we integrated aneuploidy, transcriptomic, and proteomic data from hundreds of The Cancer Genome Atlas/Clinical Proteomic Tumor Analysis Consortium tumor samples. We found a surprisingly large number of expression changes happened on other, non-aneuploid chromosomes. Moreover, we identified an association between those changes and co-complex members of proteins from aneuploid chromosomes. This co-abundance association is tightly regulated for aggregation-prone aneuploid proteins and those involved in a smaller number of complexes. On the other hand, we observed that complexes of the cellular core machinery are under functional selection to maintain their stoichiometric balance in aneuploid tumors. Ultimately, we provide evidence that those compensatory and functional maintenance mechanisms are established through post-translational control, and that the degree of success of a tumor to deal with aneuploidy-induced stoichiometric imbalance impacts the activation of cellular protein degradation programs and patient survival.

The Dynamic Instability of the Aneuploid Genome.

Proper partitioning of replicated sister chromatids at each mitosis is crucial for maintaining cell homeostasis. Errors in this process lead to aneuploidy, a condition in which daughter cells harbor genome imbalances. Importantly, aneuploid cells often experience DNA damage, which in turn could drive genome instability. This might be the product of DNA damage accumulation in micronuclei and/or a consequence of aneuploidy-induced replication stress in S-phase. Although high levels of genome instability are associated with cell cycle arrest, they can also confer a proliferative advantage in some circumstances and fuel tumor growth. Here, we review the main consequences of chromosome segregation errors on genome stability, with a special focus on the bidirectional relationship between aneuploidy and DNA damage. Also, we discuss recent findings showing how increased genome instability can provide a proliferation improvement under specific conditions, including chemotherapeutic treatments.

Biomedical omics: first insights of a new MSc degree of the University of Milan.

The advent of technologies allowing the global analysis of biological phenomena, referred to as "omics" (genomics, epigenomics, proteomics, metabolomics, microbiomics, radiomics, and radiogenomics), has revolutionized the study of human diseases and traced the path for quantitative personalized medicine. The newly inaugurated Master of Science Program in Biomedical Omics of the University of Milan, Italy, aims at addressing the unmet need to create professionals with a broad understanding of omics disciplines. The course is structured over 2 years and admits students with a bachelor's degree in biotechnology, biology, chemistry, or pharmaceutical sciences. All teaching activities are fully held in English. A total of nine students enrolled in the first academic year and attended the courses of radiomics, genomics and epigenomics, proteomics, and high-throughput screenings, and their feedback was evaluated by means of an online questionnaire. Faculty with different backgrounds were recruited according to the subject. Due to restrictions imposed by the coronavirus disease 2019 (COVID-19) pandemic, laboratory activities were temporarily suspended, while lectures, journal clubs, and examinations were mainly held online. After the end of the first semester, despite the difficulties brought on by the COVID-19 pandemic, the course overall met the expectations of the students, specifically regarding teaching effectiveness, interpersonal interactions with the lecturers, and courses organization. Future efforts will be undertaken to better calibrate the overall workload of the course and to implement the most relevant suggestions from the students together with omics science evolution in order to guarantee state-of-the-art omics teaching and to prepare future omics specialists.

Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy.

Mitotic errors lead to aneuploidy, a condition of karyotype imbalance, frequently found in cancer cells. Alterations in chromosome copy number induce a wide variety of cellular stresses, including genome instability. Here, we show that cancer cells might exploit aneuploidy-induced genome instability and the resulting gene copy-number changes to survive under conditions of selective pressure, such as chemotherapy. Resistance to chemotherapeutic drugs was dictated by the acquisition of recurrent karyotypes, indicating that gene dosage might play a role in driving chemoresistance. Thus, our study establishes a causal link between aneuploidy-driven changes in gene copy number and chemoresistance and might explain why some chemotherapies fail to succeed.

Aneuploid senescent cells activate NF-κB to promote their immune clearance by NK cells.

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non-cell-autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF-κB signaling upregulation is central to elicit this immune response. Inactivating NF-κB abolishes NK cell-mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF-κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell-mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF-κB-mediated immunogenicity.

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition.

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

Understanding Complexity of Cancer Genomes: Lessons from Errors.

Deconstructing the events leading to cancer genome rearrangements is key to understanding tumorigenesis. In a recent issue of Science, Umbreit, Zhang et al. elegantly show that complex genome evolution is the result of a cascade of events initiated by a single error during cell division.

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploid Cell Survival Relies upon Sphingolipid Homeostasis.

Aneuploidy, a hallmark of cancer cells, poses an appealing opportunity for cancer treatment and prevention strategies. Using a cell-based screen to identify small molecules that could selectively kill aneuploid cells, we identified the compound N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide monohydrochloride (DL-PDMP), an antagonist of UDP-glucose ceramide glucosyltransferase. DL-PDMP selectively inhibited proliferation of aneuploid primary mouse embryonic fibroblasts and aneuploid colorectal cancer cells. Its selective cytotoxic effects were based on further accentuating the elevated levels of ceramide, which characterize aneuploid cells, leading to increased apoptosis. We observed that DL-PDMP could also enhance the cytotoxic effects of paclitaxel, a standard-of-care chemotherapeutic agent that causes aneuploidy, in human colon cancer and mouse lymphoma cells. Our results offer pharmacologic evidence that the aneuploid state in cancer cells can be targeted selectively for therapeutic purposes, or for reducing the toxicity of taxane-based drug regimens. Cancer Res; 77(19); 5272-86. ©2017 AACR.

Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.

Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways that limit the prevalence of such cells exist? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome mis-segregation leads to further genomic instability that ultimately causes cell-cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance.

The pleiotropic deubiquitinase Ubp3 confers aneuploidy tolerance.

Aneuploidy-or an unbalanced karyotype in which whole chromosomes are gained or lost-causes reduced fitness at both the cellular and organismal levels but is also a hallmark of human cancers. Aneuploidy causes a variety of cellular stresses, including genomic instability, proteotoxic and oxidative stresses, and impaired protein trafficking. The deubiquitinase Ubp3, which was identified by a genome-wide screen for gene deletions that impair the fitness of aneuploid yeast, is a key regulator of aneuploid cell homeostasis. We show that deletion of UBP3 exacerbates both karyotype-specific phenotypes and global stresses of aneuploid cells, including oxidative and proteotoxic stress. Indeed, Ubp3 is essential for proper proteasome function in euploid cells, and deletion of this deubiquitinase leads to further proteasome-mediated proteotoxicity in aneuploid yeast. Notably, the importance of UBP3 in aneuploid cells is conserved. Depletion of the human homolog of UBP3, USP10, is detrimental to the fitness of human cells upon chromosome missegregation, and this fitness defect is accompanied by autophagy inhibition. We thus used a genome-wide screen in yeast to identify a guardian of aneuploid cell fitness conserved across species. We propose that interfering with Ubp3/USP10 function could be a productive avenue in the development of novel cancer therapeutics.

A monoclonal antibody specific for prophase phosphorylation of histone deacetylase 1: a readout for early mitotic cells.

Histone deacetylases (HDACs) are modification enzymes that regulate a plethora of biological processes. HDAC1, a crucial epigenetic modifier, is deregulated in cancer and subjected to a variety of post-translational modifications. Here, we describe the generation of a new monoclonal antibody that specifically recognizes a novel highly dynamic prophase phosphorylation of serine 406-HDAC1, providing a powerful tool for detecting early mitotic cells.

Aneuploidy triggers a TFEB-mediated lysosomal stress response.

Aneuploidy, defined as an alteration in chromosome number that is not a multiple of the haploid complement, severely affects cellular physiology. Changes in chromosome number lead to imbalances in cellular protein composition, thus disrupting cellular processes and causing proteins to misfold and aggregate. We recently reported that in mammalian cells protein aggregates are readily encapsulated within autophagosomes but are not degraded by lysosomes. This leads to a lysosomal stress response in which the transcription factor TFEB induces expression of factors needed for macroautophagy-mediated protein degradation. Our studies uncover lysosomal degradation defects as a feature of the aneuploid state, and a role for the transcription factor TFEB in the response thereto.

Aneuploidy-induced cellular stresses limit autophagic degradation.

An unbalanced karyotype, a condition known as aneuploidy, has a profound impact on cellular physiology and is a hallmark of cancer. Aneuploid cells experience a number of stresses that are caused by aneuploidy-induced proteomic changes. How the aneuploidy-associated stresses affect cells and whether cells respond to them are only beginning to be understood. Here we show that autophagosomal cargo such as protein aggregates accumulate within lysosomes in aneuploid cells. This causes a lysosomal stress response. Aneuploid cells activate the transcription factor TFEB, a master regulator of autophagic and lysosomal gene expression, thereby increasing the expression of genes needed for autophagy-mediated protein degradation. Accumulation of autophagic cargo within the lysosome and activation of TFEB-responsive genes are also observed in cells in which proteasome function is inhibited, suggesting that proteotoxic stress causes TFEB activation. Our results reveal a TFEB-mediated lysosomal stress response as a universal feature of the aneuploid state.

Short- and long-term effects of chromosome mis-segregation and aneuploidy.

Dividing cells that experience chromosome mis-segregation generate aneuploid daughter cells, which contain an incorrect number of chromosomes. Although aneuploidy interferes with the proliferation of untransformed cells, it is also, paradoxically, a hallmark of cancer, a disease defined by increased proliferative potential. These contradictory effects are also observed in mouse models of chromosome instability (CIN). CIN can inhibit and promote tumorigenesis. Recent work has provided insights into the cellular consequences of CIN and aneuploidy. Chromosome mis-segregation per se can alter the genome in many more ways than just causing the gain or loss of chromosomes. The short- and long-term effects of aneuploidy are caused by gene-specific effects and a stereotypic aneuploidy stress response. Importantly, these recent findings provide insights into the role of aneuploidy in tumorigenesis.

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B.

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.