Restriction beyond the restriction point: mitogen requirement for G(2 )passage

Cell proliferation is dependent on mitogenic signalling. When absent, normal cells cannot pass the G(1 )restriction point, resulting in cell cycle arrest. Passage through the G(1 )restriction point involves inactivation of the retinoblastoma protein family. Consequently, loss of the retinoblastoma protein family leads to loss of the G(1 )restriction point. Recent work in our lab has revealed that cells possess yet another mechanism that restricts proliferation in the absence of mitogens: arrest in the G(2 )phase of the cell cycle. Here, we discuss the similarities and differences between these restriction points and the roles of cyclin-dependent kinase inhibitors (CKIs) herein

Exploiting aneuploidy-imposed stresses and coping mechanisms to battle cancer

Aneuploidy, an irregular number of chromosomes in cells, is a hallmark feature of cancer. Aneuploidy results from chromosomal instability (CIN) and occurs in almost 90% of all tumours. While many cancers display an ongoing CIN phenotype, cells can also be aneuploid without displaying CIN. CIN drives tumour evolution as ongoing chromosomal missegregation will yield a progeny of cells with variable aneuploid karyotypes. The resulting aneuploidy is initially toxic to cells because it leads to proteotoxic and metabolic stress, cell cycle arrest, cell death, immune cell activation and further genomic instability. In order to overcome these aneuploidy-imposed stresses and adopt a malignant fate, aneuploid cancer cells must develop aneuploidy-tolerating mechanisms to cope with CIN. Aneuploidy-coping mechanisms can thus be considered as promising therapeutic targets. However, before such therapies can make it into the clinic, we first need to better understand the molecular mechanisms that are activated upon aneuploidization and the coping mechanisms that are selected for in aneuploid cancer cells. In this review, we discuss the key biological responses to aneuploidization, some of the recently uncovered aneuploidy-coping mechanisms and some strategies to exploit these in cancer therapy

Understanding How Genetic Mutations Collaborate with Genomic Instability in Cancer

Chromosomal instability is the process of mis-segregation for ongoing chromosomes, which leads to cells with an abnormal number of chromosomes, also known as an aneuploid state. Induced aneuploidy is detrimental during development and in primary cells but aneuploidy is also a hallmark of cancer cells. It is therefore believed that premalignant cells need to overcome aneuploidy-imposed stresses to become tumorigenic. Over the past decade, some aneuploidy-tolerating pathways have been identified through small-scale screens, which suggest that aneuploidy tolerance pathways can potentially be therapeutically exploited. However, to better understand the processes that lead to aneuploidy tolerance in cancer cells, large-scale and unbiased genetic screens are needed, both in euploid and aneuploid cancer models. In this review, we describe some of the currently known aneuploidy-tolerating hits, how large-scale genome-wide screens can broaden our knowledge on aneuploidy specific cancer driver genes, and how we can exploit the outcomes of these screens to improve future cancer therapy

The cGAS Paradox:Contrasting Roles for cGAS-STING Pathway in Chromosomal Instability

Chromosomal instability (CIN) is an intricate phenomenon that is often found in human cancer, characterized by persisting errors in chromosome segregation. This ongoing chromosome mis-segregation results in structural and numerical chromosomal abnormalities that have been widely described to promote tumor evolution. In addition to being a driver of tumor evolution, recent evidence demonstrates CIN to be the central node of the crosstalk between a tumor and its surrounding microenvironment, as mediated by the cGAS-STING pathway. The role that cGAS-STING signaling exerts on CIN tumors is both complex and paradoxical. On one hand, the cGAS-STING axis promotes the clearance of CIN tumors through recruitment of immune cells, thus suppressing tumor progression. On the other hand, the cGAS-STING pathway has been described to be the major regulator in the promotion of metastasis of CIN tumors. Here, we review this dual role of the cGAS-STING pathway in the context of chromosomal instability and discuss the potential therapeutic implications of cGAS-STING signaling for targeting CIN tumors

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.</p

Does Aneuploidy in the Brain Play a Role in Neurodegenerative Disease?

Aneuploidy, a state in which cells exhibit copy number changes of (parts of) chromosomes, is a hallmark of cancer cells and, when present in all cells, leads to miscarriages and congenital disorders, such as Down syndrome. In addition to these well-known roles of aneuploidy, chromosome copy number changes have also been reported in some studies to occur in neurons in healthy human brain and possibly even more in Alzheimer’s disease (AD). However, the studies of aneuploidy in the human brain are currently under debate as earlier findings, mostly based on in situ hybridization approaches, could not be reproduced by more recent single cell sequencing studies with a much higher resolution. Here, we review the various studies on the occurrence of aneuploidy in brain cells from normal individuals and Alzheimer’s patients. We discuss possible mechanisms for the origin of aneuploidy and the pros and cons of different techniques used to study aneuploidy in the brain, and we provide a future perspective

Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-gamma, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCR alpha beta) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naive T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers

Deletion of the MAD2L1 spindle assembly checkpoint gene is tolerated in mouse models of acute T-cell lymphoma and hepatocellular carcinoma.

Chromosome instability (CIN) is deleterious to normal cells because of the burden of aneuploidy. However, most human solid tumors have an abnormal karyotype implying that gain and loss of chromosomes by cancer cells confers a selective advantage. CIN can be induced in the mouse by inactivating the spindle assembly checkpoint. This is lethal in the germline but we show here that adult T cells and hepatocytes can survive conditional inactivation of the Mad2l1 SAC gene and resulting CIN. This causes rapid onset of acute lymphoblastic leukemia (T-ALL) and progressive development of hepatocellular carcinoma (HCC), both lethal diseases. The resulting DNA copy number variation and patterns of chromosome loss and gain are tumor-type specific, suggesting differential selective pressures on the two tumor cell types