H3K9me3-mediated epigenetic regulation of senescence in mice predicts outcome of lymphoma patients

Lesion-based targeting strategies underlie cancer precision medicine. However, biological principles - such as cellular senescence - remain difficult to implement in molecularly informed treatment decisions. Functional analyses in syngeneic mouse models and cross-species validation in patient datasets might uncover clinically relevant genetics of biological response programs. Here, we show that chemotherapy-exposed primary Eµ-myc transgenic lymphomas - with and without defined genetic lesions - recapitulate molecular signatures of patients with diffuse large B-cell lymphoma (DLBCL). Importantly, we interrogate the murine lymphoma capacity to senesce and its epigenetic control via the histone H3 lysine 9 (H3K9)-methyltransferase Suv(ar)39h1 and H3K9me3-active demethylases by loss- and gain-of-function genetics, and an unbiased clinical trial-like approach. A mouse-derived senescence-indicating gene signature, termed "SUVARness", as well as high-level H3K9me3 lymphoma expression, predict favorable DLBCL patient outcome. Our data support the use of functional genetics in transgenic mouse models to incorporate basic biology knowledge into cancer precision medicine in the clinic

Genotoxic stress-induced senescence

A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically and fundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscence staining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we would like to emphasize here the strength of viable fSA-β-gal to track single-cell fate in senescent populations over time

Detecting markers of therapy-induced senescence in cancer cells

Therapy-induced senescence (TIS), a lasting chemotherapy-evoked proliferative arrest of tumor cells, has gained increasing attention by cancer researchers because of its' profound biological implications, and by clinical oncologists due to its potential contribution to the long-term outcome of cancer patients post-treatment. Although both apoptosis and senescence represent therapy-inducible, ultimate cell-cycle exit programs, mediated via DNA damage response signaling, apoptotic cell death as the faster and often quantitatively more prominent tumor response has been in the scientific focus for decades. The more recently recognized TIS as another "safeguard" response of cancer cells that were never primed for or failed to execute apoptosis, not only reflects a more complex "arrest-plus-other features" cell-autonomous condition but produces non-cell-autonomous phenotypes at the tumor site, collectively impinging on tumor control and clinical outcome. Hence, TIS research is gaining pivotal interest from both a tumor biological and a therapeutic perspective, and the development of non-DNA damaging, senescence-evoking therapeutics is about to become a major research objective. In this chapter, we describe a well-characterized, genetically controlled TIS model system based on primary BCL2-expressing Emu-myc transgenic lymphoma cells harboring defined genetic lesions and provide protocols for co-staining of either senescence-associated beta-galactosidase (SA-beta-gal) activity or trimethylated lysine 9 of histone H3 (H3K9me3) together with Ki67 to detect the senescent status of therapy-exposed cancer cells

A cFLIP-flop switch for senolysis

Persistent senescent cancer cells have tumor-promoting potential, making their selective elimination a prime therapeutic objective. The death receptor inhibitor cFLIP has now been shown to counter the susceptibility of senescent cells to DR5-mediated extrinsic apoptosis, which can be therapeutically exploited

Simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated β-galactosidase (SA-β-gal), the DNA synthesis marker 5-ethynyl-2′-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells

Virus-induced senescence is driver and therapeutic target in COVID-19

Derailed cytokine and immune cell networks account for organ damage and clinical severity of COVID-19. Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and accompanied by a senescence-associated secretory phenotype (SASP), composed of pro-inflammatory cytokines, extracellular matrix-active factors and pro-coagulatory mediators. COVID-19 patients displayed markers of senescence in their airway mucosa in situ and elevated serum levels of SASP factors. Mirroring COVID-19 hallmark features such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue in vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, neutrophil extracellular trap (NET) formation as well as activation of platelets and the clotting cascade in response to supernatant of VIS cells, including SARS-CoV-2-induced senescence. Senolytics such as Navitoclax and Dasatinib/Quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-driven hamster and mouse models. Our findings mark VIS as pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest senolytic targeting of virus-infected cells as a novel treatment option against SARS-CoV-2 and perhaps other viral infections