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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., Therapy-induced senescence reflects a biological effector principle that is underrecognized in lesion-focused cancer precision medicine. Here the authors utilize mouse lymphoma genetics to functionally dissect senescence and cross-species apply a novel senescence-based prognosticator to lymphoma patients.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1–4). 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 is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5–7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections. Virus-induced senescence is a central pathogenic feature in COVID-19, and senolytics, which promote apoptosis of senescent cells, can reduce disease severity in hamsters,mice, as well as humans infected with SARS-CoV-2.

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.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16(INK4a), p21(CIP1) and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of 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 Eμ-myc transgenic lymphoma cells harboring defined genetic lesions and provide protocols for co-staining of either senescence-associated β-galactosidase (SA-β-gal) activity or trimethylated lysine 9 of histone H3 (H3K9me3) together with Ki67 to detect the senescent status of therapy-exposed cancer cells.

Introduction: Treatment decisions based on patient-specific molecular features are central to personalized cancer precision medicine. Oftentimes treatment response of an individual patient remains an issue of trial and error. Expression profiling has been utilized to study molecular subtypes of tumor entities and their impact on treatment outcome. Biological effector programs, such as cellular senescence, however, remain largely understudied. Syngeneic mouse models of cancer that can reproduce critical molecular features of human malignancies could serve as useful models to explore genetic determinants of drug sensitivity, and, likewise, to unveil molecular mechanisms of treatment resistance. Here, we focus on the involvement of therapy-induced senescence on treatment outcome in mouse models and patients diagnosed with diffuse large B-cell lymphoma (DLBCL). Methods: We present and characterize here the utilization of Eµ-myc transgenic lymphomas as a faithful model of chemoresistance and demonstrate its cross-species validity for DLBCL patients. Specifically, primary Eµ-myc lymphomas, of which we generated gene expression profiles (GEP) at diagnosis, were exposed to genotoxic therapy in vivo, and subsequently monitored regarding long-term outcome in a clinical trial-like design. Lymphoma senescence capability, a central drug effector principle, was studied in mice by unbiased approaches as well as loss- and gain-of-function genetics. Results: Investigation of DLBCL-established gene expression based subtypes related to cell-of-origin (COO - i.e. GCB/ABC subtypes) and distinct DLBCL biologies (e.g. comprehensive consensus clusters [CCC]) using machine-learning methods demonstrated their relevance in the murine platform. Moreover, our findings show an important role of histone H3 lysine 9-trimethylation (H3K9me3) for senescence induction and treatment outcome as demonstrated by shorter time to death and time to relapse of mice bearing lymphomas with engineered loss of the H3K9me3-critical methyltransferase Suv39h1 on one hand and lymphomas with genetically transferred or endogenous overexpression of H3K9-active demethylases on the other hand. Furthermore, expression levels of H3K9me3-specific demethylases stratified unmodified Eµ-myc lymphomas and DLBCL patients into two groups with superior outcome for those with lower levels. In line with these findings, DLBCL patients with high levels of the senescence-associated H3K9me3 mark in their lymphomas presented with significantly longer survival times. Further transcriptomics-based investigations of our clinical-trial like mouse model and DLBCL patients suggests the presence of a molecular network distinguishing lymphomas into a clinically superior senescence responder from an inferior non-responder group. Conclusions: Our results conclude that Eµ-myc transgenic lymphomas serve as faithful model for human DLBCL and the importance of therapy-induced senescence for treatment outcome of DLBCL patients. Our data suggest the integration of tractable, transgenic mouse models to the repertoire of functional test platforms to better implement lesion- and state-based decisions in personalized cancer precision medicine. Ongoing mouse model-based work aims at specific targeting of aberrant demethylase activities and synthetic lethal approaches to selectively eliminate potentially detrimental senescent lymphoma cells after chemotherapy to assess therapeutic long-term effects and to determine the conditions for future early-phase DLBCL clinical testing. Disclosures No relevant conflicts of interest to declare.