Senescence Program Research Articles

Loss of the transcription repressor ZHX3 induces senescence-associated gene expression and mitochondrial-nucleolar activation.

Cellular senescence is accompanied by metabolic and epigenomic remodeling, but the transcriptional mechanism of this process is unclear. Our previous RNA interference-based screen of chromatin factors found that lysine methyltransferases including SETD8 and NSD2 inhibited the senescence program in cultured fibroblasts. Here, we report that loss of the zinc finger and homeobox protein 3 (ZHX3), a ubiquitously expressed transcription repressor, induced senescence-associated gene expression and mitochondrial–nucleolar activation. Chromatin immunoprecipitation–sequencing analyses of growing cells revealed that ZHX3 was enriched at the transcription start sites of senescence-associated genes such as the cyclin-dependent kinase inhibitor (ARF-p16INK4a) gene and ribosomal RNA (rRNA) coding genes. ZHX3 expression was consistently downregulated in cells with replicative or oncogene-induced senescence. Mass spectrometry-based proteomics identified 28 proteins that interacted with ZHX3, including ATP citrate lyase and RNA metabolism proteins. Loss of ZHX3 or ZHX3-interaction partners by knockdown similarly induced the expression of p16INK4a and rRNA genes. Zhx3-knockout mice showed upregulation of p16INK4a in the testes, thymus and skeletal muscle tissues, together with relatively short survival periods in males. These data suggested that ZHX3 plays an essential role in transcriptional control to prevent cellular senescence.

Senolysis-Based Elimination of Chemotherapy-Induced Senescent Breast Cancer Cells by Quercetin Derivative with Blocked Hydroxy Groups.

Simple SummaryCellular senescence may contribute to aging and age-related diseases, and the elimination of senescent cells is considered a promising anti-aging strategy. Drug-induced senescence in cancer cells during chemotherapy may also promote a number of adverse effects. Thus, in the present study, the usefulness of three quercetin derivatives as senolytic agents was studied upon stimulation of senescence program in breast cancer cells. We have shown that quercetin derivative with blocked hydroxy groups (QD3) sensitized etoposide-induced senescent breast cancer cells to apoptotic cell death that was accompanied by a decrease in proinflammatory and HSP70-based responses. We suggest that these prosenescent and senolytic activities can be combined to design a novel anti-cancer strategy, at least, against breast cancer cells.Drug-induced senescence program may be activated both in normal and cancer cells as a consequence of chemotherapeutic treatment, leading to some adverse side effects such as senescence-associated secretory phenotype (SASP), secondary senescence, and cancer promotion. Targeted elimination of senescent cells can be achieved by drugs with senolytic activity (senolytics), for example, the plant-derived natural compound quercetin, especially when co-treated with kinase inhibitor dasatinib. In the present study, three quercetin derivatives were synthesized and tested for improved senolytic action against etoposide-induced senescent human normal mammary epithelial cells and triple-negative breast cancer cells in vitro. Transformation of catechol moiety into diphenylmethylene ketal and addition of three acetyl groups to the quercetin molecule (QD3 derivative) promoted the clearance of senescent cancer cells as judged by increased apoptosis compared to etoposide-treated cells. A QD3-mediated senolytic effect was accompanied by decreased SA-beta galactosidase activity and the levels of p27, IL-1β, IL-8, and HSP70 in cancer cells. Similar effects were not observed in senescent normal cells. In conclusion, a novel senolytic agent QD3 was described as acting against etoposide-induced senescent breast cancer cells in vitro. Thus, a new one-two punch anti-cancer strategy based on combined action of a pro-senescence anti-cancer drug and a senolytic agent is proposed.

Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

Senescence, a stable state of growth arrest, affects many physiological and pathophysiological processes, especially aging. Previous work has indicated that transcription factors (TFs) play a role in regulating senescence. However, a systematic study of regulatory TFs during replicative senescence (RS) using multi-omics analysis is still lacking. Here, we generated time-resolved RNA-seq, reduced representation bisulfite sequencing (RRBS) and ATAC-seq datasets during RS of mouse skin fibroblasts, which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome. Through integrative analysis and genetic manipulations, we found that transcription factors E2F4, TEAD1 and AP-1 are key regulators of RS. Overexpression of E2f4 improved cellular proliferative capacity, attenuated SA-β-Gal activity and changed RS-associated differentially methylated sites (DMSs). Moreover, knockdown of Tead1 attenuated SA-β-Gal activity and partially altered the RS-associated transcriptome. In addition, knockdown of Atf3, one member of AP-1 superfamily TFs, reduced Cdkn2a (p16) expression in pre-senescent fibroblasts. Taken together, the results of this study identified transcription factors regulating the senescence program through multi-omics analysis, providing potential therapeutic targets for anti-aging.

Pharmacological CDK4/6 inhibition reveals a p53‐dependent senescent state with restricted toxicity

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets.

1014 – CELLULAR SENESCENCE: AN UNINTENDED TWIST FOR GENE AND CELL THERAPY APPLICATIONS

The success of Hematopoietic Stem and Progenitor Cell (HSPC) gene therapies critically depends on the capacity to genetically engineer HSPCs without compromising their functional properties. Emerging evidence, including our own, indicates that HSPC exposure to currently available gene transfer and gene editing technologies –which require prolonged ex-vivo culture, high viral vector doses and nuclease-induced DNA double strand breaks¬– unexpectedly converge on the DNA damage response (DDR), a signaling cascade leading to cell cycle arrest. Protracted DDR impairs the hematopoietic reconstitution of gene-modified cells upon transplantation; instead, its transient inhibition significantly improves their functionality. This experimental evidence uncovers a previously unappreciated interplay between viral vector sensing and the host cell DDR machinery in human HSPCs. Yet, we have just scratched the surface of a plethora of still unexplored cellular programs potentially hampering the clonal composition and the dynamics of reconstitution upon transplantation of this primitive cell compartment. We build on the innovative concept that current gene engineering protocols may inadvertently trigger the activation of a cellular senescence program in HSPCs with both cell-autonomous and paracrine short- and long-term consequences on engineered human hematopoiesis. By employing quantitative imaging and cutting-edge genomic technologies and taking advantage of uniquely available gene therapy platforms and primary patient samples, we aim to identify the molecular determinants that promote senescence in HSPCs and design innovative hypothesis-driven strategies to mitigate senescence barriers for more effective, safer and clinically broader gene therapy applications. Our findings will unveil a new set of mechanisms controlling the biology of gene-engineered HSPCs and open novel scientific horizons for the development of innovative gene and cell therapies. The success of Hematopoietic Stem and Progenitor Cell (HSPC) gene therapies critically depends on the capacity to genetically engineer HSPCs without compromising their functional properties. Emerging evidence, including our own, indicates that HSPC exposure to currently available gene transfer and gene editing technologies –which require prolonged ex-vivo culture, high viral vector doses and nuclease-induced DNA double strand breaks¬– unexpectedly converge on the DNA damage response (DDR), a signaling cascade leading to cell cycle arrest. Protracted DDR impairs the hematopoietic reconstitution of gene-modified cells upon transplantation; instead, its transient inhibition significantly improves their functionality. This experimental evidence uncovers a previously unappreciated interplay between viral vector sensing and the host cell DDR machinery in human HSPCs. Yet, we have just scratched the surface of a plethora of still unexplored cellular programs potentially hampering the clonal composition and the dynamics of reconstitution upon transplantation of this primitive cell compartment. We build on the innovative concept that current gene engineering protocols may inadvertently trigger the activation of a cellular senescence program in HSPCs with both cell-autonomous and paracrine short- and long-term consequences on engineered human hematopoiesis. By employing quantitative imaging and cutting-edge genomic technologies and taking advantage of uniquely available gene therapy platforms and primary patient samples, we aim to identify the molecular determinants that promote senescence in HSPCs and design innovative hypothesis-driven strategies to mitigate senescence barriers for more effective, safer and clinically broader gene therapy applications. Our findings will unveil a new set of mechanisms controlling the biology of gene-engineered HSPCs and open novel scientific horizons for the development of innovative gene and cell therapies.

An Evolving Role for the Long Non-Coding RNA H19 in Aging and Senescence

The Weathering Hypothesis states BIPOC face more stressors, by which over a lifetime they are subjected to the negative consequences of stress (e.g., poorer emotional health). Using ecological momentary assessments, we examined whether subtle discrimination moderated the within-person stressor slope on positive and negative affect. We predicted emotional wellbeing would be worse at stressor moments, and those with greater discrimination experience would be more impacted by stressors. Participants were 334 diverse adults (25-65 years, Mage = 47, 63% Female) from Bronx, New York. Positive affect decreased and negative affect increased significantly at stressor moments (p<.0001). Unexpectedly, subtle discrimination was not a significant moderator for the within-person stressor slope on positive affect and negative affect. Unlike the predictions of the Weathering Hypothesis, these results show that prior discrimination experiences may not exacerbate responses to stressors and entail additional risk in daily life.

Single cell analysis reveals stochastic cell-to-cell variation in stress response and senescence program

Prior to the pandemic, public health experts argued that loneliness was among the most significant threats facing women’s health and wellbeing. As the COVID-19 pandemic brought our social lives to an abrupt pause in March, 2020, older adults were encouraged to remain isolated from friends and family. Social distancing guidelines led many older people to decrease social interactions with others. Using a community-based longitudinal study of women age 60+, we examined how changes in feelings of social connections with others influenced loneliness in October 2020 relative to prior to the pandemic (in September 2018). Our previous research has shown that psychological resilience decreases the negative consequences of major life stressors in later life. We hypothesized that women with high social consequences of the pandemic would experience increased loneliness, but resilience would buffer these effects. In line with our hypotheses, results showed that those who reported significant declines in social connectedness with others during the pandemic (i.e., high social consequences) experienced significant increases in loneliness (beta=0.125; p<0.001). Resilience, alternatively, was significantly associated with decreased loneliness (beta=-0.05; p<0.05), and buffered the social consequences of the pandemic. That is, as resilience increased, the social consequences of COVID-19 significantly declined (p<0.01), and resilience attenuated the negative consequences of high levels of social consequences of COVID-19 on loneliness, while those with high social consequences and low resilience experienced significant increases in loneliness in association with the pandemic. Based on our findings, we discuss potential clinical implications for resilience-based interventions for older adults.

An Evolving Role for the Long Non-Coding RNA H19 in Aging and Senescence

The long non-coding RNA (lncRNA) H19 is a maternally imprinted gene transcript that, in conjunction with the neighboring Igf2 gene, is critical in controlling embryonic growth. Loss of H19 results in fetal overgrowth associated with Beckwith Weidemann syndrome, while elevated H19 occurs in human cancers. In the adult, H19 functions in cancer cells where it promotes migration and is correlated with poor prognosis, and in adult stem cells where it is a key regulator of cell fate decisions during differentiation. While the function of H19 in primary somatic cells has not been defined, a reduction in the abundance of H19 has been reported during senescence in endothelial cells. Given the critical importance of H19 in cell fate decisions, it is likely that understanding the precise function of H19 in somatic cells in general and why reduced levels occur with cellular senescence will provide novel insights into both somatic cell maintenance and the senescence program. Towards this end, we examined the role of H19 in somatic cell growth using cardiac interstitial fibroblasts. Our results indicate that H19 is not only vital for somatic cell proliferation and survival, but that depletion of H19 leads to cell cycle arrest and the formation of abnormal nuclei resulting in senescent cells. We are defining both the upstream regulators of H19 and the downstream mediators of senescence following H19 depletion. Overall, these results indicate an essential role for H19 in cell cycle progression, chromatin structure, and possibly proper mitotic division.

Deciphering the Dynamic Molecular Program of Radiation-Induced Endothelial Senescence

Radiation-induced cellular senescence is a double-edged sword, acting as both a tumor suppression process limiting tumor proliferation, and a crucial process contributing to normal tissue injury. Endothelial cells play a role in normal tissue injury after radiation therapy. Recently, a study observed an accumulation of senescent endothelial cells (ECs) around radiation-induced lung focal lesions following stereotactic radiation injury in mice. However, the effect of radiation on EC senescence remains unclear because it depends on dose and fractionation, and because the senescent phenotype is heterogeneous and dynamic. Using a systems biology approach in vitro, we deciphered the dynamic senescence-associated transcriptional program induced by irradiation. Flow cytometry and single-cell RNA sequencing experiments revealed the heterogeneous senescent status of irradiated ECs and allowed to deciphered the molecular program involved in this status. We identified the Interleukin-1 signaling pathway as a key player in the radiation-induced premature senescence of ECs, as well as the endothelial-to-mesenchymal transition process, which shares strong hallmarks of senescence. Our work provides crucial information on the dynamics of the radiation-induced premature senescence process, the effect of the radiation dose, as well as the molecular program involved in the heterogeneous senescent status of ECs.

Therapy-Induced Senescence As an Anti-Cancer and Immune-Stimulatory Strategy

Acute myeloid leukaemia (AML) is the most common type of leukaemia in elderly, for which the current gold standard of treatment is chemotherapy. Recently, it has been observed that AML blasts can activate the senescence program in response to chemotherapy (Therapy Induced Senescence, TIS). Cellular senescence is a stable and terminal state of growth arrest, often caused by nuclear DNA damage, associated with the transcriptional activation of a so-called Senescence Associated Secretory Phenotype (SASP), characterized, among others, by cytokines release, reported to promote immune-surveillance. Here we show that blasts, in response to chemotherapy, accumulate DNA damage and activate the senescence program, that in turn leads to HLA molecules upregulation, making them more prone to be cleared by T-cells.To evaluate TIS in AML blasts, we started by applying chemotherapy treatment (ARA-C) in six AML cell lines with different p53 status and FAB classification, observing reduction in proliferation rate and activation of DNA damage response pathways in the absence of overt apoptosis. We then quantified Senescence-Associated β-galactosidase (SA-βgal) activity and detected induction of senescence longitudinally with different extent. Accordingly, we observed the same features when applying ARA-C ex-vivo to primary AML samples collected at diagnosis.To delve deeper into the changes associated with the establishment of senescence in primary blasts, we performed RNA-seq analysis and observed an upregulation of pro-inflammatory genes (including IL1, IL6 and IL8) along with genes involved in immunogenicity. Investigating the biological significance of the transcriptional changes observed, we first reported an increase of HLA molecules on the surface of senescent blasts, as measured by FACS analyses. This observation prompted us to study the interaction between the immune system and senescent blasts, exploiting the Mixed Lymphocyte Reaction (MLR) assay. As expected, we detected a higher T-cell activation of both CD8+ and CD4+ subpopulations, accompanied by an increase in immunological synapses events and in apoptosis induction, when co-culturing chemotherapy treated blasts with T cells.In order to uncover the molecular mechanisms involved in TIS, we disentangled the role of DNA damage and cell cycle arrest in the phenotype observed comparing AML cell lines treatment with either ARA-C or the cdk4/6 inhibitor, which causes cell cycle arrest without inducing DNA damage. We found that HLA molecules overexpression is linked to the establishment of DNA damage response, however, when comparing acute to chronic ARA-C treatment, we observed that expression levels increased with treatment duration, suggesting that this feature is necessary but not sufficient to increase AML immunogenicity. Next, taking advantage of shRNAs (delivered by lentiviral vectors), we investigated deeper into the role of cell cylcle arrest. By stably knocking down p21, a crucial cell cycle inhibitor, we observed that ARA-C treated blasts had a reduced capacity of activating T-cell. Taken together, these observations point out to a crucial role for senescence in the improved immune-based clearance observed upon ARA-C treatment of blasts. Interestingly, a retrospective analysis showed that a cohort of patients clinically considered “responders” displayed a higher SA-βgal activity, further supporting the idea that senescence establishment in AML may act as a tumour suppressor mechanism.Overall, our study provides mechanistic insights into the biological and cellular response of AML cells to TIS and presents senescence as a positive mechanism able to promote AML eradication. This opens new lines of research aimed to develop novel therapeutic approaches against AML, exploiting senescence-induced features. DisclosuresVago: Moderna Therapeutics: Research Funding; GEN-DX: Patents & Royalties.

Paradoxical implication of BAX/BAK in the persistence of tetraploid cells

Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.

DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.

A Balancing Act: p53 Activity from Tumor Suppression to Pathology and Therapeutic Implications.

TP53, encoding the p53 transcription factor, is the most frequently mutated tumor suppressor gene across all human cancer types. While p53 has long been appreciated to induce antiproliferative cell cycle arrest, apoptosis, and senescence programs in response to diverse stress signals, various studies in recent years have revealed additional important functions for p53 that likely also contribute to tumor suppression, including roles in regulating tumor metabolism, ferroptosis, signaling in the tumor microenvironment, and stem cell self-renewal/differentiation. Not only does p53 loss or mutation cause cancer, but hyperactive p53 also drives various pathologies, including developmental phenotypes, premature aging, neurodegeneration, and side effects of cancer therapies. These findings underscore the importance of balanced p53 activity and influence our thinking of how to best develop cancer therapies based on modulating the p53 pathway.

REST/NRSF deficiency impairs autophagy and leads to cellular senescence in neurons.

During aging, brain performances decline. Cellular senescence is one of the aging drivers and a key feature of a variety of human age‐related disorders. The transcriptional repressor RE1‐silencing transcription factor (REST) has been associated with aging and higher risk of neurodegenerative disorders. However, how REST contributes to the senescence program and functional impairment remains largely unknown. Here, we report that REST is essential to prevent the senescence phenotype in primary mouse neurons. REST deficiency causes failure of autophagy and loss of proteostasis, increased oxidative stress, and higher rate of cell death. Re‐establishment of autophagy reverses the main hallmarks of senescence. Our data indicate that REST has a protective role in physiological aging by regulating the autophagic flux and the senescence program in neurons, with implications for neurological disorders associated with aging.

Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis

ABSTRACTActivating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms.

DLBCL-Derived NF-κb Mutations Exploit Senescence and Immune Evasion Mechanisms to Promote Myc-Driven Lymphomagenesis

Introduction: Constitutive NF-κB activation is a hallmark feature of B-cell malignancies, including the ABC-subtype of diffuse large B-cell lymphoma (DLBCL), and is mainly due to gain-of-function mutations in components of the Toll-like receptor (MYD88) or B-cell receptor (CD79B, CARD11) pathways, inactivating mutations of cellular NF-κB suppressors (TNFAIP3/A20, BIRC3) or copy number gain (NFKBIZ), often in the presence of high Myc oncoprotein expression. In order to understand whether such genetic lesions may cooperate with Myc during lymphomagenesis, we took advantage of the Eµ- myc transgenic mouse in which aggressive B-cell lymphomas are induced by B-cell-specific overexpression of c-Myc.Methods: Eµ- myc transgenic hematopoietic stem cells (HSC) were stably transduced with GFP-expressing retroviral vectors encoding the murine orthologs of human MYD88-L265P, NFKBIZ, CD79B-ITAM-Y196H, CARD11-L244P, CARD11-L225LI or shRNA against TNFAIP3/A20 or BIRC3 prior to their transplantation into lethally irradiated syngenic C57BL/6 mice. Recipient animals were subsequently monitored for the development of a palpable lymphadenopathy, and lymphomas were subject to immunophenotyping, analysis of growth parameters and transcriptome profiling by RNA-Seq.Results : Ectopic overexpression of NFKBIZ, MYD88-L265P, CD79B-ITAM-Y196H or knockdown of TNFAIP3/A20 in Eµ- myc transgenic HSC gave rise to GFP(+) B-cell lymphomas (> 70% GFP+/B220+ cells), albeit at varying frequencies. All mutant-driven lymphomas manifested with reduced apoptotic TUNEL/B220 indices when compared to the empty-vector control cohort (ctrl. lymphomas). In contrast, neither knockdown of BIRC3 nor overexpression of the CARD11 mutants generated GFP(+) Eµ- myc lymphomas in vivo . Unexpectedly, we found senescent GFP(+) B- lymphocytes residing in the spleens of these healthy animals after transplantation. Genetic ablation of the senescence program (via allelic deletion of the histone demethylase Suv39h1 or the INK4a/ARF locus) in Eµ- myc HSC prior to transduction with the CARD11 mutants gave rise to palpable GFP(+) B-cell lymphomas (unlike the empty-vector), indicating that CARD11 missense mutations require a senescence-deficient background to promote Eµ- myc -driven lymphomagenesis.RNA-Seq unvealed downregulation of MHC class II genes as a common feature of all mutant-driven Eµ- myc lymphomas when compared to ctrl. lymphomas. Since MHC/T-cell receptor interactions are required for an effective anti-tumoral adaptive immune response, this finding suggested immune evasion mechanisms to be exploited by all genetic lesions tested to promote Myc-driven lymphomagenesis. Further supporting this notion, MYD88-L265P-driven lymphomas exhibited elevated surface expression of the immune checkpoint ligands PD-L1 and PD-L2. Strikingly, knockdown of PD-L1 alone was sufficient to significantly delay MYD88-L265P-driven lymphomagenesis in Eµ- myc HSC transplantation experiments, suggesting that PD-L1-mediated immune evasion mechanisms contribute significantly to the aggressive phenotype observed under the MyD88-L265P mutant in vivo . Furthermore, overexpression of PD-L1 in Eµ- myc HSC alone also induced GFP(+) B-cell lymphomas that displayed a striking overlap in gene expression with MyD88-L265P-driven Eµ- myc lymphomas.Conclusions: We continue to characterize the Eµ- myc transgenic mouse as a well-suited model platform to functionally dissect DLBCL-relevant oncogenic networks in general and NF-κB-activating mutations in the context of high Myc protein expression, a frequent feature of DLBCL, in particular. Our systematic in vivo analysis of different NF-κB-activating mutants - so far rather recognized as functionally interchangeable moieties - indicated that some of these genetic lesions directly cooperate with Myc (i.e. MyD88-L265P, NFKBIZ) - partly via utilizing immune evasion mechanisms - while others (e.g. CARD11 mutations) require upfront senescence-ablating lesions to license Myc-driven lymphomagenesis. Our findings might explain the pressure to select for senescence-ablating lesions like INK4a/ARF loss frequently found in human DLBCL, and could inform immune checkpoint blockade treatment strategies based on certain oncogenic/immune evasion network interconnections. DisclosuresNo relevant conflicts of interest to declare.

Indisulam Synergizes With Palbociclib to Induce Senescence Through Inhibition of CDK2 Kinase Activity

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

ELV-N32 and RvD6 isomer decrease pro-inflammatory cytokines, senescence programming, ACE2 and SARS-CoV-2-spike protein RBD binding in injured cornea

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic collapse around the world. Effective treatments to mitigate this viral infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells (HCEC) in culture challenged with IFNγ as models of the eye surface to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein to angiotensin-converting enzyme 2 (ACE2). We found that the lipid mediators, elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i) decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ-stimulated HCEC. There was also a concomitant decrease in the binding of Spike RBD with the lipid treatments. Using RNA-seq analysis, we uncovered that the lipid mediators also attenuated the expression of pro-inflammatoy cytokines participating in hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to open therapeutic avenues to counteract virus attachment and entrance to the body.

Exercise reduces circulating biomarkers of cellular senescence in humans.

Cellular senescence has emerged as a significant and potentially tractable mechanism of aging and multiple aging‐related conditions. Biomarkers of senescent cell burden, including molecular signals in circulating immune cells and the abundance of circulating senescence‐related proteins, have been associated with chronological age and clinical parameters of biological age in humans. The extent to which senescence biomarkers are affected by interventions that enhance health and function has not yet been examined. Here, we report that a 12‐week structured exercise program drives significant improvements in several performance‐based and self‐reported measures of physical function in older adults. Impressively, the expression of key markers of the senescence program, including p16, p21, cGAS, and TNFα, were significantly lowered in CD3+ T cells in response to the intervention, as were the circulating concentrations of multiple senescence‐related proteins. Moreover, partial least squares discriminant analysis showed levels of senescence‐related proteins at baseline were predictive of changes in physical function in response to the exercise intervention. Our study provides first‐in‐human evidence that biomarkers of senescent cell burden are significantly lowered by a structured exercise program and predictive of the adaptive response to exercise.

Onset of Senescence and Steatosis in Hepatocytes as a Consequence of a Shift in the Diacylglycerol/Ceramide Balance at the Plasma Membrane.

Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids’ production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.