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.

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. Disclosures Vago: 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.

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.

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.

HDAC4 degradation during senescence unleashes an epigenetic program driven by AP-1/p300 at selected enhancers and super-enhancers

Background Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. Results Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. Conclusions Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

Role of Cdkn2a in the Emery–Dreifuss Muscular Dystrophy Cardiac Phenotype

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

The Interaction of Viruses with the Cellular Senescence Response

Cellular senescence is viewed as a mechanism to prevent malignant transformation, but when it is chronic, as occurs in age-related diseases, it may have adverse effects on cancer. Therefore, targeting senescent cells is a novel therapeutic strategy against senescence-associated diseases. In addition to its role in cancer protection, cellular senescence is also considered a mechanism to control virus replication. Both interferon treatment and some viral infections can trigger cellular senescence as a way to restrict virus replication. However, activation of the cellular senescence program is linked to the alteration of different pathways, which can be exploited by some viruses to improve their replication. It is, therefore, important to understand the potential impact of senolytic agents on viral propagation. Here we focus on the relationship between virus and cellular senescence and the reported effects of senolytic compounds on virus replication.

Metabolic Regulation of the Senescence Program

Cellular senescence is a cell fate defined by an irreversible cell-cycle arrest and a pro-inflammatory secretory profile. It is a consequence of a shift in metabolism and rearrangement of chromatin. Accumulation of senescent cells is a universal hallmark of age-related pathologies suggesting these cells contribute to age-related susceptibility to disease. Here, we examine the interplay between two metabolic inhibitors of senescence: Rapamycin treatment and Methionine restriction (metR). We report that a combination of methionine restriction and rapamycin induces a metabolic reprogramming that prevents activation of the senescence program in human fibroblasts. The treated cells continue to divide at a slow rate at a high passage and lack senescence-associated markers and inflammatory cytokines. Genome-wide chromatin accessibility analysis reflects chromatin remodeling with distinctly increased accessibility of heterochromatic regions in treated cells. Further, Transcriptome-wide analysis reveals increased expression of specific methyltransferases which alter the trimethylation of H3, one of the strongest hallmarks of open chromatin. This may represent a mechanistic link between a major hallmark of senescence and nuclear events required for senescence.