Treatment-induced Senescence Research Articles

BIOM-02. DISSECTING CDK4/6 INHIBITOR RESISTANCE MECHANISMS IN RECURRENT PEDIATRIC BRAIN TUMORS WITH LONGITUDINAL CSF LIQUID BIOPSIES FROM THE SJDAWN CLINICAL TRIAL

Abstract BACKGROUND SJDAWN (NCT03434262) is a St. Jude Children’s Research Hospital phase 1 trial that assessed the safety and benefit of CDK4/6 inhibitor-based therapy in patients with relapsed CNS tumors. To capture tumor evolution dynamics and arising resistance mechanisms in response to treatment, 143 longitudinal CSF samples were collected from 34/68 (50%) patients for cell-free DNA (cfDNA) analysis. METHODS cfDNA was extracted from CSF liquid biopsies and analyzed using a novel methylation-based platform that integrates copy number, tumor classification, and deconvolution of methylation signatures into normal vs. disease fractions. Samples with measurable residual disease (MRD) were further characterized by deep whole-genome sequencing to discover mutation-driven resistance mechanisms. When available, pre- and post-SJDAWN tumor tissues were processed for single-nucleus multi-omics to track tumor evolution at single-cell resolution. RESULTS In the context of CDK4/6 inhibitor-based therapy, longitudinal cfDNA assessment revealed treatment-induced selection for subclones with focal amplification of positive cell cycle regulators CDK6, CCND2, and MYCN. Emergence of these resistance mechanisms notably preceded disease progression on MRI and CSF cytology by up to 6 months. Of the 68 patients enrolled on SJDAWN, 4 patients (3 Group 3/4, subtype III medulloblastomas and 1 anaplastic ependymoma) achieved >2-year progression-free survival. Interestingly, serial cfDNA profiling of 1 long-term survivor (Group 3/4, subtype III medulloblastoma) exhibited undetectable MRD status for >1.5 years prior to reemergence, suggesting tumor senescence by CDK4/6 inhibition may impede circulating tumor DNA (ctDNA) release kinetics. CSF liquid biopsies were further evaluated in comparison to matched tumor tissues, and changes in CSF-specific subclones were observed in response to treatment. CONCLUSIONS In addition to early MRD detection, liquid biopsies capture tumor evolution dynamics and can inform developing resistance mechanisms. Focal amplification of CDK6, CCND2, and MYCN facilitates CDK4/6 inhibitor resistance. In responders, treatment-induced senescence may inhibit ctDNA release kinetics.

PP2A activation as a new therapeutic strategy in neuroblastoma: Making sense out of senescence.

10055 Background: Neuroblastoma (NBL) is the most common extracranial solid tumor in children, accounting for 15% of all pediatric cancer deaths. The five-year survival rate for children with high-risk disease is less than 50%, with the primary reasons for mortality being refractory and relapsed disease. One hypothesis for the poor response to therapy and propensity for recurrent disease in high-risk patients is treatment-induced senescence (TIS). Protein phosphatase 2A (PP2A) is a known tumor suppressor that is downregulated in NBL. PP2A activation has been shown to reverse cellular senescence in non-cancerous pathologies, therefore we hypothesized that reactivation of PP2A using novel small molecule activators would function as a novel senotherapeutic in NBL. Methods: We employed MYCNamplified, SK-N-BE(2), and non-amplified, SK-N-AS, established human NBL cell lines. Topotecan, a common NBL chemotherapeutic, known to induce senescence, and ATUX-1215, a small molecule PP2A activator, were utilized. NBL cells were treated with topotecan and ATUX-1215 alone, as well as in combination. Colorimetric assays detected cell viability and proliferation. Immunoblotting identified proteins of interest including γ-H2AX and CCL2. TIS was detected by S-β galactosidase (Sβgal) assay. Results: We found increased expression of the senescence marker, histone γ-H2AX, and increased Sβgal-positive tumor cells supporting the ability of topotecan to generate TIS in NBL. Following treatment with PP2A activator in combination with topotecan, markers of TIS are decreased in both MYCN amplified and non-amplified NBL cells. Further, and importantly, PP2A activation reversed the effects of topotecan TIS and acted in a synergistic fashion to decrease NBL cell viability. Conclusions: Activation of PP2A with a novel small molecule ameliorates topotecan-induced TIS in NBL cells irrespective of MYCN status. These results are promising, providing a potential novel therapeutic adjunct to children who are most in need of innovative interventions.

Abstract 2961: Investigating the role of aging and treatment-induced senescence in the metastatic liver cancer tumor microenvironment

Abstract Liver senescence, marked by the progressive decline in hepatic cell function and regenerative capacity, plays a vital role in the context of aging, chronic liver diseases, and cancer progression, particularly when influenced by anti-cancer therapies. This study is designed to extensively characterize liver senescence under two specific conditions: replicative aging and therapy-induced changes. We examined liver samples from 20 individuals, covering a wide age range (young, <40 years; old, >60 years), and 16 cases of colorectal cancer (CRC) liver metastasis (ages 37-66). Our integrative approach melds single-nucleus RNA-sequencing (snRNA-seq) and ATAC-sequencing (snATAC-seq) to investigate the transcriptomic and epigenetic landscapes across a spectrum of liver cell types, including hepatocytes, cholangiocytes, stellate cells, liver sinusoidal endothelial cells, and diverse immune cells. Key findings include an upregulation of senescence markers such as CDKN1A, CDKN2A, and HMGB1, particularly in specific hepatocytes from older individuals and various cell types in the CRC liver metastasis group. Furthermore, we noted a distinctive senescence-associated secretory phenotype (SASP) across multiple immune cell types in our treated CRC liver metastasis group and upregulation of treatment-induced senescence gene signatures, such as SenCan. Moreover, hepatic stellate cells from older subjects exhibited augmented senescence-related gene signatures (SenSig) and transcription factors (SenMayo), with myofibroblast-like and FAP+ stellate cells showing a marked senescence phenotype, evidenced by elevated SASP molecule expression (CCL2, SERPINE1, GLB1, IGFBP7, IGFBP3).Incorporating advanced high-resolution imaging techniques, our study delves into the detailed morphological characteristics and spatial distribution of senescence markers within the liver in the setting of replicative aging and treatment-induced senescence. This study sheds light on the heterogeneity and intricacies of senescence-associated gene expression within liver cell populations. By combining comprehensive multiomic data with advanced high-resolution spatial profiling, we aim to decipher unique senescence phenotypes in the context of both replicative and treatment-induced senescence. Our work highlights the significance of cellular senescence in liver pathologies and cancer progression, suggesting new directions for therapeutic interventions that target these complex dynamics. Citation Format: Kelsey Gallant, Alla Karpova, Xiang Li, Michael Iglesia, Andrew Houston, Daniel Rapp, Chien-Wei Peng, Clara Liu, John Herdon, Feng Chen, Ryan Fields, Li Ding. Investigating the role of aging and treatment-induced senescence in the metastatic liver cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2961.

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.

Selected Articles from This Issue

Intratumoral electroporation of IL12-encoding plasmids (IT-pIL12-EP) enhances antitumor immunity in treated and distant tumors. However, IT-pIL12-EP outcomes are in part dictated by infiltrating T-cell composition, where higher proportions of cytotoxic T cells as opposed to more immunosuppressive immune cells predicts enhanced IT-pIL12-EP responsiveness. In an effort to increase proportions of activated cytotoxic T cells in IT-pIL12-EP-treated tumors, Han and colleagues supplemented IT-pIL12-EP with electroporation of plasmids encoding membrane-anchored anti-CD3 single-chain variable fragments (scFv). The authors found that including anti-CD3 scFv with IL12 enhances proliferation and interferon-γ (IFNγ) secretion in both CD8+ cytotoxic T cells and CD4+ T regulatory (Treg) cells, and that costimulation with membrane-bound anti-CD3 with IL12 reduces Treg suppression. Supplementing IT-pIL12-EP with plasmids encoding anti-CD3 scFv in vivo enhances proliferation and cytotoxicity of tumor antigen-specific T cells, leading to augmented antitumor immunity against B16-F10 melanoma and 4T1 mammary tumor cells. Treating human melanoma-infiltrating T cells with IL12 and anti-CD3 scFv enhances cytotoxic CD8+ expansion, IFNγ secretion, and PD-1 expression in vitro, suggesting IT-pIL12-EP and anti-CD3 scFv could augment PD-1 immune checkpoint inhibition responsiveness in melanoma patients.Tumor necrosis factor alpha (TNFα)-mediated cytotoxicity can be resisted via NF-κB activation and downstream survival signals in head and neck squamous cell carcinoma (HNSCC). Toxicities accompanying NF-κB signaling inhibition necessitate other methods of targeting the pathway. To search for novel targets affecting NF-κB signaling, Hu and colleagues performed a RNAi screen targeting the kinome and druggable genomic targets in a HNSCC cell line expressing NF-κB promoter response elements in tandem with a β-lactamase reporter gene. The screen revealed that G2/M checkpoint kinases WEE1 and cyclin dependent kinase 1 (CDK1) promote NF-κB signaling. WEE1 inhibition using AZD1775 disrupts WEE1 interactions with IκB kinase (IKK)α/β and RELA, reducing IKKα/β and RELA phosphorylation and NF-κB signaling. Correspondingly, AZD1775 augments TNFα-mediated HNSCC cell death, including in HNSCC radiotherapy in vivo. WEE1 and CDK1 expression levels and disease outcome correlations are dependent on human papillomavirus (HPV) infection status, suggesting HPV influences the signaling pathway. In addition to unveiling a novel signaling axis, this study presents a new potential way in which NF-κB can be therapeutically targeted in HNSCC.Decreased SIN3A expression in human breast cancer cell lines enhances tumorigenic phenotypes in vitro and tumor progression in vivo. However, how SIN3A abundance in breast tumors compares to its abundance in normal breast tissue is unknown, as is how SIN3A expression is regulated in breast tumors. To address these questions, Davenport and colleagues assessed SIN3A mRNA and corresponding protein levels in patients and validated these findings using public data sets (TCGA and METABRIC). The authors found that the SIN3A mRNA 3′ untranslated region harbors a sequence complementary to that of oncogenic miR-183, and that miR-183 silences SIN3A expression and enhances expression of genes that SIN3A transcriptionally represses. Accordingly, miR-183 expression negatively correlates with breast cancer patient survival and induces breast cancer cell migration and invasion in vitro and pulmonary metastasis in vivo through SIN3A downregulation. Overall, the novel miR-183-mediated regulation of SIN3A expression uncovered in this study could be used to inform metastatic breast cancer intervention strategies moving forward.Radiation and temozolomide (TMZ), the standard treatments for glioblastoma (GBM), are known to induce a non-proliferative, senescent phenotype in surviving GBM cells. However, GBM cells often eventually escape treatment-induced senescence, leading to residual tumor outgrowth and GBM recurrence. To assess whether recently discovered senolytic drugs can eliminate senescent GBM cells, Rahman and colleagues tested the effects of 10 senolytic drugs on 12 human GBM cell lines treated with radiation or TMZ. The authors found that senolytic drugs targeting BCL-XL, as well as siRNA-mediated BCL-XL abrogation, enhance senescent GBM cell death. The results of the study suggest that pharmacological BCL-XL inhibition could augment antitumor effects of radiation and TMZ treatment, and potentially inhibit residual GBM cell outgrowth and disease recurrence.

Blocking iASPP/Nrf2/M-CSF axis improves anti-cancer effect of chemotherapy-induced senescence by attenuating M2 polarization

The complex interaction between cancer cells and the immune microenvironment is a central regulator of tumor growth and the treatment response. Chemotherapy-induced senescence is accompanied by the senescence-associated secretion phenotype (SASP). However, the mechanisms underlying the regulation of the SASP remain the most poorly understood element of senescence. Here, we show that nuclear erythroid factor 2-like factor 2 (Nrf2), a master antioxidative transcription factor, accumulates upon doxorubicin-induced senescence. This is due to the increased cytoplasmic Inhibitor of Apoptosis Stimulating Protein of P53, iASPP, which binds with Keap1, interrupting Keap1/Nrf2 interaction and promoting Nrf2 stabilization and activation. Activated Nrf2 transactivates a novel target gene of SASP factor, macrophage colony-stimulating factor (M-CSF), which subsequently acts on macrophages and induces polarization from M1 to M2 via a paracrine mechanism. Genetic inhibition of iASPP-Nrf2 suppresses the growth of apoptosis-resistant xenografts, with further analysis revealing that M-CSF/M-CSFR-regulated macrophage polarization is critical for the functional outcomes delineated above. Overall, our data uncover a novel function of iASPP-Nrf2 in skewing the immune microenvironment under treatment-induced senescence. Targeting the iASPP-Nrf2 axis could be a powerful strategy for the implementation of new chemotherapy-based therapeutic opportunities.

Engineered Aging Cardiac Tissue Chip Model for Studying Cardiovascular Disease

Due to the rapidly growing number of older people worldwide and the concomitant increase in cardiovascular complications, there is an urgent need for age-related cardiac disease modeling and drug screening platforms. In the present study, we developed a cardiac tissue chip model that incorporates hemodynamic loading and mimics essential aspects of the infarcted aging heart. We induced cellular senescence in H9c2 myoblasts using low-dose doxorubicin treatment. These senescent cells were then used to engineer cardiac tissue fibers, which were subjected to hemodynamic stresses associated with pressure-volume changes in the heart. Myocardial ischemia was modeled in the engineered cardiac tissue via hypoxic treatment. Our results clearly show that acute low-dose doxorubicin treatment-induced senescence, as evidenced by morphological and molecular markers, including enlarged and flattened nuclei, DNA damage response foci, and increased expression of cell cycle inhibitor p16^INK4a, p53, and ROS. Under normal hemodynamic load, the engineered cardiac tissues demonstrated cell alignment and retained cardiac cell characteristics. Our senescent cardiac tissue model of hypoxia-induced myocardial infarction recapitulated the pathological disease hallmarks such as increased cell death and upregulated expression of ANP and BNP. In conclusion, the described methodology provides a novel approach to generate stress-induced aging cardiac cell phenotypes and engineer cardiac tissue chip models to study the cardiovascular disease pathologies associated with aging.

Abstract PS5-43: Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity

Abstract IntroductionClassically, cancer therapy approaches lead to tumor cell death or an alternative fate such as senescence. Senescent cells remain viable but without proliferation markers. Senescent cells secrete factors with diverse actions that influence surrounding cells, the extracellular matrix (ECM) and the immune system, a phenomenon termed the senescence-associated secretory phenotype (SASP). Cyclin-dependent kinase 4/6 inhibitors (CDKi) are game-changers in the therapy of metastatic hormone receptor-positive, HER2-negative breast cancer. A potential mechanism of the action of these agents is induction of senescence in breast tumor cells, going beyond cell cycle arrest. However, senescence can be studied mainly using invasive biopsies, and therefore the prevalence and importance of senescence in patients are largely unknown. Moreover, only few circulating biomarkers exist to predict activity or measure CDKi effects. In order to develop a correlative liquid biopsy for CDKi activity, we have analyzed epigenetic (methylation) changes following therapy-induced senescence in vitro. We sought to identify (un)methylated loci that will be affected by different approaches and later can be discovered in circulating cell-free DNA. MethodsWe have treated luminal breast cancer MCF-7 cells with Doxorubicin, CDKi (Palbociclib) or by irradiation. This protocol resulted in up to 80% of cells with senescence-related beta-galactosidase activity. DNA methylation was profiled using Illumina Infinium MethylationEPIC 850K BeadChip. Differentially methylated loci (mDNA) were identified using GenomeStudio and Minfi. Analyses were done using GREAT, CSGene, and Reactome databases. Methylation age was analyzed using the Horvath Methylation Calculator. Statistical significance was defined as p<0.05, q<0.1. ResultsSignificantly differentially methylated sites (in comparison to untreated cells) were revealed as following: 9111 sites in Doxorubicin treated cells, 3828 sites in Palbociclib treated cells, and 694 sites in irradiated cells. These loci comprise 1%, 0.45%, and 0.08% of analyzed methylation sites, respectively. 324 loci were similarly changed following the three treatment options ('common sites'). We found that the 'geographic' distribution of intragenic and intergenic methylation sites (5' UTR, gene body etc.) in all treated cells was similar. Gene set over-representation analysis revealed that sites associated with genes of the 'collagen metabolic process' set were significantly altered in drug-treated cells. Pathway investigation of the 324 'common sites' revealed that ECM-related 'focal adhesion assembly' is the most significant pathway involved. Specific analysis of senescence-related gene sets showed that 15.1% (76/503) of senescence genes changed after Doxorubicin treatment, 6.2% (31/503) after Palbociclib and 1% (5/503) after irradiation. Pathway analysis of these genes showed that drugs affected the 'oxidative stress-induced senescence' pathway, while irradiated cells had SASP-related genes affected.Despite the above mentioned methylation changes, age calculation based on methylation clock showed that all samples had similar age (6), regardless of manipulation. ConclusionSingle treatment of MCF-7 cells with known senescence inducers results in changes in methylation patterns. A significant number of common loci changed following all types of treatments, suggesting them as potential surrogate loci of senescence. Also, we have reproduced the well-known interplay between collagen and senescence/SASP.This preliminary data sheds light on epigenetic changes following treatment-induced senescence. Further studies are needed to validate whether these methylation changes can be found in vivo in tumors and in patients’ cell-free DNA following therapy with CDKi. Citation Format: Albert Grinshpun, Anatoli Kustanovich, Joshua Moss, Yael Gabai, Yuval Dor, Ittai Ben-Porath. Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-43.

Senescence, Necrosis, and Apoptosis Govern Circulating Cell-free DNA Release Kinetics

The kinetics of circulating cell-free DNA (cfDNA) release may provide a real-time assessment of induced cell death. However, there is a limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. Utilizing multiple invitro and invivo preclinical models, we show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Interestingly, we identify treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that can counteract its release. Necrosis is the predominant cell death mechanism that consistently contributes to cfDNA release in response to ionizing radiation, and, surprisingly, apoptosis plays a comparatively minor role in some tumors. Based on our results, we propose a model to explain cfDNA release from cells over time, with important implications for future studies.

Biological Rationale for Circulating Cell-Free DNA Release

The kinetics of circulating cell-free (cf)DNA release may provide a real-time assessment of induced cell death. However, there is limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. We utilized multiple in vitro and in vivo preclinical models to show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Using cfDNA from head and neck squamous cell carcinoma (HNSCC) cell lines and fibroblasts grown in culture, or as mouse xenografts, we identified treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that counteracts its release. Necrosis was the predominant cell death mechanism that consistently contributed to cfDNA release from HNSCC in response to ionizing radiation, while, surprisingly, apoptosis played a comparatively minor role in some tumours. Based on these results, we propose a generalizable model to explain cfDNA release from cells over time. These findings will have important implications for future studies evaluating the utility of measuring cfDNA kinetics in clinical settings.

Acquired Stem Cell Properties In Therapy-Induced Senescence Of Lymphomas and Acute Leukemias In Vitro and In Vivo

IntroductionPremature senescence is a permanent proliferative arrest that occurs in response to oncogenic signaling or DNA-damaging chemotherapy. Although tumor cell senescence has been recognized as a prognostically relevant contribution to long-term outcome post-therapy in hematological tumor models, therapeutic utilization of senescence is still hampered by an incomplete understanding of biological properties and long-term fate of senescent tumor cells in patients. Interestingly, senescence-regulating factors have recently been shown to limit reprogramming of somatic cells to pluripotency, and to protect stem cell compartments from premature exhaustion. Hence, we explore here whether cellular senescence and stemness may functionally overlap, thereby potentially equipping arrested cells with latent self-renewing potential. MethodsStem cell-related features (stem cell gene signatures, Sca-1 expression, ALDH and ABC transporter activity) were analyzed in primary apoptosis-blocked Eµ-myc transgenic B-cell lymphomas, which enter treatment-induced senescence (TIS) in response to standard antineoplastic agents. Several (e.g. Suv39h1- or p53-based) genetic models were established, in which TIS occurred in a conditional and reversible fashion. Clonogenicity, proliferative and repopulating assays were performed in vitro and in vivo, comparing individual lymphomas that grew out of senescence (“Previously Senescent”, PS) with matched lymphoma cells that equally received chemotherapy, but were incapable of entering TIS (“Never Senescent”, NS). In a mouse model of T-cell acute lymphoblastic leukemia (T-ALL), stem cell markers and tumor initiating potential were assessed in a flow-sorted non-self-renewing leukemia cell population after senescence induction by chemotherapy. Human hematological cancer cell lines and tumor samples obtained from B-cell lymphoma and acute myeloid leukemia (AML) patients were analyzed for stem cell features after exposure to senescence-inducing chemotherapy in vitro. ResultsSenescent mouse lymphomas were strongly skewed towards an increased expression of an adult tissue stem cell signature, distinct stem cell markers and functional stemness properties. Upon release from conditional senescence, PS cells resumed proliferation and rapidly exceeded the proliferative, clonogenic and tumor-initiating capacity of NS cells. Interrogation of self-renewal-relevant cascades revealed activation of and dependence on canonical Wnt signaling in senescence, as blocking of this pathway reduced the growth of PS, but not NS cells. Moreover, TIS-related stemness occurred independent of secretable factors. Strikingly, in a murine T-ALL model, temporary senescence enforcement re-programmed non-stem leukemia cells into leukemia stem cells, allowing PS bulk leukemia cells to de novo initiate leukemias in recipient mice. These results were supported by consistent findings in human hematological cancer cell lines as well as primary human B-cell lymphoma and acute myeloid leukemia samples. ConclusionsOur findings uncover senescence-associated stemness as a detrimental capability which is latently enriched for by chemotherapy in lymphoma and leukemia. The aggressive growth potential might become evident when senescent cells occasionally acquire alterations that allow them to re-enter the cell-cycle, thereby unleashing the tumor-promoting potential of a biological program so far considered to operate as a tumor-suppressive mechanism. However, targeted intervention at stemness-related signaling cascades in senescence may open novel therapeutic options for apoptosis-resistant lymphoma and leukemia. Disclosures:No relevant conflicts of interest to declare.

Senescence, a new concept in pathologic response evaluation of rectal carcinomas (RC) after neoadjuvant treatment.

e21021 Background: Available protocols of response evaluation after therapy are based on the presence of viable cells, fibrosis and necrosis. Senescence is an irreversible cell cycle arrest that can be activated by different kinds of stress like DNA damage and cytotoxic drugs. Senescent cells are histologically very similar to viable neoplasic cells and usually evaluated alike. Our hypothesis is that neoadjuvant treatment can induce senescence as part of the therapeutic response and its presence may impact the prognosis. Methods: Tumor samples of 45 consecutive patients (pts) with RC treated between sept/02 and feb/2007 with fluorouracil-based chemotherapy and neoadjuvant radiotherapy, and of 33 non-treated colon cancer pts (controls), were selected. p53 and P16-ki67 double immunostaining were retrospectively evaluated in endoscopic-pretreatment biopsies, post-therapy specimens, and controls. P16+/ki67- malignant glands were considered as senescent-like glands. SPSS v20.0 (Kruskal-Wallis, Cox regression and log-rank test) was employed for analysis. Results: After a median follow up of 60 months 13 pts had relapsed. Significant differences in the percentage of senescent-like glands were observed between treated carcinomas and their pre-treatment samples (p=0.0001) and also with the colonic-non-treated carcinomas (p=0.0001). A tendency toward a better disease free survival (DFS) (p=0.236) in those patients with more than 30% of senescent glands after treatment was observed. No differences in p53 were found between the 3 groups. However, low levels of p53 expression in pretreatment-rectal biopsies correlate with a better pathologic response (GR) (p=0.029), and better DFS (p=0.0958). Conclusions: The number of senescent-like glands increases after neoadjuvant therapy. There was a tendency for a better DFS in the pts with treatment-induced senescence. Moreover, an inverse correlation between p53 expression in pretreated endoscopic-samples and pathologic response and DFS was observed. Further and larger studies should be performed to confirm the prognostic implications of evaluating senescence markers in treated rectal carcinomas.

Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.

Oncogene- and Radiation-induced Senescence on Human Colonic Neoplasm and sm 22 as a Promising Marker

Purpose: Cellular senescence, which is a response to nonlethal stress, results in irreversible growth arrest with a distinct morphological and biochemical phenotype. Current research suggests that cellular senescence is a self-defense mechanism to suppress cancer progression. Treatment-induced senescence represents a novel functional target that may improve cancer therapy efficacy and help predict its prognosis. The aim of this study was to identify and validate useful biomarkers for detection of cellular senescence in human colorectal cancer tissue. Methods: The tissues from 36 patients who underwent colonoscopy and were found to have adenoma and adenocarcinoma at Korean cancer center hospital (KCCH). beta-galactosidase stain, western blotting and immunohistochemistry on proteins associated with cellular senescence were performed on normal mucosa, adenoma and adenocarcinoma tissue. Four rectal cancer patients who were supposed to undergo radiation therapy, level of senescence were evaluated on normal, pre-radiation cancer and post-radiation cancer tissue. This study was approved by IRB of KCCH. Results: Senescence was induced in human colorectal adenoma, whereas it was attenuated in human colorectal carcinomas in beta-galactosidase stain and p16 protein assay by western blotting. In addition, sm 22 is correlated with p16. In human rectal cancer, senescence was induced by radiation therapy. Conclusion: In conclusion, oncogene-induced senescence and radiationinduced senescence were shown in human colorectal cancer tissue, and sm 22 could be a potential marker for detection of cellular senescence induced by oncogene and radiation.

Radiation-induced cell death mechanisms

The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.

Senescence-Associated Exosome Release from Human Prostate Cancer Cells

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

Accelerated senescence: An emerging role in tumor cell response to chemotherapy and radiation

Treatment of malignancies with chemotherapeutic drugs and/or radiotherapy is designed to eliminate the disease by depriving the tumor cell of its reproductive potential. Frequently, the desired effect of cell killing is achieved through the promotion of apoptosis; however, accumulating evidence suggests that apoptosis may not be the exclusive or even primary mechanism whereby tumor cells lose their self-renewal capacity after radiation or drug treatment, particularly in the case of solid tumors. While failure to undergo apoptosis in response to chemotherapeutic drugs or radiation may represent a mechanism of drug and radiation resistance, particularly in the case of leukemias and lymphomas, it is gradually being recognized that in the case of solid tumors, loss of reproductive capacity can occur through alternative pathways including reproductive cell death or mitotic catastrophe, through autophagic cell death, and as described below, through a terminally arrested state similar to replicative senescence. Studies building upon the phenomenon of replicative senescence in normal cells approaching the limit of their reproductive potential have identified a comparable senescence-like arrest as a component of the tumor cell response to chemotherapeutic drugs and radiation. This response, which has been termed “premature senescence”, “senescence-like growth arrest”, “stress-induced premature senescence”, and “accelerated senescence”, can also result from supraphysiological mitogenic signaling, sub-optimal culture conditions, and ectopic expression of oncogenes. Here, we will use the term “accelerated senescence” in our consideration of the morphological, biochemical, and molecular aspects of treatment-induced senescence, its relationship to classical replicative senescence, its prevalence in clinical specimens and the implications of accelerated senescence for the outcome of cancer therapy.

Tumor senescence as a determinant of drug response in vivo.

It has become apparent in the last few years that induction of apoptosis is insufficient to account for the therapeutic effect of anticancer agents. Chemotherapy and radiation induce two other antiproliferative responses in tumor cells, cell death through mitotic catastrophe and terminal growth arrest through the program of senescence. Different types of tumor cells were found to develop the senescent phenotype upon drug treatment in vitro and in vivo. Cell culture studies demonstrated that this phenotype marks tumor cells that survive drug exposure but lose the ability to proliferate, and that such cells activate multiple growth-inhibitory genes. A recent study demonstrated that senescence, along with apoptosis, is a key determinant of in vivo response to chemotherapy in a transgenic mouse model of B-cell lymphoma. This review discusses the results of the latter study, as well as the differences between the genetic determinants of treatment-induced senescence in murine lymphoma and in human solid tumor cells.

Backup Plan: Senescence fights tumors when apoptosis fails (Cancer; Senescence)

Treating cancer often seems more like gambling than science. Some tumors yield to a particular chemotherapeutic agent, whereas others of the same type thrive in spite of the drug. Genetic differences among tumors are thought to underlie these divergent responses. Chemotherapy works in part by triggering cell suicide, and mutations that disrupt this process--called apoptosis--can confer drug resistance. Now, researchers present evidence that cellular senescence--a program that permanently arrests cell growth--also influences treatment outcome, a finding that might lead to better therapies. Furthermore, the discovery bolsters the possibility that senescence promotes aging. "For many years, people believed that chemotherapy drugs damaged cells, which just blew up," says Scott Lowe, a cancer geneticist at Cold Spring Harbor Laboratory in New York and the study's senior author. But researchers now know that drugs disable cancer in part by triggering apoptosis, a normal process that eliminates potentially unruly cells. The compounds might also spur cellular senescence, but scientists have long questioned whether the process even occurs in animals, says Tyler Jacks, a cancer geneticist at the Massachusetts Institute of Technology in Cambridge. Although a few researchers have found evidence for senescence in live organisms and some have proposed that it fosters aging, the process's significance remains an open question (see "More Than a Sum of Our Cells" ). To investigate the role of senescence in chemotherapy, Schmitt and colleagues bred mice that produce lymphomas with defined mutations in apoptosis and senescence genes. Although the tumors in these animals are pathologically identical, their response to cyclophosphamide (CTX) varies. "Some can be cured, and others just laugh at the drug and come back," says Lowe. The researchers transplanted the tumors into mice and treated the animals with CTX. As expected, the compound requires apoptosis to deliver its blow. After drug treatment, mice that carried tumors with functional apoptosis machinery survived longer than did animals whose tumors couldn't trigger this cell death program. But senescence partially makes up for the loss of apoptosis. Tumors with genetic blocks to both apoptosis and senescence were even more deadly than tumors with defects in apoptosis alone. The results suggest that CTX quashes tumors by exploiting senescence as well as apoptosis. This study is the first to demonstrate that senescence can influence drug treatment outcome in animals, says cancer geneticist Igor Roninson of the University of Illinois, Chicago. Jacks adds, "There's an important need to understand how cancer cells respond to therapy," and until now, scientists have focused on apoptosis. "This work highlights the idea that another process is involved," he says. If we can understand the processes chemotherapeutic agents employ to stifle cancer, we can make better drugs and use them in a way that will be more effective, says Lowe. Furthermore, the fact that senescence genes affect tumor growth in mice lends support to the idea that senescence functions in whole animals, not just cultured cells, he adds: "We hope [that] this [finding] stimulates more excitement about the senescence program as a whole and about questions of its role in aging." --Christie Aschwanden C. A. Schmitt, J. S. Fridman, M. Yang, S. Lee, E. Baranov, R. M. Hoffman, S. W. Lowe, A senescence program controlled by p53 and p16 INK4a contributes to the outcome of cancer therapy. Cell 109 , 335-346 (2002). [Abstract] [Full Text] Further Reading I. B. Roninson, E. V. Broude, B.-D. Chang, If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist. Updates 4 , 303-313 (2001). [Abstract] (Full text is not available online)

If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells.

Inhibition of the program of apoptosis has been reported to have little or no effect on clonogenic survival after treatment with drugs or radiation in several tumor cell lines. A decrease in apoptosis is compensated in such cell lines by an increase in the fractions of cells that undergo permanent growth arrest with phenotypic features of cell senescence, or die through the process of mitotic catastrophe. Most of the tested tumor cell lines have retained the capacity of normal cells to undergo accelerated senescence after treatment with DNA-interactive drugs, ionizing radiation, or cytostatic agents. p53 and p21(Waf1/Cip1/Sdi1) act as positive regulators of treatment-induced senescence, but they are not required for this response in tumor cells. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may produce secreted proteins with potential tumor-promoting activities. The expression of such proteins is mediated at least in part by the induction of p21(Waf1/Cip1/Sdi1). The other anti-proliferative response of tumor cells is mitotic catastrophe, a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Mitotic catastrophe is induced by different classes of cytotoxic agents, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Mitotic catastrophe can also develop as a consequence of aberrant reentry of tumor cells into cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced senescence and mitotic catastrophe should assist in improving the efficacy and decreasing side effects of cancer therapy.