Senescence-associated Secretory Phenotype Research Articles

PGC1α-Inducing Senomorphic Nanotherapeutics Functionalized with NKG2D-Overexpressing Cell Membranes for Intervertebral Disc Degeneration.

Cellular senescence is a significant contributor to intervertebral disc aging and degeneration. However, the application of senotherapies, such as senomorphics targeting senescence markers and the senescence-associated secretory phenotype (SASP), remains limited due to challenges in precise delivery. Given that the natural killer group 2D (NKG2D) ligands are increased on the surface of senescent nucleus pulposus (NP) cells, the NKG2D-overexpressing NP cell membranes (NNPm) are constructed, which is expected to achieve a dual targeting effect toward senescent NP cells based on homologous membrane fusion and the NKG2D-mediated immunosurveillance mechanism. Then, mesoporous silica nanoparticles carrying a peroxisome proliferator-activated receptor-ɣ coactivator 1α (PGC1α)inducer (SP) are coated with NNPm (SP@NNPm) and it is found that SP@NNPm selectively targets senescent NP cells, and the SP cores exhibit pH-responsive drug release. Moreover, SP@NNPm effectively induces PGC1α-mediated mitochondrial biogenesis and mitigates senescence-associated markers induced by oxidative stress and the SASP, thereby alleviating puncture-induced senescence and disc degeneration. This dual-targeting nanotherapeutic system represents a novel approach to delivery senomorphics for disc degeneration treatment.

Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype

Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.

Persistent epigenetic signals propel a senescence-associated secretory phenotype and trained innate immunity in CD34+ hematopoietic stem cells from diabetic patients

BackgroundDiabetes-induced trained immunity contributes to the development of atherosclerosis and its complications. This study aimed to investigate in humans whether epigenetic signals involved in immune cell activation and inflammation are initiated in hematopoietic stem/progenitor cells (HSPCs) and transferred to differentiated progeny.Methods and resultsHigh glucose (HG)-exposure of cord blood (CB)-derived HSPCs induced a senescent-associated secretory phenotype (SASP) characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27genes, upregulation of NFkB-p65 transcription factor and increased secretion of the inflammatory cytokines TNFα and IL6. Chromatin immunoprecipitation assay (ChIP) of p65 promoter revealed that H3K4me1 histone mark accumulation and methyltransferase SetD7 recruitment, along with the reduction of repressive H3K9me3 histone modification, were involved in NFkB-p65 upregulation of HG-HSPCs, as confirmed by increased RNA polymerase II engagement at gene level. The differentiation of HG-HSPCs into myeloid cells generated highly responsive monocytes, mainly composed of intermediate subsets (CD14hiCD16+), that like the cells from which they derive, were characterized by SASP features and similar epigenetic patterns at the p65 promoter. The clinical relevance of our findings was confirmed in sternal BM-derived HSPCs of T2DM patients. In line with our in vitro model, T2DM HSPCs were characterized by SASP profile and SETD7 upregulation. Additionally, they generated, after myeloid differentiation, senescent monocytes mainly composed of proinflammatory intermediates (CD14hiCD16+) characterized by H3K4me1 accumulation at NFkB-p65 promoter.ConclusionsHyperglycemia induces marked chromatin modifications in HSPCs, which, once transmitted to the cell progeny, contributes to persistent and pathogenic changes in immune cell function and composition.

ScRNA-Seq: First Atlas and Cellular Landscape of Lacrimal Sac: Implications in Primary Acquired Nasolacrimal Duct Obstruction Pathogenesis.

The aim of this study was to describe the transcriptional changes of individual cellular components in the lacrimal sac in patients with primary acquired nasolacrimal duct obstruction (PANDO) and attempt to construct the first lacrimal sac cellular atlas to elucidate the potential mechanisms that may drive the disease pathogenesis. Lacrimal sac samples were obtained intra-operatively during the endoscopic dacryocystorhinostomy (EnDCR) procedure from five patients. Single-cell RNA sequencing was performed to analyze each individual cell population including epithelial and immune cells during the early inflammatory and late inflammatory phases of the disease. Eleven cell types were identified among 25,791 cells. T cells and B cells were the cell populations with the greatest variation in cell numbers between the two phases and were involved in immune response and epithelium migration-related pathways. The present study showed that epithelial cells highly expressed the genes of senescence-associated secretory phenotype (SASP) and were involved in influencing the inflammation, neutrophil chemotaxis, and migration during the late inflammatory stage. Enhanced activity of CXCLs-CXCRs between the epithelial cells and neutrophils was noted by the cell-cell communication analysis and is suspected to play a role in inflammation by recruiting more neutrophils. The study presents a comprehensive single-cell landscape of the lacrimal sac cells in different phases of PANDO. The contribution of T cells, B cells, and epithelial cells to the inflammatory response, and construction of the intercellular signaling networks between the cells within the lacrimal sac has further enhanced the present understanding of the PANDO pathogenesis.

Enteric Nervous System Alterations in Inflammatory Bowel Disease: Perspectives and Implications

The enteric nervous system (ENS), consisting of neurons and glial cells, is situated along the gastrointestinal (GI) tract’s wall and plays a crucial role in coordinating digestive processes. Recent research suggests that the optimal functioning of the GI system relies on intricate connections between the ENS, the intestinal epithelium, the immune system, the intestinal microbiome, and the central nervous system (CNS). Inflammatory bowel disease (IBD) encompasses a group of chronic inflammatory disorders, such as Crohn’s disease (CD) and ulcerative colitis (UC), characterized by recurring inflammation and damage to the GI tract. This review explores emerging research in the dynamic field of IBD and sheds light on the potential role of ENS alterations in both the etiology and management of IBD. Specifically, we delve into IBD-induced enteric glial cell (EGC) activation and its implications for persistent enteric gliosis, elucidating how this activation disrupts GI function through alterations in the gut–brain axis (GBA). Additionally, we examine IBD-associated ENS alterations, focusing on EGC senescence and the acquisition of the senescence-associated secretory phenotype (SASP). We highlight the pivotal role of these changes in persistent GI inflammation and the recurrence of IBD. Finally, we discuss potential therapeutic interventions involving senotherapeutic agents, providing insights into potential avenues for managing IBD by targeting ENS-related mechanisms. This approach might represent a potential alternative to managing IBD and advance treatment of this multifaceted disease.

Senescent adipocytes and type 2 diabetes - current knowledge and perspective concepts.

Among civilization diseases, the number of individuals suffering from type 2 diabetes (T2DM) is expected to increase to more than a billion in less than 20 years, which is associated with, e.g., populational aging, poor diet, sedentary lifestyle, genetic predispositions, and immunological factors. T2DM affects many organs and is characterized by insulin resistance, high glucose levels, and adipocyte dysfunction, which are related to senescence. Although this type of cellular aging has beneficial biological functions, it can also act unfavorable since senescent adipocytes resist apoptosis, enhance cytokine secretion, downregulate cell identity genes, and acquire the senescence-associated secretory phenotype that renders a more oxidative environment. Opposing T2DM is possible via a wide variety of senotherapies, including senolytics and senomorphics; nevertheless, further research is advised to expand therapeutic possibilities and benefits. Consequences that ought to be deeply researched include secretory phenotype, chronic inflammation, increasing insulin resistance, as well as impairment of adipogenesis and functioning of adipocyte cells. Herein, despite reviewing T2DM and fat tissue senescence, we summarized the latest adipocyte-related anti-diabetes solutions and suggested further research directions.

Kdm6a-CNN1 axis orchestrates epigenetic control of trauma-induced spinal cord microvascular endothelial cell senescence to balance neuroinflammation for improved neurological repair

Cellular senescence assumes pivotal roles in various diseases through the secretion of proinflammatory factors. Despite extensive investigations into vascular senescence associated with aging and degenerative diseases, the molecular mechanisms governing microvascular endothelial cell senescence induced by traumatic stress, particularly its involvement in senescence-induced inflammation, remain insufficiently elucidated. In this study, we present a comprehensive demonstration and characterization of microvascular endothelial cell senescence induced by spinal cord injury (SCI). Lysine demethylase 6A (Kdm6a), commonly known as UTX, emerges as a crucial regulator of cell senescence in injured spinal cord microvascular endothelial cells (SCMECs). Upregulation of UTX induces senescence in SCMECs, leading to an amplified release of proinflammatory factors, specifically the senescence-associated secretory phenotype (SASP) components, thereby modulating the inflammatory microenvironment. Conversely, the deletion of UTX in endothelial cells shields SCMECs against senescence, mitigates the release of proinflammatory SASP factors, and promotes neurological functional recovery after SCI. UTX forms an epigenetic regulatory axis by binding to calponin 1 (CNN1), orchestrating trauma-induced SCMECs senescence and SASP secretion, thereby influencing neuroinflammation and neurological functional repair. Furthermore, local delivery of a senolytic drug reduces senescent SCMECs and suppresses proinflammatory SASP secretion, reinstating a local regenerative microenvironment and enhancing functional repair after SCI. In conclusion, targeting the UTX-CNN1 epigenetic axis to prevent trauma-induced SCMECs senescence holds the potential to inhibit SASP secretion, alleviate neuroinflammation, and provide a novel treatment strategy for SCI repair.

Abstract 2954: Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT

Abstract Introduction: Androgen Deprivation Therapy (ADT) is a highly effective treatment for prostate cancer. However, resistance to ADT develops in most patients, leading to a lethal state of castration-resistant prostate cancer (CRPC). Senescent prostate cancer (SPC) cells can arise after ADT and contribute to therapy resistance. SPCs can restore replicative function and alter the tumor microenvironment through pro-tumorigenic senescence-associated secretory phenotype (SASP). We aimed to characterize changes in SASP in prostate cancer cell lines and serum samples from patients receiving ADT. Methods: SASP factors (Luminex 100/200 System, xMAP Instruments) were analyzed in patients undergoing ADT. To correlate SASP with the sensitivity to ADT, we sought to evaluate the regulation of SASP in vitro using PCa cell lines. The PC3 (AR resistant) and LNCaP (AR sensitive) cell lines were used in the experiments. We performed an analysis of archival tumor tissue (paired) from 18 patients with or without alterations in DNA repair genes (BRCA1, BRCA2, and CDK12). We performed immunohistochemical (IHC) staining of known SASP markers, such as P16, uPAR, gamma-H2AX, and Lamin B1. Results: In a cohort of patients (n=8) with pre and post-ADT matched status, we demonstrate increased levels of SASP factors such as IL-6 (pre-ADT: 1.54±0.24 pg/ml, post-ADT: 5.23±1.3 pg/ml p=0.001), IL-7 (pre-ADT: 3.11±0.6 pg/ml, post-ADT: 13.5±2.7 pg/ml p=0.01), IL-1 alpha (pre-ADT: 1.06±0.3 pg/ml, post-ADT: 15.3±3.5 pg/ml p=0.001), IL-15 (pre-ADT: 15.6±3.1 pg/ml, post-ADT: 37.1±4.8 pg/ml p=0.002), MIF (pre-ADT: 1036±67.6pg/ml, post-ADT: 2362±55.5 pg/ml p=0.003), and IL-15 (pre-ADT: 5±2.1 pg/ml, post-ADT: 37.5±5.1 pg/ml p=0.002). In vitro, the AR-responsive LNCaP cell line, exposed to AR blockade, showed similar findings in upregulating IL-6, IL-7, and IL-15 but not the PC3 cell line. We also demonstrate measurable differences between the expression of u-PAR, gamma-2AX, p16, and loss of Lamin B1 expression in tumor specimens from patients with advanced castration-resistant prostate cancer (n=18). The expression of p16 was significantly upregulated post-treatment irrespective of DDR status (p=0.0417). Other SASP factors showed a trend in upregulation but were insignificant (u-PAR p=0.056, gamma-H2AX p=0.62, Lamin B1 p=0.72) Conclusion: These results provide evidence of systemic increases in SASP-related cytokines following treatment with ADT. The in vitro results suggest that secretion of SASP is promoted by an AR-mediated mechanism. Preliminary results from tumor specimens suggest increased senescence marker p16 expression induced by therapy. Ongoing experiments are further characterizing the senescence markers in prostate tumor specimens and correlated to treatment effect. Citation Format: Maximilian Schwermann, Matthew Hadfield, Shaolei Lu, Praveen Srinivasan, Vida Tajiknia, William MacDonald, Andrew George, Shengliang Zhang, Leiqing Zhang, Kimberly Meza, Andre De Souza, Dragan Golijanin, Elias Hyams, Galina Lagos, Sheldon Holder, Anthony Mega, John Sedivy, Howard Safran, Wafik El-Deiry, Benedito Carneiro. Androgen deprivation therapy (ADT) and senescence-associated secretory phenotype (SASP) in vitro: Correlation with SASP in tumor specimens as well as in the serum of patients after ADT [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 2954.

Abstract 3452: Senescence-associated secretory phenotype (SASP) aging index is associated with cancer risk in the Atherosclerosis Risk in Communities (ARIC) Study

Abstract Background: The accumulation of senescent cells, which release SASP proteins damaging neighboring cells, contributes to aging and age-related diseases, such as cancer. Therefore, SASP proteins could be used as biomarkers for individuals’ aging process, but no studies have tested if SASP protein-based aging index is associated with cancer risk. We created a SASP-aging index based on the 72 SASP proteins reported in Tanaka et al. [2018] and examined its association with cancer risk in the ARIC study. Methods: ARIC is an ongoing cohort of White and Black females and males initiated in 1987, followed for cancer until 2015 (median follow-up=17.3 yrs). At Visit 2 (1990-92), 5000 plasma proteins were measured using SomaScan – an aptamer-based assay in 10,834 participants (aged 46-70 yrs, 55% female, 76% White): 3,347 participants developed cancer during follow-up and 7,487 participants stayed cancer-free. In the training set of 67% randomly selected cancer-free participants, we constructed the SASP-aging index by applying Ridge regression to train the 72 SASP proteins against chronological age. The SASP-aging index was validated in the remaining 33% of cancer-free participants (test set). We calculated age acceleration as residuals after regressing the SASP-aging index on age in participants after excluding the training set. We used Cox proportional hazards regression to estimate hazard ratio (HR) and 95% confidence intervals (CI) for the risk of any type of cancer per 5 years increase in age acceleration, employing Barlow’s method [1994] to account for the exclusion of the training set. HR was adjusted for age, sex, race, center, education, BMI, smoking status, alcohol intake, aspirin use, diabetes, and eGFR. We also estimated HRs for obesity-related, smoking-related, and the most common cancers (breast, prostate, colorectal, and lung). Results: In the test set, Pearson correlation coefficient between the SASP-aging index and age was 0.50. Age acceleration was significantly associated with the risk of any type of cancer [HR (95% CI) per 5 years=1.21 (1.08-1.36)]. There was a suggestion of a higher HR for any type of cancer in females [1.26 (1.07-1.48)] than males [1.18 (1.00-1.39)] [p-interaction=0.08], but HRs were similar across race and smoking status [p-interaction>0.50]. Age acceleration was significantly associated with the risk of obesity-related cancers [1.36 (1.20-1.54)]. A significant association was also found with smoking-related cancers [1.43 (1.24-1.65)] and this association remained significant even among never smokers [1.39 (1.07- 1.82)]. Age acceleration was also significantly associated with colorectal [1.34 (1.03-1.75)] and lung cancer risk [1.63 (1.31-2.02)] but not with breast or prostate cancer risk. Conclusion: SASP protein-based aging index shows promise as a potential biomarker for cancer risk. Funding: NHLBI, NCI, NPCR Citation Format: Shuo Wang, Zexi Rao, Anne H. Blaes, Josef Coresh, Corinne E. Joshu, James S. Pankow, Bharat Thyagarajan, Peter Ganz, Elizabeth A. Platz, Weihua Guan, Anna Prizment. Senescence-associated secretory phenotype (SASP) aging index is associated with cancer risk in the Atherosclerosis Risk in Communities (ARIC) Study [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 3452.

Abstract 2969: Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models

Abstract Clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. The use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform unique in its ability to be selectively active in the context of diseased tissues, but not normal tissues. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Since our CAB technology is currently being tested as a cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. In our previous report, we demonstrated several novel senescence-specific surface antigens upregulated in senescent cells (1). CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions in in vitro binding assays. In in vitro antibody-dependent cell-mediated cytotoxicity assays, CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic condition. To study whether CAB antibody reduces senescent cell occurrence in vivo, we developed a unilateral ureteral obstruction (UUO) mouse model to induce senescent cells, fibrosis, and inflammation in mouse kidney. The mice treated with CAB antibodies had a significant reduction of senescent cells and inflammatory cells infiltrated in the renal cortex, compared to the mice treated with benchmark and isotype antibodies in UUO kidneys. Thus, the CAB antibody-based therapeutic is a promising strategy for senescent cell removal in an inflammatory microenvironment. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. (1). Chen J, Wang J, Liu H, Chang C, Boyle WJ, and Short JM. Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging. Cancer Res (2023) 83. Citation Format: Jian Chen, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, Jay M. Short. Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models [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 2969.

Abstract 2963: Dihydromyricetin is a senomorphic agent to promote therapeutic outcome of age-related conditions including cancer and Alzheimer's disease

Abstract Aging is characterized by a progressive decline of organ function and gradual rise of susceptibility to multiple chronic disorders, with cancer being a primary cause of human morbidity and mortality worldwide. Cellular senescence represents a state of permanent cell cycle arrest and contributes to chronological aging and age-related diseases including Alzheimer's disease (AD), mainly through development of a distinct secretome termed the senescence-associated secretory phenotype (SASP). By screening a natural medicinal agent library, we noticed Rattan tea extract (RTE) has a prominent antiaging potential, wherein dihydromyricetin (DMY), the most abundant natural flavonoid in RTE, played a central role in suppressing the SASP expression of senescent cells. DMY enhanced total antioxidant capacity (T-AOC), increased catalase (CAT) and glutathione peroxidase (GSH-Px) activity, upregulated the expression of HO-1, NQO1, nuclear Nrf2 and phospho-ERK (p-ERK), but decreased malondialdehyde (MDA) level in senescent cells. Furthermore, DMY regulates PI3K/Akt/mTOR signaling pathway, NLRP3 inflammasome formation, the NF-κB complex activity and ER stress-response, each responsible for a full spectrum SASP development. Transcriptomic profiling suggested that DMY can dampen the genome-wide upregulation of pro-inflammatory factors produced by senescent cells, resulting in abrogated progression of cancer malignancy including elevated proliferation, migration, invasion and chemoresistance. Importantly, combination of DMY with classic chemotherapy significantly enhanced therapeutic efficacy, causing remarkable tumor regression and extending posttreatment survival. Alzheimer's disease (AD) is marked by the presence of amyloid plaques, neurofibrillary tangles, damaged neuronal macromolecules and redox sensitive ions. We found that DMY restrains inflammatory injury in microglial cells, reduces their apoptosis and inflammation, and inhibits TLR4 and MD2 expression. Results of small molecule-protein docking and pull-down assays indicated that DMY binds MD2 to suppress the formation of TLR4 complex. In AD mice, DMY improved the cognitive disorder of animals, suppressed inflammatory injury in brain and reduced TLR4 expression. Together, DMY represents a valid natural candidate that has senomorphic activity and can be applied for treatment of malignancies or neurodegenerative disorders such as AD, pathologies that frequently arise in late life of humans. Citation Format: Qixia Xu, Zi Li, Zhirui Jiang, Judith Campisi, Yu Sun. Dihydromyricetin is a senomorphic agent to promote therapeutic outcome of age-related conditions including cancer and Alzheimer's disease [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 2963.

Abstract 5861: CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch

Abstract Background: Cyclin-dependent kinase inhibitors (CDK4/6i) have revolutionized the therapy for estrogen receptor positive (ER+) breast cancer. Despite the response to CDK4/6i, resistance and recurrence are still a clinical reality, resulting in death due to breast cancer. G1 cell cycle arrest has been considered as the main mechanism of action of CDK4/6i in luminal breast cancer. However, the response to therapy does not correlate with the expression of CDK4/6 or key cell cycle proteins. It is also unclear whether CDK4/6i-induced senescence enhances the therapeutic response or mediates development of resistance. Experimental Procedures: We have developed drug-tolerant persisters resistant to CDK4/6 inhibitors (CDK4/6i-DTPs) in a panel of estrogen receptor positive (ER+) cells to explore the molecular mechanisms associated with resistance. Using different cell phenotype assays (i.e., cell viability/cell cycle, senescence/senescence reversibility and wound healing) as well as senescence-associated secretory phenotype (SASP) human cytokine array of 105 markers, we characterized the phenotype and secretome of these CDK4/6i DTPs. Summary: CDK4/6i-DTPs induced by abemaciclib, palbociclib and ribociclib (all from Selleckchem) demonstrated resistance to these drugs and dramatically slowed growth. The percentage of senescent cells reached 30-89% of the total population for all cell lines in all three drugs, whereas control parental cells exhibited around 10%. CDK4/6i DTPs also exhibited significant cell migration, and increased lysosomal phenotype compared to their parental sensitive cells. Further analysis with human cytokine array revealed that protein levels of growth differentiation factor 15 (GDF-15) are elevated in both cell lysates and conditioned-media of CDK4/6i DTPs, whereas Serpin E1 (Plasminogen Activator Inhibitor, Type I, PAI-1) was unique to secreted CDK4/6i DTPs. Interestingly, we did not observe changes in pro-inflammatory senescence-associated secretory phenotype (SASP) markers such as interleukins. Apart from being a SASP protein, GDF-15 is a mitochondrial cytokine (mitokine), suggesting a role in mitochondrial dysfunction-associated senescence (MiDAS). HMGB1, and LMNB1 are also down-regulated in CDK4/6-DTPs supporting the MiDAS phenotype. CDK4/6i-induced MiDAS appears to be transient, as CDK4/6i-DTPs reverted to the parental phenotype in drug-free culture media 20 days after the removal of CDK4/6i's. This included growth characteristics and sensitivity to CDK4/6i. This reversion of phenotype was observed in all five cell line models tested with all three drugs. Conclusions: CDK4/6-induces a mitochondrial dysfunction-associated senescence (MiDAS). This appears to be transient in nature as withdrawal of the drug results reacquisition of parental cell phenotype and drug-sensitivity. Targeting MiDAS pathway might be critical to improve efficacy of CDK4/6 inhibitors. Citation Format: Yesim Gokmen-Polar, Yuan Gu, Ling Ruan, Sunil S. Badve. CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch [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 5861.

Abstract 511: Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC

Abstract Cell cycle inhibitors have a long history as anti-cancer therapeutics in clinical applications. Uncontrolled and infinite cell cycles are hallmarks of cancer, which enable them to proliferate rapidly and survive. To target these key features of cancer cells, cell cycle inhibitors are used to arrest their active cell cycle and induce cell death. In this study, beyond the known primary effect on cell cycle control, we identified that an expected and a secondary effect in immune modulation by the cell cycle inhibitors. Anti-mitotic agents including Paclitaxel (microtubule stabilizer), Palbociclib (CDK4/6 inhibitor) and AZD1152 and GSK1070916 (Aurora Kinase B inhibitors) can also eliminate cancer cells via an alternative mechanism - the activation of STING signaling. Our results demonstrated that these anti-mitotic agents caused DNA damage and cytosolic DNA accumulation. The cytosolic DNA was captured by a cytosolic DNA sensor DDX41,which triggered STING-TBK1-IRF3/7 pathway to upregulate secretion of pro-inflammatory senescence-associated secretory phenotype (SASP) factors in cancer cells. In addition, we revealed that transcription of DDX41 was mediated by the transcription factor known ashypoxia-inducible factor (HIF). In HCC, hypoxia induced DDX41 expression through HIF-1,thus hypoxic HCC cells were more prone to STING activation and SASP production under mitotic stress induced by cell cycle inhibitors. In the tumor microenvironment, the SASP enhanced infiltration of immune cells into the tumor core to eliminate cancer cells. We also observed the additive effects of cell cycles inhibitors (Paclitaxel, Palbociclib, and AZD1152)with anti-PD-1 mAb to arrest growth of HCC in mouse models. In summary, this study exhibited the novel immune-mediated aspect of cell cycle inhibitors in suppressing tumor growth. Our data suggested the potential combination regimen of cell cycle inhibitors and currently available immunotherapy with promising result. Citation Format: Cerise Yuen Ki Chan, Po Yee Wong, Helen Do Gai Xue, Chi Ching Goh, Jacinth Wing Sum Cheu, Aki Pui Wah Tse, Misty Shuo Zhang, Carmen Chak Lui Wong. Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC [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 511.

Abstract 5941: Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer

Abstract PARP inhibitors (PARPi) targeting poly (ADP-ribose) polymerase are the first clinically approved drugs designed to apply synthetic lethality in BRCA1 mutant/deficient cancer. However, the treatment can cause reversion of BRCA mutation which results in drug resistance. To explore new strategies to improve antitumor efficacy of PARPi, we applied PARPi in combination with a small molecule NSC59984 for cancer therapy. NSC59984 is a small molecular targeting mutant p53 degradation and activating p73 via ROS-ERK2-MDM2 pathway. The treatments with radiation or hydrogen peroxide showed that NSC59984 enhanced DNA comet tails and gamma-H2AX, correlated to reduction of rad51 foci in cancer cells. These results suggest that NSC59984 impairs DNA damage repair via abrogation of homologous recombination (HR). We further applied the combinational treatment of NSC59984 and PARPi in BRCA1 wild-type ovarian cancer cells and found that NSC59984 synergized with PARPi to reduce cell viability, inhibit colony formation, and increase cell death. The combination treatment enhanced DNA damage and correlated with reduction of BRCA1 at the protein level. These results, taken together, suggest that the induction of BRCAness causes a synthetical lethality with PARPi in BRCA1 wild-type cancer cells. Cell cycle profiling showed that the cells were arrested at the G2/M phase in response to combinational treatment. At the G2 phase arrest, the high doses of the combinational drugs led to cell death via mitotic catastrophe, and the intermediate doses induced cellular senescence defined by senescence phenotypic hallmarks including senescence-associated beta-gal staining and a secretome consistent with senescence-associated secretory phenotype (SASP). Therapy-induced senescence (TIS) in cancer cells is considered as one of the mechanisms of tumor recurrence and drug resistance. To reduce the risk of TIS in treatment, we applied senolytic treatment to target senescent cells in the combinational treatment. The senolytic drug ABT263 treatment eliminated the senescent cancer cells from the combinational treatment. ABT263, NSC59984 + PARPi combinational treatment further reduced cell viability. Our study provides a rationale for small molecular compounds targeting HR deficiency in combination with PARPi to treat BRCA1wild-type ovarian cancer cells, and additional senolytic treatment may be required to limit resistance by removal of the TIS. Citation Format: Shengliang Zhang, Lanlan Zhou, Leiqing Zhang, Maximilian Pinho-Schwermann, Benedito Carneiro, John Sedivy, Wafik S. El-Deiry. Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer [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 5941.

Abstract 2962: Chemotherapy induced senescence drives peripheral neuropathy

Abstract Chemotherapy is a mainstay of cancer therapy. Unfortunately, while chemotherapy can profoundly impact disease free survival, it is often accompanied with devastating side effects including hair loss, bone loss, cognitive impairment (referred to as "brain fog"), and peripheral neuropathies. While many of these side effects may subside after the completion of chemotherapy, some persist for a lifetime, substantially compromising the quality of life for survivors. Particularly insidious is chemotherapy-induced peripheral neuropathy (CIPN), which can impair a patient's ability to care for themselves. Shockingly, in over 30% of patients, CIPN persists even after the cessation of chemotherapy, subjecting them to a lifetime of numbness and/or pain. Paclitaxel (PTX), a common treatment for breast cancer, induces CIPN in 61-92% of patients depending on the administered dose. Unfortunately, there are currently no preventative or curative measures for CIPN resulting in side effects that persist for months or even years after the cessation of chemotherapy. Considering chemotherapy can trigger cellular senescence, leading to the release of a diverse array of factors collectively known as the senescence-associated secretory phenotype (SASP) that are capable of negatively influencing neighboring cells. To determine if senescence played a role in CIPN we treated with PTX and found that animals displayed reduced axon innervation in the paws and diminished paw withdrawal responses during the von Frey assay, characteristic symptoms of CIPN. To determine if senescent drove PTX-induced CIPN, we utilized the INKATTAC (INK) mouse, a model in which a p16INK4a promoter drives an inducible suicide transgene to eliminate p16+ senescent cells following treatment with AP20187 (AP). Employing the INK mouse, we successfully prevented CIPN by eliminating PTX-induced senescent cells. Furthermore, through bone marrow (BM) transplant studies using BM from INK mice into wildtype recipients, we failed to prevent CIPN. This result establishes that senescent immune cells, do not contribute to CIPN. In an independent approach, we found that senolytic drugs (kill senescent cells), such as dasatinib and quercetin (DQ) or ABT737 (ABT), effectively prevented and reversed CIPN in mice. Additionally, since the p38MAPK pathway has been shown to regulate SASP mRNA stability, we treated with p38 and MK2 inhibitors and found that inhibition prevented CIPN in mice. To address the mechanism behind CIPN we have carried out single cell RNA sequencing (scRNA-seq) to identify the cell population senescing in response to chemotherapy. These analyses will allow us to understand the mechanisms that drive CIPN and may lead to new treatments for patients suffering from CIPN. Citation Format: Taylor Malachowski, Ganesh Kumar Raut, Xianmin Luo, Satarupa Mullick Bagchi, Sheila Stewart. Chemotherapy induced senescence drives peripheral neuropathy [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 2962.

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 2966: Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells

Abstract Diverse conventional cancer therapies, including genotoxic agents and cell cycle or cell division-targeting drugs, can trigger cancer cell senescence. Senescent cells (SnCs) induced by cancer therapy often display unrepaired chromosomal damage but persist indefinitely. SnCs remain metabolically active and display distinct alterations in transcription, secretion, and membrane protein expression/presentation. While initially viewed as a favorable outcome and likely tumor-suppressive mechanism, therapy-induced senescence has more recently been recognized as contributing to resistance, recurrence and metastasis. Adverse effects may be mediated in part by release of inflammatory mediators as components of the senescence-associated secretory phenotype (SASP). Recent studies have argued for reconsidering the potential benefits of therapy-induced senescence, especially in modulating the tumor microenvironment to potentiate anti-tumor immunity and potentiate immunotherapy. We have explored the potential for SnCs to serve as vaccines, building upon prior findings where we demonstrated the immunogenic senescence triggered by the combination of ionizing radiation (IR) and the PARP1/2 inhibitor veliparib. We find that SnCs are avidly taken up by type I conventional, CD11c+/CD103+ dendritic cells (DCs), promoting their activation and maturation. Thereby, SnCs promote DC function in T cell priming and stimulation. The capacity of SnCs to activate DCs depends on how the SnCs are formed, with our results confirming IR+veliparib induces high immunogenicity and implicating a distinct SASP in these effects. On the other hand, blocking STING signaling limits SnC activation of DCs. In vivo experiments with the murine tumor models CT26 and 4T1 suggest that both SnCs and SnC-pulsed DCs serve as effective anti-tumor vaccines. They not only confer protective immunity against tumor engraftment but also act as therapeutic vaccines, inhibiting both primary and disseminated tumor growth, and synergize with radiotherapy and immune checkpoint blockade. While direct administration of tumor-derived SnCs to patients may raise safety concerns, pulsing autologous DCs with tumor SnCs may help drive anti-tumor immune response, offering a promising path toward personalized cancer immunotherapy. Citation Format: Yue Liu, Joanna Pagacz, Donald J. Wolfgeher, Stephen J. Kron. Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells [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 2966.

Abstract 1499: Senescent stromal cells drive lung metastases of breast cancer

Abstract In 2022 in the United States, there were nearly 300,000 women diagnosed with breast cancer (BC) with the risk increasing significantly after age 50. The overall 5-year survival rate dramatically decreases, from 99% for patients with stage I, to a devastating 25% for those with stage IV (metastatic) BC, making distal metastasis the leading cause of BC mortality. This mortality rate has not changed in decades with lungs being one of the most common sites for distal metastasis of BC. The BC microenvironment is a complex mix, consisting of tumor cells and numerous nontumor cells including distinct subpopulations of cancer-associated fibroblasts (CAFs): vascular (vCAFs), inflammatory (iCAFs), and myofibroblastic CAFs (myCAFs), which can play pro- or anti-tumor roles. Senescent fibroblasts, which accumulate with age typically express p16INK4A and can modulate the cells around them via the secretion of a cadre of pro-tumorigenic factors including cytokines, chemokines, and extracellular matrix (ECM) modulating enzymes, collectively called the senescence-associated secretory phenotype (SASP). To investigate how p16+ senescent cells impact tumorigenesis, we mated the well-characterized MMTV-PyMT mouse, to the INK-ATTAC (INK) mouse, wherein treatment with AP20187 (AP) selectively eliminates p16+ senescent cells. Preliminary data showed that elimination of p16+ aSMA+ senescent fibroblasts (senCAF) resulted in a significant decrease in primary tumors, lung tumor foci and lung metastatic burden. Further, we found that senescent CAFs (senCAFs) were the only stromal cells eliminated in the PyMT-INK mouse treated with AP, suggesting that this CAF subtype is a key contributor to tumor progression. To determine if senCAFs directly impacted lung metastasis, EO771-LG Luc mammary tumor cells were injected in the tail vein of INKATTAC mice that were treated with AP to eliminate p16+ senCAFs. We found that metastatic tumor burdens were significantly reduced in INK+ versus control mice and immunohistochemical analysis revealed that tumor burden and foci was significantly reduced, suggesting that senCAFs in the lung promoted tumor cell seeding and outgrowth. Citation Format: Anupama Melam, Jiayu Ye, Sheila A. Stewart. Senescent stromal cells drive lung metastases of breast cancer [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 1499.

Abstract 1728: Senescent cancer-associated fibroblast drives colorectal cancer progression in a METTL1-dependent manner

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. More comprehensive studies of key molecular alterations were urgent. Cancer-associated fibroblasts (CAFs) exhibit the senescence-associated secretory phenotype (SASP) which contributes to the progression of cancer through the transcriptomic reprogramming at their senescent states. N7-methylguanosine (m7G), one of the most common RNA modifications, is catalyzed by Methyltransferase-like 1 (METTL1) in human RNA. We therefore investigated the role of METTL1-mediated transcriptomic reprogramming in the crosstalk between senescent CAFs (s-CAFs) and cancer cells. Methods: The SA-β-gal staining and immunofluorescence assays were conducted to characterize the senescent cells. The transwell, wound scratch and organoids assays were preformed to test the abilities of viability, migration and invasion. The concentrations of cytokines and chemokines were determined by Luminex liquid suspension chip. The methylated RNA immunoprecipitation and dot blot assays were performed to characterize the RNA m7G levels in the transcriptome. Results: Through the activation of mTOR signaling, the expression of P16 gene was significantly up-regulated in s-CAFs which was treated by H2O2, accompanied by an increased proportion of SA-β-gal+ cells and altered expression level of METTL1 protein. Luminex liquid suspension chip analysis revealed a significant increase in SASP cytokines such as TNF-α and PDGF-BB in the conditioned media of s-CAFs. Co-culturing CRC cell and organoids with s-CAFs resulted in down-regulation of METTL1 expression and significantly enhanced its invasion and migration abilities, while organoids exhibited increased activity and proliferated more rapidly. TNF-α and PDGF-BB down-regulated the METTL1 expression in CRC cell, while inhibitors targeting TNF-α and PDGF-BB rescued this trend. Interestingly, the regulatory effect on METTL1 of CRC cell by s-CAFs which treated by rapamycin was disappeared. These results indicated that METTL1 modulates RNA m7G modifications influenced by SASP cytokines for promoting CRC metastasis flow. Conclusions: In summary, our findings illustrated that METTL1 plays a role in relaying tumor-promoting signals from s-CAFs in response to SASP cytokines. This ultimately contributes to the tumor-promoting signal flow from s-CAFs. This highlights the importance of understanding the molecular mechanisms underlying the communication between different cell types in the tumor microenvironment. In addition, it also provides a novel strategy for clinical treatment in advanced CRC. Keywords: METTL1, Epigenetic regulation, Senescent CAFs, Tumor microenvironment Citation Format: Jingrong Weng, Xiaolin Wang, Xiaoxia Liu, Yanxin Luo, Huichuan Yu. Senescent cancer-associated fibroblast drives colorectal cancer progression in a METTL1-dependent manner [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 1728.

Abstract 142: HDAC3 is required for pathognomonic features of Langerhans cell histiocytes

Abstract Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm that develops due to dysregulated dendritic cell (DC) development and most commonly affects the children with an incidence of 2.6 to 8.9 cases per million individuals per year. BRAFV600E, the most common disease-driving mutation, activates the mitogen-activated protein kinase (MAPK) pathway causing pathognomonic features that lead to the accumulation of LCH cells and formation of granulomatous lesions. These features include enhanced myelopoiesis, reduced tissue-egress and acquisition of an oncogene induced senescence-associated secretory phenotype (SASP) (marked by increased expression of anti-apoptotic proteins and inflammatory cytokines). Accumulation of LCH cells can occur in any organ causing a wide range of clinical sequelae, which is directly related to the originating cell bearing the oncogenic mutation. Frontline therapy for LCH involves combination chemotherapy and steroidal anti-inflammatories, or MAPK inhibitors, which have significant toxicity and fail to eliminate disease causing precursors. We have found that HDAC3 is required for epidermal LC homeostasis and hypothesized that LCH cells rely on similar epigenetic factors to LCs. CD11cCre LSL-BRAFV600E (BRAFV600ECD11c) mice develop severe multifocal LCH with pronounced lesion development in their livers and lungs, hepatosplenomegaly, and a reduced lifespan due to the accumulation of LCH cells. To test our hypothesis, we generated BRAFV600ECD11c HDAC3fl/fl (BRAFV600EHD3KO) mice, which produce LCH cells that harbor a conditional deletion in the deacetylase domain of HDAC3. Compared to BRAFV600ECD11c mice, BRAFV600EHD3KO mice exhibited significantly less hepatosplenomegaly, reduced lesional burden, and improved survival indicating reduced LCH disease burden. Compared to BRAFV600ECD11c, flow cytometry showed BRAFV600EHD3KO had reduced numbers of LCH cells in lungs and livers, which were more apoptotic. We also found decreased frequencies of circulating LCH cells and LCH precursors, indicating that a lack of HDAC3 activity prevents the development of LCH cells. Bone-marrow derived LCH-like cells provide a valuable drug screening tool. We treated Bone-marrow derived LCH-like cells with RGFP966, an HDAC3-specific inhibitor, increased apoptosis indicated by annexin-V and DAPI staining, reduced expression of Bcl-2, increased CCR7 expression, and decreased S6 phosphorylation (an indication of a loss of the SASP), thus inhibition of HDAC3 may prove therapeutically efficacious by abrogating pathognomonic features of LCH cells. Together, our findings identify HDAC3 as a critical epigenetic regulator LCH cell pathophysiology and HDAC3 blockade would address a great need in treatment of patients with LCH. Furthermore, our results indicate that LCH cells rely on similar epigenetic regulators as epidermal LCs despite coming from a different myeloid lineage. Citation Format: Peter Dimitrion, Jugmohit Toor, James Ge, Qiyan Wang, Carl Allen, Li Zhou, Qing-Sheng Mi. HDAC3 is required for pathognomonic features of Langerhans cell histiocytes [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 142.