Abstract Human papillomavirus (HPV)-related and HPV-unrelated oropharyngeal squamous cell carcinomas (OPCs) are distinct entities with different clinical outcomes. While p16 immunohistochemistry (IHC) is widely used as a surrogate marker for HPV-driven OPC, a subset of HPV-unrelated OPCs also overexpress p16, and the biological basis of this discordance remains unclear. Here, we performed integrated clinicopathological, transcriptomic, genomic, and functional analyses of OPCs and demonstrated that dysregulation of the p16–CDK6 axis characterizes HPV-unrelated p16-positive OPCs. Although these tumors closely resembled HPV-unrelated p16-negative OPCs in their clinicopathological and transcriptomic characteristics, they exhibited a more favorable prognosis. CDK6 was recurrently upregulated in HPV-unrelated OPC regardless of p16 status and was already detectable in high-grade dysplastic leukoplakia, suggesting that CDK6 activation is an early event in HPV-unrelated tumorigenesis. In experimental models, CDK6 overexpression induced compensatory p16 upregulation, creating selective pressure for subsequent CDKN2A inactivation. Consistent with this model, homozygous CDKN2A loss predominated in p16-negative tumors. We further identified CDKN2A frameshift mutations generating p14ARF–p16 chimeric proteins that retain p16 immunoreactivity despite functional loss of wild-type p16, revealing a previously unrecognized diagnostic pitfall of p16 IHC. These findings provide a biological framework for p16 overexpression in HPV-unrelated OPC and suggest that assessment of the p16–CDK6 axis may refine molecular classification and risk stratification beyond p16 IHC alone.

Abstract Cellular senescence contributes to aging and disease, and senolytic drugs that selectively eliminate senescent cells hold therapeutic promise. Although over 20 candidates have been reported, their relative efficacies remain unclear. Here, we systematically compared 21 senolytic agents using a Senolytic Specificity Index (SSI), identifying the Bcl-2 inhibitor ABT263 and the BET inhibitor ARV825 as most effective senolytics across fibroblast and epithelial senescence models. However, even upon extended treatment with these most potent senolytics, a proportion of senescent cells remained viable. We found that senolytic resistance was driven by maintenance of mitochondrial integrity through V-ATPase-mediated clearance of damaged mitochondria. Imposing mitochondrial stress via metabolic workload enhanced the senolytic efficacies of ABT263 and ARV825 in vitro and in mouse models, ketogenic diet adoption or SGLT2 inhibition similarly potentiated ABT263- and ARV825-induced senolysis, reducing tumour growth and metastasis. These findings suggest that mitochondrial quality control is a key determinant of resistance to ABT263- and ARV825-induced senolysis, providing a possible framework for rational combination senotherapies. Citation Format: Masahiro Wakita, Eiji Hara. Comparative analysis of senolytic drugs reveals mitochondrial determinants of efficacy and resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB287.

As Nature Aging celebrates its fifth anniversary, the journal asks some of the researchers who contributed to the journal early on to reflect on the past and the future of aging and age-related disease research, the impact of the field on human health now and in the future, and what challenges need to be addressed to ensure sustained progress.

The p16-3MR mouse model, designed to express Renilla luciferase, mRFP, and herpes simplex virus 1 thymidine kinase (HSV-TK) under the p16 INK4a promoter, has been widely used to visualize and ablate senescent cells in vivo, but our analyses revealed critical limitations. Bioluminescence signals in p16-3MR mice were extremely weak and virtually indistinguishable from those of wild-type mice injected with coelenterazine-h, indicating that previously reported signals largely reflected substrate background rather than authentic reporter expression. Signal intensity remained unchanged with aging, doxorubicin treatment, or cutaneous wound healing, failing to replicate earlier observations. Furthermore, RFP signals were undetectable in senescent fibroblasts from p16-3MR mice, and senescent cells were not eliminated by ganciclovir treatment, suggesting poor expression and lack of functional activity of the mRFP and HSV-TK transgenes. These results demonstrate functional deficiencies in all three transgenes, highlighting the importance of using wild-type controls and calling for careful reevaluation of studies employing this system.

Host survival during infection has traditionally been attributed to pathogen clearance, yet increasing evidence supports a complementary mechanism known as disease tolerance, which limits tissue damage without directly affecting pathogen burden. Here, we identify p16High immune cells as critical mediators of disease tolerance. We show that the FDA-approved BNT162b2 mRNA COVID-19 vaccine rapidly induces p16High immune subsets in mice and humans. These cells are required for protection against lipopolysaccharide-induced endotoxin shock, bacterial sepsis, and ionizing irradiation. Mechanistically, Toll-like receptor 7 (TLR7) activation or low-level STING signaling promotes p16High immune cell induction, reduces adenosine accumulation in part through nicotinamide N-methyltransferase (NNMT)-dependent regulation, and preserves tissue homeostasis. Furthermore, genetic deletion of Ifih1 enhances tonic STING activation and expands p16High immune subsets, improving resilience to severe inflammation and delaying age-related organ deterioration. Our data highlight the beneficial role of the BNT162b2 mRNA COVID-19 vaccine and Ifih1attenuation in inducing disease tolerance through protective p16High immune subsets.

Comparative biology plays a crucial role in uncovering fundamental biological mechanisms and providing evolutionary models for their variation. This approach is particularly valuable for studying aging, given the remarkable diversity in aging trajectories across the tree of life. Many evolutionary theories of aging were proposed well before the discovery of the molecular mechanisms involved, and they remain largely theoretical. Moreover, the growing number of model organisms and the expanding array of experimental and theoretical approaches used to study aging have often remained compartmentalized. As a result, integrating these diverse insights into a unified framework has become increasingly important. As a step toward this goal, this field perspective outlines general biological mechanisms that help explain the variability in aging patterns and longevity across the animal kingdom.

The discovery of the senescence-associated secretory phenotype (SASP) has reshaped our understanding of cellular senescence, shifting its role from a solely tumor-suppressive mechanism to a potential driver of chronic inflammation and age-related diseases. Accordingly, senolytic drugs, which selectively eliminate senescent cells, have garnered considerable interest due to promising preclinical studies. However, concerns remain regarding the reproducibility and generalizability of these findings. In this cross-laboratory study, we rigorously tested the senolytic efficacy of a GLS1 inhibitor and an anti-PD-1 antibody—agents previously reported to reduce the burden of p16 INK4a -positive senescent cells and improve health outcomes in aged mice. Contrary to earlier reports, our study demonstrates that neither GLS1 inhibition nor PD-1 blockade significantly reduced p16 INK4a -positive cell burden or improved aging-related health parameters. Although we do not seek to discredit prior work, our results underscore the need for rigorous design, standardized protocols, and independent validation to ensure reliable senolytics before clinical translation.

Intrahepatic cholangiocarcinoma (iCCA) is a primary liver cancer with poor prognosis. Pan-cancer genomic analyses have suggested that polyploidization contributes to cancer progression, by promoting chromosomal instability and malignant evolution. However, its impact on iCCA remains unexplored, partly due to the abundance of stroma and the limited cohort size. This study assessed tumor cell ploidy at the single-cell level via chromosome FISH to examine its association with intratumoral heterogeneity and effects on iCCA pathogenesis. Both iCCA and hepatocellular carcinoma exhibited intratumoral heterogeneity in tumor cell ploidy, with a strong correlation between median ploidy and heterogeneity levels. Polyploid iCCA demonstrated aggressive features including high invasiveness, increased proliferation, and poor differentiation. Tumor cells in polyploid iCCA overexpressed IL6, potentially driving tumor aggressiveness, while suppressing anti-tumor immunity. These findings highlight ploidy alterations as determinants of iCCA pathogenesis and suggest that targeting ploidy could offer a novel approach to precision medicine for iCCA.

BACKGROUND & AIMS:Pancreatic ductal adenocarcinoma is a highly aggressive malignancy characterized by a fibroblast-rich tumor microenvironment. Cancer-associated fibroblasts closely interact with tumor cells and play a pivotal role in cancer pathogenesis. Single-cell analyses have identified distinct cancer-associated fibroblast subsets that exert either tumor-promoting or tumor-suppressive effects in pancreatic ductal adenocarcinoma. Senescent cancer-associated fibroblasts have recently been linked to poor prognosis through their senescence-associated secretory phenotype. However, the dynamics of senescent cancer-associated fibroblast induction and their spatial distribution in pancreatic ductal adenocarcinoma remain largely unclear. This study aimed to investigate the heterogeneity and spatial organization of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma, with a specific focus on the induction and localization of senescent cancer-associated fibroblasts. METHODS:We performed immunostaining and spatial transcriptomic analyses covering unbiased regions of tumor architecture to map cancer-associated fibroblast subpopulations and characterize senescent cancer-associated fibroblast induction and localization. RESULTS:Senescent cancer-associated fibroblasts were found to accumulate preferentially at the gross tumor edge, and their abundance was associated with poor patient prognosis. Chemotherapy further promoted senescence in myofibroblastic cancer-associated fibroblasts within the tumor core, resulting in an increased population of senescent cancer-associated fibroblasts. Spatial transcriptomic profiling identified gene signatures specific to senescent cancer-associated fibroblasts and revealed their distinct interactions with neighboring cells. Notably, senescent cancer-associated fibroblasts exhibited enhanced transforming growth factor-β signaling activity and upregulation of downstream genes, such as CCN2 and PLAU, which may contribute to tumor proliferation and invasion. CONCLUSIONS:This study reveals that senescent cancer-associated fibroblasts preferentially localize near the gross tumor edge and could impact tumor progression through their senescence-associated secretory phenotype. These findings highlight the spatial plasticity of pancreatic cancer-associated fibroblasts and underscore the pathological significance of senescent cancer-associated fibroblasts in human pancreatic ductal adenocarcinoma.

Cellular senescence contributes to aging and disease, and senolytic drugs that selectively eliminate senescent cells hold therapeutic promise. Although over 20 candidates have been reported, their relative efficacies remain unclear. Here we systematically compared 21 senolytic agents using a senolytic specificity index, identifying the Bcl-2 inhibitor ABT263 and the BET inhibitor ARV825 as most effective senolytics across fibroblast and epithelial senescence models. However, even upon extended treatment with these most potent senolytics, a proportion of senescent cells remained viable. We found that senolytic resistance was driven by maintenance of mitochondrial integrity through V-ATPase-mediated clearance of damaged mitochondria. Imposing mitochondrial stress via metabolic workload enhanced the senolytic efficacies of ABT263 and ARV825 in vitro, and in mouse models, ketogenic diet adoption or SGLT2 inhibition similarly potentiated ABT263-induced and ARV825-induced senolysis, reducing metastasis and tumor growth. These findings suggest that mitochondrial quality control is a key determinant of resistance to ABT263-induced and ARV825-induced senolysis, providing a possible framework for rational combination senotherapies.

Abstract Persistent hyposmia is a hallmark of post COVID-19 conditions, yet the mechanisms sustaining olfactory dysfunction after viral clearance remain poorly understood. Here, using mouse models of SARS-CoV-2 infection, we show that virus-induced senescence-like changes in uninfected olfactory mucosal fibroblasts persist long after viral clearance and drive prolonged olfactory dysfunction. These senescence-like cells secrete SASP factors, including IFNγ, CXCL9, and CXCL11, thereby recruiting γδ T cells to the olfactory mucosa. The accumulated γδ T cells produce excessive IL-17A, which acts on IL-17 receptor A expressed on olfactory sensory neurons, leading to sustained impairment of their function. Genetic ablation of senescence pathways (p16/p21 double knockout), pharmacological elimination of senescent cells with the senolytic drug ABT263, or olfactory neuron-specific deletion of IL-17 receptor A each significantly alleviate prolonged olfactory dysfunction. These findings identify a senescence–γδ T cell–IL-17A axis as a key driver of prolonged hyposmia following SARS-CoV-2 infection in mice.