Linking CCND1 Expression in Senescent Cells and DNA Damage and Inflammation Highlights a Therapeutic Target for Aging

By Stuart P. Atkinson

Composite overview of Rajesh et al. implicating CCND1 in senescence, DNA damage, and aging.
Composite overview of Rajesh et al. implicating CCND1 in senescence, DNA damage, and aging. (f) from figure 1: CCND1 protein over-expression in senescent nuclei. (k, l) from figure 2: response of senesence and secretory phenotype (SASP) genes and interferon-stimulated genes (ISGs) to irradiation with and without Palbociclib. (a) from figure 3: COMET assay measuring DNA damage when exposing senescent cells to irradiation with and without Palbociclib. (iO from figure 4: DNA damage assays in the presence of siRNA vs CDKN2A, with and without Palbociclib.

Cell Senescence: Chronic Inflammation, Chromatin Fragmentation, and the Paradox of CCND1

Cell senescence - a stable form of cell-cycle arrest triggered by stressors that include DNA damage, oncogene activation, and the shortening of telomeres significantly contributes to the aging process and the onset of associated diseases by driving chronic inflammation through the senescence-associated secretory phenotype (SASP) and the expression of interferon-stimulated genes (ISGs) (Copp et al., Kuilman et al., and Cecco et al.). Furthermore, the persistence of DNA damage-mediated signaling promotes cytoplasmic chromatin fragment formation and cyclic GMPAMP synthase (cGAS)stimulator of interferon genes (STING) pathway activation to sustain the SASP and systemic chronic inflammation (Miller et al.).

Researchers from the laboratory of Peter D. Adams (Sanford Burnham Prebys) sought to identify the molecular drivers maintaining the chronic inflammatory state of senescent cells to improve our understanding of age-related dysfunction and hence inform the future development of ameliorative therapeutic strategies. Their recent preprint article (Rajesh et al.) focused on Cyclin D1 (CCND1) - a regulator of G1 cell cycle progression whose expression level undergoes a paradoxical elevation in senescent cells despite proliferative arrest (Lucibello et al., Fukami-Kobayashi and Mitsui, and Fukami et al.). Interestingly, the findings of this exciting new study now reveal a novel role for CCND1 and its kinase partner Cyclin-dependent kinase 6 (CDK6) in regulating DNA damage and inflammation during senescence and normal aging, which highlights this cell cycle regulator as a promising therapeutic target for age-associated disorders.

While this study employed bulk transcriptomic assays and analyzed single-cell RNA sequencing data, the authors did not complement these approaches using epigenetic assays that can describe the chromatin landscape of single cells while simultaneously measuring RNA expression. Could the additional integration of simultaneous profiling of transcriptomics and epigenetics afforded by applying Paired-Tag technology from Epigenome Technologies further our understanding of cellular senescence and the role of CCND1 and move us closer to identifying treatment avenues for a host of aging-associated disorders?

Part of Firuge 1 from Rajesh, et al.
Figure 1 from Rajesh, et al: CCND1 is elevated in senescence and marks cells with canonical senescent features. (a) RNA-seq of IMR90 fibroblasts 10 days after ionizing radiation (IR) shows CCND1 among a small subset of proliferation-associated genes upregulated in senescence. (b) Western blot of time-course of cells exposed to IR, demonstrating accumulation of cyclin D1. (c) Genome browser tracks show CCND1 upregulation in replication-induced and oncogene-induced senescence.

Defining a New Aging Target CCND1-mediated DNA Damage and Inflammation

The in vitro section of this fascinating study employed human fibroblasts induced to senesce via exposure to ionizing radiation. The authors discovered that induced senescence prompted an increase in CCND1 at the mRNA and protein levels; furthermore, experiments employing small interfering RNA-mediated knockdown and pharmacological inhibition revealed the requirement for CCND1 and CDK6 for the SASP and ISG expression. Next, the team revealed that CCND1 and CDK6 functioned to promote DNA damage and cytoplasmic chromatin fragment accumulation - the precursors to the SASP induction and ISG expression via cytosolic DNA signaling (cGASSTING activation) - in senescent human fibroblasts, but that p53-CDKN1A and chromatin-associated factors such as KIF4A and KIF5B could restrain these outcomes. KIF4A, previously reported as playing roles in chromatin compaction and DNA repair (Wu et al.), may support sustained DNA damage and inflammatory signaling thanks to altered repair dynamics in senescent versus proliferating cells. Meanwhile, KIF5B, previously implicated in facilitating DNA double-strand break repair via the formation of nuclear envelope tubules (Shokrollahi, Stanic, and Hundal et al.), may suggest a role for CCND1CDK6 in influencing chromatin architecture through kinesin-mediated transport mechanisms.

The in vivo section of the study first assessed hepatocytes isolated from young and old mice, which revealed a significant age-associated increase in Ccnd1 mRNA levels and the localization of Ccnd1 protein to non-proliferating hepatocytes from old livers, suggesting their status as senescent. Spatial transcriptomics performed on livers from young (4-month) and old (22-month) mice revealed increasing Ccnd1 mRNA levels with age in hepatocytes alongside the expression of senescence-associated genes and ISGs. Hepatocyte-specific Ccnd1 knockout in 17-month-old mice revealed decreased levels of Ccnd1 (mRNA and protein), DNA damage indicators, inflammation-associated gene/ISG expression, and cytoplasmic chromatin fragments after three weeks. Overall, the authors provided evidence that age-associated Ccnd1 expression in hepatocytes occurs in senescent cells in the aged mouse liver, where this cell cycle-associated factor functions to sustain DNA damage and inflammatory gene expression. Finally, the authors evaluated the impact of Cdk6 inhibition in aged mice via palbociclib treatment in young and old mice; overall, they confirmed target engagement and observed reduced ISG expression. Interestingly, these experiments also highlighted the ability of Cdk6 inhibition to protect against age-related increases in frailty scores (i.e., gait disorders, hearing loss, vestibular disturbance, and vision impairment) when compared to non-treated mice. Overall, these exciting findings suggested that targeting Ccnd1-Cdk6 could reduce inflammation-associated gene expression and protect against age-associated motor and physiological decline in aging mice.

Part of Figure 2 from Rajesh, et al.
Figure 2 from Rajesh, et al.: CCND1 and CDK6 are required to sustain inflammatory and interferon gene expression and senescence. (i) Heatmap of all differentially expressed genes across Palbociclib conditions. (j) Enrichment analysis of the Palbociclib-suppressed cluster shows reduced interferon-α response. (m) qPCR validation of SASP and ISG repression following Palbociclib treatment; fold changes relative to DMSO control.

Can Targeting CCND1-CDK6 Protect Against Aging-associated Physiological Decline

Overall, this fascinating study provide robust evidence for a senescence-associated function of the canonical cell-cycle regulator CCND1 and its kinase partner CDK6 linked to sustained DNA damage signaling, cytoplasmic chromatin fragment accumulation, and persistent inflammatory responses associated with non-proliferative senescent phenotypes during aging and, as such, highlights new opportunities for therapeutic intervention to mitigate age-related pathology and preserve tissue function during aging. The authors do note the need for future studies, which include the exploration of the molecular mechanisms underlying CCND1-mediated chromatin dynamics, CCND1 interactions with kinesins, how CDKN1A mechanistically constrains CCND1CDK6 activity, and how CCND1CDK6 axis modulation impacts multiple tissues across varying aging contexts.

Part of Figure 5 from Rajesh, et al.
Figure 5 from Rajesh, et al.: CDK4/6 inhibition reduces inflammatory gene expression and preserves function in aged mice. (f) Longitudinal rotarod trajectories over the two-month treatment course showing progressive improvement in Palbociclib-treated aged mice compared vehicle-treated aged mice.

Can Simultaneous Single-cell Transcriptomic and Epigenetic Analyses Extend these Findings?

While this study did not involve epigenetic analyses, an understanding of the single-cell gene regulatory programs active in senescing/senescent cells (mediated by specific epigenetic profiles) and their relation to the transcriptomic profiles of the same single cell may provide an detailed description of the aging process and reveal more about the role of CCND1 and CDK6. Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays while avoiding the need for cell sorting. The implementation of Paired-Tag technology may enable these researchers to define the molecular roots of cell senescence and identify the critical regulatory elements controlling this age-related process. These details may represent the key to the development of novel treatment strategies for various aging-related disorders that may promote a healthier aging process.