Targeting CyclinD1-CDK6 to Mitigate Senescence-Driven Inflammation and Age-Associated Functional Decline

Abstract

Cellular senescence contributes to aging and age-related diseases by driving chronic inflammation through the Senescence Associated Secretory Phenotype (SASP) and interferon-stimulated genes (ISGs). Cyclin D1 (CCND1), a key cell cycle regulator, is paradoxically upregulated in these non-proliferating cells. We show that CCND1 and its kinase partner CDK6 drive SASP and ISG expression in senescent cells by promoting DNA damage accumulation. This leads to the formation of cytoplasmic chromatin fragments (CCFs) that activate pro-inflammatory CGAS-STING signaling. The tumor suppressor p53 (TP53) and its target p21 (CDKN2A) antagonize this CCND1-CDK6-dependent DNA damage accumulation pathway to suppress the SASP. In aged mouse livers, senescent hepatocytes show increased Ccnd1 expression. Hepatocyte-specific Ccnd1 knockout or treatment with the Cdk4/6 inhibitor Palbociclib reduces DNA damage and ISGs in aged mouse liver. Notably, Palbociclib also suppresses frailty and improves physical performance of aged mice. These findings reveal a novel role for CCND1/CDK6 in regulating DNA damage and inflammation in senescence and aging, highlighting it as a promising therapeutic target.

Figure 1.. 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 time course (0–11 days post-IR) shows progressive accumulation of cyclin D1 protein in senescent cells. Ponceau staining was used as a loading control c, Genome browser tracks show CCND1 upregulation in replication-induced and oncogene-induced senescence. d, Western blot of senescent IMR90s shows increased CCND1 and CCND2, decreased CDK6, and no change in CDK4. Phosphorylated pRB (ppRB) and total pRB were both reduced. Senescence markers CCNA2, CCNB1 and LMNB1 also decreased, while CDKN1A and phospho-p65 (pp65) were increased. Total p65 was unchanged. Ponceau staining was used as a loading control. e, Representative immunofluorescence images of proliferating (Pro) and senescent (Sen) IMR90s. In senescent cells, CCND1 localizes to nuclei that are EdU-negative, ppRB–negative, and express high CDKN1A and IL-8. CCND1⁺ cells also exhibit cytoplasmic chromatin fragments (CCFs; γH2AX/DAPI-positive puncta), enlarged nuclei, and reduced Lamin B1 at the nuclear periphery. f, Quantification of: percentage of total nuclei that are CCND1⁺; percentage of CCND1⁺ nuclei that are EdU⁺, ppRB⁺ or CDKN1A⁺; number of CCFs per nucleus; and nuclear area. Each dot represents an independent biological replicate (separate irradiation). For immunofluorescence quantifications, each dot is the average of ≥3 technical replicates from the same irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using t-test. P < 0.05 was considered significant.

Figure 2.. CCND1 and CDK6 are required to sustain inflammatory and interferon gene expression in senescence

a, Experimental design for siRNA knockdown experiment: IMR90 fibroblasts were induced senescent by ionizing radiation (IR) and transfected with siRNAs targeting CCND1, CDK4, CDK6, or both CDK4 and CDK6. Non-targeting controls (siNT1, siNT2) and proliferating (non-irradiated) cells were included. b, Experimental design for Palbociclib experiment: IMR90 fibroblasts were induced senescent by IR and treated with DMSO or Palbociclib. Proliferating (non-irradiated) controls were included. c–d, Western blots validating knockdown of CCND1, CDK4 and CDK6. e, Heatmap of all differentially expressed genes across siRNA conditions. A red box highlights a senescence-associated gene cluster selectively suppressed by CCND1 or CDK6 knockdown. f, Top enriched pathways in the red-box cluster include interferon-α response, TNFα signaling via NFκB, and interferon-γ response. g–h, Expression of SASP and ISG genes that are differentially expressed between proliferating cells and senescent siNT controls. i, Heatmap of all differentially expressed genes across Palbociclib conditions, with a red box indicating a cluster suppressed by Palbociclib treatment. j, Enrichment analysis of the Palbociclib-suppressed cluster shows reduced interferon-α response, TNFα signaling via NFκB, and interferon-γ response — the same top pathways as in f. k–l, Expression of SASP and ISG genes differentially expressed between proliferating and senescent DMSO-treated controls, showing suppression with Palbociclib. m–n, qPCR validation of SASP and ISG repression following Palbociclib treatment. Gene expression values are shown as fold change relative to senescent DMSO-treated controls, normalized to the geometric mean of GAPDH and RPL13. Each biological replicate represents an independent irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. P < 0.05 was considered significant.

Figure 3 |. CCND1–CDK6 promotes DNA damage and cytoplasmic chromatin fragment (CCF) accumulation in senescent cells.

a, Representative comet assay images and quantification of comet tail length and olive moment in IR-induced senescent IMR90 fibroblasts, showing reduced DNA damage with Palbociclib treatment. b, Western blots show decreased 53BP1 and γH2AX protein levels following Palbociclib; Ponceau staining was used as a loading control. c, Representative immunofluorescence images and quantification of nuclear γH2AX intensity and CCF frequency, both reduced in Palbociclib-treated senescent cells. d, ELISA for 2′3′-cGAMP shows reduced cGAS–STING activation after Palbociclib. e–f, Immunoprecipitation–mass spectrometry (IP–MS) using two CCND1 antibodies identifies top CCND1 interactors in senescent IMR90s, shown as ranked bar plots of log10(LFQ+1) intensity comparing D1 IP versus IgG control. g, Co-immunoprecipitation confirms interaction between CCND1 and CDKN1A in senescent cells. h, Knockdown of CDKN1A increases nuclear γH2AX and CCF formation. i, Palbociclib rescues the elevated DNA damage and CCFs caused by CDKN1A knockdown. j, Representative immunofluorescence images and quantification showing that KIF4A knockdown reduces CCF frequency. k, qPCR analysis showing that KIF4A knockdown suppresses SASP and ISG gene expression. Expression values are shown as fold change relative to non-targeting control siRNA in senescent cells, normalized to the geometric mean of GAPDH and RPL13. Each biological replicate represents an independent irradiation. For immunofluorescence quantifications, each dot reflects the average of ≥3 technical replicates per irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using one-way ANOVA. P < 0.05 was considered significant.

Figure 4 |. CCND1 is elevated in aged hepatocytes and drives DNA damage and inflammation in vivo.

a, qPCR analysis of isolated hepatocytes from young (4–5-month-old) and old (23-month-old) mice showing increased Ccnd1 expression with age. Expression values are shown as fold change relative to young and normalized to the geometric mean of GAPDH and HPRT. b, Immunofluorescence staining and quantification of liver sections from young (4-month-old) and old (21-month-old) mice showing increased CCND1 expression in hepatocyte nuclei of aged livers. CCND1 signal was observed predominantly in Ki67-negative cells, indicating non-dividing hepatocytes. c, CosMx spatial transcriptomics of livers from young (4-month-old) and old (22-month-old) mice showing elevated Ccnd1 transcript levels in hepatocytes of aged livers. d, CosMx spatial transcriptomics showing that Ccnd1-positive hepatocytes in 22-month-old livers are enriched for SenMayo senescence-associated genes compared to Ccnd1-negative hepatocytes. e, Western blot analysis of liver lysates from 17-month-old mice 3 weeks after hepatocyte-specific Ccnd1 knockout using AAV8–TBG–saCas9, showing marked reduction in CCND1 protein levels and γH2AX, confirming knockout efficiency and decreased DNA damage; Ponceau staining was used as a loading control. f, Bulk RNA-seq of liver tissue from 22-month-old mice showing broad suppression of inflammatory gene sets following Ccnd1 knockout. g, Heatmap showing downregulation of interferon-stimulated genes (ISGs) that are typically elevated with age in livers of 22-month-old mice. h, Immunofluorescence analysis of liver sections from 22-month-old mice showing reduced cytoplasmic γH2AX puncta and a trend toward fewer cytoplasmic chromatin fragments (CCFs; overlapping γH2AX- and dsDNA-positive puncta in the cytoplasm, example shown with arrow) in Ccnd1-knockout livers. Each biological replicate represents a separate animal. Error bars denote mean ± s.d. Statistical analysis was performed using Welch’s t-test. For qPCR comparisons, Mann–Whitney U test was used. P < 0.05 was considered significant.

Figure 5 |. CDK4/6 inhibition reduces inflammatory gene expression and preserves function in aged mice.

a, Western blot of spleen lysates from aged mice treated with Palbociclib, showing reduced phosphorylated pRB (ppRB), confirming target engagement; Ponceau staining was used as a loading control. b–c, qPCR analysis of liver tissue from two short-term Palbociclib dosing cohorts. Mice received 64 mg/kg Palbociclib in autoclaved H₂O via oral gavage (200 μL) either daily (aged 25 months, young controls 8 months) or three times per week (aged 23 months, young controls 6 months) for 3 weeks. Both regimens showed a trend toward reduced interferon-stimulated gene (ISG) expression in Palbociclib-treated aged mice relative to age-matched controls. Expression values are shown as fold change relative to aged vehicle-treated mice and normalized to the geometric mean of GAPDH and HPRT. d, Body weight over the course of the long-term treatment study, showing no significant difference between Palbociclib- and vehicle-treated aged mice. e, Rotarod test performance at baseline and after two months of Palbociclib treatment in 18-month-old mice, demonstrating improved motor coordination and endurance in treated animals, comparable to 4-month-old young controls. f, Longitudinal rotarod trajectories over the two-month treatment course showing progressive improvement in Palbociclib-treated aged mice compared vehicle-treated aged mice. g, Subcomponent analysis of frailty scores showing the strongest Palbociclib-associated improvements in gait, hearing, vestibular function, and vision. h, Frailty index at the end of the study: Palbociclib-treated aged mice maintained stable frailty scores, whereas vehicle-treated aged mice showed increased frailty over time. i, Longitudinal frailty index trajectories showing divergence between vehicle and Palbociclib-treated groups across the treatment period. Each biological replicate represents a separate animal. Error bars denote mean ± SEM. Statistical analysis for longitudinal and paired measurements was performed using two-way repeated measures ANOVA. P < 0.05 was considered significant.