p300 inhibition delays premature cellular senescence

Abstract

Cellular senescence represents a permanent state of cell cycle arrest, also observed in neurodegenerative disorders. As p300 has been identified as an epigenetic driver of replicative senescence, we aimed to investigate whether in vitro p300 inhibition could rescue the stress-induced premature senescence (SIPS) phenotype. We exploited 2D and 3D (brain organoids) in vitro models of SIPS using two different stressor agents. In addition, we combined the treatment with a p300 inhibitor and validated p300 role in SIPS by analyzing different senescence markers and the transcriptome in our models. Interestingly, p300 inhibition can counteract the DNA damage and SIPS phenotype, detecting a dysregulation of gene expression and protein translation associated with the senescence program. These findings highlight both the molecular mechanisms underlying senescence and p300 as a possible pharmacological target. Thus, targeting p300 and, by extension, senescent cells could represent a promising therapeutic strategy for age-related diseases such as neurodegenerative disorders.

Fig. 1. EP300^mut LCLs are less sensitive to DNA damage induced by SIPS agents.

a Representative flow cytometry panel for γH2AX (FITC-A) and cleaved Caspase 3 (APC-A) evaluation of LCLs derived from one individual (RSTS39) of the cohort with germline pathogenic variants in EP300 (EP300^mut) and from one individual (HD2) of the control cohort (HD) both untreated (CTRL), treated with hydrogen peroxide (H₂O₂), doxorubicin (DOXO) or its vehicle (DMSO); for each cell line there are four quadrants showing percentage of cells negative for both APC and FITC signals (lower left, quadrant LL), positive for APC and negative for FITC signal (upper left quadrant, UL), positive for both (upper right quadrant, UR) or negative for APC and positive for FITC signal (lower right quadrant, LR). b Flow cytometry analysis showing percentage of cells positive to γH2AX and not to cleaved Caspase 3 (% γH2AX⁺/Cas-3^- cells, on Y axis) on EP300^mut and HD lines untreated (CTRL, dark gray), treated with hydrogen peroxide (H₂O₂, blue), DMSO (gray stripes) or doxorubicin (DOXO, plum); each dot represents the mean of triplicates of n = 4 cell lines and values are expressed as means ± SD. c mRNA relative expression of senescence markers CDKN1A and GLB1 in LCLs bearing a pathogenic variant in EP300 (EP300^mut) and controls (HD) LCLs exposed to SIPS agents such as hydrogen peroxide (H₂O₂ in blue) or doxorubicin (DOXO in plum), expressed as fold change calculated on their respective controls (untreated in dark gray and treated with DMSO in gray stripes); dots express triplicates of the four lines (n = 12) and values are expressed as means ± SEM. Representative western blot showing total protein abundance of senescent markers p21 and p53 normalized on GAPDH (d) and replicates analysis (e) on LCLs with pathogenic variant in EP300 (EP300^mut) and controls (HD) LCLs not treated (CTRL, dark gray) or exposed to hydrogen peroxide (H₂O₂ in blue) or doxorubicin (DOXO in plum); dots represent n = 4 replicates and values are expressed as means ± SEM. Statistical analysis was performed using two-tailed Student t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 2. p300 inhibition by CCS1477 protects HD LCLs from DNA damage induced by SIPS agents.

a Acetylation of lysine 27 of histone 3 (H3K27Ac) normalized on unmodified lysine 4 of histone 3 (H3K4) (H3K27Ac/H3K4, on Y-axis) in LCLs derived from three healthy donors (HD) not treated (CTRL, dark gray bar), treated with vehicle (DMSO, gray striped bar) or with CCS1477 (CCS, green bar), assessed by AlphaLISA assay; dots show n = 3 biological triplicates and values are expressed as means ± SEM. b mRNA relative expression of known p300 transcriptional targets MYC and POLD2 in all healthy donors LCLs (HD) untreated (CTRL, dark gray bar), treated with vehicle (DMSO, gray striped bar) or with CCS1477 (CCS, green bar); dots express triplicates of the four lines (n = 12) and values are expressed as means ± SEM. c Representative flow cytometry panel for γH2AX (FITC-A) and cleaved Caspase 3 (APC-A) evaluation of one control LCL (HD1) treated with hydrogen peroxide alone (H₂O₂) or combined with CCS1477 (CCS + H₂O₂), treated with its vehicle (CCS + H₂O), with doxorubicin alone (DOXO) or combined with CCS1477 (CCS + DOXO) or treated with its vehicle (CCS + DMSO); for each cell line there are four quadrants showing percentage of cells negative for both APC and FITC signals (lower left, quadrant LL), positive for APC and negative for FITC signal (upper left quadrant, UL), positive for both (upper right quadrant, UR) or negative for APC and positive for FITC signal (lower right quadrant, LR). d Flow cytometry analysis showing percentage of cells positive for γH2AX and not to cleaved Caspase 3 (% γH2AX⁺/Cas-3^- cells, on Y axis) on controls lines (HD) treated with hydrogen peroxide alone (H₂O₂, blue bar) or combined with CCS1477 (CCS + H₂O₂, light blue bar), treated with its vehicle (CCS + H₂O, gray bar), with doxorubicin alone (DOXO, plum bar) or combined with CCS1477 (CCS + DOXO, pink bar) or treated with its vehicle (CCS + DMSO, gray striped bar); each dot represents the mean of triplicates of n = 4 cell lines and values are expressed as means ± SD. e mRNA relative expression of senescence markers CDKN1A, IL6 and GLB1 in controls LCLs (HD) exposed to SIPS agents such as hydrogen peroxide (H₂O₂ in blue) or doxorubicin (DOXO in plum) alone or combined with CCS1477 (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar), expressed as fold change calculated on their respective controls (untreated, treated with DMSO, CCS + H₂O or CCS + DMSO which values are represented by the dotted line); dots express triplicates of the four lines (n = 12) and values are expressed as means ± SEM. Representative western blot showing total protein abundance of senescent markers p21 and p53 normalized on GAPDH (f) and replicates analysis (g) on controls LCLs (HD) untreated (CTRL, dark gray bar) or exposed to hydrogen peroxide (H₂O₂, blue bar), doxorubicin (DOXO, plum bar), CCS1477 (CCS, gray striped bar) or SIPS agents in combination with CCS (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar); dots represent n = 4 replicates and values are expressed as means ± SEM. Statistical analysis was performed using two-tailed Student t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 3. p300 inhibition by CCS1477 rescues the senescent-like phenotype induced by SIPS agents in iNeurons.

a Characterization of the three main differentiation stages of iNeurons (hiPSC-NGN2, Pre-neurons and iNeurons) by brightfield acquisitions at 10x magnification (upper panel) and by immunofluorescence experiments acquired using a confocal microscope with 60x magnification (bottom panel): confocal images show hiPSCs tagged with OCT4 (green signal) and NANOG (red signal), Pre-neurons with NESTIN (red signal) and SOX2 (white signal), iNeurons with CTIP (red signal) and TUJ1 (white signal), all nuclei with DAPI (blue signal), while endogenous GFP due to inducible differentiation (eGFP, green signal) was detected in both Pre-Neurons and iNeurons. b Representative panels of SA-β-Gal staining on D23 iNeurons untreated (CTRL), treated with CCS1477 (CCS), with SIPS agents alone (H₂O₂ or DOXO) or in combination with CCS (CCS+H₂O₂or CCS + DOXO); brightfield images acquired at 20x magnification show cells positive for staining as blue-colored. c Analysis of iNeurons percentage positive to SA-β-Gal (% β-Gal+ cells, on Y-axis) untreated (CTRL, dark gray bar), treated with CCS (gray striped bar), with H₂O₂-treatments (H₂O₂, blue bar; CCS + H₂O₂, light blue bar) or DOXO-treatments (DOXO, plum bar; CCS + DOXO pink bar); dots show mean of blind fields counts of n = 3 biological replicates and values are expressed as means ± SD. d mRNA relative expression of senescence markers CDKN1A, LMNB1, IL6 and GLB1 in iNeurons exposed to SIPS agents alone (H₂O₂, blue bar or DOXO, plum bar) or combined with CCS1477 (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar), expressed as fold change calculated on their respective controls (untreated CTRL, dark gray bar and CCS, gray striped bar); dots express biological triplicates (n = 9) and values are expressed as means ± SEM. Representative western blots showing total protein abundance of senescent markers p21 and p53 normalized to TUJ1 (e) and replicates analysis (f) on iNeurons (iN D23) untreated (CTRL, dark gray bar) or exposed to H₂O₂ (blue bar), DOXO (plum bar), CCS (gray striped bar) or SIPS agents in combination with CCS (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar); dots represent at least n = 4 replicates and values are expressed as means ± SEM. Statistical analysis was performed using a two-tailed Student t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 4. p300 inhibition by CCS1477 rescues the senescent-like phenotype induced by SIPS agents in Brain Organoids (BOs).

a Scheme of organoid differentiation protocol starting from hiPSC to d65-organoid. b Brightfield images of brain organoids at different stages of differentiation at 4× and 10× magnification. c Characterization of brain organoids at d65 by immunofluorescence experiments; c’,c” 20 μm brain organoid sections immunolabelled with anti-NESTIN antibody (green) and anti-TUJ1 antibody (red) (c’), and anti-CITIP (green), anti-MAP2 (red) and anti-TUJ1 (white) (c”) at 10X, 20X and 40X magnification; nuclei were counterstained with DAPI; boxes indicate areas shown at higher magnification below to the corresponding panel; images were acquired by confocal microscope with 10×, 20× and 40× magnification with scale bars 200 μm (10×), 100 μm (20×), 50 μm (40x). d mRNA relative expression of senescence markers CDKN1A, GLB1, IL6 and IL8 in BOs exposed to SIPS agents alone (H₂O₂, blue bar or DOXO, plum bar) or combined with CCS1477 (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar), expressed as fold change calculated on their respective controls (untreated CTRL, dark gray bar and CCS, gray striped bar); dots express biological triplicates (n = 9) and values are expressed as means ± SEM. Representative western blot showing total protein abundance of senescent markers p21 and p53 normalized on TUJ1 (e) and replicates analysis (f) on BOs (BO D65) untreated (CTRL, dark gray bar) or exposed to H₂O₂ (blue bar), DOXO (plum bar), CCS (gray striped bar) or SIPS agents in combination with CCS (CCS + H₂O₂, light blue bar; CCS + DOXO, pink bar); dots represent n = 4 replicates and values are expressed as means ± SEM. Statistical analysis was performed using two-tailed Student t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 5. p300 inhibition by CCS1477 rescues the transcriptional senescent cascade induced by SIPS agents in Brain Organoids (BOs).

a Heatmaps of whole transcriptome RNA-seq expression data showing top 25 DEGs in biological triplicates of BOs (marked with I, II, III) each treated with SIPS agents alone (H₂O₂ or DOXO) or combined with CCS1477 (CCS + H₂O₂ or CCS + DOXO); comparison of H₂O₂-treated and DOXO-treated BOs are shown on the left and on the right panels respectively; DEGs were selected based on nominal p < 0.05 and absolute foldchange > 1.5. b PCA plots of transcriptomic analysis on biological triplicates of BOs (marked with I, II, III) each treated with CCS1477 (CCS), SIPS agents alone (H₂O₂ or DOXO) or combined with CCS1477 (CCS + H₂O₂ or CCS + DOXO); comparison between CCS with H₂O₂-treated and DOXO-treated BOs are shown on the left and on the right plots respectively; on axes is shown the percentage of each principal component (PC1 on x-axis, PC2 on y-axis) that explains the population variance.