EP300 deficiency leads to chronic replication stress mediated by defective replication fork protection

Abstract

Mutations in the global transcriptional activator EP300/KAT3B are being reported in aggressive malignancies. However, the mechanistic contribution of EP300 dysregulation to cancer is currently unknown. While EP300 has been implicated in regulating cell cycle and DNA replication, the role of EP300 in maintaining replication fork integrity has not been studied. Here, using EP300-mutated adult T-cell leukemia/lymphoma cells and an EP300-selective degrader, we reveal that EP300 loss leads to pronounced dysregulations in DNA replication dynamics and persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to replisome pausing. EP300 deficiency results in a prominent defect in fork protection resulting in the accumulation of single-stranded DNA gaps. Importantly, we find that the loss of EP300 results in decreased expression of BRCA2 protein leading to sensitivity to treatments that are cytotoxic to BRCA-deficient cancers. Overall, we demonstrate that EP300-mutated cells recapitulate features of BRCA-deficient cancers.

Fig. 1. EP300-deficient cells have aberrant cell cycle dynamics, spontaneous DNA damage, increased sensitivity to replication stress, and persistent checkpoint activation.

A, B Cell doubling time calculated every 48 h from three independent cultures of untreated (A) and 500 nM JQAD1-treated (B) EP300WT ATLL or non-ATLL B-lymphocyte cells; for all experiments, data are presented as mean values +/− SEM; for (A), N = 5 and for (B), N = 3 (C) Flow cytometry-based cell cycle analysis of untreated EP300WT (top) and EP300Mut (bottom) NA-ATLL cells. Cell cycle progression was monitored by co-staining for 5-ethynyl-2’-deoxyuridine (EdU) and propidium iodide (PI). D Analysis of the number of phospho- H2AX (phosphorylation of Histone H2AX at Ser139) foci per cell nuclei in EP300Mut and EP300WT NA-ATLL cells treated with 0.4uM Aphidicolin (APH) overnight (O/N), N = 3. E Measurement of DNA single-strand breaks by alkaline Comet assay in EP300Mut and EP300WT cells treated with 0.4uM APH (O/N). Comet tail lengths were measured using the OpenComet plugin as part of the ImageJ software, N = 3. F Left: Expression levels of phospho-ATR in EP300WT/Mut NA-ATLL cells in the absence of exogenous replication inhibition. Right: Relative protein expression of pATR in EP300Mut and WT NA-ATLL cells, N = 2. G Time course experiment to measure recovery of NA-ATLL EP300Mut/WT cells from 2 mM Hydroxyurea treatment (4 h) after release into drug-free media over the course of 48 hours. Cells were collected at eight time points (0, 4, 8, 12, 24, 28, 32, 48 hours) and expression levels of phospho-RPA Ser4/8 and phospho-Histone H2AX Ser139 were measured by western blotting. Expression levels of Vinculin were used as a loading control for phospho-H2AX, and pRPA was measured relative to RPA32, N = 2. H Quantification of % AnnexinV/PI +ve cells assessed by flow cytometry in EP300Mut, EP300WT NA-ATLL cells +/− 500nMJQAD1 (EP300-specific PROTAC degrader) and +/− 2 mM HU (4 h), N = 3. For all experiments, data are presented as mean values +/− SD. P-values were determined by a two-tailed Student T-test. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. N represents three experimental replicates from independent cultures of cells as indicated. Source data are provided as a Source Data file.

Fig. 2. EP300-mutated cells have aberrant replication dynamics associated with increased origin firing.

A Locus map of a 375 kb region in the CFS-FRA6E obtained by PmeI digestion. The region includes the fragility core of CFS-FRA6E (pink line – 162 kb). The FISH probes that identify the segment are labeled in blue. Top: Locus map of the PmeI-digested FRA6E segment. Bottom: Aligned photomicrograph images of labeled DNA molecules from the DNA Replication program at CFS-FRA6E in (B) J-ATLL EP300WT (J2); (C) J-ATLL EP300Mut cells (J1); (D) NA-ATLL EP300WT cells (NA1); (E) NA-ATLL EP300Mut (NA2) cells; (F) NA-ATLL EP300WT treated with 500 nM JQAD1 for 48 h. The yellow arrows indicate the sites along the molecules where the IdU transitioned to CldU. The molecules are arranged in the following order: molecules with initiation events, molecules with 3’ to 5’ travelling forks, molecules with 5’ to 3’ travelling forks, and molecules with termination events. G Percentage of molecules with initiation sites in FRA6E in J-ATLL EP300WT, J-ATLL EP300Mut, NA-ATLL EP300WT, NA-ATLL EP300Mut and NA-ATLL EP300WT cells + 500nMJQAD1 (48 h), N = 2. Data are presented as mean values +/− SD. P-values were determined by a two-tailed, Student T-test. The p-values are indicated as follows: * <0.03, ** <0.0021. Source data are provided as a Source Data file.

Fig. 3. EP300 deficiency results in genome-wide replication stalling and fork collapse, leading to the accumulation of extensive ssDNA gaps.

A Schematic model depicting replication fork stalling, fork protection, and nucleolytic degradation, (Created in BioRender. Madireddy, A. (2025), is licensed under CC BY 4.0, https://BioRender.com/xtm4wme). B DNA fiber analysis of 50uM hydroxyurea (HU) (60 min) treated EP300WT (NA1, NA4), EP300Mut (NA2, NA3) and EP300WT NA-ATLL (NA1, NA4) cells treated with negative control (S, S) stereoisomer or with JQAD1 (500 nM for 48 h) treatment to assess replication fork stalling. The fork rate (CldU/IdU ratio) is indicated. N = 3. C DNA fiber analysis measuring nucleolytic degradation after 4 mM HU treatment in EP300WT, EP300Mut NA-ATLL cells. The fork rate (CldU/IdU ratio) is indicated, N = 3. D Analysis of the number of single-stranded gaps (ssDNA)/Iododeoxyuridine (IdU) foci (green) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with JQAD1 (500 nM). ssDNA gaps are measured by IdU incorporation under non-denaturing conditions. Representative images are shown on the right, N = 3. E Analysis of number of pRPA Ser4/8 foci (red) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with 500 nM JQAD1. Representative images are shown on the right, N = 3. F Protein expression of phospho-RPA Ser4/8 in the presence or absence of EP300, presented as a fold change quantified from the immunoblot in Supp. Fig. S3D. G Analysis of the number of single-stranded gaps/breaks (ssDNA)/Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 50uM Mirin (4 h) (Mre11 nuclease inhibitor), N = 3. H Analysis of the number of single-stranded gaps/breaks (ssDNA)/ Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 20uM C5 (24 h) (DNA2 nuclease inhibitor), N = 3. For all experiments, data are presented as mean values +/− SD. P-values were determined by a two-tailed Student T-test. P-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm. N represents three experimental replicates from independent cultures of cells. Source data are provided as a Source Data file.

Fig. 4. EP300-deficient cells have a prominent defect in downstream fork restart machinery.

A Expression levels of BRCA2 protein from WCE in EP300WT and EP300Mut NA-ATLL cells treated with 2 mM HU (4 h), by immunoblotting. Expression levels of Vinculin were used as a loading control. B Expression levels of BRCA2 protein in the NA4 EP300WT NA-ATLL cell lines treated with negative control (S, S) stereoisomer compared to cells 3,5,7 days after 500 nM JQAD1 treatment (48 h). Expression levels of LaminB1 were used as a loading control. C Expression levels of BRCA2 protein in the NA1 EP300WT NA-ATLL cell line with negative control (S, S) stereoisomer compared to cells 5,7 days after 500 nM JQAD1 treatment (48 h) and NA2 EP300Mut cell line 5 days after JQAD1 treatment. Expression levels of LaminB1 were used as a loading control. D Relative expression of BRCA2 protein between EP300WT and EP300Mut cells. E Analysis of the number of BRCA2 foci (green) per EDU-positive (red) cell nucleus (DAPI, blue) in EP300WT (NA1, NA4), and EP300Mut (NA2, NA3) cells exposed to 0.4 uM APH (O/N). Representative images are shown on the right, N = 3. F Analysis of the number of RAD51 foci (red) per cell nucleus (DAPI, blue) in EP300WT (NA1, NA4) +/− 0.4 uM APH (O/N), EP300Mut +/− 0.4 uM APH (O/N) and EP300WT (NA4) treated with 500 nM JQAD1 (48 h). Representative images are shown on the right, N = 3. G Volcano plot comparing gene (mRNA) expression levels, quantified and presented as a fold change in EP300WT (NA1, NA4) and EP300Mut (NA2, NA3) cell lines after RNA-seq analysis. H Analysis of the number of POLD3 foci (green) per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h). Representative images are shown on the right, N = 3. I Quantification of the percentage of AnnexinV/PI +ve cells assessed by flow cytometry in EP300Mut, EP300WT NA-ATLL cells +/− REV1i (10uM JHRE06 for 24 h) and +/− PARPi (10uM Olaparib for 72 h), N = 3. J Quantification of percentage of AnnexinV/PI +ve cells assessed by flow cytometry in EP300Mut, EP300WT NA-ATLL cells +/− POLQi (20uM ART558 for 72 h) and +/− PARPi (10uM Olaparib for 72 h). Representative images are shown on the right, N = 3. K Quantification of the percentage of AnnexinV/PI +ve cells assessed by flow cytometry in EP300Mut (NA2), EP300WT (NA1) cells +/− POLQi (20uM ART558 for 72 h) and +/− PARPi (10 uM Olaparib for 72 h). For all experiments, data are presented as mean values +/- SEM. P-values were determined by a two-tailed Student T-test. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm. N represents experimental replicates from independent cultures of cells. Source data are provided as a Source Data file.

Fig. 5. EP300 mutated cells have under-replicated DNA that results in inherited DNA lesions in G1.

A Analysis of number of FANCD2 twin-foci (green) per mitotic cell nucleus (DAPI, blue) in EP300WT and EP300Mut cells exposed to 0.4uM Aphidicolin (APH) overnight (O/N), representative images are shown on the right hand most corner, N = 2. B Analysis of number of EdU foci (red), sandwiched between FANCD2 twin-foci, per mitotic cell nucleus (DAPI, blue) in EP300WT and EP300Mut cells exposed to 0.4 uM APH overnight (O/N), representative images are shown on the right, N = 2. C Analysis of the percentage of pRPA (red) positive micronuclei in EP300Wt and EP300Mut cells, in the absence of any inhibitor treatments. Representative images are on the right, for samples J1, J2, NA1, and NA3, N = 4, and for NA2, N = 5. D Percentage of FANCD2 (green) positive micronuclei in EP300WT and EP300Mut cells, in the absence of any inhibitor treatments. Representative images are on the right, N = 2. E Analysis of the number of 53BP1 nuclear bodies positive in G1 cells (cyclin A negative) in EP300WT and EP300Mut cells exposed to 0.4uM APH overnight (O/N), representative images are shown on the right, For J1, J2, NA1, and NA2 (UT) N = 3 and for NA2 (APH) and NA3, N = 4. For all experiments, data are presented as mean values +/− SD. P-values were determined by a two-tailed Student’s T-test. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm. N represents two/three experimental replicates from independent cultures of cells. Source data are provided as a Source Data file.

Fig. 6. EP300-mutated cancers closely resemble BRCA-deficient tumors.

(Top) In EP300WT cells, replication fork stalling in response to replication inhibition (depletion of nucleotide pools, DNA polymerase inhibition, oncogene activation), results in fork reversal at nascently synthesized DNA followed by RAD51 loading and the efficient restart of stalled forks in the presence of downstream effectors such as BRCA1/BRCA2/ PALB2, thus maintaining genome stability. (Bottom) However, in EP300Mut cells, elevated endogenous replication stress results in dormant origin firing at common fragile sites and increased genome-wide replication pausing. While fork regression followed by RAD51 leading occurs in these cells, defective downstream effectors, such as BRCA2, lead to excessive nucleolytic degradation at regressed forks, resulting in increased ssDNA at collapsed forks. This in turn, triggers POLD3-mediated replication restart during the S-phase, collectively mimicking phenotypes observed in BRCA-deficient cancers. Upon transitioning to G2/M, EP300-deficient cells rely on POLD3-dependent MiDAS to overcome under-replicated DNA, which is largely insufficient to repair the excessive persistent damage, resulting in miss-segregation of DNA during mitosis to daughter cells in G1. “EP300 mutated cancers closely resemble BRCA-deficient tumors,”. Created in BioRender. Madireddy, A. (2025), is licensed under CC BY 4.0, https://BioRender.com/9lfufms.