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Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress, Mediated by Defective Fork Protection at Stalled Replication Forks in Adult T-Cell Leukemia/Lymphoma
Angelica Barreto-Galvez1, Mrunmai Niljikar1, B. Hilda Ye, PhD2, Advaitha Madireddy, PhD1,31Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA;
2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA;
3Department of Pediatrics Hematology/Oncology, Robert Wood Johnson Medical School, New Brunswick, NJ, USA
Background: Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). In a recent retrospective analysis of a single center cohort of Caribbean/American ATLL (NA-ATLL), the median overall survival (OS) was only 6.9 months, worse than the OS outcome in the largest ATLL cohort from Japan (J-ATLL) of ~ 1 year. Further analysis revealed that 20% of NA-ATLL patients had mutations in the EP300 gene, and that ATLL patients with epigenetic mutations had worse prognosis as compared to those without these mutations. The mechanistic contribution of p300 dysregulation to aggressive disease presentation in NA-ATLL is currently unknown.
EP300/CBP are global transcriptional coactivators that catalyze the addition of acetyl groups to lysine residues on histones and non-histone proteins. Reports suggest that the independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA repair. While CBP downregulation results in changes to genes involved in antigen presentation, and terminal B-cell differentiation. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Almost all functional studies, in the literature, either examine p300 and CBP together or assess the importance of p300 to DNA repair since studies look at response to DNA damaging agents such as UV irradiation or ionizing radiation. For example, histone acetylation at double strand breaks by p300/CBP has been shown to recruit DNA repair proteins to chromatin. In addition, numerous studies show that p300/CBP-mediated acetylation of proteins involved in DNA replication/repair can stimulate or inhibit their activities in reconstituted systems. Despite the suggested role for EP300 in DNA replication, it is not known whether inactivating mutations in EP300 spontaneously induce endogenous replication stress. Importantly, functional studies analyzing how these EP300Mut cells overcome replication fork collapse in the presence of replicative inhibitors such as aphidicolin and hydroxyurea have not been performed.
Aim and Methods: In this study, we sought to characterize the independent role of p300 in maintaining DNA replication integrity by studying patient-derived North American ATLL cell lines, that carry loss-of-function somatic mutations in EP300 (but not CBP). These novel ATLL cell line models provide a powerful tool to examine genotype to phenotype correlations associated with EP300 deficiency. To directly establish causal connections, we also took advantage of a highly selective EP300-specific PROTAC degrader that does not target CPB (a common problem with commercial EP300 inhibitors) and has limited toxicity in vivo. To address p300’s role in DNA replication, we analyzed replication integrity at the genome-wide level by DNA fiber analysis and a powerful locus specific single molecule assay called SMARD to assess replication dynamics at the locus-specific level. In addition, we analyzed cell cycle progression and sensitivity of p300 deficient cells to replicative inhibitors by flow cytometry and evaluated the chromatin recruitment of a number of replication fork protection and restart proteins by immune staining. To understand the consequences of s-phase defects in G2/M phase of the cell cycle, we quantified the presence and repair of under-replicated DNA in mitosis by Mitotic DNA synthesis. Finally, we assessed the prevalence of mitotic segregation defects by analyzing micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in p300-deficient cells.
Results: Here, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells.
Conclusions: We demonstrate that the DNA replication dynamics of EP300-mutated cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart, resulting in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.
Speakers
Advaitha Madireddy