Abstract: PD12-07
Sources of Funding: National Institutes of Health Grant R01CA108646 (NCI)

Introduction

Hemorrhagic cystitis is a highly-morbid inflammatory bladder disease associated with nitrogen mustard alkylating agents, most notably cyclophosphamide. Acrolein, a reactive oxygen species metabolite of cyclophosphamide, has been shown to silence DNA damage repair genes via global methylation pathways. 8-oxoguanine DNA glycosylase (Ogg1) is one such silenced base excision repair enzyme that can restore DNA integrity. Subsequent inflammation from the accumulation of DNA damage results in bladder smooth muscle pyroptotic cell death. We hypothesized that reversing inflammasome-induced imprinting and gene silencing in the bladder smooth muscle could prevent hemorrhagic cystitis.

Methods

Experiments were carried out using cultured detrusor fibroblasts, B6 wild-type mice, and Ogg1 knockout mice. Hemorrhagic cystitis was induced with either cyclophosphamide or acrolein. Mesna, the current standard of care treatment to prevent hemorrhagic cystitis; Nicotinamide, a vitamin B-3 analog shown to ameliroate bladder inflammation; and suberoylanilide hydroxamic acid, a histone deacetylase (HDAC) inhibitor with anti-inflamatory properties, were added to treatment groups. Harvested tissues and cells were subjected to bisulfite sequencing and chromatin immunoprecipitation analysis to evaluate DNA methylation patterns and epigenetic imprinting.

Results

There was enhanced recruitment of Dnmt1 and Dnmt3b to the Ogg1 promoter in acrolein treated bladder fibroblasts as demonstrated by the pattern of CpG-island methylation and resultant bisulfite sequencing. Accumulation of reactive oxygen species with spontaneous pyroptotic signaling was found in Ogg1 knockout detrusor cells. Suberoylanilide hydroxamic acid restored Ogg1 expression to physiologic levels moreso than either nicotinamide or Mesna in all hemorrhagic cystitis models. Additionally, suberoylanilide hydroxamic acid restored histologically-visible cyclophosphamide-induced bladder damage to that of normal untreated control mice.

Conclusions

The pattern of epigenetic imprinting induced by inflammation suggests a novel therapeutic target for the treatment of hemorrhagic cystitis. HDAC inhibitors can reactivate Ogg1 expression by altering DNA methylation through Dnmt3B regulation. More broadly, the data suggest that re-programming epigenetic imprinting could limit the inflammatory process induced by not only cyclophosphamide but by a multitude of other toxic insults as well.

Funding

National Institutes of Health Grant R01CA108646 (NCI)