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SIRT1 Inhibitors Mitigate Radiation-Induced GI Syndrome by Enhancing Intestinal Stem Cell Survival

Acute radiation syndrome (ARS) affects the haematopoietic system (HP) and GI tract following exposure to high doses of radiation. Currently, there are no FDA approved countermeasures for the treatment of radiation-induced GI syndrome (RIGS). This is despite increasing concern about the risk of radiological accidents or terrorism.

Investigation of ARS progression in mice demonstrates that ARS can be induced with 12-15Gy abdominal irradiation or approximately 9Gy whole-body irradiation. The latter exposure results in intestinal stem cell damage and crypt survival responses rather than a full GI syndrome. At doses under 12Gy, fatalities occur due to GI syndrome, but death due to HP failure can be prevented by bone marrow transplant. Lethal doses of radiation deplete the stem cell pool at the bottom of the crypts of Lieberkühn, leading to impaired crypt-villus regeneration and loss of mucosal function and integrity. These effects result in diarrhoea, dehydration, nausea, anorexia, and systemic infection, which can lead to septic shock and death.

GI syndrome occurs via activation of p53 in the GI epithelium resulting in PUMA-induced apoptosis and depletion of intestinal stem cells (ISCs). p53 is also required to induce p21, which directs intestinal regeneration and resolves DNA damage and replication stress. Lysine acetylation of p53 occurs in response to radiation and DNA damage, this induces apoptosis and prevents p53 being degraded by MDM2. Sirtuin 1 (SIRT1), activated by caloric restriction and other stresses, deacetylates p53 resulting in p53 degradation by MDM2.

In this study, the authors investigated the effects of SIRT1 inhibitors on crypt regeneration and intestinal stem cell survival in Lgr5 +ve intestinal organoids.

Ex vivo crypt organoids were formed from intestinal crypts extracted from C57BL/6J mice and placed in matrigel. Radiation exposure resulted in a dose-dependent decrease in survival of crypt organoids with an LD50 of approximately 6Gy.

24hr post 8Gy irradiation crypt organoids were treated with nicotinamide (NAM), a SIRT1 inhibitor. This treatment significantly improved the survival of the organoids compared to untreated controls, with bFGF used as a positive control.

Further analysis identified a window between 12 and 30hrs post-irradiation where NAM could prevent organoid death with a peak at 24hrs demonstrating that NAM is a mitigator of the radiation response but not a protector. A precursor of NAD, nicotinamide mononucleotide (NMN), did not have a protective effect suggesting that the response is not due to increased NAD bioavailability.

Western blots also demonstrated that SIRT1 levels were not changed by radiation treatment.

A selection of SIRT1 inhibitors, EX527, Sirtinol, Inauhzin and Salemide also demonstrated similar mitigation of radiation in crypt organoids. Treatment with SIRT2 inhibitors Thiomyristoyl, SirReal2 and AGK2 did not affect crypt organoid survival after irradiation.

In mice irradiated abdominally with 13, 14 and 15 Gy, peritoneal injection of 1000mg/kg NAM protected mice from death due to acute GI radiation syndrome. A single 14Gy dose of subtotal body irradiation (SBI) led to complete depletion of crypts in untreated mice. A single dose of NAM 24hrs post-treatment increased mouse survival, 56.25% compared to 6.25% of controls. This was confirmed using EX527, where 30% of mice survived compared to 0% of controls.

Using a microcolony formation assay to assess regenerating crypts, NAM treatment increased the number of regenerating crypts compared to controls. NAM treatment also resulted in full GI mucosa recovery and no GI syndrome lethality.

The effects of SIRT1 inhibitors on ISC survival in vivo was assessed using Lgr5-LacZ transgenic mice. Significantly higher numbers of LacZ+ ISC were detected in mice that were treated with NAM or EX527 post-irradiation compared to untreated controls. The findings were confirmed in an Lgr5-GFP transgenic mouse model.

Western blot analysis of small intestine epithelial organoids treated with NAM after irradiation demonstrated increased p53 acetylation (K379) and enhanced expression of p21. No changes were observed for p53(K12) acetylation and PUMA expression was unchanged.

In SIRT1 KO epithelial organoids treated with radiation, the mitigation effect of NAM on organoid survival was lost. A similar effect was observed in p53 KO organoids. SIRT1 KO organoids were also more radiosensitive than WT controls.

Edu staining was used to identify and quantify mitotic cells in the crypts treated with irradiation and NAM. There were much fewer dividing crypts in NAM treated mice compared to controls 36 hours post-irradiation. More residing crypt cells were detected in NAM treated mice compared to controls, suggesting that inhibition of SIRT1 activates p53 inducing a cell cycle arrest that improves ISC survival after irradiation.

NAM treatment showed very similar radio-protective effects in human ileum organoids.

In this study, the authors demonstrated that SIRT1 inhibitors were able to mitigate some of the effects of acute radiation exposure. SIRT1 inhibitors prevented GI syndrome and protected the ISC from being depleted following abdominal irradiation. Further analysis revealed that the effects were most likely due to increased p53 acetylation and upregulation of p53 targets such as p21 resulting in cell cycle arrest rather than inducing PUMA-mediated apoptosis. The authors have also assessed CBP300 inhibitors in these models having important implications in the development of countermeasures for radiation-induced injuries.

Reference

Fu et al, 2021. SIRT1 inhibitors mitigate radiation-induced GI syndrome by enhancing intestinal-stem-cell survival. Cancer Letters, 501, 20-30
10.1016/j.canlet.2020.12.034

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Epistem's expertise and history in radiation biology has been applied to the evaluation of potential mitigators of Acute Radiation Syndrome (ARS) to aid the development of medical countermeasures. Epistem has over 10 years of experience in the evaluation of ARS mitigators for NIAID and other US Government departments. Models are provided with variable levels of bone marrow shielding and types of medical management to mimic a variety of radiation exposures and clinical support measures.

Epistem offers validated small intestinal in vitro organoid models derived from a variety of species, including human, canine and rodents. Our organoid cultures display cellular architecture that closely resembles that observed in vivo. Cultures mimic the stem cell niche allowing cell proliferation and differentiation to occur. The established organoid protocols allow MOA determination and provide in vitro to in vivo biological and PD linkages through the following readouts:

  • Viability assays (visual or with MTS)
  • Histology/IHC or RNAscope to assess the effect on specific protein expression including LGR5
  • Gene expression (NGS, Microarray or qRT-PCR) to evaluate the effect of treatment on expression of signalling pathways genes
  • FACS analysis to identify specific cell populations affected
  • ELISA to identify and quantify cytokines of interest within organoid's spend media

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