Normal stem cells from tissues often exhibiting radiation injury are highly radiosensitive and exhibit a muted DNA damage response, in contrast to differentiated progeny. These radioresponses can be attributed to unique epigenetic regulation in stem cells, identifying potential therapeutic targets for radioprotection.

SummaryDynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.

Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.

Radiation therapy is often associated with normal tissue injury as an undesired side effect. This is promoted by the depletion of normal stem cells and a decreased regenerative capacity of the affected organs. To reduce normal tissue injury during radiotherapy protective treatments have to be developed that improve stem cell survival without protecting tumor cells. In this study we investigate the acetylation and methylation of histone 3 at lysine 9 (H3K9), that is responsive to DNA damage and uniquely regulated in stem cells, as potential target for the development of radioprotective therapeutics. We analyzed stem cells in mouse tissue niches in vivo, murine embryonic stem cells in culture and neural stem cells freshly isolated from newborn mouse pups. We investigate repair factors, histone modifications and apoptosis pathways by immunofluorescence staining, western blotting and flow cytometry after X-ray irradiation or micro-irradiation of individual cells. H3K9 modifying enzymes are targeted with inhibitors, activators, RNA interference or overexpression by plasmid transfection and the effect on stem cells’ radiosensitivity evaluated. We show that stem cells are highly radio-sensitive and have reduced DNA repair with low activation of DNA damage factors like ATM. We screened for stem cell specific levels of histone modifications that are responsive to DNA damage and discovered that stem cells of adult tissue niches like brain and testis as well as murine embryonic and neuronal stem cells show very high levels of H3K9ac, while H3K9me3 levels are decreased. In tumor cells H3K9 acetylation (H3K9ac) was shown to be reduced after damage induction, while H3K9 tri-methylation (H3K9me3) is increased to fully activate the repair factor ATM. We hypothesize that in stem cells, an inept transition between H3K9 acetylation and tri-methylation prevents robust activation of ATM leading to high radiosensitivity. In agreement with this hypothesis, we find reduced down-regulation of H3K9ac in stem cells at the DNA damage sites. To transiently improve the H3K9ac/me3 switch in stem cells in order to enhance activation of DNA repair factors and stem cell survival after irradiation, we aim to transiently reduce H3K9 acetylation by targeting the responsible acetyltransferases and deacetylases. We show expression levels of H3K9 modifying enzymes in stem cells before and after irradiation as well as preliminary results of the effect of down- or up-regulation of H3K9 modifying enzymes on stem cell radiosensitivity. We conclude that elevated H3K9 acetylation and reduced H3K9 tri-methylation levels in stem cells and their responsible H3K9 modifying enzymes are potential targets to improve ATM activation and stem cell survival after irradiation. Citation Format: Barbara Meyer, Keith M. Jacobs, Suyash Raj, Cheri L. Zobel, Dennis E. Hallahan, Girdhar G. Sharma. The role of H3K9 acetylation and tri-methylation in stem cell radiosensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2015-3299

Side effects resulting from ionizing radiation (IR) during cancer radiotherapy often involve normal tissue injury including depletion of tissue regenerative capacity. Utilizing multiple in vivo tissue niches and cell culture models, we demonstrate that normal stem cells are highly radiosensitive while differentiated progeny are radioresistant, and IR-induced apoptosis of stem cells is observed broadly throughout the cell cycle independent of proliferation status. Our study demonstrates that normal stem cells fail to activate the DDR, do not display IR-induced foci in vivo as well as in culture, and exhibit severely attenuated DNA repair. Despite normal sensing of DNA breaks, recruitment/ retention of repair factors at DNA break sites is repressed in stem cells. Stem cells are unable to eliminate constitutive phosphorylation of H2AX-Y142 around break sites, abetting stem cells toward an apoptotic instead of repair pathway. The abrogated DDR in normal stem cells is associated with a constitutively enhanced histone-3 lysine-56 acetylation, which epigenetically contributes to muted retention of repair factors. Reinforced PP2A phosphatase expression was also identified as substantial regulators of stem cell radiosensitivity and transient inhibition of phosphatase activity restores stem cell survival. We thus identify pluralistic interacting molecular and epigenetic mechanisms that collectively control and impart IR hypersensitive phenotype to the normal stem cells. These insights will be crucial in development of prevention and intervention therapeutic strategies to minimize IR-induced stem cell dropout and increase the efficacy of radiotherapy. Citation Format: Keith M. Jacobs, Sandeep Misri, Barbara Meyer, Suyash Raj, Cheri L. Zobel, Barry P. Sleckman, Dennis E. Hallahan, Girdhar G. Sharma. Concerted epigenetic and signaling mechanisms regulate normal stem cell radiosensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2015-3300