Purpose/Objective(s) Establish a novel therapeutic strategy for the management of ovarian cancer with LR-IL-22 gavage, an intestinal radioprotector, through its pivotal role in modifying the tumor microenvironment and subsequently improving the survival of female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer. Materials/Methods To assess differential gene expression following irradiation and LR-IL-22 gavage, we performed RNAseq analyses using Lgr5+GFP+ mice. Male and female mice were divided into four groups: control (0 Gy), 12 Gy total body irradiation (TBI), 12 Gy TBI + LR, and 12 Gy TBI + LR-IL-22. Twenty-four hours after TBI, mice received LR or LR-IL-22 gavage. In a separate experiment with female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer, flow analysis and immunohistochemistry were used to assess the tumor microenvironment following fractionated whole abdomen irradiation (WAI) using 6 Gy × 4 fractions with or without LR-IL-22 gavage. Results We observed that intestinal stem cell gene expression varied widely following exposure to TBI. Genes were either upregulated or downregulated following irradiation, and many were solely rescued by the genetically engineered probiotic LR-IL-22. In fact, 135 genes were uniquely rescued and thus downregulated by LR-IL-22, compared to 43 genes uniquely rescued and thus upregulated by LR-IL-22 following TBI. Moreover, the addition of fractionated WAI to LR-IL-22 not only induced PD-L1 protein expression in ovarian cancer cells and mobilized CD8+ T cells, but potentially reduced tumor growth rate through the migration of LR bacteria into the 2F8cis ovarian tumors. Conclusion Our data illustrates the effectiveness of LR-IL-22 as a radiation mitigator that manifests its role by modifying both, gene expression and the tumor microenvironment, following irradiation. Indeed, irradiation induced differential gene expression in intestinal stem cells, and significantly upregulated CD8+ T cells infiltrate in 2F8cis tumors, giving them an immunological ‘‘hot’’ phenotype which positively correlated with PD-L1 expression. This forms the basis for combining WAI with targeted therapies, including immune checkpoint inhibitors, in advanced or recurrent epithelial ovarian cancer.

LR-IFN-β is both a feasible and effective radiation mitigator that could potentially improve the management of ovarian cancer patients. Furthermore, the subsequent addition of platinum/taxane-based chemotherapy to the combination of WAI and LR-IFN-β should reduce tumor volume while protecting the intestine and thus improve overall survival in ovarian cancer patients.

Oral administration (gavage) of a second-generation probiotic, Lactobacillus reuteri (L. reuteri), that releases interleukin-22 (LR-IL-22) at 24 h after total-body irradiation (TBI) mitigates damage to the intestine. We determined that LR-IL-22 also mitigates partial-body irradiation (PBI) and whole-abdomen irradiation (WAI). Irradiation can be an effective treatment for ovarian cancer, but its use is limited by intestinal toxicity. Strategies to mitigate toxicity are important and can revitalize this modality to treat ovarian cancer. In the present studies, we evaluated whether LR-IL-22 facilitates fractionated WAI in female C57BL/6 mice with disseminated ovarian cancer given a single fraction of either 15.75 Gy or 19.75 Gy or 4 daily fractions of 6 Gy or 6.5 Gy. Mice receiving single or multiple administrations of LR-IL-22 during WAI showed improved intestinal barrier integrity (P = 0.0167), reduced levels of radiation-induced intestinal cytokines including KC/CXCL1 (P = 0.002) and IFN-γ (P = 0.0024), and reduced levels of plasma, Eotaxin/CCL11 (P = 0.0088). LR-IL-22 significantly preserved the numbers of Lgr5+GFP+ intestinal stem cells (P = 0.0010) and improved survival (P < 0.0343). Female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer that received LR-IL-22 during 6.5 Gy WAI in 4 fractions had reduced tumor burden, less intestinal toxicity, and improved 30-day survival. Furthermore, LR-IL-22 facilitated WAI when added to Paclitaxel and Carboplatin chemotherapy and further increased survival. Oral administration (gavage) of LR-IL-22 is a potentially valuable intestinal radioprotector, which can facilitate therapeutic WAI for widespread intra-abdominal ovarian cancer.

The data indicate that sorted irradiated red senescent tdTOMp16 stromal cells can be isolated as a pure non-dividing population and induce fibrotic markers in target cells in non-contact transwell cultures. This unique system can now be used to define the mechanism of senescent cell-induced radiation fibrosis, and the effects of senolytic drugs.

We sought to determine whether specific taxa in the communities of the C57BL/6J mouse intestinal microbiome were associated with survival after the LD50/30 dose of total body irradiation (TBI), and if administration of a second-generation probiotic producing anti-inflammatory Interleukin-22 (IL-22) mitigated Gastrointestinal Syndrome inducing doses of total body irradiation (TBI). Female C57BL/6J mice were irradiated to LD50/30 9.25 Gy TBI, or 19.75 Gy whole abdominal irradiation, and evaluated for primary endpoint of survival and secondary endpoint of expression of inflammatory proteins and bone marrow CFU-GEMMs per 104 cells. Daily collected fecal samples were analyzed for 16sRNA associated with 15 major taxa in the intestinal microbiome. Second-generation probiotic Lactobacillus reuteri or Escherichia-coli producing IL-22 (LR-IL-22 or E. coli-IL-22, respectively) was administered to subgroups by gavage at 24 hours after irradiation. The results were compared to subcutaneous administration of IL-22 (n = 12). Thirty-day TBI survivors of 9.25 Gy had at day 14 a predictive increased relative abundance of Lactobacillus, Roseburia, and Akkermansia, compared to other taxa, as did radiation mitigator treated (G-CSF or JP4-039) mice. Administration of LR-IL-22 at 24 hours after irradiation increased survival (p = 0.0144), as did E. coli-IL-22. GFP-IL-22 fusion protein producing probiotics showed uptake in intestinal villi at 2 h after gavage and clearance by day 5. At 24 hrs. after 9.25 Gy TBI, mice with 4 antibiotic-cleared intestinal microbiomes (5 weeks administration in drinking water of vancomycin, neomycin trisulfate, metronidazole, and ampicillin) had increased survival when treated with E. coli-IL-22, (p < 0.0001). On day 5 after 9.25 Gy TBI, the intestine and bone marrow were isolated. Luminex assay showed significantly decreased inflammatory proteins in mice gavaged with LR-IL-22, and bone marrow had significantly increased CFU-GEMMs per 104 cells compared to 9.25 Gy only (15.1 ± 1.1 and 9.1 ± 1.5, respectively, p = 0.0351). Whole abdomen irradiation to 19.75 Gy followed by gavage at 24 hrs. of LR-IL-22, or E. coli-IL-22 significantly increased survival (p = 0.0138, 0.0473). These data indicate that the relative abundance of specific taxa in the intestinal microbiome correlates with survival after total body irradiation. Furthermore, gavage of LR-IL-22 improves survival after GI Syndrome inducing doses of irradiation. Elucidation of the molecular mechanism of interaction of pro-survival microbiome communities with intestinal stem cells and regenerating crypts should identify new targets for intestinal radiation protection and mitigation.

Ocular melanoma is treated by placing a plaque containing radioactive seeds behind the eye for 72 hr. This has been a successful method for treating ocular melanoma; but unfortunately, approximately 50% of these patients develop radiation retinopathy leading to blindness. JP4-039 is a mitochondrial targeted nitroxide which has been demonstrated in mice to protect against total body irradiation and protect the oral cavity from radiation induced oral mucositis (Rwigema et al, IJROBP 80:860-868, 2011 and Berhane et al, Radiation Research, 182:35-49, 2014). To demonstrate that JP4-039 can protect the retina from irradiation, the eyes of Balb/C mice were injected with JP4-039 in 30% cyclodextrin and irradiated twenty minutes later to 20 Gy to the eye. The eyes were examined for the percent of the retina showing apoptosis. Balb/C mice were anesthetized with isoflurane. A 27 gauge needle was placed in the anterior chamber of the eye to remove some of the vitreous fluid. Bodipy-JP4-039 dissolved in 30% cyclodextrin was pulled up in a 1 ml syringe. A 31 gauge needle was placed on the syringe and 20 ul of JP4-039 was injected into the vitreous cavity. Mice were sacrificed at 0, 15, 60 or 120 minutes after injection, with the eye removed and fixed in 2% paraformaldehyde for 1 hr and then placed in 30% sucrose. The eyes were sectioned, examined using a fluorescent microscope and the fluorescence measured. In separate experiments, JP4-039 was injected into the Balb/C eye and irradiated to 20 Gy thirty minutes later. The eye irradiation was done using 6 MV electrons on a Varian True Beam irradiator with a 5 mm cone and 1 cm of bolus covering the eye. Eighteen hours later the mice were sacrificed, the eye removed and fixed as described above. The eyes were sectioned, stained for apoptosis using a tunnel assay and the percent of retinal cells undergoing apoptosis was determined. At 15 minutes following injection of bodipy-JP4-039, there was an increase in fluorescence in the retina from 5083 ± 390 to 9875 ± 436 (p < 0.0001). By 60 minutes no fluorescence was detected above background in the retina of the eye. Control mice or control mice injected with JP4-039 had 1.0 ± 0.4 or 1.2 ± 0.4% of the cells in the retina displayed apoptosis. Mice irradiated to 20 Gy had a significant increase in the percent of the apoptotic cells with 49.0 ± 2.7% of the cells showing apoptosis (p < 0.0001) which is decreased by the intraocular injection of JP4-039 to 35.8 ± 2.5% (p = 0.0066). Intraocular injections of JP4-039 in 30% cyclodextrin resulted in uptake of the JP4-039 by the retina. Following 20 Gy to the eye, there is a significant increase in apoptosis in the retina which is decreased by the intraocular injections of JP4-039 before irradiation. Further investigation is warranted to demonstrate that JP4-039 can protect the eye during radiotherapy for ocular melanoma.

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse or 3) human cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice (n=12) received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P. saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at days 1, 14, and 21 post-irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender-mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell ASCs demonstrated significant down regulation in lower level cells of pro-fibrotic genes (including collagen 1-4, and TGF β) in unirradiated acutely irradiated and irradiation- fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson's Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. Single 1 x 106 ASCs injection day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.

Fanconi anemia (FA) is an inherited disorder that is associated with anemia, congenital abnormalities and radiosensitivity. Marrow transplanted and non-transplanted FA patients have an increased incidence of malignancy, in particular oral cavity squamous cell carcinomas. Some FA patients are sensitive to radiation therapy due to a mutation in one or more of the 23 genes in the FA pathway3. Radiation protective agents and normal tissue sparing proton therapy may help treat these patients safely. We tested the combination of JP4-039, a GS-nitroxide radiation damage mitigator, and proton therapy in a mouse model of FA. In vitro: Fanca-/- and Fanca+/+ bone marrow stromal cells were pre-treated with 10 uM JP4-039 and irradiated with either proton or photons (0-10 CGE/Gy) followed by clonogenic survival and β-Galactosidase senescence analysis. In vivo: Fanca-/- and Fanca+/+ mice were pretreated with water (100uL), 4-AT/Miglyol (0.4 mg), Miglyol (vehicle control) or JP4-40 (0.4 mg) 10 minutes prior to oropharyngeal irradiation with either protons or photons (0 or 30CGE/Gy) followed by sacrifice and oral cavity ulceration scoring, distant hematopoietic suppression, and RT-PCR. Fanca-/- cells treated with proton therapy had a D0 = 2.11 ± 0.17 CGE; while Fanca+/+ cells had a D0 = 3.28 ± 0.10 CGE (P = 0.0027). Pre-treatment with JP4-039 was radioprotective in Fanca-/- and Fanca+/+. Following proton treatment, Fanca-/- cells had lower β-galactosidase positive clonogens compared to Fanca+/+ (10% vs. 28%, P < 0.05). Following photon treatment, Fanca-/- cells had higher β-galactosidase positive clonogens compared to Fanca+/+ (17% vs. 7%, P < 0.05). Pretreatment with JP4-039 reduced oral cavity ulceration among Fanca-/- (6.5% vs. 26.5%, P < 0.0001) and Fanca+/+ (9% vs. 23.5%, P < 0.0001) mice irradiated to 30CGE. Oral cavity transcripts Nfkb, Ap1, Sp1, and Nrf2 were increased following proton radiation, compared to unirradiated tissue, and was mitigated by pretreatment with JP4-039. Proton-induced changes in cytokine (TGF-β and IL-1α), oxidative stress response (Sod2), and radiation response-related (Gadd45, Rad51, p21 and p53) transcripts were also observed. Proton therapy also resulted in distant marrow suppression in Fanca+/+ and Fanca-/- mice. Abscopal bone marrow suppression was reduced with JP4-039 pretreatment. JP4-039 was able to protect Fanca-/- and Fanca+/+ cells from both proton and photon radiation. In our in vivo model, oral JP4-039 reduced the degree of oral mucositis following a large single fraction of 30CGE to the head and neck region of both Fanca-/- and Fanca+/+ mice. These data support future clinical trials to evaluate the use of intraoral JP4-049 among FA patients receiving radiation therapy and indicate that more research is required to understand the differences between photon and proton irradiation.

Irradiation of cells results in induction of several programmed death pathways such as apoptosis, necroptosis, and ferroptosis. Injection of one mitigator may block one of the death pathways, but not the other pathways which can still lead into death of the cells. Maximal mitigation may require multiple mitigators administered at the appropriate time which can block different pathways. Previously, we demonstrated that the administration of JP4-039 at 24 hr after irradiation and necrostatin-1 at 72 hr resulted in increased survival than when given individually (Steinman, Radiation Research, 189: 68-83, 2018). We now describe the mitigation of irradiation damage using a combination of an anti-apoptosis drug (JP4-039), an anti-necroptosis drug (necrostatin) and anti-ferroptosis drug (baicalein). C57BL/6NTac female mice at 8 weeks of age were irradiated to 9.25 Gy total body irradiation. Mice were injected with either JP4-039 (20 mg/kg) or baicalein (50 mg/kg) at 24 hr or necrostatin-1 (1.65 mg/kg) at 48 hr after irradiation. Combinations of the three drugs were also administered with JP4-039 and Baicalein given at 24 hr after irradiation and necrostatin-1 at 72 hr. Mice were injected with one drug, two drugs or all three drugs. In other experiments, mice were sacrificed at 0, 1, 2, 3 or 4 days after irradiation and samples of the bone marrow from the femur, ileum, lungs and blood plasma were collected. The samples were analyzed for gene expression for 33 pro-inflammatory genes using a Luminex assay. Mice irradiated to 9.25 Gy had a 5% survival at day 30 following irradiation. Mice injected with JP4-039, necrostatin-1, or Baicalein had an increased survival of 25% (p = 0.0432, 0.2816 or 0.1120, respectively). Combinations of necrostatin-1 and Baicalein resulted in a survival of 40% (p = 0.0359), JP4-039 and necrostatin had a survival of 50% (p = 0.0062), and JP4-039 and Baicalein had a survival of 60% (p = 0.0064). The best survival followed the combined administration of all three drugs with a survival of 75% (p = 0.0002). Luminex assays demonstrated a decreased expression of inflammatory genes in mice administered the three mitigators following irradiation. Administration of three drugs blocking three different programed cell death pathways following irradiation mitigated irradiation-induced damage resulting in increased survival and decreased inflammatory gene expression. Further investigations of using these drugs is warranted.

Total body irradiation (TBI) can cause multiple organ failure, including bone marrow and gastrointestinal (GI) tract failure. The gut microbiota is closely involved in most, if not all, aspects of normal host physiology, from nutritional status to environmental stress response. It is not yet known if radiation-induced changes in the gut microbiota can impact recovery after TBI. In this study, we investigated changes in gut microbiome in multiple mouse strains after TBI. Based on these results, we tested the hypothesis that gavage of the anaerobe Akkermansia muciniphilia would increase survival after TBI. Microbiome profiling: 20x C57BL/6J, 10x BALB/c and 10x sv129 mice, all male and 8-week- old, received the LD50/30 TBI dose of 9.25 Gy. Fecal pellets were collected on day of irradiation and on days 5, 10 and 15 after TBI. Bacterial 16S rRNA gene sequences in each sample were amplified, sequenced on the Illumina MiSeq, and analyzed with QIIME. qPCR was performed with A. muciniphilia specific primers and Applied Biosystems PowerUp SYBR Green Master Mix. Antibiotic treatment and Akkermansia gavage: 20x C57BL/6J mice, all male and 8-week-old, were placed on drinking water with ampicillin, vancomycin, neomycin trisulfate and metronidazole for 4 weeks, and one day later, 10 mice were gavaged with 2x10ˆ8 CFU A. muciniphilia and 10 with PBS control. Then 5 of each group received TBI dose of 9.25 Gy one day later. DSS colitis model: 2% dextran sodium sulfate (DSS;40 – 50 kDa) was put into drinking water for 4 days. Radiation induced alterations in the gut microbiota: All radiated mice experienced a collapse of bacterial diversity, regardless of strain or sex. DNA sequencing results and confirmatory qPCR indicated that the abundance of A. muciniphila increased dramatically after TBI, as early as day 2 after TBI. qPCR with specific primers further confirmed this finding. To determine if this result was specific to radiation injury, we also assessed microbiome changes in the DSS colitis model and observed a similarly sharp increase in Akkermansia abundance after injury. Therapeutic potential of A. muciniphila: In a separate TBI experiment, antibiotic-treated mice receiving A. muciniphila one day prior to TBI exhibited increased 30-day survival (5 out of 5 mice survived) compared to mice receiving a sham gavage (0 out of 5 survived). TBI dramatically shifts mouse gut microbiota, with rapid enrichment of A. muciniphila, an anaerobe with widely recognized beneficial properties. The increase in A. muciniphila likely represents the normal response to gut mucosal injury, since other gut damage models also facilitate its proliferation. These results suggest that supplementation of A. muciniphila or other probiotics can improve outcomes after radiation injury by augmenting the endogenous stress response. Supported by NIH/NIAID grants U19AI067773 and U19-A168021.

Total body irradiation (TBI) initiates an inflammatory response that includes multi-factorial and multi-stage mechanisms. Intestinal epithelium is an early target for radiation induced damage. High levels of iPLA2Y expression is commonly associated with the accumulation of pro-inflammatory oxygenated octadecanoids and eicosanoids that cause mitochondrial dysregulation. Here we employed an in vivo TBI model of inflammation and demonstrated by LC-ESI-MS/MS that mitochondrial phospholipids (CL/CLox) and lipid mediators are essential drivers of the inflammatory responses. We further demonstrate that suppression of their formation by a specific inhibitor of iPLA2-gamma, R-BEL, is highly protective against TBI-induced injury associated with excessive activation of pro-inflammatory processes. C57BL/6NHsd female mice were irradiated to a dose of 9.25 Gy total body irradiation (TBI). At 24 hr after irradiation a subgroup of mice were injected intravenously with R-BEL (6 mg/kg in 10% cremphor el, 10% ethanol and 80% water). On days 0 to 7 after irradiation 5 mice per group were sacrificed, ileum isolated with half fixed for histology and half snap frozen. The ileum was homogenized, lipids extracted and pre-separated by SPE. FFA and PL were analyzed after reverse phase LC-ESI-MS using a hybrid-quadrupole-orbitrap mass spectrometer and a mass spectrometer. Lipids were filtered by retention time and confirmed by MS/MS analysis. Total body irradiation revealed significant damage of the mouse ileum epithelium as evidenced by confocal microscopy. We performed redox lipidomics analysis of the diversified classes of phospholipids, FFA and lipid oxidation products. We demonstrated that TBI induces a decrease in the total content of CL, particularly species containing polyunsaturated fatty acids (PUFA). We demonstrated that TBI resulted in the hydrolysis of polyunsaturated CLs as well as oxygenated CLs, particularly 9- and 13-HODE-cadiolipins. We were able to detect the formation of various molecular species of monolyso-CLs and release of free fatty acids, including pro- and anti-inflammatory lipid mediators. Significantly increased levels of iPLA2Y in the intestinal epithelial cells of mice after TBI was accompanied by the accumulation of free AA and AA-derived pro-inflammatory lipid mediators (HXA3 and LTB4). Survival of mice in the TBI-induced injury model was significantly enhanced in the presence of R-BEL. Moreover, R-BEL was able to markedly decrease the level of the iPLA2Y expression and block TBI-induced accumulation of pro-inflammatory mediators, HXA3 and LTB4. R-BEL markedly decreased the iPLA2Y expression and blocked accumulation of pro-inflammatory mediators in the ileum after TBI thus mitigating irradiation induced damage.

Ionizing irradiation induced senescence may be a mechanism underlying late effects through the senescence-associated secretory phenotype or loss of proliferation. We sought to determine the effect of the Fanconi Anemia (Fanca-/-) genotype and MMS350 on irradiation-induced senescence. Fanca+/+, 129Sv+/+ (WT), and 129Sv Fanca-/- bone marrow stromal cells were irradiated to doses up to 10Gy, grown in the presence or absence of 400 μM MMS350, and assayed at day 3. Cells were plated at sub-confluence, then irradiated using a cesium irradiator. Senescence was determined by chromogenic beta-galactosidase assay (percent positive b-gal). 129Sv+/+ and 129Sv Fanca-/- mice received total body irradiation (TBI) using a Mark I Gamma Cell Irradiator. Subgroups of TBI and control mice were maintained on regular water or with 400uM MMS350 (Kalash, et al., Radiat Res, 180:474, 2013). Subgroups were sacrificed at 7days or 1 year after irradiation. Organs were immediately sectioned and scored for b-gal at 40x magnification. Statistical analysis was carried out using student’s t-test. Control non-irradiated cells showed no significant effect of genotype on senescence-associated b-gal staining at baseline (0.23% +/- 0.23% in WT vs 0.23% +/- 0.23% in Fanca-/-). By day 3 after 10Gy, b-gal staining was significantly induced in both WT (3.06% +/- 1.08%, p=0.007) and Fanca-/- cell lines (10.5% +/- 1.17%, p=0.001). Fanca-/- showed significantly higher induction compared to WT (p<0.0001). Growth in MMS350 after 10Gy reduced senescence in Fanca-/- cell lines (8.12% +/- 1.2%, p=0.04). Seven days after 7Gy TBI, the spleen showed a significant increase in b-gal staining in both WT (0.74 +/- 0.07 at 0Gy vs 1.81 +/- 0.39 at 7Gy, p=0.020) and in Fanca-/- (0.38 +/- 0.20 at 0Gy vs 1.5 +/- 0.45 at 7Gy, p=0.029). At 1 year, WT showed a higher baseline staining compared to Fanca-/- in spleen (1.43 +/- 0.10 in WT vs 0.83 +/- 0.14 in Fanca-/-, p=0.025). In contrast, 7.5 Gy caused a significant decrease in b-gal in WT at 1 yr (0.81 +/- 0.12, p=0.036) with little effect of MMS350. In Fanca-/- spleen, there was an increase at 1 yr (1.58 +/- 0.13 after 7.5 Gy, p=0.0009), which was reduced by MMS350 (1.17 +/- 0.13). The brain showed no significant effect of time or genotype between groups. Fanca-/- bone marrow stromal cell lines showed greater irradiation induction of senescence over time compared to WT. MMS350 caused a reduction in the senescence in Fanca-/-. In vivo, the spleen showed a significant radiation induction in Fanca-/- and WT at 7 days. In contrast, WT spleen showed decreased senescence at 1 year after irradiation, while Fanca-/- spleen showed an increase. In the Fanca-/- mice, MMS350 reduced senescence at 1 year after irradiation.

Effective mitigation of ionizing irradiation toxicity in the civilian or military setting must be rapidly deployable in a formulation suitable for self-administration. The mitochondrial targeted GS-nitroxide (JP4-039) has been demonstrated to be highly effective in total body irradiation (TBI) mitigation in animal models (Rwigema, IJROBP, 80(3): 860-869, 2011, Goff, In Vivo, 25: 315-324, 2011, Steinman, Radiation Research, 189(1): 68-83, 2018), and is effective when delivered as late as 72 h after TBI. The drug has been shown to mitigate ionizing irradiation damage to the bone marrow and the gastrointestinal tract, and it is effective in organ specific (partial body) irradiation protection and mitigation (Berhane, Radiation Research, 182: 35-49, 2014, Shinde, Radiation Research, 185: 134-150, 2016). JP4-039 has also been shown to be effective for mitigation and treatment of beta irradiation (fallout) skin damage (Brand, J Investigative Derm, 137(3): 576-586, 2017). Pharmacokinetics (PK) experiments demonstrated clearance of JP4-039 after I.V. administration with a half-life of 10 min when administered I.V. (Christner, J Pharm & Biomed Analysis, S0731-7085(17): 32605-5, 2017). We evaluated the feasibility of intra-muscular injection of JP4-039 in each of three commercially available formulations (Captisol, 2-hydroxypropyl-B-cyclodextrin (Cyclodextrin), and Miglyol-812-N). C57BL/6J male and female mice received the LD50/30 TBI dose of 9.25 Gy, and 24 h later were administered 10 mg/kg JP4-039 in 100 μmL I.M. formulation. Significant radiation mitigation was demonstrated when JP4-039 was administered in each formulation (Table) (n=12 per group). There was no significant mitigation seen in mice given each formulation alone (n=12 per group). Abstract SU_40_2393; Table 1Survival Increase After Total Body Irradiation (TBI)FormulationLD50/30 Survival Increase at Day 30 After 9.25 GyMiglyol-812-N/JP4-039p = 0.0088Cyclodextrin/JP4-039p = 0.0015Captisol/JP4-039p = 0.0247 Open table in a new tab Satisfactory stability of JP4-039 in the most straightforward formulation using Miglyol-812-N was observed at room temperature, and this must now be assessed over a wide range of temperatures and storage intervals. The effectiveness in I.M. administration makes JP4-039 suitable for both civilian and military applications.

ALS is a fatal neuromuscular disease caused by the progressive nerve degeneration of motor neurons in the brain and spinal cord. While the cause of ALS is unclear, 10 to 15% of patients have a familial history of the disease, while the majority of cases (85 – 95%) develop the disease sporadically. There are nearly forty genetic mutations associated with ALS, the second most common of which is a mutation in the Superoxide Dismutase-1 (SOD1) gene. The well-established SOD1G93A mouse model, which replicates ALS in human patients, and shows progressive muscle wasting, paralysis, and death. Current antioxidant therapies, biological response modifiers, cytokine inhibitors, cytokine receptor antagonists, and an introduction into the spinal cord of neurotrophic growth factors or mesenchymal stem cells engineered to produce neuroleukines have been largely unsuccessful. Since most ALS patients are diagnosed after onset of symptoms, we carried out experiments with 90 day old SOD1G93A mice demonstrating level 1 paralysis. Mice received 9.0 Gy total body irradiation (TBI) Cesium source 340 cGy per minute, and intravenous bone marrow transplantation with 1 x 106 C57BL/6 GFP+ donor bone marrow. Control C57BL/6NTac mice were transplanted at the same age with SOD1G93A ALS bone marrow. Spinal cord sections were analyzed by single photon, confocal, ribbon-scanning microscopy for bone marrow origin cells. Blood/brain barrier permeability was analyzed by I.V. perfusion with 0.2 μm Fluorospheres (F8810, Invitrogen). The results demonstrated significant prolongation of paralysis free survival in bone marrow transplanted SOD1G93A mice from 100 to over 250 days (p=0.0018). In contrast, control mice transplanted with SOD1G93A bone marrow showed no evidence of paralysis. Marrow transplanted SOD1G93A mice demonstrated bone marrow origin, GFP+ M2 microglial cells surrounding degenerating anterior horn motor neurons at days 120, 200, and as late as day 280. Blood/brain barrier permeability was detected in SOD1G93A, but not control mice, or bone marrow transplanted SOD1G93A mice. Total body irradiation and bone marrow transplantation is a potentially valuable therapeutic option for treating ALS. Further studies are required to determine whether whole spine or segmental spine irradiation, brain and/or spine irradiation, or TBI will provide a therapeutic effect in the absence of marrow transplantation. The subset of donor bone marrow cells (hematopoietic stem cells, committed granulocyte/macrophage progenitors, or mesenchymal stem cells) required to provide the therapeutic effect must also be determined.

The increasing incidence of oral cavity squamous cell carcinomas in radiosensitive Fanconi Anemia (FA) patients suggested that protons might minimize normal tissue toxicity. We tested the radiation protector/mitigator, JP4-039, with Proton irradiated mesenchymal stem cell lines derived from Fanca-/- mouse marrow. Fanca-/- and control Fanca+/+ bone marrow stromal cell lines were irradiated to doses of 0 – 10 Gy via pencil beam scanning proton therapy system. Subgroups of cultures were treated with 100uM JP4-039 for 12 h prior to irradiation and after plating for clonogenic survival curves. Parallel cultures were irradiated using a JL Shepherd Model 68A cesium irradiator at 340 cGy per minute, subgroups treated with JP4-039 in a similar protocol. Cells were plated in 4 well limbro plates and incubated for 10 days in a CO2 tissue culture incubator. Colonies of greater than 50 cells were stained with crystal violet and counted. Data from triplicate experiments was analyzed using Linear Quadratic and Single Hit, Multiple Target model. Cultures irradiated to 5 or 10 Gy using proton or gamma irradiation were plated in T25 flasks and stained 10 days later for β-Galactosidase. Proton irradiation revealed radiosensitivity of Fanca-/- bone marrow stromal cell lines compared to Fanca+/+ cell lines (D0 = 2.11 ± 0.17, vs D0 = 3.28 ± 0.10 p = 0.0027, ñ = 1.1 ± 0.1, vs ñ = 1.0 ± 0.1, respectively). Fanca-/- cells were also radiosensitive to gamma irradiation, (D0 = 2.08 ± 0.17, vs D0 = 3.36 ± 0.05 p = 0.0017, ñ = 2.0 ± 0.5, ñ = 1.3 ± 0.3, respectively). JP4-039 provided significant radiation protection for both Fanca-/- and Fanca+/+ cell lines against both proton irradiation (D0 = 3.01 ± 0.19 p = 0.0125, ñ = 1.0 ± 0.1, vs D0 = 3.84 ± 0.11 p = 0.0196, ñ = 1.0 ± 0.1), and gamma irradiation ((D0 = 3.81 ± 0.72 p = 0.0423, ñ = 1.2 ± 0.2; D0 = 3.70 ± 0.06 p = 0.0133 p = 0.0133, ñ = 1.7 ± 0.7). Following proton irradiation, Fanca+/+ cells showed greater senescence compared to Fanca-/- cells: 28% vs 10% β-galactosidase positive (p < 0.05). In contrast, gamma irradiation of Fanca+/+ compared to Fanca-/- cells showed decreased senescence (7% compared to 17% (p < 0.05). Fanca-/- bone marrow stromal cell lines are radiosensitive to proton and gamma irradiation, but significantly protected by JP4-039. The data support intra-oral JP4-039 for Fanconi Anemia patients receiving proton radiotherapy for oral squamous cell carcinoma.

Total body irradiation to 3 Gy of E13.5 pregnant C57BL/6NTac mice reduces survival to 9.5 ± 5.6% of the pups at birth. Administration of the radiation mitigator JP4-039 twenty-four hours after irradiation on E14.5 increases pup survival to 54 ± 12% at weaning (p = 0.0023 compared to 3 Gy controls). Fetal Mice were tested to determine the cause of death. Pregnant mice were irradiated to 3 Gy total body irradiation on E13.5. Some of the mice were injected intravenously on E14.5 (24 hr after irradiation) with 20 mg/kg JP4-039 in F14 emulsion. In some experiments, the pregnant mice were sacrificed on the day before birth, the pups and placentas removed, weighed, and fixed in 10% formalin. In other experiments the pups were sacrificed within 4 hours of birth, or followed for survival. Necropsies were performed and all organs were collected including the brain, heart, lungs, liver, spleen, intestine, kidneys, and bone marrow. Organs were fixed in 10% formalin were sectioned, stained with H&E and examined microscopically. Placentas and pups removed from 3 Gy mothers or 3 Gy + JP4-039 mothers were measured. Pups from 3 Gy and 3 Gy + JP4-039 at birth were smaller (0.958 ± 0.028 g and 2.02 ± 0.04 cm in length and 0.837 ± 0.0014 g and 1.99 ± 0.03 cm in length, respectively) compared to nonirradiated pups (1.089 ± 0.023 g, p = 0.0015 and 0.0015, respectively, and 2.37 ± 0.02 cm in length, p < 0.0001). The placentas of the irradiated pups and pups treated with JP4-039 were also smaller than the control placentas (0.120 ± 0.004 g, 0.123 ± 0.005 g vs 0.140 ± 0.006 g, respectively, p = 0.0097 and 0.0553). By 5 days after birth the irradiated pups treated with JP4-039 at E14.5 had gained weight (2.45 g ± 0.11 gm) and were not significantly different than the control nonirradiated pups (2.70 ± 0.09 g). However, the weight of the pups from 3 Gy irradiated mothers was significantly decreased compared to the JP4-039 treated pups and the control pups (1.64 ± 0.04 g, p = 0.0168 and 0.0001, respectively). Necropsy of the pups on the day of birth revealed that pups from the 3 Gy irradiated mothers had decreased development of the cerebrum and cerebellum. The pups treated with JP4-039 also had decreased development of the cerebrum but not to the extent found in the irradiation controls. Fifty percent of the irradiation controls that survived through weaning developed hydrocephalus. In contrast pups from the 3 Gy irradiated mothers treated with JP4-039 that survived to adulthood developed no hydrocephaly. The JP4-039 treated females were infertile while males were fertile. Irradiation to 3 Gy of pregnant C57BL/6NTac mice on E13.5 decreased pup survival and retarded development of fetal cerebral cortex and cerebellum. There was hydrocephaly in mice surviving to adulthood. Treatment of the irradiated mice with JP4-039 on E14.5 increased survival, and had better developed cerebra and cerebella with no hydrocephaly. Therefore, treatment with JP4-039 protects the developing fetal brain from 3 Gy total body irradiation.

Skin is the largest human organ, and it provides a first line of defense that includes physical, chemical, and immune mechanisms to combat environmental stress. Radiation is a prevalent environmental stressor. Radiation-induced skin damage ranges from photoaging and cutaneous carcinogenesis caused by UV exposure, to treatment-limiting radiation dermatitis associated with radiotherapy, to cutaneous radiation syndrome, a frequently fatal consequence of exposures from nuclear accidents. The major mechanism of skin injury common to these exposures is radiation-induced oxidative stress. Efforts to prevent or mitigate radiation damage have included development of antioxidants capable of reducing reactive oxygen species. Mitochondria are particularly susceptible to oxidative stress, and mitochondrial-dependent apoptosis plays a major role in radiation-induced tissue damage. We reasoned that targeting a redox cycling nitroxide to mitochondria could prevent reactive oxygen species accumulation, limiting downstream oxidative damage and preserving mitochondrial function. Here we show that in both mouse and human skin, topical application of a mitochondrially targeted antioxidant prevents and mitigates radiation-induced skin damage characterized by clinical dermatitis, loss of barrier function, inflammation, and fibrosis. Further, damage mitigation is associated with reduced apoptosis, preservation of the skin's antioxidant capacity, and reduction of irreversible DNA and protein oxidation associated with oxidative stress.

Senescent cells accumulate in tissues affected by aging, ionizing irradiation, and loss of telomere length and other sources of DNA damage signaling. FA mice deficient in the Fancd2 protein display reduced homologous recombination and increased nonhomologous end joining for repair of DNA double strand breaks. Studies also show a role for Fancd2 in the ALT pathway of telomere maintenance. Fancd2-/- bone marrow stromal cell lines are radiosensitive compared to Fancd2+/+ marrow stromal cells (Do = 1.4 ± 0.1 Gy vs 1.6 ± 0.1 Gy, p = 0.0124), and by comet assay where Fancd2-/- cells display slower DNA repair after irradiation (p < 0.0001) (Berhane, et. al., Radiation Research 181:76-89, 2014). We investigated whether Fancd2-/- cells were more susceptible to induction of senescence. Fancd2+/+ control and Fancd2-/- murine bone marrow stromal cell lines were analyzed for irradiation induced senescence. Bone marrow stromal cell lines derived from long term bone marrow cultures of Fancd2+/+, and Fancd2-/- marrow (129/Sv) were plated in 6 well tissue culture plates and irradiated to 5 or 10 Gy. At 24, or 48 hr after irradiation, cells were stained for senescence biomarkers B-Gal and p21. Fancd2+/+ and Fancd2-/- bone marrow stromal cells stained for percent B-Gal positive cells following 0, 5 or 10 Gy at 24 and 48 hr after irradiation showed that Fancd2-/- cells had increased B-Gal staining compared to Fancd2+/+ cells at 0 Gy (10 ± 1% vs 1 ± 1%, p<0.0001). At 24 and 48 hr after 5 and 10 Gy Fancd2-/- cells had increased B-Gal staining (14 ± 1% at 24 hr after 5 Gy, 18 ± 1% at 24 hr after 10 Gy, 18 ± 1% at 48 hr after 5 Gy and 23 ± 1% at 48 hr after 10 Gy, p < 0.0123 compared to 0 Gy). In contrast increased B-Gal staining was delayed in FancA+/+ cells to 48 hr after both 5 and 10 Gy (7 ± 4% at 24 hr after 5 Gy, 3 ± 1% at 48 hr after 10 Gy, 3 ± 1% at 48 hr after 5 Gy, 4 ± 1% at 48 hr after 10 Gy, p = 0.0027 and < 0.0001 comparing 5 and 10 Gy at 48 hr to 0 Gy, respectively). The percent of nonirradiated Fancd2-/- cells positive for p21 was statistically increased compared to Fancd2+/+ cells (16 ± 2% vs 4 ± 1%, p = 0.0007). Both control Fancd2+/+ and Fancd2-/- cells had increased p21 positive cells at 24 and 48 hr following 5 or 10 Gy (p < 0.05) was always greater in Fancd2-/- cells . At 24 and 48 hrs after 5 Gy, Fancd2-/- cells had 45 ± 5% and 39 ± 1% positive p21 cells compared to 9 ± 1% and 5 ± 1% for Fancd2+/+ cells (p < 0.0001). At 24 and 48 hours after 10 Gy the percent of Fancd2-/- cells positive for p21 was 58 ± 3% and 48 ± 5%, respectively, compared to 11 ± 1% and 7 ± 1%, respectively, for Fancd2+/+ cells (p < 0.0001). The radiosensitivity of Fancd2-/- bone marrow stromal cells increases senescence in vitro as measured by both B-Gal and p21. Study of FA tissues in vivo after TBI should reveal organ specific acceleration of senescence and provide insight into late irradiation effects in marrow transplanted FA patients.