The acute lethality of total-body irradiation (TBI) involves damage to multiple organs, including bone marrow and intestine. Ionizing radiation mitigators that are effective when delivered 24 h or later after TBI include the anti-apoptotic drug, JP4-039 and the anti-necroptotic drug, necrostatin-1. In contrast to effective delivery of JP4-039 at 24 h after TBI, necrostatin-1 is most effective when delivery is delayed until 48 h, a time that correlates with the elevation of necroptosis-inducing inflammatory cytokines and necroptosis-induced serine phosphorylation of receptor-interacting serine/threonine-protein kinase-3 (RIP3) in tissues. The goal of this work was to determine whether administration of JP4-039 influenced the optimal delivery time for necrostatin-1. We measured daily levels of 33 proteins in plasma compared to intestine and bone marrow of C57BL/6NTac female mice over a 7-day time period after 9.25 Gy TBI (LD). Protein responses to TBI in plasma were different from those measured in intestine or bone marrow. In mice that were given JP4-039 at 24 h after TBI, we delayed necrostatin-1 delivery for 72 h after TBI based on measured delay in RIP-3 kinase elevation in marrow and intestine. Sequential delivery of these two radiation mitigator drugs significantly increased survival compared to single drug administration.

Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2 mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg (B6) and the most prevalent human genotype Fanca (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-β-resistant SMAD3 (129/Sv) mice and double-knockout SMAD3 Fancd2 (129/Sv) mice treated daily with TGF-β receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-β mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg and Fanca mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.

Total body irradiation (TBI) leads to dose- and tissue-specific lethality. In the current study, we demonstrate that a mitochondrion-targeted nitroxide JP4-039 given once 24 hours after 9–10 Gy TBI significantly improves mouse survival, and the recovery of intestinal barrier, differentiation and stem cell functions. The GI-protective effects are associated with rapid and selective induction of tight junction proteins and cytokines including TGF-β, IL-10, IL-17a, IL-22 and Notch signaling long before bone marrow depletion. However, no change was observed in crypt death or the expression of prototypic pro-inflammatory cytokines such as TNF-α, IL-6 or IL-1β. Surprisingly, bone marrow transplantation (BMT) performed 24 hours after TBI improves intestinal barrier and stem cell recovery with induction of IL-10, IL-17a, IL-22, and Notch signaling. Further, BMT-rescued TBI survivors display increased intestinal permeability, impaired ISC function and proliferation, but not obvious intestinal inflammation or increased epithelial death. These findings identify intestinal epithelium as a novel target of radiation mitigation, and potential strategies to enhance ISC recovery and regeneration after accidental or medical exposures.

Local tumor ablation can be achieved by physical energy-based therapies, such as, irradiation, ultrasound, microwave and radiofrequency. Although tumor antigens released from ablated tumors could induce an anti-tumoral immunity, antigen presentation from ablated tumors are not efficient in inducing a strong systemic immunity. Recently, we demonstrated that immunopriming by LOFU, followed by hypofractionated radiation, reversed immunological tolerance of T cells in draining lymph nodes, induced systemic anti-tumoral immunity, and produced local, regional and metastatic control of murine melanoma (J. Immunol. 196(4):1964Immunol. 196(4): -1976). LOFU uses low energy (w500 W/cm 2 ) and operating frequency of 1 MHz to induce membrane perturbation and mild hyperthermia at the focal zone, without ablating the tissue. Here, we investigate the effect of LOFU on gene expression and cell surface immunomodulation of murine tumor cells. Materials/Methods: LOFU parameters were set at 3-5W of total acoustic output power, 50% duty cycle, 1.5 seconds, and 1 mm spacing (TIPS, Philips Research). After LOFU treatment of a cell pellet, cells were cultured for various time points for RNA sequencing, qPCR, immunofluorescence and flow cytometry studies. Tumor cell lines included human DU145 prostate cancer cells and mouse RM1 and TPSA23 prostate cancer, Lewis lung carcinoma (3LL) and breast cancer (4T1). TPSA23 was derived from TRAMPC1 which was genetically engineered to express human PSA. Statistical evaluation distinguished gene expression changes by 1.5-fold, with false detection rate (FDR) <0.05. Wilcoxon rank sum was used to detect statistical significance in gene expression from RT-PCR results (p<0.05). Results: Gene expression studies (RNAseq and qRT-PCR) demonstrate that LOFU (3W) induced key genes involved in the unfolded protein response (UPR) pathway. Compared to untreated DU145 cells, LOFU induced ERN1 three-folds, HSPA1B and HSPH1 by 8.79 folds and HSP105/110 by 6.18-fold within 6-24 hrs. In separate experiments, there was a significant increase in Hspa1 RNA expression (132-fold) and CHOP expression (2.5 fold) 24 h post-LOFU, in RM-1 and 4T1 cells, respectively. Flow cytometry and immunofluorescence in 3LL, 4T1 and RM1 cells demonstrated the translocation of stress proteins, BiP, calreticulin and HSP70 to the cell surface. There was induction of cell surface death receptors (CD40 and Fas), immune checkpoint inhibitors (PD-L1 and PD-L2) and class I MHC in 3LL, 4T1 and TPSA23 cell lines. Conclusion: These studies demonstrate that LOFU induces expression of genes involved in UPR and endoplasmic stress response. Translocation of stress chaperones to the cell surface along with increased expression of MHC I and death receptors contribute to the immunomodulatory properties of LOFU.