Intraoral Mitochondrial-Targeted GS Nitroxide JP4-039 Ameliorates Radiation-Induced Mucositis in Orthotopic Tumor-Bearing Fanconi Anemia (FA) (Fancd2-/-) Mice.
Authors: Shinde Ashwin , Rhieu Byung Han , Berhane Hebist , Guinan Eva C. , Parmar Kalindi , Wang Hong , Franicola Darcy , Zhang Xichen , Li Song , Dixon Tracy M. , Greenberger Joel S. 
Published: October 2019

Abstract
Fanconi anemia (FA) patients suffer from bone marrow failure, hematologic malignancies and late onset of squamous cell carcinoma. Some FA patients are highly sensitive to irradiation due to cellular DNA repair defect. Very severe radiation-induced mucositis can be dose limiting for these FA patients. We tested intraoral delivery of the mitochondrial-targeted antioxidant, JP4-039, in a novel F15 emulsion, to reduce mucositis in irradiated FA mice, namely FancD2-deficient mice, with orthotopic TC-1 squamous cell carcinoma cell line derived tumors. TC-1 cells (1 x 106) were injected into the left cheek of Fancd2+/+, Fancd2+/-, and Fancd2-/- mice. Once tumors were palpable, mice were treated with fractionated irradiation of 8 Gy x 4 days to the oral cavity. Subgroups of mice (N=4/group) received intraoral JP4-039/F15 (100 ul containing 4 mg/ml of JP4-039 via a feeding tube placed in the oral cavity) 10 minutes prior to irradiation, F15 alone or irradiation alone. Tumor size was measured daily. Five days after the last radiation dose, mice were sacrificed, tumors and tongue tissue removed for histopathology, and gene transcripts quantitated by real time polymerase chain reaction (RT-PCR). Intraoral JP4-039/F15 prior to 8 Gy x 4 significantly decreased oral cavity ulceration (p < 0.001). Following 8 Gy x 4, F15-JP4-039 treated Fancd2-/- knockout mice had 47.8 ± 11.1% of the tongue ulcerated compared to 81.7 ± 11.6% ulceration in irradiated control mice (p < 0.001). Results in F15-JP4-039 treated irradiated heterozygotes and wild-type Fancd2+/+ mice (34.1 ± 21.2 or 16.9 ± 12.6% ulceration, respectively) were also improved compared to control-irradiated mice (88.3 ± 8.0 or 76.3 ± 17.2% ulceration, respectively) (p < 0.001 for all groups). In contrast, F15 alone did not reduce ulceration. Fancd2-/- F15-JP4-039 treated mice still displayed increased ulceration compared to Fancd2+/+ mice (47.8 ± 11.1% and 16.9 ± 12.6%, respectively, p < 0.0001), Both single fraction and fractionated irradiation controlled tumors at 5 days in Fancd2+/+, Fancd2+/-, and Fancd2-/- mice (0.4 ± 0.3, 0.05 ± 0.05, or 0.1 ± 0.1 mm3, respectively) compared to nonirradiated controls (2.1 ± 0.4, 1.9 ± 0.5, or 3.1 ± 0.1 mm3, respectively, p ≤ 0.027). Tumor bearing Fancd2-/- mice had reduction in irradiation induced normal oral tissue NFKB, MnSOD, p21, and IL1a transcripts, while tumor bearing Fancd2+/+ and Fancd2+/- mice showed elevated transcripts for irradiation induced TGFB, Nrf2, Gadd45, IL1a, and p-21 (p<0.0412 and p<0.0041, respectively) compared to a nonirradiated tumor bearing mouse. In conclusion; an antioxidant intraoral JP4-039 protects normal tissue in irradiated Fancd2-/- as well as wild-type mice without associated tumor protection. JP4-039 therefore can be useful agent during radiation therapy of squamous cell carcinoma in Fanconi anemia patients.

Disclosures
No relevant conflicts of interest to declare.