The rationale of this study stems from the concern of a radiation-induced accident or terrorist-mediated nuclear attack resulting in large populations of people exposed to nonlethal radiation doses or after a course of definitive radiation therapy which could substantially increase the risk for cancer induction after exposure. Currently, there are no safe and effective interventions to reduce this increased cancer risk to humans. We have tested the hypothesis that the mTOR inhibitor, rapamycin, administered in the diet of mice would reduce or delay radiation-induced cancer when given after radiation exposure. A total-body irradiation (TBI) of 3 Gy was administered to female C3H/Hen mice. Immediately after TBI, along with untreated control groups, animals were placed on chow containing different concentrations of encapsulated rapamycin (14, 40, 140 mg/kg chow). Animals remained on the respective control or rapamycin diets and were followed for their entire lifespan (total of 795 mice). The endpoint for the study was tumor formation (not to exceed 1 cm) or until the animal reached a humane endpoint at which time the animal was euthanized and evaluated for the presence of tumors (pathology evaluated on all animals). Kaplan-Meier survival curves revealed that all three concentrations of rapamycin afforded a significant survival advantage by delaying the time at which tumors appeared and reduction of the incidence of certain tumor types such as hepatocellular carcinomas. The survival advantage was dependent on the rapamycin concentration used. Further, there was a survival advantage when delaying the rapamycin chow by 1 month after TBI. Rapamycin is FDA-approved for human use and could be considered for use in individuals exposed to nonlethal TBI from a nuclear accident or attack or after significant therapeutic doses for cancer treatment.

Kaplan-Meier survival plots for mice receiving 0 Gy or 5.4 Gy TBI.

Supplementary Figure S5. Representative western blot of two independent experiments characterizing the impact PF-05212384 has on ATM and DNA-PKc autophosphorylation and total protein levels as a function PF-05212384 (10 μM) treatment time following irradiation (7.5 Gy) in exponentially growing UMSCC1 and UMSCC46 HNSCC cells (A).

experimental methods and all supplementary figure legends

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Temporal profiles of 4 metabolic features identified by random forest analysis of control versus IR treated mice (taken from Table 1).

Temporal profiles of a few selected metabolites.

Comparison of metabolite profiles of 5.4 Gy (IR) to 0 Gy (Control) using 525 samples (Control = 141 from 17 mice, IR = 384 from 51 mice).

Distribution of neoplasm types between Control (0 Gy) and irradiated (5.4 Gy) mice (excluding outliers) ; Number of feature samples for various groups at different time intervals within 1 year ; Retention times and m/z ratios of quality controls and their standard errors ; Summary of Random Forest Classification ; Significant features (FDR<4.2%) in Control vs. IR comparison at any one of the four time groups ; Features Correlating with Pattern Matching from IRH, IRB, and IRS Neoplasms .

Figure S4. (A) Pilot H460 xenograft study combining Abemaciclib and fractionated radiation (2 Gy x 3). Once tumors reached size of 200 mm3, the tumor bearing legs were exposed to local fractionated radiation (2 Gy) on Monday, Wednesday and Friday followed by two daily gavages of Abemaciclib (100 mg/kg) on Monday and Tuesday. Two doses of Abemaciclib per day was not well tolerated and subsequently mice where given single Abemaciclib doses on Wed-Fri. The combination of XRT and Abemaciclib resulted in modest enhancement of tumor radiosensitivity. (B) Western blot analysis of H460 xenograft showing CDK4/6 target inhibition. Expression of RB/E2F target cyclin A is shown. Changes in p-ERK1/2 and p-S6 is also shown for xenografts receiving Abemaciclib treatment for week 1 and week 2 post fractionated radiation. (C) H460 xenograft showing enhanced tumor regrowth delay by daily administration of 100mg/kg body weight of Abemaciclib through oral gavage for 5 days immediately after 2 Gyx3 IR dose administered every alternate day indicated as week 1. The drug dosing was continued for another 5 days indicated as week 2. The significance for tumor regrowth delay was measured when tumor size reached 800mm3. The statistical significance is derived from 8 animals per group.

Six patterns of relative IRB, IRH and IRS levels considered for template matching.

Legend for Supplementary Figures S1-S5.

<p>PDF file - 125KB, Supplemental Table 1. CBA Tumor Spectrum of Major Malignant HN and Non-HN Supplemental Table 2. C3H Major Tumor Spectrum of of Major Malignant HN and Non-HN Supplemental Table 3. Incidence and Mean Survival of CBA with Hepatocellular Tumors Supplemental Table 4. Incidence and Mean Survival of C3H with Hepatocellular Tumors Supplemental Table 5. Ratio of HN to Non-HN Supplemental Table 6. Relative Hazard (RH) Ratios for CBA Mice Supplemental Table 7. Relative Hazard (RH) Ratios for C3H Mice Supplemental Table 8. Gene Expression and Onotology Classification</p>

Supplementary Data from In vitro and In vivo Radiation Sensitization of Human Tumor Cells by a Novel Checkpoint Kinase Inhibitor, AZD7762

Figure S2. (A) Immunoblot analysis showing expression of DNA damage repair proteins in H1299 cells post-IR and Abemaciclib treatment. (B) Immunoblot analysis showing target inhibition in H1975 cells post-IR and Abemaciclib treatment (C) Immunofluorescence expression of p-RB (Ser811/780, shown in green) for H460 cells treated with Abemaciclib for 4 hour and 24 hour. Nucleus was stained with DAPI, 4',6-diamidino-2-phenylindole (show in blue). (D and E) Flow cytometry analysis showing percentage (%) of cells positive for p-HH3 (Ser10, 1:100 dilution) expression (D) and AnnexinV (1:100) +PI (propidium iodide, 5ug/ml) positive (E) post Abemaciclib+IR treatment for 24 hours.

Figure S1. Radiation survival curve of H460 cells treated with Palbociclib (A) Ribociclib (B) and Abemaciclib (C) at a dose of 1µM and 10µM administered either 24 hour pre-or post for indicated dose (Gy) of radiation exposure. (D) Full dose survival curve of H460 cells treated with 10µM of Abemaciclib 24 hr pre-IR exposure.

Supplementary Figures 1-3 from Gene Expression Profiling of Breast, Prostate, and Glioma Cells following Single versus Fractionated Doses of Radiation

excel file table showing details of microsatellite markers used for cell line authentication

Supplementary Table S1. Senescence pathway directed RT-PCR genomic expression analysis of UMSCC1 cells treated with 24 hour PF-05212384 (10 M) or DMSO (10 M) followed by irradiation (10 Gy) or mock irradiation.

Supplementary Figure S6. Heat map of individual mRNA absolute Ct obtained from senescence pathway directed RT-PCR of UMSCC1 cells treated with 24 hour PF-05212384 (10 μM) or DMSO (10 μM) followed by irradiation (10 Gy) or mock irradiation.