Margaret A. Connors

Supplemental Figure 1 shows bar graph of quantification of cells that have >25 gH2AX foci per cell in M15 cell line, after treatment with peposertib and radiation

Supplementary Materials and Data show the cell culture and western blot data

Supplemental Figure 2 shows bar graph of quantification of cells that have >25 gH2Ax foci per cell in M27 cell line, after treatment with peposertib and radiation

Top row: Correlation of Normalized Enrichment Scores (NES) and viability of IR+peposertib normalized to IR in Homologous Recombination (HR), Microhomology-mediated End Joining (MMEJ), Non-Homologous End Joining (NHEJ), and mRNA PRKDC rpkm in four PDX GBM lines (GBM 6, 10, 22, and 76). Bottom row: Same data but split into TP53 wildtype lines (TP53wt, GBM10 and GBM76) vs. TP53 mutant lines (TP53mut, GBM 6 and 22) with IR and IR+peposertib data normalized to DMSO.

Standardization curve of peposertib (M3814) concentration as measured by MALDI in normal and tumor tissue.

A, Immunoflourescence of 53BP1 foci in U251 cells 1- and 24-hours after indicated treatments. B, PRKDC methylation scores in indicated GBM PDX lines. C, Somatic mutations and Copy Number Variations in GBM PDX lines tested in vivo in key GBM pathways. D, Western blot confirmation of DNA-PK knock down by shRNA (shPRKDC) or non-targeting shRNA (shNT).

Supplementary Data S2: derivation of modified biologically effective dose model.

Supplementary Figure S2 demonstrates agreement between AUC computed from simulated and measured profiles.

Ataxia Telangiectasia–mutated (ATM) inhibitors are being developed as radiosensitizers to improve the antitumor effects of radiotherapy, but ATM inhibition can also radiosensitize normal tissues. Therefore, understanding the elevated risk of normal tissue toxicities is critical for radiosensitizer development. This study focused on modeling the relationship between acute mucosal toxicity, radiation dose, fractionation schedule, and radiosensitizer exposure. The ATM inhibitor WSD0628 was combined with single or fractionated doses of radiation delivered to the oral cavity or esophagus of Friend Leukemia virus B (FVB) mice. The potentiation by WSD0628 was quantified by a sensitizer enhancement ratio (SER), which describes the changes in radiation tolerance for radiation combined with WSD0628 relative to radiation-only regimens. WSD0628 profoundly enhanced radiation-induced acute oral and esophageal toxicities. For oral mucosal toxicity, the enhancement by WSD0628 with 3 fractions of radiation resulted in an SER ranging from 1.3 (0.25 mg/kg) to 3.1 (7.5 mg/kg). For the 7.5 mg/kg combination, the SER increased with increasing number of fractions from 2.2 (1 fraction) to 4.3 (7 fractions) for oral toxicity and from 2.2 (1 fraction) to 3.6 (3 fractions) for esophageal toxicity, which reflects a loss of the normal tissue sparing benefit of fractionated radiation. These findings were used to develop a modified biologically effective dose model to determine alternative radiation schedules with or without WSD0628 that result in similar levels of toxicity. Successful radiosensitizer dose escalation to a maximally effective therapeutic dose will require careful deliberation of tumor site and reduction of radiation dose volume limits for organs at risk.

Supplementary Figure S1 includes repeats of immunofluorescence images and an example of H&E staining from untreated oral mucosa.