2788 - Bragg Peak Proton Radiotherapy Demonstrates Differing Radiobiological Phenotype on Phosphoproteomic Analysis

D. K. Ebner¹, J. Kloeber¹, Y. Han¹, Q. Zhou¹, N. Remmes¹, X. Wu², J. Zhong³, A. Olson¹, A. Pandey³, Z. Lou⁴, and R. W. Mutter¹; ¹Department of Radiation Oncology, Mayo Clinic, Rochester, MN, ²Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, ³Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, ⁴Department of Medical Oncology, Mayo Clinic, Rochester, MN

Purpose/Objective(s): Proton radiotherapy (PRT) enables the delivery of dose to a target without significant exit dose, offering a key physical distribution difference in comparison with X-ray radiotherapy (XRT). PRT additionally delivers increased linear energy transfer (LET) at the distal edge of the beam path, a property providing enhanced radiobiologic effect. The etiology of these radiobiological differences remain unclear; here, phosphoproteomic analysis is employed to evaluate differences between low and high LET PRT in vitro. Materials/

Methods: hTERT-RPE1 cells were treated with 8 Gy photon-equivalent doses of entrance or Bragg Peak PRT, representing low-LET and high-LET radiobiological treatments, respectively. This was accomplished using a 76.8 MeV spot scanning proton beam, a mini pyramid filter, and 25 mm range shifter in the nozzle, with four cell culture 6-well plates fitted in a phantom such that wells were exposed to either low LET (2.2 keV/µm) or high LET (7.0 keV/µm) portions of the proton beam. Cells were harvested at 1h, 4h, 8h and 24h timepoints. Phosphoproteomic analysis was performed through isolation of (phospho)peptides and 18-plex tandem mass tag labeling through an optimized institutional platform. These sample sets were computationally analyzed for changes in phosphopeptide signaling.
Results: A total of 13782 phosphosites were detected across all samples. Analysis of phosphopeptide changes across timepoints revealed a core set of shared peptide changes seen after both entrance and Bragg Peak proton radiation. However, it also revealed distinct groups of peptides for entrance vs Bragg Peak protons. For high-LET Bragg Peak protons, AKT1 and PLK1 substrates were upregulated at one hour, whereas CSNK2A1 and CSNK2A2 substrates were upregulated in low-LET protons. At 4h, high-LET protons exhibit upregulation of PRKDC substrates vs. low-LET proton upregulation of CSNK2A1 and AURKB substrates; at both 4h and 8h, high-LET proton demonstrates upregulated CDK4 and CDC7 substrates with low-LET protons showing upregulated BUB1B and PLK1 substrates. At all timepoints, high-LET proton demonstrated decline in BUB1B and BUB1 substrates, while low-LET proton demonstrated decreased ABL1 and MAPK8.
Conclusion: Low- and high-LET proton radiotherapy induce distinct phosphopeptide changes. High-LET protons appear to promote stronger activity of key S-phase kinases CDK4 and CDC7 while low-LET protons more strongly activate mitotic-type checkpoints involving BUB1B and PLK1. Correlation with transcriptomics is ongoing.