IADR Abstract Archives
PTRIP13: Prospective Predictive Marker of Radiation Response in HNSCC
Objectives: With almost 70% of patients with head and neck cancers presenting with late-stage disease, aggressive treatment modalities, including radiation, are utilized to achieve disease control. Whilst the benefits of radiation are indisputable, site-specific side effects and damage to adjacent normal tissue can significantly affect the patient’s quality of life. Moreover, ~30% of patients develop radiation-resistant tumors. Hence, identifying a predictive marker of treatment response during the planning phase could optimize treatment selection, reduce side effects, and improve outcomes. Previously we showed that phosphorylation of Thyroid Receptor Interacting Protein (TRIP13) at Tyrosine 56 mediates radiation resistance by enhancing DNA repair. The main obejective of this work is to assess pTRIP13 as a predictive marker of response to radiation.
Methods: A site-specific antibody detecting phosphorylation of TRIP13 at Y56F was developed. Oligopeptides were generated at EGFR kinase consensus target sites. Peptide slot blots were used to assess antibody specificity for relevant phosphor-sites. Sensitivity to detect protein by immunoblot and immunoprecipitation was investigated. Tumor growth was monitored in athymic nude mice injected with UM-SCC-1-TRIP13 or UM-SCC-1-Y56F. Specificity for detection of radiation sensitivity or resistance was investigated by performing immunohistochemistry on tissue sections from irradiated or non-irradiated tumors. DenseNet CNN classifiers on HALO-AI™ was used to analyze staining.
Results: Phospho-Y56 TRIP13 antibody reproducibly detected phosphorylation at the target site on slot blot assays, immunoblot, and immunoprecipitation. In pre-clinical models, tumors with the Y56F mutation in TRIP13 were smaller and associated with better survival than tumors with wild type TRIP13. Immunohistochemistry supported these findings; radiation increased the ratio of phospho-TRIP13 to total TRIP13 in both tumor groups; but staining was significantly less in mutant than wild type tumors.
Conclusions: These data support investigation of the utility of pTRIP13 in patient samples to interrogate its significance as a predictive marker of response to radiation.
Methods: A site-specific antibody detecting phosphorylation of TRIP13 at Y56F was developed. Oligopeptides were generated at EGFR kinase consensus target sites. Peptide slot blots were used to assess antibody specificity for relevant phosphor-sites. Sensitivity to detect protein by immunoblot and immunoprecipitation was investigated. Tumor growth was monitored in athymic nude mice injected with UM-SCC-1-TRIP13 or UM-SCC-1-Y56F. Specificity for detection of radiation sensitivity or resistance was investigated by performing immunohistochemistry on tissue sections from irradiated or non-irradiated tumors. DenseNet CNN classifiers on HALO-AI™ was used to analyze staining.
Results: Phospho-Y56 TRIP13 antibody reproducibly detected phosphorylation at the target site on slot blot assays, immunoblot, and immunoprecipitation. In pre-clinical models, tumors with the Y56F mutation in TRIP13 were smaller and associated with better survival than tumors with wild type TRIP13. Immunohistochemistry supported these findings; radiation increased the ratio of phospho-TRIP13 to total TRIP13 in both tumor groups; but staining was significantly less in mutant than wild type tumors.
Conclusions: These data support investigation of the utility of pTRIP13 in patient samples to interrogate its significance as a predictive marker of response to radiation.