A Zebrafish Drug Screen Repositions FDA-Approved Compounds for Radiochemotherapy

Abstract

The apoptosic response to ionizing radiation (IR) is dependent on functional p53. As up to 50% of tumors harbor mutations in TP53, many cancer types are thought to be resistant to chemoradiation treatments. Small molecules that specifically radiosensitize tumors to IR while maintaining minimal toxicity in normal cells may enhance the efficacy of radiation therapy while minimizing adverse side effects. However, radiosensitization must occur in the context of mutant TP53 in order to target therapeutically resistant tumor cells. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. Our screen identified 13 compounds that demonstrate specific radiosensitizing effects in irradiated p53 mutant embryos after 15 Gy IR while showing no toxicity in non-irradiated embryos. Acridine orange staining to label apoptotic or necrotic cells identified 2 compounds that induced cell death specifically in combination with radiation. The putative targets of these candidate radiosensitizers were assembled using a combination of Similarity Ensemble Approach (SEA) searches with Structure Activity Relationship (SAR) analysis. To determine if the compounds identified in fish have relevance to human tumors, they were screened for survival after IR using a resazurin cell viability assay in a panel of cell lines, specifically focusing on HNSCC. Two compounds specifically led to increased cell death in p53 mutant fish after radiation. Compound A is a veterinary antihelminth with multiple analogs that are approved for human use. This compound is particular amenable to target prediction with SEA and SAR, yielding putative targets PIN1, ERBB2, and NMDZ1. Compound B is an aromatase inhibitor whose current indication includes ER-positive breast cancer. Neither compound has been previously indicated as a radiosensitizer and both have shown minimal toxicity to embryos in the absence of radiation. In addition, compound B has showed efficacy as a radiosensitizer in HeLa and CAL27 cell lines. We have identified two candidate radiosensitizers in p53 mutant zebrafish to IR with a large therapeutic window, indicating promising repurposing possibilities. Further study is required to identify the mechanism of action of how these compounds sensitize in the context of TP53 mutation.