Deficiencies in the Fanconi anemia or homologous recombination pathway enhance the antitumor effects of the hypoxia-activated prodrug CP-506.

The novel hypoxia-activated prodrug CP-506 selectively targets the hypoxic, treatment-resistant tumor microenvironment. Given the alkylating effector metabolites of CP-506, we hypothesized that defects in interstrand crosslink (ICL) and double-strand break repair influence treatment efficacy. In vitro and in vivo isogenic cancer models proficient or deficient in the Fanconi anemia (FA), homologous recombination (HR), or non-homologous end joining (NHEJ) pathway were used to assess CP-506-induced cytotoxicity and DNA damage. Viability and clonogenic assays demonstrated enhanced sensitivity to CP-506 in FA- or HR-deficient cells compared to parental cells, which was confirmed by spheroid growth inhibition studies. In vivo, CP-506 caused greater enhancement ratios in FA- and HR-deficient xenografts versus parental controls (p < 0.0001) but not in NHEJ-deficient xenografts (p = 0.18). Mechanistically, CP-506 increased γH2AX expression (1.9- to 9.3-fold) in FA- and HR-deficient cells and xenografts, whereas NHEJ-deficient models showed a 0.5-fold reduction. Alkaline comet assays confirmed CP-506-induced ICLs and DNA strand breaks but did not explain the differential therapeutic responses among isogenic cancer cells. These data indicate that deficiencies within FA or HR, but not NHEJ or nucleotide excision repair (NER), determine CP-506 sensitivity, consistent with a synthetic-lethal interaction. Therefore, tumor hypoxia and DNA repair status are key biomarkers for stratifying patients in CP-506 clinical trials.