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

## TL;DR

This study shows that tumors with defects in specific DNA repair pathways respond better to a new cancer drug called CP-506, especially in low-oxygen environments.

## Contribution

The study identifies Fanconi anemia and homologous recombination pathway deficiencies as biomarkers for CP-506 efficacy in hypoxic tumors.

## Key findings

- FA- or HR-deficient cells and xenografts showed increased sensitivity to CP-506 compared to parental controls.
- CP-506 increased γH2AX expression in FA- and HR-deficient models but not in NHEJ-deficient models.
- Deficiencies in FA or HR, but not NHEJ or NER, determine CP-506 sensitivity through a synthetic-lethal interaction.

## Abstract

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.

This study identifies deficiencies in the Fanconi anemia or homologous recombination DNA repair pathways, alongside the presence of tumor hypoxia, as key complementary biomarkers for therapeutic efficacy and thus for stratifying patients most likely to benefit from CP-506 treatment.