The UVA-induced single-base mutational signature of CX-5461 in human cells

Drug-related UVA-induced photoreactions have been reported for several therapeutic compounds, including fluoroquinolones. CX-5461 is a clinical stage quinolone-derived anti-cancer small molecule with documented UVA-sensitizing activity. Here, we compared, by bulk and clonal whole-genome sequencing under extraneous light-protected conditions, the mutational signatures in human retinal pigment epithelial cells (RPE1) exposed to UVA, CX-5461, or co-exposed to UVA and CX-5461. Treatment with CX-5461 or UVA alone resulted in a low single-base mutation burden and background-like mutational profiles. In contrast, bulk sequencing of human cells co-exposed to UVA and CX-5461 had a markedly higher mutation burden characterized by T > A and T > C substitutions. Furthermore, single-cell clonal expansion and sequencing of CX-5461 alone, UVA alone, or CX-5461+UVA treatments confirmed that the pattern was only observed when cells were exposed to both UVA and CX-5461. The CX-5461+UVA-associated SBS signature we report arises only when CX-5461-treated cells are exposed to UVA, and is not observed when CX-5461-treated cells are shielded from light. We do not observe strong single-base mutagenic activity of CX-5461 alone, under light-protected conditions. Our data define a human SBS mutagenesis signature for CX-5461 potentiated UVA mutations and emphasize the need for appropriate controls and light-exposure precautions when studying SBS activity of known photosensitizer molecules.