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

Abstract
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.

Introduction
CX-5461 is a clinical-stage anti-cancer small molecule evaluated in clinical trials as a G4 stabilizer [1] and as an RNA Pol I inhibitor [2]. The first-in-human clinical trial of CX-5461 was conducted in patients with advanced haematological cancers, where it demonstrated pharmacological inhibition of RNA Pol I. CX-5461 is clastogenic, inducing single and double strand breaks in DNA. In solid tumours, CX-5461 showed anti-cancer activity in patients with homologous recombination repair-deficient (HRD) tumours [3], with genetic evidence of action through the HR deficient state. Both trials also identified phototoxicity as a clinically relevant toxicity, which can be managed with photoprotection measures.

CX-5461 has quinolone structural features and quinolone/fluoroquinolone compounds have been previously shown to exhibit UVA-mediated phototoxicity (wavebands centred at 335 and 365 nm [4, 5]). UVA (wavelengths 320–400 nm) comprises >95% of the solar UV radiation reaching the Earth’s surface, whereas UVB (280–320 nm) accounts for the remainder because most UVB and essentially all UVC (wavelengths below 280 nm) are removed by the ozone layer. This makes UVA exposure clinically relevant for trials involving photosensitizing quinolone compounds. Cyclobutane pyrimidine dimer (CPD) adducts, reactive oxygen species, and DNA damage accumulate in cultured cell lines and model organisms treated with CX-5461 and irradiated with UVA [3]. These effects were not reproduced in human cell lines by structurally unrelated G4 ligands such as PDS, BRACO-19, TMPYP4, or PhenDC3. This is consistent with the UVA-photosensitization tendency of CX-5461 being a class effect associated with its quinolone-derived structure and not a general feature of G4 ligands.

Although G4 DNA damage effects of CX-5461 have been reported in cell lines and model organisms, the UV mutation spectrum of CX-5461 requires further clarification. An early C. elegans study reported a UV-associated single-base mutational signature consisting of T > N mutations. However, the human mutational spectrum of UVA+CX has not been clearly documented in human cells. Here, we report the single and double base substitution and indel signatures of CX-5461 in human RPE1 cells exposed to UVA. Joint UVA+CX-5461 exposure resulted in a bulk whole-genome sequencing (WGS) detectable mutational burden, with a distinct mutational pattern characterized by T > A and T > C mutations. Exposure to CX-5461 alone with careful photoprotection measures does not yield a detectable single-base substitution (SBS) signature above the measurable background of bulk or cloned single cells.