Derivation of doxorubicin mutational signatures in cancer

Doxorubicin is a chemotherapeutic agent used for the treatment of solid and hematopoietic malignancies. Doxorubicin acts as a DNA intercalator and inhibits the topoisomerase II enzyme, leading to double-stranded breaks. We hypothesize that doxorubicin mutagenesis leads to a nonrandom pattern of somatic mutations on the genome, termed a mutational signature. We expose cells to doxorubicin using two different methodologies: (1) continuous exposure of a constant dose of doxorubicin and whole-genome sequencing of single-cell clones (previously published methodology) and (2) pulsed exposure of escalating doses of doxorubicin and whole-genome sequencing of a bulk population. We first confirm that the methodologies do not yield differing results through previously published cisplatin-exposed single-cell clone genome data compared with the dose escalation and bulk genome data. We then derive two doxorubicin signatures. The first, SBS-doxorubicin-1, is defined by an abundance of C > A mutations. The second, SBS-doxorubicin-2, is defined by a higher occurrence of T > C mutations. We then assess for the presence of our signatures within the genomes of patients treated with a doxorubicin-containing regimen. Across 10 bulk immune sorted samples from three patients with follicular lymphoma/diffuse large B cell lymphoma, as well as two metastatic samples from one patient with renal medullary carcinoma, we are able to detect both signatures in 83% of samples, with remaining samples harboring either signature. In summary, we experimentally derive two doxorubicin mutational signatures and demonstrate their presence in patient samples.