Topoisomerase 1 inhibition in MYC-driven cancer promotes aberrant R-loop accumulation to induce synthetic lethality.

MYC is a key regulator of gene transcription and is dysregulated in many human cancers. As targeting MYC directly is challenging, an alternative strategy is to identify the specific proteins or processes required for MYC to function as a potent cancer driver that can be targeted to result in synthetic lethality. The goal of this study was to identify novel actionable targets that can be exploited to trigger MYC-driven cancer cell death. To this end, we performed a genome-wide CRISPR knockout screen using an isogenic pair of breast cancer cell lines, in which MYC dysregulation is the switch from benign to transformed tumor growth. Proteins that regulate R-loops were identified as a potential class of synthetic lethal targets. Dysregulated MYC elevated both global transcription and coincident R-loop accumulation. Topoisomerase 1 (TOP1), a regulator of R-loops by DNA topology, was validated to be a vulnerability in cells with high MYC activity. Genetic knockdown of TOP1 in MYC-transformed cells resulted in reduced colony formation compared to control cells, demonstrating synthetic lethality. Overexpression of RNaseH1, a riboendonuclease that specifically degrades R-loops, rescued the reduction in clonogenicity induced by TOP1 deficiency, demonstrating that this vulnerability was driven by aberrant R-loop accumulation. Genetic and pharmacological TOP1 inhibition selectively reduced the fitness of MYC-transformed tumors in vivo. Finally, drug response to TOP1 inhibitors (i.e., topotecan, SN-38) was significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids. In summary, these findings highlight TOP1 as a promising target for MYC-driven cancers.