Mechanism of Topoisomerase 2 inhibitor tolerance uncovers a fundamental histone H1 biogenesis pathway
Topoisomerase 2 (TOP2) inhibitors (TOP2i) are mainstay cancer chemotherapeutic agents that undermine genome integrity by stabilizing TOP2-DNA complexes. Nevertheless, their clinical deployment is associated with toxicity, acquired resistance and secondary malignancies, necessitating the development of improved treatment strategies. From genome-scale CRISPR-Cas9 screens, we discovered that the uncharacterized human protein CRAMP1 and H1 linker histones display synthetic lethal interactions with TOP2i. Although loss of CRAMP1 selectively hypersensitizes cells to TOP2i but not other genotoxic agents, we show that CRAMP1 does not function as a conventional repair factor for TOP2-DNA complexes. Instead, CRAMP1 defines an H1-specific histone biogenesis factor, and we demonstrate that reduced histone H1 abundance underlies TOP2i hypersensitivity in CRAMP1-deficient cells. Mechanistically, CRAMP1 acts as a transcription factor for both replicative and non-replicative H1 genes, driven by its concurrent targeting to histone gene loci and H1-specific promoter motifs. We establish that defective H1 biogenesis induces TOP2i hypersensitization via a novel paradigm uncoupled from DNA repair, increasing cellular demand for TOP2 activity through extensive formation of novel TOP2 substrates due to chromatin decompaction, thereby lowering the threshold for TOP2i-induced cytotoxicity. Collectively, through revealing CRAMP1-dependent H1 synthesis as an essential prerequisite for TOP2i resistance, our findings elucidate the mechanistic basis of linker histone biogenesis in human cells and provide a framework for delineating H1 functions in chromatin biology. Overall design: CC-seq-v2 maps of the genome-wide distribution of Top2ccs in CRAMP1 WT U2OS, CRAMP1-KO U2OS, and CRAMP1-KO U2OS complemented with GFP-CRAMP1. The experiment was conducted in quadruplicate.