The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Histone H2AX has a role in suppressing genomic instability and cancer. However, the mechanisms by which it performs these functions are poorly understood. After DNA breakage, H2AX is phosphorylated on serine 139 in chromatin near the break. We show here that H2AX serine 139 enforces efficient homologous recombinational repair of a chromosomal double-strand break (DSB) by using the sister chromatid as a template. BRCA1, Rad51, and CHK2 contribute to recombinational repair, in part independently of H2AX. H2AX(-/-) cells show increased use of single-strand annealing, an error-prone deletional mechanism of DSB repair. Therefore, the chromatin response around a chromosomal DSB, in which H2AX serine 139 phosphorylation plays a central role, "shapes" the repair process in favor of potentially error-free interchromatid homologous recombination at the expense of error-prone repair. H2AX phosphorylation may help set up a favorable disposition between sister chromatids.

Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.