A Common Chk1-Dependent Phenotype of DNA Double-Strand Break Suppression in Two Distinct Radioresistant Cancer Types

Abstract

Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. In a panel of 10 breast cancer cell lines with/ without TN receptor status, we screened with small molecule inhibitors against Chk1 and related kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using a previously established ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was explored in TNBC and lung adenocarcinoma patients. In vitro and patient biopsy studies identified a positive association between TN hormone receptor status and radiosensitization by Chk1 inhibition. Dose enhancement factors (SF10%) were 1.3-1.7. Interestingly, lower numbers of IR-induced DSBs were observed in TNBC when compared to non-TNBC cells, correlating with their observed radioresistance. Unexpectedly, we found that Chk1 suppressed IR-induced DSBs in these cells by ∼1.5-fold, which was dependent on the H3K9me3/S10p mark, previously found to indicate radioresistance in KRAS mutant lung cancer. The effects of Chk1 inhibition seen in TNBC were reproduced in KRAS mutant but not wild-type cells. Consistent with our functional studies we observed co-expression of genes in this Chk1 pathway in TNBC and KRAS mutant lung cancers. Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of these challenging tumor types.