nu-7441 and olaparib

Understanding differences in DNA double-strand break (DSB) repair between tumor and normal tissues would provide a rationale for developing DNA repair-targeted cancer therapy. Here, using knock-in mouse models for measuring the efficiency of two DSB repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ), we demonstrated that both pathways are up-regulated in hepatocellular carcinoma (HCC) compared with adjacent normal tissues due to altered expression of DNA repair factors, including PARP1 and DNA-PKcs. Surprisingly, inhibiting PARP1 with olaparib abrogated HR repair in HCC. Mechanistically, inhibiting PARP1 suppressed the clearance of nucleosomes at DNA damage sites by blocking the recruitment of ALC1 to DSB sites, thereby inhibiting RPA2 and RAD51 recruitment. Importantly, combining olaparib with NU7441, a DNA-PKcs inhibitor that blocks NHEJ in HCC, synergistically suppressed HCC growth in both mice and HCC patient-derived-xenograft models. Our results suggest the combined inhibition of both HR and NHEJ as a potential therapy for HCC.

Topics: Animals; Carcinoma, Hepatocellular; Chromones; DNA Breaks, Double-Stranded; DNA Damage; DNA End-Joining Repair; DNA Repair; DNA-Binding Proteins; Drug Therapy, Combination; Gene Knock-In Techniques; Homologous Recombination; Humans; Liver Neoplasms; Mice; Mice, Nude; Morpholines; Phthalazines; Piperazines; Poly (ADP-Ribose) Polymerase-1; Recombinational DNA Repair; Xenograft Model Antitumor Assays

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Cell Cycle Proteins; Cells, Cultured; Chromones; DNA Breaks, Double-Stranded; DNA-Activated Protein Kinase; DNA-Binding Proteins; Embryonic Stem Cells; Endodeoxyribonucleases; Histones; Homologous Recombination; Mice; Morpholines; Phosphorylation; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proteins; Radiation, Ionizing; RNA, Untranslated; Serine; Tumor Suppressor Proteins