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posted on 2023-10-16, 14:20 authored by Helena Folly-Kossi, Joshua D. Graves, Lidija A. Wilhelms Garan, Fang-Tsyr Lin, Weei-Chin Lin DNA2 inhibitor d16 synergizes with PARPis in cancer cells. A, BT549, MDAH-2774, and MDA-MB-468 cells were treated with increasing concentrations of d16, RU, or both as indicated. MTT assay was performed 72 hours following treatment. B, CI values were calculated (in brackets) and Fa-CI plots were generated using CompuSyn software. CI < 1 indicates synergism. The isobolograms for each CI analysis are shown in Supplementary Fig. S7A. C, MDAH-2774 and BT549 were treated with d16 (10 µmol/L), talazoparib (Tala; 2.5 µmol/L) or both for 72 hours, and cell viability was measured by MTT assay. D, MDAH-2774, BT549, and C33A cells stably expressing either a scrambled shRNA (shScr) or an shRNA against mutp53 (shp53) were treated with increasing concentrations of d16, RU, or both as indicated for 72 hours followed by MTT assay. The data shown represent mean ± SD (n = 3 biological replicates). P values are *, P < 0.05 and ***, P < 0.001 indicating statistically significant difference (two-tailed t test). Expression of mutp53 in these stable cell lines is shown in Fig. 4B. The detailed Fa-CI plots are shown in Supplementary Fig. S7B. E–G, Cpd 16 and PARP1/2 inhibitor talazoparib synergistically induce apoptosis in breast cancer cells. MDA-MB-468 cells were treated with increasing concentrations of d16, talazoparib, or both as indicated for 21 hours. Active caspase-3/7 was determined by Caspase-Glo 3/7 Assay and was normalized to protein concentrations. Data shown are the mean ± SE from three independent experiments. CI values, Fa-CI plots and Isobologram were generated using CompuSyn software. CI < 1 indicates synergism.
Funding
HHS | National Institutes of Health (NIH)
U.S. Department of Defense (DOD)
Rivkin Center for Ovarian Cancer (Rivkin Center)
History
ARTICLE ABSTRACT
The tumor suppressor p53 promotes tumor-suppressive activities including cell-cycle inhibition, apoptosis, senescence, autophagy, and DNA repair. However, somatic mutations in the TP53 gene are one of the most common alterations in human cancers. We previously showed that mutant p53 (mutp53) can bind TopBP1, an ATR activator, to attenuate its ATR-activating function. A partially defective ATR function caused by mutp53 makes cancer cells more vulnerable to inhibitors of other TopBP1-independent ATR activators, such as DNA2. DNA2 plays a role in homologous recombination (HR) repair by resecting DNA ends in double-strand breaks and preparing them for invasion of homologous duplex. Here we identify a new DNA2 inhibitor, namely d16, and show that d16 exhibits anticancer activities and overcomes chemotherapy resistance in mutp53-bearing cancers. Similar to DNA2 depletion, d16 treatment results in cell-cycle arrest mainly at S-phase. Moreover, reexpression of mutp53 in a p53-null cancer cell line makes cells more vulnerable to d16-mediated inhibition of ATR activity. As d16 also inhibits HR, a combination of d16 and PARP inhibitors displays synergistic induction of cell death. DNA2 is often overexpressed in cancer, particularly in cancer cells harboring mutp53. Overexpression of DNA2 is associated with poor outcome in ovarian cancer. Overall, our results provide a rationale to target DNA2 as a new synthetic lethality approach in mutp53-bearing cancers, and further extend the benefit of PARP inhibitors beyond BRCA-mutated cancers.
This study identifies a new DNA2 inhibitor as a synthetic lethal targeted therapy for mutp53-harboring cancers, and provides a new therapeutic strategy by combining DNA2 inhibitors with PARP inhibitors for these cancers.
