Under this regimen, tumor cells are in constant exposure to PARPi

Under this regimen, tumor cells are in constant exposure to PARPi. sustained PARPi therapy in the medical center. Importantly, PARPi-induced senescence renders ovarian and breast malignancy cells transiently susceptible to second-phase synthetic lethal approaches targeting the senescence state using Diosmetin senolytic drugs. The combination of PARPi and a senolytic is effective in preclinical models of ovarian and breast cancer suggesting that coupling these synthetic lethalities provides a rational approach to their clinical use and may together be more effective in limiting resistance. mutations and have high rates of copy number anomalies23C26. In particular, OV4453 carries a mutation that is likely responsible for PARPi sensitivity4,23. Real-time imaging confirmed dose-dependent Olaparib-mediated inhibition of cell proliferation in which higher concentrations were required for two cell lines and IC50 were consistent with those obtained using clonogenic assays (Fig.?1a, Supplementary Fig.?1A). Interestingly, live-cell imaging revealed that inhibition of cell proliferation was not accompanied by significant cell detachment. This was confirmed by correspondingly small increases in total cumulative cell death/apoptosis, as only 20C40% of cells were cumulatively AnnexinV and/or DRAQ7 positive 6 days after treatment initiation, even at the highest Olaparib concentrations (Fig.?1b, Supplementary Fig.?1B). However, real-time images revealed treatment-associated changes in cell morphology, including cell enlargement that started at day 3 and became more pronounced at day Diosmetin 6 (Supplementary Fig.?1C), suggesting a senescence cell fate response. Open in a separate windows Fig. 1 Olaparib induces a senescence-like phenotype in HGSOC cell lines. a Cell proliferation curves of HGSOC H2B-GFP cell Diosmetin lines exposed to increasing concentrations of Olaparib. b, c HGSOC lifeless cells analyzed by circulation cytometry (b) and SAgal positive HGSOC cells (c) following 6 days treatment with selected Olaparib concentrations (Supplementary Fig.?1A). d HGSOC cell morphology analyzed by circulation cytometry following 6 days of treatment Nr2f1 with Olaparib IC50 concentrations (observe Supplementary Fig.?1A, E for details). e, f Levels of IL-6 (e), IL-8 (f) were measured by ELISA assay following 6 days treatment with Olaparib IC50 concentrations. g Quantity of -H2AX foci per nucleus in HGSOC cells lines following 6 days of treatment with Olaparib IC50 concentrations. h, i Analysis of 8-h (h) or 24-h (i) EdU pulse after 6 days exposure of HGSOC cells to Olaparib IC50 concentrations. j Circulation cytometry analysis of cell cycle populations following 6 days exposure of HGSOC cells to Olaparib IC50 concentrations. Data in (a) Diosmetin are representative curves of at least three impartial experiments. For all the data, the mean??SEM of three indie experiments is shown. Data were analyzed using the two-tail Student test. *Denotes mutant status22, which was confirmed for HGSOC cells in this study23C26. Therefore, increased levels of the direct p53 transcriptional target p21 are unexpected. However, p53-impartial activation of p21 has been reported during embryonic- and oncogene-induced senescence33 and following overexpression of the Chk2 DDR kinase in epithelial malignancy cells34. To test whether a Chk2-p21 pathway similarly regulates PARPi-induced proliferation arrest in HGSOC cells, we verified the Chk2 (test. *Denotes test. *Denotes test. * Denotes test. * Denotes mutations in this type of malignancy40. Olaparib doseCresponse curves for mutant triple unfavorable breast malignancy (TNBC) MDA-MB-231 cells41 revealed a concentration-dependent inhibition of cell proliferation that was in a IC50-intermediate range when compared to HGSOC cells (Fig.?6a, IC50: 2.92??0.17?M). As in HGSOC cells, Olaparib induced a senescence-like phenotype in MDA-MB-231 cells, including a very low cumulative cell death rate even at concentrations above the IC50 (Fig.?6b, Supplementary Fig.?11A), a significant increase in SAgal positive cells (Fig.?6c, Supplementary Fig.?11B), and a clear cell enlargement even at a lower concentration (2.5?M) (Supplementary Fig.?11C, D). Short and long EdU pulse-labeling assays revealed a dose dependent decrease in DNA synthesis at day 6 in Olaparib-treated TNBC cells (Fig.?6d), indicating an apparent and stable SAPA in MDA-MB-231 cells. This was confirmed by cell cycle analysis at 6 days post-treatment showing an accumulation at the G2/M phase of the cell cycle (Fig.?6e, Supplementary Fig.?11E). Furthermore, gene-expression analysis exhibited that p21, CHK2, IL-6, IL-8, and BCL-XL were significantly upregulated in TNBC cells treated with Olaparib for 3 and 6 days (Fig.?6f, g). Thus, PARPi induced a significant senescent-like state with cell cycle arrest in TNBC cells. Importantly, a combination therapy of Olaparib at IC50 or higher doses with the senolytics ABT-263, A-1155463, and to a lesser extent PPL experienced synergistic killing effects (Fig.?6hCk, Supplementary Fig.?12ACD), suggesting that this senescence-like state induced by PARPi therapy is common to ovarian and breast cancer cells and can be similarly targeted. Open in a separate windows Fig. 6 Olaparib induces a targetable senescence-like phenotype in a TNBC cell collection. a Proliferation response.

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