Checkpoint Kinase 1 Pharmacological Inhibition Synergizes with DNA-Damaging Agents and Overcomes Platinum Resistance in Basal-Like Breast Cancer

Abstract

Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells

“…In this study, MCF 10A was applied as non-cancerous control for the MDA-MB-231 cells, which is a breast adenocarcinoma lineage. Cisplatin was shown to inhibit 50% of the growth of MDA-MB-231 at 6 µM 63 , which is a similar value to the ones we observed for MTT, NR or the phenothiazinium dyes. To reinforce the reproducibility of our results, we tested the LLC-MK2 cell lineage, which is widely used in cytotoxicity assays 65 , 66 , including phenothiazinium dyes 48 .…”

“…Several studies have reported the use of cisplatin for in vitro assays in several cell lines 61,62 . Our results indicated three patterns of susceptibility to cisplatin, according to cell line: MCF 10A is a line applied as resistance control (for mammary cancer) to cisplatin, with IC 50 concentrations between 5 and 26 µM 63,64 . In this study, MCF 10A was applied as non-cancerous control for the MDA-MB-231 cells, which is a breast adenocarcinoma lineage.…”

Role of protein phosphorylation in cell signaling, disease, and the intervention therapy

“…Prexasertib has a good application value in ovarian neoplasms because there is evidence of cytotoxic T‐cell activation in the blood of patients when it is combined with PD‐L1 inhibitors 234 . Rabusertib showed a good synergistic activity in platinum‐resistant breast cancer cells 235 …”

Expanding the Use of PARP Inhibitors as Monotherapy and in Combination in Triple-Negative Breast Cancer

“…Therefore, the combination of an ATM inhibitor and PARPi led to extensive DNA damage, activating the G2 damage checkpoint kinase cascade, allowing entry into mitosis, and resulting in mitotic and post-mitotic cell death [81]. The inhibition of CHK1 kinase, which is a key component of checkpoint mediate cell cycle arrest, also demonstrated synergy with olaparib in basallike breast cancer cells [82]. A phase I clinical trial further investigated the combination of CHK1 and olaparib and demonstrated preliminary antitumor activity in the context of BRCA MUT patients with high-grade serous ovarian cancer with a previous progression on PARPi alone [83].…”