Berzosertib (VE-822) has been used in trials studying the treatment of Ovarian Neoplasms, Ovarian Serous Tumor, Adult Solid Neoplasm, Advanced Solid Tumor, and Advanced Solid Neoplasm, among others.

Compared to the individual effects of XTR, the combination of 60 mg/kg VE-822 and XTR doubled the time required for tumors to reach 600 mm³ in mice carrying PSN-1 and MiaPaCa-2 tumors. This co-administration significantly prolonged tumor growth delay in PSN-1 tumor-bearing mice compared to the combination of gemcitabine and XRT1, highlighting VE-822's effectiveness. Additionally, the combination of 60 mg/kg VE-822 and XRT1 resulted in a 44% increase in tumor uptake versus XRT1 alone, demonstrating that VE-822 enhances the persistence of DNA damage caused by XRT through increased γH2AX phosphorylation. Furthermore, VE-822 inhibits the phosphorylation of serine 345-Chk1 in PSN-1 tumors in mice, indicating its potential role in cancer treatment strategies.

At a concentration of 80 nM, VE-822 alone increased the proportion of MiaPaCa-2 and PSN-1 cells remaining in the G1 phase and eliminated enriched G2/M phase fractions containing XRT in these cells. VE-822's sole application had limited impact, but its combination with XRT and/or gemcitabine significantly enhanced both early and late apoptosis in PSN-1 cells. VE-822 also heightened tumor response to agents causing DNA damage associated with (pChk1 Ser345) blockade. Furthermore, VE-822 (80 nM) attenuated the ATR signaling pathway, reducing tumor cell survival rates in response to XRT and gemcitabine. However, in normal cells, although 80 nM VE-822 weakened the ATR signaling pathway, it did not increase the killing ability of radiation and gemcitabine against these cells.

A375 cells are pre-treated with 1 μM JNK-IN-8 for the indicated amounts of time. Remove the medium and wash 3 times with PBS. Resuspend the cell pellet with 1 mL Lysis Buffer (1% NP-40, 1% CHAPS, 25 mM Tris, 150 mM NaCl, Phosphatase Inhibitor Cocktail, and Protease Inhibitor Cocktail). Rotate end-to-end for 30 min at 4°C. Lysates are cleared by centrifugation at 14000 rpm for 15 min in the Eppendorf. The cleared lysates gel filtered into Kinase Buffer (0.1% NP-40, 20 mM HEPES, 150 mM NaCl, Phosphatase Inhibitor Cocktail, Protease Inhibitor Cocktail) using Bio-Rad 10DG colums. The total protein concentration of the gel-filtered lysate should be around 5-15 mg/mL. Cell lysate is labeled with the probe from ActivX at 5 μM for 1 hour. Samples are reduced with DTT, and cysteines are blocked with iodoacetamide and gel filtered to remove excess reagents and exchange the buffer. Add 1 volume of 2X Binding Buffer (2% Triton-100, 1% NP-40, 2 mM EDTA, 2X PBS) and 50 μL streptavidin bead slurry and rotate end-to-end for 2 hours, centrifuge at 7000 rpm for 2 min. Wash 3 times with 1X Binding Buffer and 3 times with PBS. Add 30 μL 1X sample buffer to beads, heat samples at 95°C for 10 min. Run samples on an SDS-PAGE gel at 110V. After transferred, the membrane is immunoblotted with JNK antibody[1].

VE-822 is dissolved in DMSO and stored, and then diluted with appropriate media before use[1]. Gemcitabine (10 nM) is added 24 h pre-XRT and is replaced with fresh medium before addition of VE-822. PSN-1 cells are treated with VE-822 (80 nM) for 1 h before, through to 18 h after, XRT (6 Gy). Apoptosis is analyzed 48 h after XRT by flow cytometry using an Annexin V-FITC kit with PI[1].