Abstract
Background: Relapsed/Refractory diffuse large B-cell lymphoma (DLBCL) patients had a poor prognosis, especially those relapsing within 12 months of completion therapy. Clinically, there is a need to identify and target pathways associated with acquired treatment resistance. Using rituximab-chemotherapy resistant DLBCL cell lines generated in our laboratory, we demonstrated the acquirement of rituximab resistance is associated with changes in the cell cycle distribution (S phase arrest) and expression of cell cycle regulatory proteins (cyclin B, CDC2, CDK7, Wee1, checkpoint kinase 1 [CHK1] and checkpoint kinase 2 [CHK2]). CHK1 and 2 are serine/threonine kinases that control: 1) G2/M phase transition in response of DNA damage, 2) stabilization of DNA replication fork and 3) coordinate the mitosis. Inhibition of CHKs is an attractive strategy in cancer medicine. CHK1 inhibitors are been studied in phase I clinical trials in solid tumor malignancies (i.e. breast, pancreas or lung cancer). We hypothesized that CHK inhibition by Prexasertib has effective anti-tumor activity in DLBCL pre-clinical models. Methods: We used a panel of DLBCL cell lines including rituximab sensitive (Raji, RL, DHL4, DHL6, TMD8, RIVA, and U2932), rituximab resistant (Raji 4RH and RL 4RH), and double hit DLBCL (DOH2, ROS50 and VAL). In addition, experiments were conducted in primary tumor cells isolated from lymphoma patients. Cells were exposed to prexasertib alone or in combination with chemotherapy agents. Cell viability were determined by Presto Blue assay. IC50 was calculated by Graphpad software and coefficient of synergy was calculated using the CalcuSyn software. Subsequently, cells were expose to prexasertib and changes in cell cycle distribution and apoptosis were determined by propidium iodide staining (Pi), annexing V/PI staining and by Caspase3/7 activity detection assay. Changes in mitochondrial potential were determined by DiOC6 staining. Induction of DNA double strain break was investigated by western blot probed with phosphory-H2A.X antibody. At molecular basis, downstream pathway activities ( Mcl-1, phosphor-GSK3β, phosphory-H3, and wee1) were determined by western blot. Results: Prexasertib exhibited a dose dependent anti-lymphoma activity in all all cell lines tested including DHL cell lines. The IC50 of the cells ranged from 11nM to 2.8uM at 72 hours. Additive/synergistic effects were observed by combining prexasertib and doxorubicin, etoposide, vincristine, gemcitabine and proteasome inhibitors. At the dosage of 10nM and 25nM, prexasertib induced cell cycle arrest at G2/M phase. It also induced low membrane potential, caspse3/7 activation and apoptosis in all DLBCL cell lines. Interestingly, western blot also showed CHK inhibitor at 10nM was able to reduce Mcl-1 and p-GSK 3beta. Conclusion: Our data suggests that targeting checkpoint kinase is a promising therapeutic strategy in the lymphoma, including in double hit lymphoma pre-clinical models. In vivo studies are planned. Our data supports the clinical development of prexasertib in DLBCL. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant)
Disclosures
No relevant conflicts of interest to declare.
DNA repair inhibitors sensitize cells differently to high and low LET radiation
