P13.06. A evaluation of BBB permeable WEE1 and CHK1 inhibitors in combination with standard of care for the treatment of Glioblastoma

BACKGROUND Glioblastoma (GBM) is a highly aggressive intracranial malignancy of unmet clinical need. Current therapies such as temozolomide (TMZ) chemotherapy exert suboptimal efficacy, necessitating new therapies such as those targeting nuclear kinases, Wee1 and Chk1. Wee1 and Chk1 are master regulators of S phase and G2/M arrest ensuring DNA replication and genomic stability. Wee1 via inhibitory phosphorylation of CDK1 regulates the intra-S phase checkpoint and G2/M arrest, while Chk1 via Cdc25 inhibition controls mitotic entry and replication fork regulation in S phase. Previous studies combining commercially available Wee1 inhibitor AZD1775 and Chk1 inhibitor AZD7762 showed synergism in melanoma cells. However, a prerequisite of GBM therapies is blood-brain barrier (BBB) penetrance for drugs, thus we assessed synergism in combinations of TMZ with novel Wee1 and Chk1 inhibitors specifically developed for BBB penetrance to deduce their potential for use in GBM treatment. MATERIAL AND METHODS Primary patient-derived G7 GBM cells grown in stem-enriched conditions were used in medium-throughput ClonoScreen3D assays. 180 cells per well were seeded into Matrigel-coated 3D-Alvetex 96-well plates and incubated for 20 hours prior to drug treatment. Cells were incubated for 13 days before incubation with MTT reagent for manual colony quantification. Drugs tested were TMZ (0-3 µM); Chk1i-a (0-0.31 µM); Chk1i-b (0-0.31 µM); and Wee1i-a (0-0.31 µM) in a half-log serial dilution. RESULTS Treatment of G7 cells with Chk1i-b showed the highest single-agent activity (EC50=73 nM, confidence interval (CI)=0.677-0.774), followed by Chk1i-a (EC50 = 125 nM, CI=0.393-0.805) and TMZ (EC50=849 nM, CI=0.042-0.826) as assessed by clonogenic survival assay (CSA). TMZ+Chk1i-a showed the strongest synergism in combination in G7 cells (mean combination index score (CIS)=0.49), followed by Chk1i-a+Wee1i-a (mean CIS=0.52), and Chk1i-b+Wee1i-a (mean CIS=0.74). Wee1i-a+Chk1i-a caused the highest reduction in clonogenicity measured via CSA, while TMZ+Chk1i-a showed the strongest synergism in combination. CONCLUSION Our novel BBB-permeable Wee1 and Chk1 inhibitors show synergism inducing higher cell kill in patient-derived G7 GBM stem-like cells. Combinations will next be tested with radiation to deduce radiosensitisation effects. With synergism observed between novel BBB-penetrant Wee1 and Chk1 inhibitors, these drugs show potential in becoming viable new therapies for GBM treatment.