P15.02 Loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression

Abstract

Abstract BACKGROUND The prognosis for newly diagnosed adult glioblastoma multiforme (GBM) patients is poor even after standard therapy with a median survival of approximately 14–15 months. The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) efficiently counteracts formation of the most lethal DNA adducts by temozolomide (TMZ) chemotherapy, and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has previously been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling together with SMARCA5 in the WICH complex) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its ability to regulate the DDR in GBMs, is so far not known. In this study, we investigated whether loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. MATERIAL AND METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. For each cell line, three knockout clones were chosen for further investigation. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding WSTF-expressing control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p <0.0001, U-251: Clone 7/35.1/70.2 vs control: p <0.0001/p= 0.0107/p= 0.0119). Furthermore, two out of three clones of T98G and U-251 (T98G Clone 13 and 128 vs control, p <0.0001, U-251 Clone 7 vs control, p= 0.0062; clone 70.2, p= 0.0052) showed significantly reduced plating efficiency compared to control cells. CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.