Introduction: Glioblastoma (GBM) is the most commonly diagnosed primary brain tumor in adults. The 14.6 months median survival period of GBM patients is still palliative due to resistance to the first-line chemotherapeutic agent temozolomide (TMZ).
Methods: The cell growth inhibition effect was assessed using the SRB assay. The mRNA expression levels were examined using RT-qPCR. The protein expression levels were determined using Western blot analysis. The DNA repair by non-homologous end-joining (NHEJ) was quantified using NHEJ reporter assay. The TMZ-induced apoptosis was detected by the Caspase 3/7 activity kit. The DNA binding activity in cells was determined using chromatin fractionation assay. The 53BP1 inhibitor was identified using virtual screening followed by Western blot analysis. The synergy between TMZ and 53BP1 inhibitor in vivo was analyzed using a xenograft mouse model.
Results: We found that non-homologous end joining (NHEJ), which is one of the major DNA double-strand break repair pathways, participates in acquired TMZ-resistance in GBM. Canonical NHEJ key factors, XLF and 53BP1, are upregulated in TMZ-resistant GBM cells. Depletion of XLF or 53BP1 in TMZ-resistant cells significantly improve the potency of TMZ against GBM cell growth. Importantly, we identified a small molecule HSU2018 to inhibit 53BP1 at nanomolar concentration. The combination of HSU2018 and TMZ generates excellent synergy for cell growth inhibition in TMZ-resistant GBM cells and xenograft.
Conclusion: Our data suggest that NHEJ is a novel mechanism contributing to TMZ-resistance, and its key factors may serve as potential targets for improving chemotherapy in TMZ-resistant GBM.
Keywords: glioblastoma, temozolomide, XLF, 53BP1, non-homologous end joining, chemoresistance, 53BP1 inhibitor