PO-021 Inhibition of MK2 stabilises wild-type P53 and improves temozolomide efficacy in glioblastoma

Abstract

Introduction Temozolomide (TMZ) remains the first line therapy for glioblastoma since its approval in 2005. TMZ efficacy is marginal as patients succumb to the disease within two years, urging the need for new therapeutic approaches. Frequent genetic alterations in the DNA damage machinery of most cancer cells opens a window of possible therapeutic vulnerability. Tumours lacking p53 rely heavily on the G2/M cell cycle checkpoint for DNA repair, due to the loss of a functional G1 checkpoint. Therefore, inhibiting the G2/M checkpoint allows for synthetic lethality, as these cells are forced to continue through the cycle with unrepaired DNA ultimately resulting in cell death. It was previously reported that in the absence of p53, cells depend on the checkpoint kinase MK2 for cell cycle arrest and survival after DNA damage by doxorubicin. However, it is currently unknown if targeting MK2 is a viable therapeutic approach for glioblastoma therapy in conjunction with TMZ. Thus, we aimed to investigate the effect of inhibiting MK2 and whether it will improve the effectiveness of TMZ in glioblastoma. Material and methods Inhibition of MK2 was conducted using siRNA, CRISPR-Cas9 and pharmacological inhibitors. Signalling pathways, stability and nuclear retention of proteins was determined by Western blotting. Cell cycle arrest was analysed using FUCCI transfected cells and flow cytometry. Cellular senescence was visualised by staining the presence of b-galactosidase activity. Glioblastoma cell survival was assessed by colony formation and 3D spheroid tumour growth. Results and discussions Our data demonstrates that inhibition of MK2 increases the stability and expression of p53, indirectly by reducing the phosphorylation of its ligase MDM2. This suggests that MK2 plays a role in regulating p53, as inhibition of MK2 increased the levels of active p53 inside the nucleus. We also found that the combination of TMZ and MK2 inhibition did not induce G2/M arrest as expected, but G0/G1 arrest. This increase of cells in G0/G1 led to cellular senescence through the reactivation of the p53-p21 pathway. Moreover, we found that MK2 inhibition enhances the efficacy of TMZ by attenuating long-term clonal and 3D spheroid growth in p53 proficient cells, unlike previously described. Conclusion The combination of MK2 inhibition alongside TMZ provides a new therapeutic strategy to enhance the effectiveness of current chemotherapy in p53 proficient tumours.