Abstract Diffuse Midline Gliomas (DMG) are universally fatal paediatric brain tumours with no curative treatment. ACT001 is a brain-penetrant parthenolide derivative that has demonstrated anti-tumour activity in preclinical models and a Phase I clinical trial (ACTRN12618001934235). Although ACT001 modulates the NF-κB1, STAT32, and PI3K/Akt3 pathways in glioblastoma, its mechanism in DMG remains unclear. This study aimed to define ACT001’s mechanisms in DMG and identify rational therapeutic combinations. We previously demonstrated that ACT001 induces oxidative stress and apoptosis in DMG cells. Multi-omics analyses in this study revealed that ACT001 alters pathways associated with oxidative stress, unfolded protein response and NF-κB signalling. Validation by qPCR and Western blot confirmed upregulation of oxidative stress markers (including NQO1, TrxR, HO-1), alongside reduced NF-κB-p65 expression and its downstream anti-apoptotic target, Mcl-1. Induction of apoptosis was confirmed by flow cytometry and increased levels of cleaved-PARP protein. To overcome resistance and enhance therapeutic efficacy, high-throughput combination screening identified 13 agents that synergise with ACT001. Mcl-1 inhibitors (MIK665 and AZD5991) showed the strongest synergy consistent with ACT001-mediated suppression of anti-apoptotic signalling. The combination markedly enhanced cytotoxicity across genetically diverse DMG cell lines, with no adverse effects against normal cells. Mechanistically, RNA sequencing and proteomic analyses confirmed that the combination further potentiates ACT001’s impact on the unfolded protein response, endoplasmic reticulum stress, apoptotic pathways, and NF-κB signalling. In vivo, ACT001, the Mcl-1 inhibitor S63845, and their combination each extended survival without evidence of synergy in orthotopic DMG models and reduced Ki67-positive tumour cells. Additional in vivo studies are ongoing to further define the therapeutic potential of this combination. Collectively, these findings indicate that ACT001 is a multi-targeted agent that disrupts NF-κB and apoptotic signalling while promoting oxidative stress and the unfolded protein response. Combining ACT001 with inhibitors of anti-apoptotic proteins represents a promising therapeutic strategy for patients with DMG. 1. Li, Q., Sun, Y., Liu, B., Li, J., Hao, X., Ge, W., Zhang, X., Bao, S., Gong, J., Jiang, Z. and Qiu, C., 2020. ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth. Journal of Molecular Medicine, 98, pp.263-277. 2. Tong, L., Li, J., Li, Q., Wang, X., Medikonda, R., Zhao, T., Li, T., Ma, H., Yi, L., Liu, P. and Xie, Y., 2020. ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma. Theranostics, 10(13), p.5943. 3. Xi, X., Liu, N., Wang, Q., Chu, Y., Yin, Z., Ding, Y. and Lu, Y., 2019. ACT001, a novel PAI-1 inhibitor, exerts synergistic effects in combination with cisplatin by inhibiting PI3K/AKT pathway in glioma. Cell Death & Disease, 10(10), p.757.
Lawrence et al. (Tue,) studied this question.