Targeted inhibition of Bcl-xL following radiation reduces tumourigenesis in preclinical models of H3K27M-altered diffuse midline glioma

Abstract Background: Diffuse midline gliomas (DMGs) with histone H3K27M mutations represent a devastating paediatric brain cancer characterized by abysmal prognosis and limited treatment options. The only approved treatment is radiotherapy (RT), but most of the tumours relapse with fatal consequences. In this study, we sought to investigate whether irradiation leads to senescence induction and explore the efficacy of senolytics against DMG. Methods: We have characterised the senescent phenotype of five genetically heterogeneous H3K27M-altered human DMG cell lines, combining cellular and/or molecular approaches. The sensitivity of senescent cells to Bcl-xL inhibition has been demonstrated in dose/response curves in vitro and in a PDX model of DMG. Results: Here, we show that ionizing radiation induces senescence and SASP responses in both TP53 mutant and wild-type H3K27M-altered human DMG cell lines. We identify Navitoclax as a potent senolytic agent that selectively targets senescent DMG cells into apoptosis by inhibiting Bcl-xL. Related compounds, such as a proteolysis-targeting chimera (PROTAC)-mediated Bcl-xL degradation and a galacto-conjugated form of Navitoclax also show an effective senolytic activity in senescent cancer cells. Finally, we show that a combination therapy of irradiation and Navitoclax results in reduced tumor burden and increased mouse survival in an orthotopic xenograft DMG model. Conclusion: These results offer a rationale for further clinical development of senolytic therapies as part of multimodal treatment approaches for DMG patients/ DIPG cell lines, were irradiated, with sham (0Gy), 6 Gy and 24 Gy, following 24 hours, and 5 days RNA isolated from the cells and subsequent sequencing. Bulk RNA-seq

摘要 背景：伴组蛋白H3K27M突变的弥漫性中线胶质瘤（Diffuse midline gliomas, DMGs）是一类毁灭性儿童脑癌，预后极差且治疗手段有限。目前唯一获批的治疗方案为放疗（radiotherapy, RT），但多数肿瘤仍会复发并导致患者死亡。本研究旨在探究放疗是否可诱导细胞衰老，并探索抗衰老药物（senolytics）针对DMG的治疗效果。
方法：本研究对5株基因异质性的H3K27M突变人源DMG细胞系的衰老表型进行了表征，结合了细胞生物学与/或分子生物学实验手段。通过体外剂量-反应曲线以及DMG患者来源的异种移植（patient-derived xenograft, PDX）模型，验证了衰老细胞对Bcl-xL抑制的敏感性。
结果：本研究证实，电离辐射可在TP53突变型及野生型H3K27M突变人源DMG细胞系中诱导细胞衰老及衰老相关分泌表型（senescence-associated secretory phenotype, SASP）应答。我们鉴定出Navitoclax是一种强效抗衰老药物，可通过抑制Bcl-xL选择性诱导衰老的DMG细胞发生凋亡。相关化合物，如蛋白降解靶向嵌合体（proteolysis-targeting chimera, PROTAC）介导的Bcl-xL降解剂以及半乳糖缀合形式的Navitoclax，在衰老癌细胞中同样表现出有效的抗衰老活性。最后，我们发现，在原位异种移植DMG模型中，放疗联合Navitoclax治疗可降低肿瘤负荷并延长小鼠生存期。
结论：本研究结果为将抗衰老疗法作为DMG患者/弥漫内生型桥脑胶质瘤（DIPG）细胞系多模态治疗方案的组成部分，开展进一步临床开发提供了理论依据。针对DIPG细胞系，本研究分别施以假照射（0Gy）、6Gy及24Gy辐射，于照射后24小时及5天收集细胞RNA并开展后续测序实验，采用批量RNA测序（Bulk RNA-seq）技术。