MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31^comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8’s FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

MAD2 是一种纺锤体组装检查点蛋白，参与有丝分裂检查点复合体的形成，从而阻止有丝分裂进展。RNF8 是一种已确定的 DNA 损伤反应蛋白，与有丝分裂检查点调节有关，但其确切作用仍知之甚少。在这里，RNF8 邻近蛋白质组学揭示了 RNF8-MAD2 在产生有丝分裂检查点信号中的作用。具体来说，RNF8 与一小部分 p31^彗星竞争，通过其 RING 结构域与 MAD2 的闭合构象异构体结合，而 CAMK2D 充当分子支架，通过其 p-Thr287 和 RNF8 之间的瞬时/弱相互作用来浓缩 RNF8-MAD2 复合物。 FHA 域。因此，RNF8 过表达会以 FHA 和 RING 依赖性方式损害神经胶质瘤干细胞 (GSC) 有丝分裂进展。重要的是，RNF8 低表达与神经胶质瘤预后较差相关，而 RNF8 过度表达会阻碍 GSC 致瘤性。最后，我们使用化学生物学方法鉴定了模拟 RNF8 过表达的 PLK1 抑制剂，并证明了 PLK1/HSP90 抑制剂组合可以协同减少 GSC 增殖和干细胞性。因此，我们的研究揭示了一种先前未被识别的 CAMK2D-RNF8-MAD2 复合物在调节与神经胶质瘤相关的有丝分裂检查点方面的作用，该复合物在治疗上是可靶向的。