An increasing number of glioma-related studies have been conducted on multiple aspects of IDH1 mutations, which have been identified as the most prevalent molecular alteration (found in > 70% of cases) in LGGs(3). Previous studies have suggested that glioma patients harbouring mIDH1 have more favourable outcomes than those harbouring wild-type IDH, a phenomenon that is linked to the gain-of-function phenotype resulting from this mutation(13, 34, 35). However, the molecular mechanisms underlying the difference in prognosis between IDH1-mutant and IDH1-WT gliomas remain unclear. Here, we investigated whether this difference can be attributed to differences in the therapeutic response, which could be represented by drug sensitivity and resistance in glioma patients. Currently, exploring promising therapeutic strategies that can enhance drug efficacy or minimize drug resistance in gliomas is the main objective of research.
Since TMZ is a standard chemotherapeutic drug that improves the survival of patients with glioma, our current work focuses on the differences in TMZ sensitivity between patients with IDH1-mutant glioma and those with IDH1-WT glioma, as well as on the identification of key elements involved in regulating the response to TMZ in IDH1-mutant gliomas, with the purpose of revealing novel therapeutic options. TMZ, a DNA-methylating agent, induces DSBs and cell death by efficiently penetrating the blood–tumour barrier(36). As is well-known, the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is closely associated with the therapeutic efficacy of TMZ and the prognosis of glioma patients(37). Recent studies found that IDH-mutant glioblastoma patients tend to have higher levels of MGMT promoter methylation and lower MGMT mRNA expression, which could increase the response to TMZ treatment(38). The methylation pattern may be linked to IDH mutations and metabolic changes. However, the predictive value of MGMT methylation for TMZ sensitivity may be influenced by other molecular factors, such as chromosomal variations, DNA alterations, RNA expression profiles, and immune microenvironment(39–41). Therefore, further studies are needed to elucidate the correlation and specific mechanisms between MGMT promoter methylation status and TMZ sensitivity in gliomas.
In this study, we primarily focused on the impact and potential mechanisms of mIDH1 protein on TMZ-induced glioma cell cycle, apoptosis, DNA damage, and related processes. First, the results of a series of in vitro and in vivo experiments indicated that IDH1-mutated glioma cells exhibited greater TMZ sensitivity than IDH1-WT glioma cells, although the association between IDH1 mutation and TMZ sensitivity remains controversial. Specifically, available studies have suggested that mIDH1 enhances TMZ sensitivity via ATM-related pathways(42). Similarly, studies have shown that mIDH1 in glioma can increase the cytotoxic effect of TMZ in a dose- and time-dependent manner due to the TMZ-dependent accumulation of reactive oxygen species (ROS)(43). However, contrasting findings have indicated that the expression of mIDH1 confers TMZ resistance rather than TMZ sensitivity on glioma cells by upregulating the HR-associated protein RAD51(14). Moreover, a previous study demonstrated that overexpression of the IDH1-R132H protein suppressed the growth and migration of glioma cells, resulting in radiosensitivity but not TMZ sensitivity(44). In general, although different conclusions about the effects of IDH1 mutation on TMZ sensitivity were drawn in the above studies, we can still speculate that mIDH1 affects the response to TMZ because of dysfunction of DDR, abnormal ROS levels or epigenetic dysregulation of gene expression. However, few studies have focused on the effect of mIDH1 on TMZ responsiveness from the perspective of proteomics, i.e., targeting mIDH1 itself. Herein, we investigated the protein-related mechanisms underlying the relationship between mIDH1 and the TMZ response via IP-MS analysis and identified the protein binding to mIDH1, i.e., RPA1, which is involved in DNA replication and the DDR process.
A key intermediary protein involved in maintaining genome integrity, RPA1 has been reported to trigger the DNA damage response (DDR) cascade following toxic cellular injury(45, 46). Notably, a growing body of research suggested that RPA1 functions as an oncogene. A recent study showed that high expression of RPA1 in gastric cancer can promote tumour cell growth and increase resistance to replication stress(47). Additionally, RPA1 has been shown to promote the proliferation, migration, and invasion of liver cancer cells both in vivo and in vitro, and its overexpression is positively correlated with poor prognosis in liver cancer patients(48). Consistent with these observations, we found that the mRNA and protein levels of RPA1 were elevated in glioma and correlated with poor prognosis in glioma patients, suggesting that RPA1 might promote gliomagenesis. In addition, we found that the RPA1 expression level effectively predicted the sensitivity of glioma cells to TMZ and that high expression of RPA1 may be associated with TMZ resistance. Moreover, studies have focused on the relationship between RPA1-mediated DNA repair and the response of patients with tumours to radiotherapy and chemotherapy. When chemotherapeutic drugs or radiation therapy induce DSBs in tumour cell DNA, the homologous recombination repair (HRR) pathway is activated. A recent study showed that the interaction of the mitochondrial protein HIGD1A with RPA1 activated the DNA damage-dependent chromatin association of the RAD9-RAD1-HUS1 complex, leading to DNA damage checkpoint pathway activation and eventually promoting HR and radio/chemosensitivity(49). Another study demonstrated that the interplay between the tumour suppressors PTEN and RPA1 facilitated RPA1 accumulation on replication forks to regulate the DNA replication process(50). These results verified that the roles of RPA1 could be influenced through its physical or functional interactions with other proteins, contributing to regulating the DDR process. Moreover, previous studies have indicated that RPA1 induces the progression of malignancies and impairs radiosensitivity in oesophageal cancer(51). Decreased expression of RPA1 was shown to enhance cell sensitivity to oxaliplatin-based chemotherapy in colorectal cancer(52). However, whether RPA1 can regulate the sensitivity of glioma cells to TMZ – the chemotherapeutic drug most commonly used in glioma – and the possible regulatory mechanism involved remain obscure. In this study, we demonstrated that the overexpression of RPA1 reversed the DNA damage caused by TMZ, while the inhibition of RPA1 increased TMZ-induced apoptosis and DNA damage in vivo and in vitro. These findings provided insight into combining the RPA1 inhibitor HAMNO with TMZ to enhance the therapeutic effects of TMZ. Interestingly, a similar enhanced response to TMZ was found in IHD1-mutated cells and HAMNO-treated cells. Hence, we hypothesized that RPA1 dysfunction occurs in IDH1-mutant glioma cells. To confirm this hypothesis, we performed an EMSAs to compare the ssDNA-binding ability of RPA1 between IDH1-mutated and IDH1 wild-type glioma cells, and the results indicated a decreased binding affinity of RPA1 and ssDNA in IDH1-mutated glioma cells treated with TMZ. Given that the DBD-A domain of RPA1 interacts with both mIDH1 and ssDNA, it is possible that the mIDH1-RPA1 and RPA1-ssDNA complexes bound via the DBD-A domain of RPA1 have a competitive relationship. RPA1 consists of four DNA binding domains with high affinity but with differential dynamics. Studies have demonstrated that RAD52, an RPA1-interacting protein, can stabilize the binding of RPA1 and ssDNA by regulating the dynamics of the DBD-D domain(53). Moreover, a recent study showed that lamin-associated protein (LAP2α) can physically interact with RPA1 via its DBD-A domain, promoting RPA1-ssDNA binding and maintaining genome stability(54). Then, we further verified the diminished ssDNA binding affinity upon deletion of the RPA1 DBD-A domain. Consequently, we proposed that mIDH1 could impair the ability of RPA1 to bind to ssDNA via its interaction with RPA1 in glioma cells.
As the initial step of HRR, the binding of RPA1 to ssDNA protects DSBs from the formation of secondary structures, which interfere with the process of DNA repair. Then, the complex activates a series of interrelated pathways, including the cell cycle checkpoint-related ATR/CHK1 pathway, which is a major pathway required for DSB repair(55). ATR, activated by RPA-coated ssDNA, plays a role in activating the S-phase checkpoint, consequently allowing DNA repair and hindering premature mitotic progression(56). Then, the downstream regulator CHK1 is phosphorylated and causes G2/M arrest(57). In line with this observation, a recent study showed that the activation of ATR/CHK1 was significantly associated with senescence triggered by TMZ, which led to G2/M arrest in glioma cells(58). Moreover, actin-like 6A (ACTL6A) was found to mediate DNA replication to reduce DNA damage via the ATR-CHK1 pathway, resulting in the promotion of glioma cell growth(59). Considering the above observations, we speculated that RPA1 plays crucial roles in TMZ-treated glioma cells via the ATR-CHK1 pathway. Therefore, we measured the protein levels of ATR/CHK1 in RPA1-overexpressing glioma cells and HAMNO-treated glioma cells. Overexpressing RPA1 contributed to the phosphorylation of ATR and CHK1, while inhibiting RPA1 expression led to the inactivation of ATR and CHK1, suggesting the regulatory mechanism by which RPA1 promotes HR in glioma cells upon TMZ treatment. Collectively, our results showed that the mIDH1-RPA1 interaction weakened the ability of RPA1 to load onto ssDNA, and such a defect in RPA1 loading could result in failure to activate the ATR/CHK1 pathway, ultimately promoting TMZ sensitivity in IDH1-mutant glioma cells.
As shown in Fig. 8, the findings from our study revealed that G2/M arrest, increased apoptosis and increased DNA damage resulted from TMZ treatment in IDH1-mutant glioma cells, which exhibited more obvious TMZ-induced cytotoxicity. The proposed reason for these effects is that mIDH1 interacts with RPA1 and inhibits its binding to ssDNA, resulting in suppression of the HR process and increased sensitivity of IDH1-mutant glioma cells to TMZ. In addition, our research provides the basis for the clinical proposal of combination therapy with TMZ and the RPA1 inhibitor HAMNO, which has a significant synergistic inhibitory effect on IDH-mutated glioma.