Abstract
Glioblastoma is the most common and most aggressive primary brain tumor in adults and is currently incurable. Glioblastomas revert a poor prognosis despite multimodal first line treatment comprising surgical tumor resection followed by radiochemotherapy with the DNA-methylating agent, temozolomide. Ultimately, the vast majority of glioblastomas progress within the first year following diagnosis due to resistance against temozolomide, limiting therapeutic options and mounting into nearly universal mortality of patients. Among other DNA modifications, temozolomide causes methylation of the O6-position of guanine (O6-MetG), a base lesion that is highly toxic for replicative cells, but which is efficiently repaired by O6-methylguanine-DNA methyltransferase (MGMT). In glioblastoma, spontaneous silencing of the MGMT gene by promoter methylation is frequent and renders tumor cells susceptible to temozolomide treatment (Hegi et al. 2005). Temozolomide sensitivity depends on the DNA mismatch repair (MMR) pathway, which recognizes O6-MetG lesions and triggers cell cycle arrest or cell death upon temozolomide exposure (D’Atri et al. 1998). MMR protects genomic DNA against mutagenesis by detecting and initiating the repair of base mismatches and damaged bases on DNA, which are produced during normal DNA replication or exposure to DNA damaging agents. Defective MMR function leads to alkylating therapy resistance and an increased mutation frequency in various human cancers. At diagnosis, MMR deficiency is rare in glioblastomas, but during temozolomide treatment nearly one third of glioblastomas acquire MMR defects, mainly by mutation of the MutS homolog 6 (MSH6) gene (Cahill et al. 2007) to mediate temozolomide therapeutic failure. The literature on the role of MMR mutations in mediating temozolomide resistance is extensive, however, the clinical implication of MMR mutations on disease progression and radiotherapy response remains under-explored. Recent retrospective clinical studies suggest that MMR deficiency is associated with poor post-recurrence survival in recurrent gliomas (Touat et al. 2020, Suwala et al. 2021). Whether unfavorable survival of patients with MMR-deficient gliomas can be attributed to temozolomide resistance alone, or whether de novo oncogenic mutations contribute to accelerated tumor progression, is a matter of debate. In this project, we developed immunocompetent RCAS/tv-a glioblastoma mouse models that recapitulate the genotype of MMR-deficient recurrent glioblastoma. Oncogenic drivers of the most frequent chromosomal aberrations found in glioblastomas were modeled, including aberrant platelet-derived growth factor (PDGF) signaling (chromosome 7 gain) and mutation or loss phosphate and tensin homolog (PTEN) (chromosome 10 loss) and Cyclin dependent kinase inhibitor 2A (CDKN2A) co-deletion (Ozawa et al. 2014). Additionally, we generated triple expression vectors to model altered tumor protein 53 (TP53) signaling, MMR loss-of-function and MGMT promoter methylation. In this MMR-deficient glioblastoma model, Msh6 depletion impaired survival and promoted tumor growth in mice in the absence of treatment. Msh6-deficient tumors exhibited an increased number of proliferating cells. In vitro, MSH6 (Msh6) loss accelerated cell proliferation in vitro in human and murine glioblastoma cell lines. Immunohistochemistry of tumor tissues revealed an increase in the proportion of G2 and M phase cells along with a reduction in the number of S phase cells in Msh6-deficient tumors compared to their Msh6 proficient counteracts. In line with these findings, MSH6-deficient glioblastoma cells progressed more rapidly through the S phase and S/G2 boundary. We hypothesized that MSH6-deficient cells fail to detect replication-associated base mismatches thereby reducing activation of the intra-S and S/G2 checkpoints mediated by ATR-CHK1 that normally delay S phase and cell cycle progression to gain time for DNA repair. The effect of Msh6 loss on cell proliferation was not completely recapitulated by depletion of the Msh6 binding partner, MutS homolog 2 (Msh2), or by depletion of the MMR protein Msh3. Msh6 loss, but not MutS homolog 3 (Msh3) loss, restored radio-sensitivity of radio resistant p53-deficient murine glioblastoma. In vitro, Msh6 loss led to the accumulation of persistent DNA double-strand breaks (DSB) following ionizing irradiation (IR), suggesting defective repair of IR-induced DSB. Depletion of Msh2 resulted in an even stronger increase of radio-sensitivity in vivo and in vitro compared to Msh6 loss. Msh2 can form two distinct MMR heterodimers by interacting either with Msh6 or Msh3. The Msh2/Msh3 complex mediates efficient DSB repair by homologous recombination (HR) when the DNA replication fork encounters DNA damage and collapses. We hypothesized that Msh2/Msh3 complex has a similar role in enhancing IR-induced DSB repair efficiency by HR. Classical non homologous end-joining (c-NHEJ), the second major DSB pathway in eucaryotic cells, is the most proficient pathway to repair IR-induced DSB, while HR plays an accessory role in repairing secondary DSB which arises through the conversion of single strand breaks to DSB during DNA replication. MSH6 has been suggested to enhance c-NHEJ efficiency by interacting with key c-NHEJ effector proteins. The dual impact of MSH2 loss on HR and c NHEJ through destabilization of the MSH2/MSH3 and MSH2/MSH6 complexes, respectively, may explain the dramatic impact of Msh2 depletion on tumor cell radio sensitivity which was not recapitulated by Msh3 or Msh6 depletion alone. Taken together, this study uncovers a non-canonical function of MSH6 in regulating cell cycle duration and DSB repair. Deficiency in MSH6 accelerates tumor growth but increases radio-sensitivity, probably by impairing c-NHEJ-mediated DSB repair, independently of its canonical role in mediating alkylating therapy resistance and mutagenesis. Our results suggest that the poor clinical outcome observed in patients with MMR-deficient gliomas may be improved by targeting specific vulnerabilities of this subtype, notably by combinational treatment with radiotherapy and HR inhibitors.
Lourman, Roxanne