DDRugging glioblastoma: understanding and targeting the DNA damage response to improve future therapies

Abstract

Glioblastoma is the most frequently diagnosed type of primary brain tumour in adults. These aggressive tumours are characterised by inherent treatment resistance and disease progression, contributing to ~ 190 000 brain tumour-related deaths globally each year. Current therapeutic interventions consist of surgical resection followed by radiotherapy and temozolomide chemotherapy, but average survival is typically around 1 year, with < 10% of patients surviving more than 5 years. Recently, a fourth treatment modality of intermediate-frequency low-intensity electric fields [called tumour-treating fields (TTFields)] was clinically approved for glioblastoma in some countries after it was found to increase median overall survival rates by ~ 5 months in a phase III randomised clinical trial. However, beyond these treatments, attempts to establish more effective therapies have yielded little improvement in survival for patients over the last 50 years. This is in contrast to many other types of cancer and highlights glioblastoma as a recognised tumour of unmet clinical need. Previous work has revealed that glioblastomas contain stem cell-like subpopulations that exhibit heightened expression of DNA damage response (DDR) factors, contributing to therapy resistance and disease relapse. Given that radiotherapy, chemotherapy and TTFields-based therapies all impact DDR mechanisms, this Review will focus on our current knowledge of the role of the DDR in glioblastoma biology and treatment. We also discuss the potential of effective multimodal targeting of the DDR combined with standard-of-care therapies, as well as emerging therapeutic targets, in providing much-needed improvements in survival rates for patients.

Keywords: DNA damage response; chemotherapy; glioblastoma; radiotherapy; synthetic lethality; tumour-treating fields.

Fig. 1

The role of ATM and ATR in cell cycle regulation following DNA damage. The processes of cell division (mitosis, M phase) and DNA synthesis (S phase) are separated by two important gap phases (G1 and G2). Progression of mitotic cells through the cell cycle is controlled by periodic accumulation and destruction of the aptly named cyclin‐dependent kinases (CDKs) and cyclins. Inappropriate progression through phases of the cell cycle is prevented by three main checkpoints (G1/S, intra‐S and G2/M checkpoints; dashed red lines). Following DNA damage, checkpoint activation is critical to provide ample time and recruit the necessary machinery required to maintain genomic integrity. Checkpoint activation: DNA double‐strand breaks (DSBs) activate the apical DNA damage response (DDR) kinase ataxia telangiectasia mutated (ATM), which can influence all three major cell cycle checkpoints via the phosphorylation of checkpoint kinase 2 (CHK2) and subsequent downstream signalling. In contrast, ataxia telangiectasia and Rad3‐related kinase (ATR) is activated by the presence of replication protein A (RPA)‐coated single‐stranded DNA (ssDNA) and contributes to maintenance of the intra‐S phase and G2/M checkpoints via phosphorylation of checkpoint kinase 1 (CHK1) and subsequent downstream signalling as indicated. G1/S checkpoint: Phosphorylation of p53 by CHK2 and ATM directly (arrow not shown) results in a reduction in the binding of mouse double minute 2 homolog (MDM2) to p53 and p53 activation, promoting its nuclear accumulation and stabilisation. Subsequently, elevated p53 levels promote increased transcription of p21, which inhibits CDK2–cyclin‐E activity, resulting in prevention of progression to S phase. Intra‐S checkpoint: Within S phase, the activation of cell division cycle 25 (CDC25) phosphatases predominantly by prevention of cell division cycle 45 (CDC45) loading onto replication origins (preventing subsequent DNA replication) primarily via the ATR–CHK1 axis, but also via ATM‐CHK2‐mediated phosphorylation of CDC25A, can instigate an intra‐S checkpoint in response to replication stress or other perturbations to optimal DNA synthesis, permitting a slowing of DNA replication. G2/M checkpoint: Both ATM‐ and ATR‐mediated phosphorylation of CHK2 and CHK1, respectively, lead to the phosphorylation of CDC25C phosphatases, which influence the G2/M checkpoint via interaction with the cyclinB1–CDK1 complex. This figure is adapted, with permission, from Ref. [227].

Fig. 2

The effects of clinically approved therapies on the DNA damage response (DDR) and novel strategies to enhance efficacy of current standard‐of‐care treatments. Schematic representation of the main DNA damage lesions (in blue italic) induced by therapies approved for clinical use to treat glioblastoma and associated DDR mechanisms. For each approved treatment, putative strategies to enhance therapeutic efficacy through targeting relevant DDR mechanism(s) are indicated. (A) Radiotherapy: generates large amounts of DNA single‐strand breaks (SSBs) and double‐strand breaks (DSBs), which activate ATR and ATM, respectively. DSB repair is then predominantly undertaken by either nonhomologous end joining (NHEJ), which is available throughout the cell cycle but compromises fidelity, or homologous recombination (HR) DNA repair, which provides a high‐fidelity repair mechanism, but is only available during S and G2 phases of the cell cycle due to the requirement for a sister chromatid. SSB repair relies on PARP1 to detect SSBs and facilitate the recruitment of XRCC1. However, the presence of strand breaks also leads to stalling of DNA replication forks, which depend on the functions of ATR and proteins within the Fanconi anaemia pathway (FAP) for stability and replication restart. Consequently, a strong scientific rationale exists supporting inhibition of either ATM (ATMi), ATR (ATRi), PARP1 (PARPi) or the FAP (FAPi) to enhance the efficacy of radiotherapy. (B) Temozolomide: produces an array of methylation lesions including N3‐methyladenine (N3MeA) and N7‐methylguanine (N7MeG), which are substrates for effective removal via DNA base excision repair (BER), and O6‐methylguanine (O6MeG), which is removed directly by the enzyme MGMT in a suicide reaction. Hypermethylation of the MGMT gene promoter region leads to reduced MGMT expression, shifting the balance in favour of persistent O6MeG. O6MeG can act as a miscoding base during DNA replication, leading to a corresponding C‐to‐T transversion within the complementary DNA strand. If O6MeG is not successfully excised by the mismatch repair (MMR) DNA repair machinery, it endures as a perpetually miscoding base, instigating ‘futile cycles’ of MMR with consequent stalling of DNA replication forks or DSBs. (C) Tumour‐treating fields (TTFields): may negatively impact FAP and HR‐mediated DNA repair processes. TTFields‐induced ‘BRCAness’ (reflecting a relative HR deficiency) provides a compelling rationale to combine this therapeutic modality with PARPi, or potentially FAPi, ATRi or even ATMi. (D) Carmustine (BCNU) – Gliadel^® wafers: provide local delivery of this bidirectional DNA alkylating agent, leading to the generation of DNA interstrand crosslinks which impede DNA replication during S phase. This leads to activation of the FAP, within which monoubiquitination of FANCD2 within the FANCD2‐I complex is a key quantifiable step. Activated FANCD2‐I coalesces as foci at sites of DNA damage and acts as a master regulator of downstream DNA repair, recruiting proteins involved in nucleotide excision repair (NER), translesion synthesis (TLS) and HR. Interplay with associated DDR mechanisms, for example ATM and ATR, leads to the phosphorylation of multiple FAP proteins (examples indicated), providing a rationale for the use of non‐FAP DDR inhibitors (e.g. ATRi or ATMi) to sensitise to crosslinking chemotherapy, and for the concept of combining multiple DDR inhibitors (including FAPi) to potentially maximise therapeutic enhancement.

Fig. 3

An approach for cancer‐selective killing through multimodality targeting of interconnected DNA damage response (DDR) pathways. A schematic representation of simultaneous targeting of multiple interconnected DDR processes to achieve cancer‐selective killing. Left – a simplified network schematic of key DDR proteins illustrating the complexity of intra‐ and interpathway protein–protein interactions within the global DDR. This complexity provides a degree of functional redundancy in DDR processes, which is likely to afford therapeutic resistance to current DNA damaging therapies. Right – due to the loss of functionality within some DDR pathways during carcinogenesis, cancerous cells often demonstrate overreliance on a reduced subset of DDR processes for cell survival. Where inhibition of a single DDR pathway may not be sufficient to provide synthetic lethality or substantial cancer cell killing, targeting multiple DDR processes simultaneously may overwhelm the remaining functional DDR leading to exquisitely potent cancer cell killing. However, by virtue of their complete repertoire of fully functional DDR processes, normal cells might continue to avoid significant toxicity associated with multi‐DDR‐targeting strategies (e.g. PARPi in noncancerous breast tissue that exhibits normal BRCA1/2 expression/function).