The role of MRNIP in replication fork stabilisation and DSB repair

Genome instability eg: a high mutation rate and/or the presence of chromosomal aberrations - can cause cancer, promote disease progression and drive the genetic variation underpinning therapeutic resistance. Since genomic DNA is constantly under threat from various sources of DNA damage, the cell has evolved elegant repair mechanisms to deal specifically with each type of lesion encountered. The most dangerous DNA lesions are Double-Strand Breaks (DSBs) - these are repaired either by inaccurate rejoining or by high-fidelity Homologous Recombination (HR), a process that uses the intact sister DNA sequence as a template to copy back the correct DNA code. Among the crucial players in DSB repair by HR are the tumour suppressors BRCA1/2 - found mutated in breast, ovarian and prostate cancers. Indeed, cancer-specific DNA repair defects also provide opportunities to implement precision medicine strategies, notably evidenced by the recent success of PARP inhibitors in the treatment of BRCA-deficient cancers.

All cells must copy their DNA before they can divide and during DNA replication the genome is particularly vulnerable. DNA replication is carried out by a complex molecular machine that unwinds the two DNA strands, forming a fork-like structure akin to a partially done zipper. Many chemotherapy drugs work by altering DNA structure and causing the zipper to get stuck. If the zipper can be freed and zipped up, then the cell can proceed normally - but if it cannot, the zipper is prone to breakage. In cellular terms, this entails fork collapse, damaged DNA and cell death. The overall response to chemotherapy is determined by the ability of the cancer cell to deal with this scenario.

It has become apparent that replication forks undergo a process of physical reversal upon encountering replication stresses or other genotoxic insults, the newly-made DNA reforms into a four-way structure described as 'chicken foot-like'. Recent work demonstrates crucial roles for several HR proteins including BRCA2, BRCA1, and RAD51 in promoting the stability of reversed forks. BRCA2 stabilises the fork by loading RAD51 onto the reversed arm - it is now apparent that RAD51 loading prevents genome instability induced by aberrant 'chewing' of the fork DNA by the nucleases MRE11 and EXO1. This function of BRCA2 is independent of its known role in DSB repair by HR. Despite significant advances in understanding the biology of the reversed fork, how MRE11 nuclease activity is regulated at reversed forks to prevent genome instability is poorly understood.

We identified an uncharacterised protein called MRNIP (MRE11-RAD50-NBS1-Interacting Protein) as a novel factor that promotes DSB repair by HR. Our ongoing studies show that MRNIP binds to and stabilises replication forks, promoting fork progression, genome stability, and resistance to multiple chemotherapies. Loss of MRNIP results in marked MRE11-dependent replication fork degradation - overall our data points to a novel important genome stability mechanism. The enzyme PARP1 recruits MRE11 to replication forks - our initial data suggests that PARP may also recruit MRNIP - indeed, it is logical that regulators of MRE11 are co-recruited by PARP to prevent aberrant degradation. Our goals are to elucidate the mechanisms via which MRNIP promotes fork stability, as well as the DSB response to ionising radiation, to analyse MRNIP structure, and to develop assays to test how MRNIP influences MRE11 activity against DNA.

We will also assess MRNIP levels in cancer tissues - MRNIP is underexpressed in some cancers including ovarian adenocarcinoma (11% of cancers), and thus an in-depth study of MRNIP levels could yield information leading to biomarker-based treatment strategies or prognostic indication. Should we identify a subset of MRNIP-deficient cancers, we will screen for genes that are required for survival of MRNIP-deficient cells, thus identifying potential novel targets for therapeutic intervention.

Who will benefit from this research / innovation?

Given worldwide cancer incidence, patient beneficiaries are likely to be widespread and global. Long-term benefits may include improved health outcomes, extended life and enhanced well-being. Economic beneficiaries may include companies involved in development of existing or novel targeted therapies, as well as health providers like the NHS, who benefit as improved treatment leads to reduced hospital admission times and a decrease in funds wasted on sub-optimal therapeutic regimens. Clinical and non-clinical cancer researchers and basic cell biologists will benefit from increased mechanistic understanding of the replication stress and DNA break responses to radiation and some forms of chemotherapy.

How will they benefit from this research/innovation?

Our long-term goal is to gain an in-depth understanding of how MRNIP regulates events at stalled replication forks (which can be caused by multiple chemotherapies including platinum compounds and alkylating agents) and DNA DSBs generated by ionising radiation, where of course the implications for radiotherapy are direct. Indeed, replication stress is a response triggered in many cancers by oncogene activation or loss of tumour suppressors, that results in elevated levels of spontaneous DNA damage and genome instability, which is a core early driver of tumour formation and progression. Our approach will involve a combination of cell-based loss-of-function studies, structural biology and in vitro assays. The data produced is of inherent value to cell biologists studying genome maintenance mechanisms, and will allow us to confidently define the role of MRNIP in the context of existing DNA repair and fork protection mechanisms.

Precision medicine strategies are being increasing employed globally in the successful identification of subsets of individuals at increased risk of disease, and the development of novel targeted therapies to treat patient cohorts following genetic analysis. Here, we demonstrate that MRNIP loss results in sensitivity to multiple chemotherapies including DNA-PK inhibitors. Further assessment of MRNIP expression in cancer could identify subsets of cancers with BRCA-ness ie: that have normal BRCA proteins, but exhibit a BRCA-like phenotype in terms of therapeutic response. Therefore, our data may inform prognostic indication and identify subsets of patients likely to respond to particular DNA-damaging chemotherapies. Cancers deficient in MRNIP may be treatable with DNA-PK inhibitors, either as single-agents or in combination with radiotherapy or other treatments. We will attempt to identify novel opportunities for therapeutic intervention based on MRNIP status or via novel insight into DNA repair and fork protection mechanisms.

We will analyse also MRNIP status in cancer tissue samples, providing the first profile of MRNIP expression in tumours. Since DNA repair and replication fork stabilisation are important determinants of cancer cell therapeutic resistance, our research may lead to future novel personalised medicine strategies based on MRNIP status. Screening for genes that are synthetic lethal with MRNIP will identify novel targets for therapeutic intervention, leading in turn to drug development or repurposing, and eventual clinical evaluation. Our stuctural work may also reveal a feature of MRNIP or a binding interface that in turn could be targeted with small molecules to chemo- and radiosensitise cancer cells, improving patient outcomes.

We are still constantly discovering novel roles for well-studied proteins, as well as ascribing functions to hitherto uncharacterised ones. Until we fully appreciate the roles of such factors in genome stability and cancer pathogenesis, our understanding will be incomplete. In turn, aspects of disease will remain unknown, potential personalised medicine strategies will be missed, and the associated economic and health benefits will go unrecognised