Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double-stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of disrupted meiotic cDNA1 (DMC1), a homolog of RAD51, the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. Although targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of CHK1 and expression of p21^CIP1/WAF1, and increased RPA foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices.Glioblastomas (GBMs) rank among the deadliest of all human cancers, with only modest improvement in patient survival over recent decades. More than 12 000 GBM patients are diagnosed annually in the United States.^{1, 2} Despite aggressive treatment consisting of maximal safe surgical resection, concurrent radiotherapy and chemotherapy, and adjuvant chemotherapy, median survival remains dismal at 12–15 months.^{3, 4} Although numerous molecular targets have been identified in GBM, no molecularly targeted therapy has demonstrated a survival benefit. Radiotherapy remains the cornerstone of post-surgical GBM therapy with modest additional benefit offered by concurrent administration of the oral methylator, temozolomide. However, radioresistance and tumor recurrence is universal in GBM.^{4, 5, 6} Radiation also damages non-neoplastic brain tissue, resulting in cognitive impairment and decreased quality-of-life.⁷ Focal high-dose radiation reduces toxicity to non-neoplastic tissue, but tumor invasion into normal brain regions limits the survival benefit of highly focused radiotherapy techniques, like gamma knife and proton beam, establishing a need for improved combinatorial treatments, such as radiosensitizers.^{8, 9} To date, no radiosensitizer has successfully increased survival with acceptable toxicity in a clinical trial. Based on this background, we sought novel molecular targets that mediate responses to genotoxic stress and have limited function in normal cells.During mitosis, cells inspect the integrity of their DNA and repair replication errors through cell-state and error-specific mechanisms.¹⁰ Unrepaired or large regions of DNA damage overwhelm replication mechanisms to induce cell death.^{10, 11} DNA double-strand breaks (DSBs) are detrimental as they cause large-scale chromosomal rearrangements.¹⁰ The homologous recombination (HR) pathway is primarily used to repair DSBs during S- and G₂-phases, providing access to both sister and homologous chromosomes as repair templates.^{7, 12} RADiation sensitive 51 (RAD51) is a key recombinase important in HR and replication fork maintenance, functioning in both mitotic and meiotic cells.^{7, 12, 13, 14, 15} Phosphorylated RAD51 replaces replication protein A (RPA) upon DNA loading.¹⁶ Recombination mediated by RAD51 with the intact DNA template strand results in a relatively error-free repair.¹²In contrast to mitosis, germ cells undergoing meiosis actively generate genetic diversity through induction of programmed DSBs, which are repaired through HR.^{17, 18, 19} In meiotic HR, RAD51 functions in conjunction with the meiosis-specific recombinase, disrupted meiotic cDNA1 (DMC1). RAD51 and DMC1 are loaded onto DNA by a meiosis-specific accessory protein complex, homologous-pairing protein 2 (HOP2)–meiotic nuclear divisions 1 (MND1), to promote homologous strand invasion and dissociation-loop (D-loop) formation.^{20, 21} D-loops formed using the DMC1–RAD51 complex are more resistant to dissociation as opposed to D-loops formed by RAD51 alone, increasing the likelihood of DNA crossover events.²⁰ In addition, DMC1-directed crossovers preferentially utilize the homologous chromosome further increasing genetic variation.²²GBM cells commonly harbor genetic lesions that promote unrestrained proliferation but also stimulate genotoxic stress responses. Neoplastic cells do not require perfect fidelity of repair. In fact, dysfunctional repair accelerates genetic evolution of clones, but cancer cells must acquire mechanisms to bypass cell death or senescence in response to exogenous stressors.^{11, 23} Radiotherapy targets proliferating cancer cells by production of reactive oxygen species, leading to generation of DSBs and activation of the DNA damage response (DDR) pathway.^{11, 24} DSBs generated as a result of ionizing radiation (IR) are repaired through HR or non-homologous end joining (NHEJ).^{7, 12, 25, 26} Terminally differentiated neurons are post-mitotic and rely on NHEJ as a means to repair DNA DSBs. Therefore, inhibition of the NHEJ pathway may result in unfavorable normal neural cell toxicity.²⁶The HR pathway is an attractive target as it is linked to increased genetic variation and loss of heterozygosity (LOH).^{12, 27} Multiple HR checkpoints have been proposed as potential therapeutic targets for GBM.^{28, 29, 30, 31} Although the prognostic value of RAD51 expression in GBM is unresolved,^{29, 32, 33} RAD51 is consistently elevated in GBM compared with normal brain.³³ Reducing RAD51 expression radiosensitizes GBM cells,²⁹ but may have a limited therapeutic index because of the potentially toxic effects on non-neoplastic cells. In this study, we investigated the aberrant activity of meiotic HR regulators in glioma, focusing on the meiosis-specific DMC1. Activation of meiotic repair genes in neoplastic cells selectively provides tumor cells with a repair mechanism to evade cell death caused by DNA damage, yet increase genetic diversity to drive clonal evolution.