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Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability. When a cell divides to make more cells, it duplicates its DNA to pass on an identical set of genes to the new cell. Copying DNA – also known as DNA replication – is a complex process that involves several steps. First, the double helix gradually unwinds and unzips to separate the DNA strands. This creates a molecule known as the ‘replication fork’. Then, copies of each strand are created and proofread for errors. Eventually, the strands are sealed back together so that the helices contain one old and one new part. But sometimes errors sneak in during DNA replication, which can lead to mutations that may cause cancer. The higher the number of mutations, the bigger the chance is that the cancer becomes aggressive and resistant to therapy. Some of the most common mutations found in tumors happen in a protein called p53. This protein is known to stop tumors from growing by selectively killing cells with mutations. When p53 is faulty, mutant cells no longer die and can grow uncontrollably to form tumors. However, its killing abilities do not fully explain how p53 protects cells from accumulating mutations that can cause cancer, and until now, it was not known if p53 also had any other roles. Now, Schlacher et al. discovered that p53 can protect the DNA from mutations. The experiments used normal cells and cancer cells from humans and mice, in which p53 was either blocked or modified. The experiments revealed that p53 plays an important role during DNA replication. When p53 is ‘healthy’, it binds to the replication fork. This ensures that replication restarts properly after it has passed faulty patches of the DNA. The p53 protein also helps to organize the proteins involved in DNA replication. When p53 was absent or mutated, the DNA-repair protein that usually binds to the fork failed to attach properly. Instead, other proteins prone to make mutations took over the replication fork and created a pattern of mutations commonly found in tumors resistant to treatment. A next step will be to investigate p53’s role at damaged DNA replication forks and how it interacts with other proteins involved in DNA replication. To fully understand all roles that p53 plays in preventing tumor growth can help to find new ways to treat tumors with p53 defects or tumors that have become resistant to treatment.

Apoptosis, autophagy, necrosis and cellular senescence are key responses of cells that were exposed to genotoxicants. The types of DNA damage triggering these responses and their interrelationship are largely unknown. Here we studied these responses in glioma cells treated with the methylating agent temozolomide (TMZ), which is a first-line chemotherapeutic for this malignancy. We show that upon TMZ treatment cells undergo autophagy, senescence and apoptosis in a specific time-dependent manner. Necrosis was only marginally induced. All these effects were completely abrogated in isogenic glioma cells expressing O(6)-methylguanine-DNA methyltransferase (MGMT), indicating that a single type of DNA lesion, O(6)-methylguanine (O(6)MeG), is able to trigger all these responses. Studies with mismatch repair mutants and MSH6, Rad51 and ATM knockdowns revealed that autophagy induced by O(6)MeG requires mismatch repair and ATM, and is counteracted by homologous recombination. We further show that autophagy, which precedes apoptosis, is a survival mechanism as its inhibition greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses (<100 µM). Cellular senescence increases with post-exposure time and, similar to autophagy, precedes apoptosis. If autophagy was abrogated, TMZ-induced senescence was reduced. Therefore, we propose that autophagy triggered by O(6)MeG adducts is a survival mechanism that stimulates cells to undergo senescence rather than apoptosis. Overall, the data revealed that a specific DNA adduct, O(6)MeG, has the capability of triggering autophagy, senescence and apoptosis and that the decision between survival and death is determined by the balance of players involved. The data also suggests that inhibition of autophagy may ameliorate the therapeutic outcome of TMZ-based cancer therapy.

RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5’ RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations. In this study, the authors present structures and functional analyses for the RAD51C-XRCC3 tumor suppressor complex, providing insights into recurrent mutations in cancer and Fanconi Anemia patients that uncover distinct DNA replication fork protection, restart and reversal regions.

The prototypic cancer-predisposition disease Fanconi Anemia (FA) is identified by biallelic mutations in any one of twenty-three FANC genes. Puzzlingly, inactivation of one Fanc gene alone in mice fails to faithfully model the pleiotropic human disease without additional external stress. Here we find that FA patients frequently display FANC co-mutations. Combining exemplary homozygous hypomorphic Brca2/Fancd1 and Rad51c/Fanco mutations in mice phenocopies human FA with bone marrow failure, rapid death by cancer, cellular cancer-drug hypersensitivity and severe replication instability. These grave phenotypes contrast the unremarkable phenotypes seen in mice with single gene-function inactivation, revealing an unexpected synergism between Fanc mutations. Beyond FA, breast cancer-genome analysis confirms that polygenic FANC tumor-mutations correlate with lower survival, expanding our understanding of FANC genes beyond an epistatic FA-pathway. Collectively, the data establish a polygenic replication stress concept as a testable principle, whereby co-occurrence of a distinct second gene mutation amplifies and drives endogenous replication stress, genome instability and disease. Tomaszowski et al show that co-mutations in Brca2 and Rad51c synergistically drive cancer and developmental disease, which was unexpected given their epistatic DNA repair roles, and expands our understanding of their tumor suppressive functions.

The DNA repair protein O 6 -methylguanine-DNA-methyltransferase (MGMT) is a key determinant of cancer resistance. The MGMT inhibitors O 6 -benzylguanine (O 6 BG) and O 6 -(4-bromothenyl)guanine (O 6 BTG) failed to enhance the therapeutic response due to toxic side effects when applied in combination with alkylating chemotherapeutics, indicating a need of inhibitor targeting. We assessed MGMT targeting that relies on conjugating the inhibitors O 6 BG and O 6 BTG to ß-D-glucose, resulting in O 6 BG-Glu and O 6 BTG-Glu, respectively. This targeting strategy was selected by taking advantage of high demand of glucose in cancers. Contrary to our expectation, the uptake of O 6 BG-Glu and O 6 BTG-Glu was not dependent on glucose transporters. Instead, it seems that after membrane binding the conjugates are taken up via flippases, which normally transport phospholipids. This membrane binding is the consequence of the amphiphilic character of the conjugates, which at higher concentrations lead to the formation of micelle-like particles in aqueous solution. The unusual uptake mechanism of the conjugates highlights the importance of proper linker selection for a successful ligand-based drug delivery strategy. We also demonstrate that proteins of the P4-Type ATPase family are involved in the transport of the glucose conjugates. The findings are not only important for MGMT inhibitor targeting, but also for other amphiphilic drugs.

In order to prioritize available immune therapeutics, immune profiling across glioma grades was conducted, followed by preclinical determinations of therapeutic effect in immune-competent mice harboring gliomas. T cells and myeloid cells were isolated from the blood of healthy donors and the blood and tumors from patients with glioma and profiled for the expression of immunomodulatory targets with an available therapeutic. Murine glioma models were used to assess therapeutic efficacy of agents targeting the most frequently expressed immune targets. In patients with glioma, the A2aR/CD73/CD39 pathway was most frequently expressed, followed by the PD-1 pathway. CD73 expression was upregulated on immune cells by 2-hydroxyglutarate in IDH1 mutant glioma patients. In murine glioma models, adenosine receptor inhibitors demonstrated a modest therapeutic response; however, the addition of other inhibitors of the adenosine pathway did not further enhance this therapeutic effect. Although adenosine receptor inhibitors could recover immunological effector functions in T cells, immune recovery was impaired in the presence of gliomas, indicating that irreversible immune exhaustion limits the effectiveness of adenosine pathway inhibitors in patients with glioma. This study illustrates vetting steps that should be considered before clinical trial implementation for immunotherapy-resistant cancers, including testing an agent's ability to restore immunological function in the context of intended use.

Abstract Fanconi Anemia (FA) is a prototypic genetic disease signified by heterogeneous phenotypes including cancer, bone marrow failure, short stature, congenital abnormalities, infertility, sub-mendelian birth rate, genome instability and high cellular sensitivity to cancer therapeutics1-4. Clinical diagnosis is confirmed by identifying biallelic, homo- or hemizygous mutations in any one of twenty-three FANC genes1,5. Puzzlingly, inactivation of one single Fanc gene in mice fails to faithfully model the human disease manifestations6-8. We here delineate a preclinical Fanc mouse model with mutations in two genes, Fancd1/Brca2 and Fanco/Rad51c, that recapitulates the severity and heterogeneity of the human disease manifestations including death by cancer at young age. Surprisingly, these grave phenotypes cannot be explained by the sum of phenotypes seen in mice with single gene inactivation, which are unremarkable. In contrast to expectations from classic epistasis analysis of genetic pathways, the data instead reveal an unexpected functional synergism of polygenic Fanc mutations. Importantly in humans, whole exome sequencing uncovers that FANC co-mutation in addition to the identified inactivating FANC gene mutation is a frequent event in FA patients. Collectively, the data establish a concept of polygenic stress as an important contributor to disease manifestations, with implications for molecular diagnostics. Competing Interest Statement The authors have declared no competing interest.

Anemia of inflammation (AI) is a common comorbidity associated with obesity, diabetes, cardiac disease, aging, and during anti-cancer therapies. Mounting evidence illustrates that males are disproportionally affected by AI, but not why. Here we demonstrate a molecular cause for a sex-bias in inflammation. The data shows that mitochondrial DNA (mtDNA) instability induced by dietary stress causes anemia associated with inflamed macrophages and improper iron recycling in mice. These phenotypes are enhanced in mice with mutations in , which predisposes to the progeroid disease Fanconi Anemia. The data reveals a striking sex-bias whereby females are protected. We find that estrogen acts as a mitochondrial antioxidant that reduces diet-induced oxidative stress, mtDNA replication instability and the distinctively mtDNA-dependent unphosphorylated STAT1 response. Consequently, treatment of male mutant mice with estrogen or mitochondrial antioxidants suppresses the inflammation-induced anemia. Collectively, this study uncovers estrogen-responsive mtDNA replication instability as a cause for sex-specific inflammatory responses and molecular driver for AI.

Classically, p53 tumor-suppressor acts in transcription, apoptosis, and cell-cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks driving genomic instability independent of transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POL pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53 suppression of replication genome instability.