FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.

Germline mutations in the mismatch repair ( MM R) genes MSH2 , MSH6 , MLH1 and PMS2 are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1 , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR in vivo : cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to N -methyl- N -nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness

Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.

Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. In some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or “hypermutation.” As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of the SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic E. coli, strain E22. SOS -inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing emergence of resistance to new antimicrobial drugs.

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.

ETNK1 Mutations in Atypical Chronic Myeloid Leukemia Induce a Mutator Phenotype That Can be Reverted with Phosphoethanolamine

ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p<0.0001). Since ROS are responsible for DNA oxidative damage, we firstly generated ChIP-Seq data for ETNK1 mutated cells using an antibody raised against the oxoguanine (oxoG) and we compared oxoG signal against the wild-type cell line, to assess whether ETNK1 mutations could cause accumulation of DNA lesions. This analysis revealed a significant increase in oxoG in mutated cells, compared to WT (p=0.018). Then, we investigated if these lesions were driving the onset of a mutator phenotype by applying the 6-thioguanine (6-TG) resistance assays to our cell models, showing that in the mutated cells there was a 5.4 fold increase in colony number compared to the WT line (p<0.0001). Moreover, we investigated if the ROS-mediated genotoxic insult operating in ETNK1-mutated lines could be also associated with an increase in DNA double-strand breaks. Comparison of ETNK1-N244S and ETNK1-WT lines revealed a sharp increase in the number of γH2AX foci (2.52 fold increase; p=0.0002) in the former. At this point, we hypothesized that the decreased P-Et concentration in ETNK1-mutated cells could be responsible for the increased mitochondrial activity. ETNK1-N244S cells treated with P-Et showed a complete restoration of the normal mitochondrial membrane potential and generation of ROS. Moreover, the mutator phenotype was reverted by P-Et treatment, supporting the hypothesis of a direct involvement of P-Et in the induction of DNA damage. To dissect the mechanism by which P-Et intracellular levels were able to control mitochondria activity, we isolated the mitochondrial oxidative phosphorylation complexes I to IV and we measured the activity of each complex in absence/presence of increasing P-Et concentrations. This approach revealed a profound, dose-dependent decrease in redox activity for mitochondrial complex II (P-Et 10μM: 1.80 fold decrease; p=0.0012; P-Et 20μM: 7.40 fold decrease; p<0.0001; P-Et 50μM: 28.85 fold decrease; p<0.0001) and virtually no effect for the other three complexes, indicating that P-Et controls mitochondria potential through direct inhibition of complex II. To gain insight into the specific mechanism by which P-Et could repress complex II, we analyzed its activity in competition assays in presence of both P-Et and increasing concentration of succinate, the endogenous substrate of succinate dehydrogenase (SDH), showing that succinate supplementation was able to restore the normal SDH activity starting from 50µM. Taken globally, these data suggest that P-Et acts as a competitive inhibitor of succinate for SDH activity. In line with these data, automatic docking of P-Et inside the SDH catalytic domain confirmed that P-Et can occupy the succinate binding site in an energetically favorable conformation, mimicking succinate. In conclusion, the reduced activity of mutant ETNK1 leads to the accumulation of new mutations through the reduced competition of P-Et with succinate, increased mitochondrial activity and ROS production. This mechanism can be blocked, at least in vitro, by P-Et supplementation, suppressing the accumulation of new mutations mediated by the ETNK1-dependent mutator phenotype. In vivo studies will address the therapeutic potential of P-Et. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Spontaneous Polyploids and Antimutators Compete During the Evolution of Saccharomyces cerevisiae Mutator Cells

Mutations affecting DNA polymerase exonuclease domains or mismatch repair (MMR) generate “mutator” phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In Saccharomyces cerevisiae haploid yeast, simultaneous defects in polymerase proofreading and MMR select for “antimutator” mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this “error-induced extinction” and routinely outcompete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting polymerase δ proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests that there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intratumoral genetic diversity for future adaptation.

Recurrent mutations in topoisomerase 2a cause a novel mutator phenotype in human cancers

Topoisomerases are essential for genome stability. Here, we link the p.K743N mutation in topoisomerase TOP2A to a previously undescribed mutator phenotype in human cancers. This phenotype primarily generates a distinctive pattern of duplications of 2 to 4 base pairs and deletions of 6 to 8 base pairs, which we call ID_TOP2A. All tumors carrying the TOP2A p.K743N mutation showed ID_TOP2A, which was absent in all of 12,269 other tumors. We also report evidence of structural variation associated with TOP2A p.K743N. All tumors with ID_TOP2A mutagenesis had several indels in known cancer genes, including frameshift mutations in PTEN and TP53 and an in-frame activating mutation in BRAF. Thus, ID_TOP2A mutagenesis almost certainly contributed to tumorigenesis in these tumors. This is the first report of topoisomerase-associated mutagenesis in human cancers, and sheds further light on TOP2A’s role in genome maintenance. We also postulate that tumors showing ID_TOP2A mutagenesis might be especially sensitive to topoisomerase inhibitors.