Hormonal Contraceptive Use and Risk of Depression Among Young Women With Attention-Deficit/Hyperactivity Disorder.

OBJECTIVE: Women with attention-deficit/hyperactivity disorder (ADHD) have an increased risk of becoming teenage mothers. Adverse effects of hormonal contraception (HC), including depression, may affect adherence to user-dependent contraception and increase the risk for unplanned pregnancies and teenage births in women with ADHD. The current study analyzed whether girls and young women with ADHD are at increased risk for depression during HC use compared with women without ADHD. METHOD: A linkage of Swedish national registers covering 29,767 girls and young women with ADHD aged 15 to 24 years and 763,146 without ADHD provided measures of ADHD and depression diagnoses (International Classification of Diseases [ICD] code) and prescription of stimulant medication, HC, and antidepressant medication (Anatomical Therapeutic Chemical [ATC] code). Cox regression models applying an interaction term (ADHD diagnosis × HC use) evaluated the excess risk of HC-induced depression in women with ADHD. RESULTS: Women with ADHD had a 3-fold higher risk of developing depression, irrespective of HC use (adjusted hazard ratio [aHR] = 3.69, 95% CI = 3.60-3.78). Oral combined HC users with ADHD had a 5 times higher risk of depression compared with women without ADHD who were not using oral combined HC (aHR = 5.19, 95% CI = 4.94-5.47), and a 6 times higher risk in comparison with women without ADHD who were on oral combined HC (aHR = 6.10 (95% CI = 5.79-6.43). The corresponding risk of depression in women with ADHD who used a progestogen-only pill (aHR = 5.00, 95% CI = 4.56-5.49). The risk of developing depression when using non-oral HC was similarly moderately increased in both groups. CONCLUSION: Girls and young women with ADHD have an increased risk of developing depression when using oral HC compared with their unaffected peers. Information on risks with HCs as well as potential benefits with long-acting reversible contraceptives needs to be an integrated part of the shared decision making and contraception counseling for young women with ADHD.

Towards individualised contraceptive counselling: clinical and reproductive factors associated with self-reported hormonal contraceptive-induced adverse mood symptoms.

OBJECTIVE: The study aim was to establish which demographic, clinical, reproductive and psychiatric factors are associated with self-reported hormonal contraceptive (HC)-induced adverse mood symptoms. STUDY DESIGN: We compiled baseline data from two Swedish studies: one cross-sectional study on combined oral contraceptive (COC)-induced adverse mood symptoms (n=118) and one randomised controlled trial on adverse mood symptoms on COC (n=184). Both included women eligible for COC use, aged over 18 years. All women answered a questionnaire on HC use and associated mood problems. The Mini-International Neuropsychiatric Interview (M.I.N.I.) was used to capture mood and anxiety disorders. Women who acknowledged HC-induced adverse mood symptoms, ongoing or previously (n=145), were compared with women without any such experience (n=157). RESULTS: Compared with women without self-reported HC-induced adverse mood symptoms, women with these symptoms were younger at HC start (adjusted odds ratio (aOR) 0.83, 95% CI 0.72 to 0.95), had more often undergone induced abortion (OR 3.36, 95% CI 1.57 to 7.23), more often suffered from an ongoing minor depressive disorder (n=12 vs n=0) and had more often experienced any previous mental health problem (aOR 1.90, 95% CI 1.01 to 3.59). CONCLUSIONS: In line with previous research, this study suggests that women with previous or ongoing mental health problems and women who are younger at HC start are more likely to experience HC-induced adverse mood symptoms. Former and current mental health should be addressed at contraceptive counselling, and ongoing mental health disorders should be adequately treated. IMPLICATIONS: This study adds valuable knowledge for identification of women susceptible to HC-induced adverse mood symptoms. It should facilitate the assessment of whether or not a woman has an increased risk of such symptoms, and thus enable clinicians to adopt a more personalised approach to contraceptive counselling.

Use of an estradiol-based combined oral contraceptives has no influence on attentional bias or depressive symptoms in healthy women.

Combined oral contraceptive (COC) use is associated with small, albeit significant, increases in mental symptom scores, predominantly irritability, depressed mood, and anxiety. Yet, randomized prospective trials are needed to better characterize the women at risk for COC-induced negative mood change. Thus, the primary aim of this sub-study to a placebo-controlled randomized trial was to determine whether COC use influences emotional interference by negative and positive stimuli. Secondly, we wanted to evaluate what factors would predict depressive symptoms at the end of the trial, taking personality factors, history of mental disorders and other demographic factors into account. Sixty-nine women were included, randomized to three cycles of treatment with a COC (1.5 mg estradiol and 2.5 mg nomegestrolacetate) or placebo. An emotional verbal Stroop task was used to measure interference of emotional stimuli, in which participants were asked to only name the color of a presented word, while ignoring the meaning of the word. Four different word categories were used; neutral, positive, depression, and anxiety. For the second aim of the study, rating on the Montgomery-Åsberg Depression Rating Scale during the final days of the trial was used as outcome. We found no interaction between emotional verbal Stroop word category and treatment, indicating that COC treatment did not evoke any differences in emotional interference to the three word categories. Significant predictors for depressive symptoms at the end of the trial were trait anxiety at baseline and prior adverse mood effects by hormonal contraceptive use. Treatment (i.e. whether women had been treated with the COC or placebo) did not play a role in predicting depression scores at the end of the trial. In conclusion, we found no evidence that combined oral contraceptive use is associated with impaired cognitive-emotional processing. Instead, the main predictors of self-rated depression at the end of the trial were baseline trait anxiety and previous mental symptoms during hormonal contraceptive use.

Association of Attention-Deficit/Hyperactivity Disorder With Teenage Birth Among Women and Girls in Sweden.

Importance: Attention-deficit/hyperactivity disorder (ADHD) is associated with a plethora of adverse health outcomes throughout life. While Swedish specialized youth clinics have carefully and successfully targeted risk of unplanned pregnancies in adolescents, important risk groups, such as women and girls with ADHD, might not be identified or appropriately assisted by these interventions. Objectives: To determine whether women and girls with ADHD are associated with increased risk of teenage birth compared with their unaffected peers and to examine the association of ADHD with risk factors for adverse obstetric and perinatal outcomes, such as smoking, underweight or overweight, and substance use disorder. Design, Setting, and Participants: This nationwide cohort study included data from 6 national longitudinal population-based registries in Sweden. All nulliparous women and girls who gave birth in Sweden between January 1, 2007, and December 31, 2014, were included. Data analyses were conducted from October 7, 2018, to February 8, 2019. Exposures: Women and girls treated with stimulant or nonstimulant medication for ADHD (Anatomic Therapeutic Chemical classification code N06BA) in the Swedish Prescribed Drug Register between July 1, 2005, and December 31, 2014. Main Outcomes and Measures: Maternal age at birth. Secondary outcome measures were body mass index, smoking habits, and psychiatric comorbidities. Results: Among 384 103 nulliparous women and girls aged 12 to 50 years who gave birth between 2007 and 2014 included in the study, 6410 (1.7%) (mean [SD] age, 25.0 [5.5] years) were identified as having ADHD. The remaining 377 693 women and girls without ADHD (mean [SD] age, 28.5 [5.1] years) served as the control group. Teenage deliveries were more common among women and girls with ADHD than among women and girls without ADHD (15.3% vs 2.8%; odds ratio [OR], 6.23 [95% CI, 5.80-6.68]). Compared with women and girls without ADHD, those with ADHD were more likely to present with risk factors for adverse obstetric and perinatal outcomes, including smoking during the third trimester (OR, 6.88 [95% CI, 6.45-7.34]), body mass index less than 18.50 (OR, 1.29 [95% CI, 1.12-1.49]), body mass index more than 40.00 (OR, 2.01 [95% CI, 1.60-2.52]), and alcohol and substance use disorder (OR, 20.25 [95% CI, 18.74-21.88]). Conclusions and Relevance: This study found that women and girls with ADHD were associated with an increased risk of giving birth as teenagers compared with their unaffected peers. The results suggest that standard of care for women and girls with ADHD should include active efforts to prevent teenage pregnancies.

Sexual function and combined oral contraceptives - a randomised, placebo-controlled trial.

OBJECTIVE: The effect of combined oral contraceptives (COC) on female sexuality has long been a matter of discussion, but placebo-controlled studies are lacking. Thus, the aim of the present study was to investigate if an estradiol-containing COC influences sexual function. DESIGN: Investigator-initiated, randomised, double-blinded, placebo-controlled clinical trial where 202 healthy women were randomized to a combined oral contraceptive (1.5 mg estradiol and 2.5 mg nomegestrol acetate) or placebo for three treatment cycles. METHODS: Sexual function at baseline and during the last week of the final treatment cycle was evaluated by the McCoy Female Sexuality Questionnaire. Serum and hair testosterone levels were assessed at the same time points. RESULTS: Compared to placebo, COC use was associated with a small decrease in sexual interest (COC median change score: -2.0; interquartile range (IQR): -5.0-0.5 vs. placebo: -1.0; IQR: -3.0-2.0, p = 0.019), which remained following adjustment for change in self-rated depressive symptoms B = -0.80 ± 0.30, Wald = 7.08, p = 0.008. However, the proportion of women who reported a clinically relevant deterioration in sexual interest did not differ between COC or placebo users (COC 18 (22.2%) vs. placebo 16 (17.8%), p = 0.47). Change in other measured aspects of sexual function as well as total score of sexual function did not differ between the two treatments. CONCLUSIONS: This study suggests that use of estradiol-based combined oral contraceptives is associated with reduced sexual interest. However, the changes are minute, and probably not of clinical relevance.

Ongoing or previous mental disorders predispose to adverse mood reporting during combined oral contraceptive use.

PURPOSE: Previous studies have emphasised that women with pre-existing mood disorders are more inclined to discontinue hormonal contraceptive use. However, few studies have examined the effects of combined oral contraceptives (COC) on mood in women with previous or ongoing mental disorders. MATERIALS AND METHODS: This is a supplementary analysis of an investigator-initiated, double-blinded, randomised clinical trial during which 202 women were treated with either a COC (1.5 mg estradiol and 2.5 mg nomegestrolacetate) or placebo during three treatment cycles. The Mini International Neuropsychiatric Interview was used to collect information on previous or ongoing mental disorders. The primary outcome measure was the total change score in five mood symptoms on the Daily Record of Severity of Problems (DRSP) scale in the intermenstrual phase of the treatment cycle. RESULTS: Women with ongoing or previous mood, anxiety or eating disorders allocated to COC had higher total DRSP Δ-scores during the intermenstrual phase of the treatment cycle in comparison with corresponding women randomised to placebo, mean difference 1.3 (95% CI 0.3-2.3). In contrast, among women without mental health problems, no difference in total DRSP Δ-scores between COC- and placebo users was noted. Women with a risk use of alcohol who were randomised to the COC had higher total DRSP Δ-scores than women randomised to placebo, mean difference 2.1 (CI 95% 1.0-3.2). CONCLUSIONS: Women with ongoing or previous mental disorders or risk use of alcohol have greater risk of COC-induced mood symptoms. This may be worth noting during family planning and contraceptive counselling.

Combined oral contraceptive use is associated with both improvement and worsening of mood in the different phases of the treatment cycle-A double-blind, placebo-controlled randomized trial.

OBJECTIVE: Ever since the introduction of combined oral contraception (COC), one of the major reasons for discontinuing the pill use has been mood-related side effects. Moreover, women who discontinue the pill turn to less effective methods whereby the probability of an unintended conception increases. Approximately 4-10% of COC users complain of depressed mood, irritability or increased anxiety, but drug-related causality has been difficult to prove. Given the lack of randomized controlled trials in this area, we aimed to prospectively estimate the severity of adverse mood in COC users that would be as representative of general users as possible. METHODS: This investigator-initiated, multi-center, randomized, double-blinded, placebo-controlled study included 202 healthy women. Women were randomized to a COC (1.5mg estradiol and 2.5mg nomegestrolacetate) or placebo for three treatment cycles. Main outcome measure was the Daily Record of Severity of Problems (DRSP), which was filled out daily during one baseline cycle and the final treatment cycle. RESULTS: Results from 84 women in the COC group and 94 women in the placebo group were analysed. COC use was associated with small, but statistically significant, increases in mean anxiety (0.22; 95% CI: 0.07-0.37, p=0.003), irritability (0.23; 95% CI: 0.07-0.38, p=0.012), and mood swings scores (0.15; 95% CI: 0.00-0.31, p=0.047) during the intermenstrual phase, but a significant premenstrual improvement in depression (-0.33; 95% CI: -0.62 to -0.05, p=0.049). Secondary analyses showed that women with previous adverse hormonal contraceptive experience reported significantly greater mood worsening in the intermenstrual phase in comparison with healthy women, p<0.05. The proportion of women who reported a clinically relevant mood deterioration did not differ between those allocated to COC (24.1%) or placebo (17.0%), p=0.262. CONCLUSION: COC use is associated with small but statistically significant mood side effects in the intermenstrual phase. These findings are driven by a subgroup of women who clearly suffer from COC-related side effects. However, positive mood effects are noted in the premenstrual phase and the proportion of women with clinically relevant mood worsening did not differ between treatment groups.

A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair.

To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation.

MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Exposure to polycyclic aromatic hydrocarbons in women from Poland, Serbia and Italy--relation between PAH metabolite excretion, DNA damage, diet and genotype (the EU DIEPHY project).

Exposure of the general population to polycyclic aromatic hydrocarbons (PAH) is ubiquitous. The aim of this study was to analyze biomarkers associated with the uptake of PAH in 428 non-smoking women from Lodz (Poland), Viterbo (Italy), Belgrade (Serbia) and from the Pancevo area, where the petrochemical complex was destroyed by the air raids in 1999. Urinary excretion of PAH metabolites was lowest in Italian women, intermediary for Serbian and highest in Polish women, who predominantly excreted hydroxy phenanthrenes as metabolites of phenanthrene. Bulky DNA adduct levels were highest in Italian and Polish women. Genotype or PAH ambient air levels could not explain the dissimilarities between the study groups with respect to biomarker patterns, which probably reflected differences in life style-associated factors.

Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates γ-histone 2AX (γ-H2AX) levels upon ionizing radiation.

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

PTEN deletion in prostate cancer cells does not associate with loss of RAD51 function: implications for radiotherapy and chemotherapy.

PURPOSE: PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. EXPERIMENTAL DESIGN: Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN(+/+), DU145-PTEN(+/-), PC3-PTEN(-/-)) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. RESULTS: PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H(2)O(2), and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. CONCLUSIONS: These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials.

DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells.

Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.

Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice.

The protective action in C57BL/6J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen®) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.

Identification of the MMS22L-TONSL complex that promotes homologous recombination.

Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.

6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance.

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2.

DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

Homologous recombination repair is essential for repair of vosaroxin-induced DNA double-strand breaks.

Vosaroxin (formerly voreloxin) is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective double-strand breaks (DSB), G2 arrest and apoptosis. Objective responses and complete remissions were observed in phase 2 studies of vosaroxin in patients with solid and hematologic malignancies, and responses were seen in patients whose cancers were resistant to anthracyclines. The quinolone-based scaffold differentiates vosaroxin from the anthracyclines and anthracenediones, broadly used DNA intercalating topoisomerase II poisons. Here we report that vosaroxin induces a cell cycle specific pattern of DNA damage and repair that is distinct from the anthracycline, doxorubicin. Both drugs stall replication and preferentially induce DNA damage in replicating cells, with damage in G2 / M > S >> G1. However, detectable replication fork collapse, as evidenced by DNA fragmentation and long tract recombination during S phase, is induced only by doxorubicin. Furthermore, vosaroxin induces less overall DNA fragmentation. Homologous recombination repair (HRR) is critical for recovery from DNA damage induced by both agents, identifying the potential to clinically exploit synthetic lethality.

The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links.

Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.