Citations

Abstract Purpose: Inhibitors of the Ataxia telangiectasia and Rad3-related (ATR) protein kinase, a critical component of the DNA damage repair response, have synergistic anti-cancer activity with platinum compounds in preclinical models. We therefore conducted a Phase 2 study of the ATR inhibitor berzosertib+carboplatin vs. docetaxel+carboplatin in metastatic castration-resistant prostate cancer. Patients and Methods: Patients previously treated with at least one androgen receptor pathway inhibitor and taxane underwent mandatory biopsy and were randomized 1:1 to receive Arm A (docetaxel 60 mg/m2+carboplatin AUC4 day 1; or carboplatin AUC5 if not a docetaxel candidate) or Arm B (berzosertib 90 mg/m2 days 2,9+carboplatin AUC5 day 1) every 21 days. The primary endpoint was overall response rate (ORR; ≥50% PSA decline or radiographic response). Results: Of 73 randomized patients, 65 received protocol treatment: 34 on Arm A (26 docetaxel+carboplatin; 8 carboplatin alone) and 31 on Arm B. Thirteen patients (38%) in Arm A and 20 (65%) in Arm B had ≥Grade 3 treatment-related adverse events (TrAEs). ORR was 15% in Arm A (5/34; 5/26[19%] with docetaxel+carboplatin) and 0% in Arm B (0/31). At interim analysis after 65 of planned 130 patients were treated, enrollment was halted due to futility. Homologous recombination repair deficiency status from tissue and blood did not correlate with clinical outcomes, but ATM-deficiency or high levels of phoshpo-KAP1 detected in tissue was associated with clinical benefit across both arms of the trial. Conclusions: Berzosertib+carboplatin demonstrated lower ORR and more frequent ≥grade 3 TrAEs compared to docetaxel+carboplatin in this heavily pre-treated, biomarker-unselected population. NCT03517969

Importance Risk-reducing salpingo-oophorectomy (RRSO) is an effective option to prevent ovarian cancer in high-risk individuals, but adverse effects from premature menopause are a barrier to uptake. Interval salpingectomy with delayed oophorectomy (ISDO) may be an effective alternative. Objective To evaluate the association of ISDO vs RRSO and sexual function and menopausal symptoms. Design, Setting, and Participants This prospective, multicenter, nonrandomized (patient preference) clinical trial was performed among Women Choosing Surgical Prevention participants with a pathogenic or likely pathogenic germline variant conferring an increased risk for ovarian cancer. Patients from 9 US centers were enrolled from June 2, 2016, to October 28, 2020; chose ISDO or RRSO; and were followed up for 24 months. The analysis was performed on November 13, 2024. Interventions Participant-selected RRSO or ISDO. Main Outcomes and Measures Changes in sexual function and menopausal symptoms from baseline to 6, 12, and 24 months. Inverse probability weighting was applied to achieve covariate balance across groups. Results The analytic sample included 334 female participants (mean [95% CI] age at consent, 39.2 [38.7-.9.7] years) who underwent RRSO (n = 165) or ISDO (n = 169) and completed 6-month questionnaires. Significantly more participants experienced clinically meaningful worsening of sexual function at 6 months compared with baseline after RRSO vs ISDO (33.1% [39 of 118] vs 16.8% [17 of 101], respectively; odds ratio, 2.00 [95% CI, 1.20-3.35]) and at 12 months (39.2% [38 of 97] vs 18.3% [17 of 93], respectively; odds ratio, 1.69 [95% CI, 1.09-2.63]). This finding was no longer significant at 24 months. When restricted to the RRSO group who took hormone replacement therapy, there was no significant worsening of total sexual function scores or menopausal symptoms compared with baseline. No interval ovarian cancers were reported. Conclusions and Relevance This nonrandomized clinical trial comparing RRSO with ISDO found that ISDO was less likely to impair sexual function, which addresses an important barrier to surgical prevention in high-risk patients in premenopause. After RRSO, use of hormone replacement therapy mitigated menopausal symptoms and prevented decline in sexual function. More information is needed about the safety of ISDO. Trial Registration ClinicalTrials.gov Identifier: NCT02760849

Integration of the high-risk human papillomavirus 16 (HPV16) genome into the host chromosome, frequently driven by microhomology-mediated end joining (MMEJ), is a critical step in the carcinogenesis of HPV-associated tumors. However, the mechanisms by which viral oncoproteins manipulate the error-prone MMEJ pathway remain poorly defined. Here, we demonstrate that the HPV16 E6 oncoprotein upregulates MMEJ to facilitate viral genome integration. This heightened MMEJ activity is driven by a marked increase in the protein levels of DNA polymerase theta (PolΘ), a central enzyme of the MMEJ pathway. Mechanistically, we show that the elevation of PolΘ levels in response to HPV16 E6 expression is dependent on the host E3 ubiquitin ligase UBE3A/E6AP but is independent of p53 degradation. E6 redirects UBE3A to enhance the ubiquitination and degradation of RAD23A, a shuttle protein required for delivering polyubiquitinated PolΘ to the proteasome. Consequently, the loss of functional RAD23A phenocopies the effect of HPV16 E6, leading to PolΘ protein stabilization and increased MMEJ activity. By elucidating the E6-UBE3A-RAD23A-PolΘ axis, our findings reveal a mechanism through which HPV manipulates the host DNA repair machinery to promote its integration and oncogenic potential.

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1 ) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta ( POLQ ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1–induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1–overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

SUMMARY Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLΘ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLΘ expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POL0 mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS. Highlights - Ewing sarcoma tumors have a deficiency in POLθ expression and a corresponding loss of MMEJ activity - EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA. - The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POLθ expression - The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Fanconi Anemia, Homologous Recombination or Non-Homologous End-Joining - Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers

Oncoprotein-driven human cancers exhibit replication stress (RS), leading to an increase in DNA double-strand breaks (DSB). These DSBs are repaired by one of the three main pathways - homologous recombination repair (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ). Cyclin D1 (CCND1) is one of the most overexpressed oncoproteins and is universally overexpressed in mantle cell lymphoma (MCL). While CCND1 overexpression drives cells into the S-phase, its specific effects on RS and DSB repair pathways are poorly understood. Here, we investigated the impact of CCND1 overexpression on RS and DSB repair mechanisms in MCL. First, the expression level of CCND1 positively correlated with heightened RS, as indicated by elevated p-RPA-S33 and p-RPA S8 levels, and increased DNA damage, assessed by g-H2AX, especially in the mitotic phase, across diverse cell models, including MCL. Since CCND1-overexpressed cells proliferate rapidly, we hypothesized that some DNA repair pathways might be upregulated to meet the demand for repairing the excess RS-driven DSBs. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon CCND1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Furthermore, CRISPR interference (CRISPRi)-mediated knockdown of CCND1 in MCL cells led to a significant reduction in MMEJ activity. This finding suggests the specificity of CCND1 in mediating the upregulation of the MMEJ pathway. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLQ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression, suggesting the crucial role of MMEJ in DSB repair processes under CCND1-overexpressed conditions. Sincethe MMEJ pathwayplays a pivotal role in repairing DSB during mitosis, we assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition (ART558 and novobiocin) of POLQ in MCL cells led to increased RS and increased unrepaired DNA damage during mitosis, ultimately leading to chromosomal instability, mitotic catastrophe, and apoptosis. Additionally, the deficiency of POLQ in MCL cells resulted in increased levels of single-strand DNA, another critical biomarker for RS. Second, we found that ATM deficiency, the second most common genetic alteration after CCND1 overexpression in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient CCND1-overexpressed cells. When POLQ was inhibited, a significant antitumor effect was evident in MCL cells, including ibrutinib-resistant MCL, both in vitro and in vivo, and this effect was enhanced by ATM deficiency. Further, simultaneous inhibition of both ATM (AZD0156) and POLQ was synthetic lethal in MCL cell lines and primary tumor cells from patients with MCL. Mechanistically, we observed the direct binding of CCND1 to the POLQ promoter and a corresponding upregulation of POLQ mRNA in CCND1-overexpressing cells. Consistently, a significant overexpression of POLQ mRNA was observed in primary MCL cells, compared to other non-Hodgkin lymphoma cells without CCND1 overexpression. Moreover, the positive correlation between POLQ and CCND1 expression was further validated using syngeneic B-lymphoma cells with and without CCND1 overexpression. Importantly, CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. In addition, ATM deficiency showed a further increase of endogenous POLQ expression and foci formation in cells with CCND1 overexpression, suggesting ATM negatively regulates the MMEJ pathway, especially in the CCND1 overexpressed background. In summary, CCND1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. Within MCL, POLQ is crucial in mitigating the adversities of CCND1-driven RS. Our data suggest that targeting the MMEJ pathway via POLQ inhibition, particularly in the presence of CCND1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in oncoprotein-driven hematologic malignancies such as MCL.

PURPOSE The multicenter, open-label, randomized phase 2 NCI-9944 study ( NCT02595892 ) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.

Abstract DNA damage response (DDR) mechanisms are critical to maintenance of overall genomic stability, and their dysfunction can contribute to oncogenesis. Significant advances in our understanding of DDR pathways have raised the possibility of developing therapies that exploit these processes. In this expert-driven consensus review, we examine mechanisms of response to DNA damage, progress in development of DDR inhibitors in IDH-wild-type glioblastoma and IDH-mutant gliomas, and other important considerations such as biomarker development, preclinical models, combination therapies, mechanisms of resistance and clinical trial design considerations.

Importance Requiring personalized genetic counseling may introduce barriers to cancer risk assessment, but it is unknown whether omitting counseling could increase distress. Objective To assess whether omitting pretest and/or posttest genetic counseling would increase distress during remote testing. Design, Setting, and Participants Making Genetic Testing Accessible (MAGENTA) was a 4-arm, randomized noninferiority trial testing the effects of individualized pretest and/or posttest genetic counseling on participant distress 3 and 12 months posttest. Participants were recruited via social and traditional media, and enrollment occurred between April 27, 2017, and September 29, 2020. Participants were women aged 30 years or older, English-speaking, US residents, and had access to the internet and a health care professional. Previous cancer genetic testing or counseling was exclusionary. In the family history cohort, participants had a personal or family history of breast or ovarian cancer. In the familial pathogenic variant (PV) cohort, participants reported 1 biological relative with a PV in an actionable cancer susceptibility gene. Data analysis was performed between December 13, 2020, and May 31, 2023. Intervention Participants completed baseline questionnaires, watched an educational video, and were randomized to 1 of 4 arms: the control arm with pretest and/or posttest genetic counseling, or 1 of 3 study arms without pretest and posttest counseling. Genetic counseling was provided by phone appointments and testing was done using home-delivered saliva kits. Main Outcomes and Measures The primary outcome was participant distress measured by the Impact of Event Scale 3 months after receiving the results. Secondary outcomes included completion of testing, anxiety, depression, and decisional regret. Results A total of 3839 women (median age, 44 years [range 22-91 years]), most of whom were non-Hispanic White and college educated, were randomized, 3125 in the family history and 714 in the familial PV cohorts. In the primary analysis in the family history cohort, all experimental arms were noninferior for distress at 3 months. There were no statistically significant differences in anxiety, depression, or decisional regret at 3 months. The highest completion rates were seen in the 2 arms without pretest counseling. Conclusions and Relevance In the MAGENTA clinical trial, omitting individualized pretest counseling for all participants and posttest counseling for those without PV during remote genetic testing was not inferior with regard to posttest distress, providing an alternative care model for genetic risk assessment. Trial Registration ClinicalTrials.gov Identifier: NCT02993068