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In a trial of patients with high grade serous ovarian cancer (HGSOC), addition of the ATR inhibitor berzosertib to gemcitabine improved progression free survival (PFS) compared to gemcitabine alone but biomarkers predictive of treatment are lacking. Here we report a candidate biomarker of response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in HGSOC ovarian cancer. Patients with replication stress (RS)-high tumors (n = 27), defined as harboring at least one genomic RS alteration related to loss of RB pathway regulation and/or oncogene-induced replication stress achieve significantly prolonged PFS (HR = 0.38, 90% CI, 0.17–0.86) on gemcitabine monotherapy compared to those with tumors without such alterations (defined as RS-low, n = 30). However, addition of berzosertib to gemcitabine benefits only patients with RS-low tumors (gemcitabine/berzosertib HR 0.34, 90% CI, 0.13–0.86) and not patients with RS-high tumors (HR 1.11, 90% CI, 0.47–2.62). Our findings support the notion that the exacerbation of RS by gemcitabine monotherapy is adequate for lethality in RS-high tumors. Conversely, for RS-low tumors addition of berzosertib-mediated ATR inhibition to gemcitabine is necessary for lethality to occur. Independent prospective validation of this biomarker is required. A randomized phase 2 study recently showed that the addition of ATR inhibitor berzosertib to gemcitabine improved PFS compared to gemcitabine alone in patients with ovarian cancer. In this preplanned exploratory study, the authors demonstrate that a genomic biomarker of replication-stress is associated with outcome to gemcitabine alone and may predict which patients benefit from addition of the ATR inhibitor berzosertib.

Metastatic castration-resistant prostate cancer remains incurable and is particularly aggressive in patients with alterations in DNA damage repair genes involved directly or indirectly in homologous recombination repair (HRR). In the primary analysis of TALAPRO-2, talazoparib plus enzalutamide significantly improved radiographic progression-free survival (rPFS) versus enzalutamide plus placebo in patients with metastatic castration-resistant prostate cancer harbouring HRR gene alterations. At primary analysis, overall survival was immature. Here we report final prespecified overall survival analysis, updated rPFS, safety, and patient-reported outcomes in the HRR-deficient cohort of TALAPRO-2. TALAPRO-2 is an ongoing international, randomised, double-blind, placebo-controlled phase 3 trial. The HRR-deficient cohort included randomly assigned patients from 142 hospitals, cancer centres, and medical centres in 26 countries; the study included men aged at least 18 years (≥20 years in Japan) with asymptomatic or mildly symptomatic metastatic castration-resistant prostate cancer, progressive disease at study entry, and no previous life-prolonging systemic therapy for castration-resistant prostate cancer, but were receiving ongoing androgen deprivation therapy. Patients were prospectively assessed for tumour HRR gene alterations and randomly assigned (1:1) to once-daily oral talazoparib 0·5 mg plus enzalutamide 160 mg or enzalutamide plus placebo stratified by prior treatment (yes vs no) for castration-sensitive disease. The sponsor, patients, and investigators were masked to talazoparib or placebo, whereas enzalutamide was open label. The primary endpoint was rPFS (time from randomisation to radiographic progression or death, whichever occurred first) by blinded independent central review, and overall survival (time from randomisation to death due to any cause) was a key alpha-protected secondary endpoint, both assessed in the intention-to-treat population. Follow-up for overall survival was intended to continue until the planned final analysis. For statistical significance at the final overall survival analysis, the two-sided p value from the stratified log-rank test needed to be 0·024 or less based on a group sequential design with O'Brien–Fleming spending function. Safety was assessed in patients who had received at least one study drug dose. The trial is registered with ClinicalTrials.gov, NCT03395197. Between Dec 18, 2018, and Jan 20, 2022, 399 patients with HRR-deficient metastatic castration-resistant prostate cancer were randomly assigned (200 [50%] to talazoparib plus enzalutamide and 199 [50%] to enzalutamide plus placebo). At a median follow-up of 44·2 months (IQR 36·0–50·8), treatment with talazoparib plus enzalutamide resulted in a statistically significant improvement in overall survival versus enzalutamide (hazard ratio [HR] 0·62 [95% CI 0·48–0·81]; two-sided p=0·0005); median overall survival 45·1 months (95% CI 35·4–not reached) in the talazoparib group versus 31·1 months (27·3–35·4) in the control group. In the subgroup of patients with BRCA1/2 alterations (n=155 [39%]), median overall survival was not reached for talazoparib plus enzalutamide versus 28·5 months for enzalutamide (HR 0·50 [95% CI 0·32–0·78]; p=0·0017); 4-year overall survival rates were 53% in the talazoparib group versus 23% in the control group. In patients without BRCA1/2 alterations (n=244 [61%]), median overall survival was 42·4 months for talazoparib plus enzalutamide versus 32·6 months for enzalutamide (HR 0·73 [95% CI 0·52–1·02]; p=0·066). Updated rPFS favoured talazoparib plus enzalutamide versus enzalutamide (HR 0·47 [95% CI 0·36–0·61]; p<0·0001; median rPFS 30·7 vs 12·3 months). No new safety signals were identified; most common adverse events of grade 3 or higher with talazoparib plus enzalutamide were anaemia (86 [43%] patients) and neutropenia (39 [20%] patients). Talazoparib plus enzalutamide resulted in statistically significant and clinically meaningful improvement in survival versus enzalutamide plus placebo, further supporting this combination as a standard of care in HRR-deficient metastatic castration-resistant prostate cancer. Pfizer.

In the phase 3 TALAPRO-2 trial, talazoparib plus enzalutamide significantly improved radiographic progression-free survival compared with placebo plus enzalutamide in men with metastatic castration-resistant prostate cancer harbouring alterations in genes involved in homologous recombination repair (HRR). We aimed to assess patient-reported outcomes in patients with HRR-deficient metastatic castration-resistant prostate cancer in TALAPRO-2. TALAPRO-2 is a randomised, double-blind, placebo-controlled, phase 3 trial conducted at 223 hospitals, cancer centres, and medical centres in 26 countries worldwide. Eligible participants were male patients aged 18 years or older (≥20 years in Japan) who were receiving ongoing androgen deprivation therapy, had asymptomatic or mildly symptomatic metastatic castration-resistant prostate cancer, an Eastern Cooperative Oncology Group performance status of 0 or 1, and had not received previous life-prolonging systemic therapy for castration-resistant prostate cancer or metastatic castration-resistant prostate cancer. Patients with HRR gene alterations were randomly assigned (1:1) using a centralised interactive web response system and a permuted block size of 4 to oral talazoparib 0·5 mg once daily or placebo, plus oral enzalutamide 160 mg once daily, stratified by previous second-generation androgen receptor pathway inhibitor (abiraterone or orteronel) or docetaxel (yes vs no) in the castration-sensitive setting. The sponsor, patients, and investigators were masked to allocation of talazoparib or placebo; enzalutamide was open-label. The primary endpoint was radiographic progression-free survival by blinded independent central review and has been reported previously. Patient-reported outcomes were assessed as secondary outcomes in the patient-reported outcomes population, which comprised patients from the intention-to-treat population with a baseline patient-reported outcome assessment and at least one post-baseline patient-reported outcome assessment. Patient-reported outcomes included time to definitive deterioration in global health status/quality of life (GHS/QoL) per European Organisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) and prostate cancer-specific urinary symptoms per EORTC Quality of Life Questionnaire-Prostate (QLQ-PR25), and time to deterioration in pain symptoms per Brief Pain Inventory-Short Form (BPI-SF). Mean change from baseline in GHS/QoL, overall cancer and prostate cancer-specific functioning and symptoms (per EORTC QLQ-C30 and QLQ-PR25), in pain symptoms per BPI-SF, and in general health status per EQ-5D-5L were also patient-reported secondary outcomes. This study is registered with ClinicalTrials.gov, NCT03395197, and is ongoing. Between Dec 18, 2018, and Jan 20, 2022, 399 patients with HRR-deficient metastatic castration-resistant prostate cancer were enrolled and randomly assigned, of whom 197 assigned to talazoparib plus enzalutamide and 197 assigned to placebo plus enzalutamide were included in the patient-reported outcome population. Median follow-up was 22·2 months (IQR 13·8–27·7) in the talazoparib plus enzalutamide group and 20·2 months (13·5–26·6) for the placebo plus enzalutamide group. Median time to definitive deterioration of GHS/QoL was longer in the talazoparib plus enzalutamide group (27·1 months [95% CI 21·2–non-estimable]) than in the placebo plus enzalutamide group (19·3 months [16·6–23·0]; hazard ratio [HR] 0·69 [95% CI 0·49–0·97]; two-sided p=0·032). Median time to definitive deterioration in urinary symptoms was also longer in the talazoparib plus enzalutamide group (non-estimable [95% CI 32·2–non-estimable]) than in the placebo plus enzalutamide group (30·2 months [24·6–non-estimable; HR 0·56 [0·34–0·93]; two-sided p=0·022). Median time to deterioration in pain symptoms was non-estimable for both treatment groups (HR 0·58 [0·33–1·01]; two-sided p=0·051). Changes from baseline in worst pain in the past 24 h (BPI-SF, question three) and in general health status (EQ-5D-5L) also favoured talazoparib plus enzalutamide versus placebo plus enzalutamide, although the differences were not clinically meaningful. Between-group differences in mean changes from baseline in GHS/QoL, functioning, and symptoms per EORTC QLQ-C30 did not reach the clinically meaningful threshold of 10 or more points, although physical, emotional, and cognitive functioning and pain favoured talazoparib plus enzalutamide. Similarly, differences in mean changes from baseline for urinary and bowel symptoms per EORTC QLQ-PR25 favoured talazoparib plus enzalutamide, but were not clinically meaningful. The demonstrated delays in definitive deterioration in GHS/QoL, urinary symptoms, and other functioning and symptom scales with talazoparib plus enzalutamide compared with placebo plus enzalutamide in patients with HRR-deficient metastatic castration-resistant prostate cancer provide insight that might inform clinical decisions for these patients. Pfizer.

The tumour suppressor breast cancer type 1 susceptibility protein (BRCA1) promotes DNA double-strand break (DSB) repair by homologous recombination and protects DNA replication forks from attrition. BRCA1 partners with BRCA1-associated RING domain protein 1 (BARD1) and other tumour suppressor proteins to mediate the initial nucleolytic resection of DNA lesions and the recruitment and regulation of the recombinase RAD51. The discovery of the opposing functions of BRCA1 and the p53-binding protein 1 (53BP1)-associated complex in DNA resection sheds light on how BRCA1 influences the choice of homologous recombination over non-homologous end joining and potentially other mutagenic pathways of DSB repair. Understanding the functional crosstalk between BRCA1–BARD1 and its cofactors and antagonists will illuminate the molecular basis of cancers that arise from a deficiency or misregulation of chromosome damage repair and replication fork maintenance. Such knowledge will also be valuable for understanding acquired tumour resistance to poly(ADP-ribose) polymerase (PARP) inhibitors and other therapeutics and for the development of new treatments. In this Review, we discuss recent advances in elucidating the mechanisms by which BRCA1–BARD1 functions in DNA repair, replication fork maintenance and tumour suppression, and its therapeutic relevance.BRCA1 and its partner BARD1 support repair of double-strand breaks by homologous recombination and protect replication forks from damage. Recent studies have improved our understanding of the molecular mechanisms of these tumour-suppressive functions of BRCA1–BARD1 and how they are subverted in therapy-resistant cancers.

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

The efficiency of homology-directed genome editing with CRISPR-Cas9 is boosted through improved design of donor DNA. Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower 1 , 2 . Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3′ end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer 1 , 2 . Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with 39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.1% of tumour samples contain ecDNA. We reveal a pattern highly indicative of tissue-context-based selection for ecDNAs, linking their genomic content to their tissue of origin. We show that not only is ecDNA a mechanism for amplification of driver oncogenes, but it also a mechanism that frequently amplifies immunomodulatory and inflammatory genes, such as those that modulate lymphocyte-mediated immunity and immune effector processes. Moreover, ecDNAs carrying immunomodulatory genes are associated with reduced tumour T cell infiltration. We identify ecDNAs bearing only enhancers, promoters and lncRNA elements, suggesting the combinatorial power of interactions between ecDNAs in trans . We also identify intrinsic and environmental mutational processes linked to ecDNA, including those linked to its formation, such as tobacco exposure, and progression, such as homologous recombination repair deficiency. Clinically, ecDNA detection was associated with tumour stage, more prevalent after targeted therapy and cytotoxic treatments, and associated with metastases and shorter overall survival. These results shed light on why ecDNA is a substantial clinical problem that can cooperatively drive tumour growth signals, alter transcriptional landscapes and suppress the immune system. A study examines the diversity of extrachromosomal DNA elements in cancer, and provides details on the frequency and origin of extrachromosomal DNA and its role in the development of different types of cancer.

CYREN is a direct inhibitor of classical non-homologous end joining that promotes error-free repair by homologous recombination during the S and G2 phases of the cell cycle. Ku succumbs to a CYREN call A broken DNA molecule can be repaired in two distinct ways: homologous recombination, or classical non-homologous end joining (cNHEJ). Both processes function in the S and G2 phases of the cell cycle, but why cNHEJ is not always used is unknown. Jan Karlseder and colleagues have found that cNHEJ is suppressed in S and G2 by CYREN (cell cycle regulator of NHEJ), which binds to the Ku complex to inhibit the initial steps of end joining. The absence of CYREN results in genomic instability. CYREN thus biases repair in S and G2, when sister chromatids are available, to a recombination process that, unlike cNHEJ, is error-free. Classical non-homologous end joining 1 (cNHEJ) and homologous recombination 2 compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1 , a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes. Germline BRCA1 loss-of-function variants are associated with predisposition to early-onset breast and ovarian cancer; here the authors use CRISPR/Cas9 genome editing to functionally assess thousands of BRCA1 variants in order to facilitate the clinical interpretation of these variants.

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. Whole-genome sequencing of tumours from 560 breast cancer cases provides a comprehensive genome-wide view of recurrent somatic mutations and mutation frequencies across both protein coding and non-coding regions; several mutational signatures in these cancer genomes are associated with BRCA1 or BRCA2 function and defective homologous-recombination-based DNA repair. Mutational signatures of breast cancers This study reports whole-genome sequencing of tumours and normal tissue from 560 breast cancer cases, providing a comprehensive genome-wide view of recurrent somatic mutations and mutation frequencies across both protein coding and non-coding regions. The authors analyse mutational signatures in these cancer genomes, including a new investigation of rearrangement mutational processes, and find several that are associated with BRCA1 or BRCA2 function and defective homologous-recombination-based DNA repair. They also find mutational signatures showing distinct DNA replication strand biases.